SGDEPARTMENT OF HEALTH AND HUMAN SERVICES

 

FOOD AND DRUG ADMINISTRATION

 

CENTER FOR FOOD SAFETY AND APPLIED NUTRITION

 

 

 

 

 

 

 

 

 

CONTAMINANTS AND NATURAL TOXICANTS SUBCOMMITTEE

 

OF THE FOOD ADVISORY COMMITTEE

 

 

 

VOLUME I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tuesday, March 18, 2003

 

8:00 a.m.

 

 

 

 

 

 

 

4700 River Road

Riverdale, Maryland

 

PARTICIPANTS

 

Francis Fredrick Busta, Ph.D. Co-Chair

James E. Heubi. M.D. Co-Chair

 

MEMBERS

 

Alex D.W. Acholonu, Ph.D.

Lawrence J. Fischer, Ph.D.

Marion H. Fuller, D.V.M.

Lawrence N. Kuzminski, Ph.D.

Ken Lee, Ph.D.

 

TEMPORARY VOTING MEMBERS

 

James Anderson, Ph.D.

Robert D. Baker, M.D., Ph.D.

Larry R. Beuchat, Ph.D.

Henry M. Blumberg, M.D., Ph.D.

Margaret E. Briley, Ph.D., R.D.,L.D..

Laurie J. Moyer-Mileur, Ph.D., R.D., C.D.

Marguerite A. Neill, M.D.

Virginia A. Stallings, M.D.

Phillip Tarr, M.D.

Patti Thureen, M.D.

 

 

NON-VOTING INDUSTRY REPRESENTATIVE

 

     Toby L. Simon, M.D.

 

TEMPORARY VOTING MEMBERS

 

     R. Bruce Tompkin, Ph.D.

C O N T E N T S

PAGE

 

Welcome, Introduction and Charges                     5

     Christine J. Taylor, FDA

 

Administrative Issues                               16

     Jeanne Latham, FDA

 

Remarks by Chairpersons                             21

 

Presentations by Guest Speakers

 

Current Marketing and Use of Powdered

  Infant Formula             27

     Sue Ann Anderson, FDA

 

Tennessee Investigation        48

     Matthew Kuehnert, CDC [via speakerphone]

 

Other Relevant Investigations          88

     Karl Klontz, FDA

 

Clinical Consequences of E. Sakazakii Infections      121

     John Alexander, FDA

 

General Microbiology - Ecology, Pathogenicity,

  Subtyping, Etc.     171

     Maria Nazarowec-White, Canada

 

Microbial Detection--Clinical and Food        197

     Don Burr, FDA

 

Resistance--Thermal and Other      221

     Robert Buchanan, FDA

 

FDA Field Survey of Powdered Formula

  Manufacturing      246

     Don Zink, FDA

 

Public Comments/Industry Speakers

     Jon A. Vanderhoof     320

     Les Smoot     329

     Russell Merritt     347

     Jatinder Bhatia, M.D.     351

 

Preliminary Subcommittee Discussion on Clinical Presentations                    388


P R O C E E D I N G S

Welcome, Introduction and Charges

     DR. BUSTA: Good morning.  We're here to convene the Contaminants and Natural Toxicants Subcommittee Meeting on Enterobacter sakazakii Contamination in Powdered Infant Formula.

     The first presentation this morning will be by Christine Taylor, who is the director of this group.  And without going any farther, I will just turn this over directly to Christine from the start.

     DR. TAYLOR:  Welcome and good morning. I am Christine Taylor, Director of the Office of Nutritional Products, Labeling and Dietary Supplements. Thank you for all for agreeing to participate in the advisory subcommittee meeting. You are an illustrious group of experts and we are looking forward to your discussions today and tomorrow.

     My job here today is to set the stage for the more in-depth presentations and white papers you will soon hear and to set the stage for the regulatory context that leads us to you ask you questions about Enterobacter sakazakii and powdered infant formula. As I am sure most of you know, FDA's mission is ensure a safe and adequate food supply. And recent findings have led us to ask questions about E. sak and powdered infant formula.

     I think it's important to recognize that within the Center we view not only the regulatory issue of powdered infant formula, but also it raises some interesting microbial questions.  So, in pulling together this effort, many factors and groups within the Center were involved.

     I think the first point I want to make is a kind of tried and true one, which is that obviously one of the reasons we're here is that FDA has the mission of ensuring a safe and adequate food supply, and certainly infant formula falls into that.  And the short summary--the one sentence reason for being here--is that recent findings have raised questions about Enterobacter sakazakii encountered in infant formula.

     [Slide.]

     Clearly, in addressing this issue, FDA needed extensive scientific input.  It needed to be of the microbiological kind, the physiological and clinical kinds, particularly in the world of infants, and the manufacturing and processing of powdered infant formula.   This required a diverse set of expertise, and the subcommittee that you see sitting amongst yourselves, before yourselves and in front of yourselves really include quite a wide range of expertise, including temporary voting members to round out expertise.  And Ms. Latham later on will clarify various members' roles.

     Could I have the next slide?

     [Slide.]

     As I've mentioned before, our focus on infant formula.  Infant formula is a  product regulated by the Food and Drug Administration.   And currently, in-place GMPs do not specify microbiological testing for E. sak. Infant formula, as probably many if not all of you know, comes in two forms:  powdered and liquid, and the powdered, which is the subject of this advisory committee meeting, in terms of dollar sales, is about half of the infant formula sold.

     Just a little bit of regulatory context: there is an Infant Formula Act.  It's part of the Food, Drug and Cosmetic Act.  And this particular set of regulatory specifications does indicate that manufacturers must retain records pertaining to the microbiological quality and purity of their product.  I do want to point out in the next slide, however, that Infant formula good manufacturing practices, while proposed, have not yet been finalized.  And, more specifically, there are no current GMP regulations for powdered products.  Many of you may know that liquid formula comes under the Novak and Tennessee provisions, but powdered formula have no such provisions.

     FDA has proposed GMPs to include practices to minimize opportunity for microbial contamination.

     Next slide.

     [Slide.]

     You'll note that despite listing a number of microbial issues, the agency did not specify, nor has it specified, provisions for Enterobacter sakazakii

     In short, what I'll try to do in the next few moments is tell the story of Enterobacter sakazakii in powdered infant formula, and then briefly go over the charges to the committee; the questions that FDA is asking.

     At that point, I will turn the meeting over to your two co-chairs.  The Committee is chaired by two very prestigious individuals, and they will do a presentation of background information--a series of white papers--and then proceed to an open comment period, followed by a discussion set of recommendations.

     The staring point for the Enterobacter sakazakii was in April of 2001, an infant in Tennessee died of meningitis with E. sak. It was a premature infant in a neonatal intensive care unit or NICU. The Centers for Disease Control and Prevention investigated the situation and determined a link between the infant's death and E. sak in the powdered formula fed to the infant in the NICU.  Because infant formula manufacturing is under the purview of  FDA, the CDC notified FDA.  In response to the CDC input, FDA undertook several activities. The Infant formula--what was left of it on the market--was recalled.  And, in addition,  FDA conducted outreach to health care professionals. The agency began to examine products and ingredients used to make this formula, and manufacturing, for the presence of E. sak, and we . conducted some related research.

     In the area of outreach, we obviously focused on health care professionals in hospitalized settings, because the incident at hand did occur in the NICU.  We drafted and disseminated very widely a letter to health professionals.  We created and posted an FDA "talk paper" on our website.  And then in addition, CDC itself published its findings in reported the Enterobacter sakazakii in the MMWR.

     In our letter to health professionals we emphasized certain things.   Powdered infant formula was a potential source of E. sak infections in infants, and we pointed out that the risk increased with prematurity and any underlying medical conditions.  We  indicated that low levels of E. sak can lead to infection and we also highlighted the CDC findings in Tennessee regarding the fatal meningitis case.   In addition, we recommended that powdered infant formulas not be used in the NICU, unless there was no alternative available.  Oftentimes powdered formulas are specialized formulas, and some physicians have indicated alternatives wouldn't be available  And we also provided suggestions for reducing infection risks.  These included the use of boiling water and certain feeding and related practices in hospitals.

     We did, later on, revise  this letter to health professionals, and we focused largely on the question of boiling water preparation because possible problems associated with the loss of heat-sensitive nutrients, the physical changes to the formula, and perhaps even inadequate E. sak eradication, as well as  injury to persons preparing the formula.  And this was done in October of this year.

     A second part, in addition in addition to our letter and outreach to help health care professionals, involved FDA's actually going into plants and testing infant formula, as well as infant formula ingredients..  Our purpose then was to look for E. sak in powdered infant formula products and the raw ingredients.  We sampled  all domestic powdered infant formula facilities as well as  all contract and manufacturing facilities. And, in addition, for transparency and clarity, we also posted the analytical method we were using on our website.

     FDA, as a result of this sampling, did find some positive low levels in both finished product and in the raw ingredients.  It's been in positive finished products that we have identified that have already been marketed, and there were two such products that were recalled voluntarily by the manufacturers, and no known cases of E. sak infection resulted from the consumption of these products is known.

     In addition, we did conduct some related research.  As I've mentioned, we worked on the method for protecting from Enterobacter sakazakii in powdered formula.  We also did various studies on heat [inaudible due to technical difficulty].  And also we conducted some studies to show effects of water temperature of nutrient levels in infant formula, which appear

at this point to not have been finalized.

     In addition, there were related activities in response to the FDA outreach.  Pediatric hospitals and laboratories also [inaudible due to technical difficulty].

     The industry itself conducted analyses and investigations, and as probably many of you know they worked hard not to distribute products that were found possibly to contain this particular strain.

     I'm here largely to give you the story of why we're here, and what you'll find out after the opening of this meeting is the specifics of much of what I've discussed, ranging from the specifics on the Tennessee case, to the FDA efforts in product sampling.  And so, with your indulgence, I'll simply give an overview for now to you that later on this morning there will be a variety of white-papers presented, designed to give you the specifics on available information once we get the final documents.

     We'll begin with the current marketing and release of powdered infant formula, fun through the overview of the Tennessee investigation and other relevant investigations, in Enterobacter sakazakii.  We'll provide for you information on the clinical consequences of the E. sak investigation, as well as some of the general microbiology and detection and [inaudible due to technical difficulty], and then end with the findings from our previous survey in the infant formula industry.

     Given all of that, there are two basic charges to this particular subcommittee.  The first charge is a request to characterize the infants at risk, relative to Enterobacter sakazakii in powdered infant formula; if there is a risk, to identify the populations of infants at risk, with due consideration to whatever factors are relevant to morbidity, other related areas, such as age, on immune status.

     The second charge to the subcommittee begins: "If there is a meaningful risk, how can this risk be addressed?    What intervention strategy can be used in infant formula processing plans?  Are there other intervention strategies?  We've taken a look at the possibility of labeling and directions for use.  Is it possible, based on available information, to specify allowable lower levels of microbial detection of E. sak in infant formula, and do allowable levels vary by risk characteristics of the infant.  And then, finally, what are the critical knowledge gaps and research priorities.

     Those are basically the two charges to the subcommittee.

     There is a final point relative to our efforts to finalize the infant formula final rule on GMPs.  The agency will be reopening the comment period for this final rule.  Any issues that may come from the discussions today and tomorrow that would be relevant to the final rule can be commented upon by interested parties at the time of that comment period.

     Again, I want to mention that I'm so pleased and very proud of the fact that the agency was able to convince the important people here at the table to come and discuss the details of this very interesting issue, and also emphasize that I am telling an initial story, and many of you might have questions about, in terms of either the outbreak in Tennessee or the FDA's activities, will be both addressed and answered by others following me.

     And my role--I'm setting the regulatory context about FDA's responsibility in the area of Infant formula, and will end right there.

     Thank you all again.  I'm happy to take questions.

     DR. BUSTA: Any questions or clarification from the committee?

     [No response.]

     DR. TAYLOR: I also, sitting at the table here, will answer questions that may come up.  Thank you.

     DR. BUSTA: The next item on the agenda is the administrative issues, and Jeanne Latham will cover those.

Administrative Issues

     MS. LATHAM: Good morning.  Is this on?  I'm Jeanne Latham.  I will be replacing Dr. Henry Kim as the Executive Secretary of the FDA Contaminants and Natural Toxicants Subcommittee of the Food Advisory Committee for the purposes of this meeting.

     I want to welcome everybody, and I'd like to read the conflict of interest statement for the record.

     By the authority granted under the Food Advisory Committee Charter of July 202, the following individuals have been appointed as temporary voting members by Joseph A. Leavitt, Director, Center for Food Safety and Applied Nutrition: James Anderson, Robert Baker, Larry Beuchat, Henry Blumberg, Margaret Briley, James Heubi, Laurie Moyer-Mileur, Marguerite Neill, Virginia Stallings, and Patti Thureen.  Dr. Phillip Tarr is a special government employee to FDA Center for Drug Evaluation and Research, and is serving as a temporary voting member under the authority of Lender Ari Skledeny, Associate Commissioner for External Relations.

     The issues to be discussed at this meeting are issues of broad applicability.  Unlike issues in which a particular sponsor's product is discussed, the matters at issue do not have a unique impact on any particular product or manufacturer, but may have widespread implications with respect to all infant formulas and their manufacturers.  To determine if any conflicts of interest exist, the committee participants have been screened for interest in companies that make infant formula.  As a result of this review, in accordance with Title 19 of the U.S. Code, Section 208(b)(3), Dr. Virginia Stallings has been granted a particular matter of general applicability waiver that permits her to participate fully in the matters at issue.  A copy of the waiver statement may be obtained by submitting a written request to the agency's Freedom of Information Office, Room 12A30 of the Parklawn Building.

     We would also like to note for the record that Dr. R. Bruce Tompkin is participating at this meeting as the acting Industry Representative and a non-voting member of the committee.  In the event that the discussions involve any other issues not already on the agenda for which FDA participants have a financial interest, the participant's involvement and their exclusion will be noted for the record.

     With respect to all other participants, we ask in the interest of fairness that they address any current or previous financial involvement with any firm which makes infant formula.

     In addition, I'd like to take a few moments to very briefly refresh everyone's memory about Advisory Committee operations, particularly the roles and responsibilities of the Food Advisory Committee members and FDA staff.  The committee members have been provided with a copy of a Committee Member Guide to FDA Advisory Committees, and there are copies of the member guide available at the registration desk for anyone who may be interested.  The Member Guide is in need of updating, but by and large it provides a good operational overview.

     As a scientific regulatory agency, FDA needs access to highly qualified expert external advisors who can provide scientific and technical advise.  Thus, FDA uses advisory committees to supplement the agency's internal expertise, and help the agency staff stay current with state-of-the-art technology.  Committee members participate in an advisory capacity, and final decisions are ultimately made by agency officials.

     With respect to the committee members, the chair of the committee--or, for purposes of this committee, the co-chairs--preside at and conduct the meeting, and they may ask the FDA staff for clarification at any time during the meeting.  Each committee member contributes their unique scientific and technical background, education and experience to the committee process.  The standing committee members--and, in this case, that is the Contaminants and Natural Toxicants Subcommittee--generally are voting members, including a consumer representative who is voting, and an industry representative who is non-voting, as specified in the charter.

     All voting members of advisory committees are appointed by the Commissioner, and must be cleared as special government employees under FDA's conflict of interest regulations.  Other advisory committee participants may include consultants, experts and guest speakers.  At this meeting, the consultants are temporary voting members with expertise infant formula and pediatrics, who will participate in the discussions of the committee.  The guest speakers will present pertinent information to the committee, and will not participate in committee discussions or vote.

     Additionally, the advisory committee process encourages public interaction with the agency in arriving at decisions.  Not only is there a consumer representative--in this case, a voting member--appointed to serve on the committee, but the public is invited to appear before the committee during the open public comment period.

     FDA participates in a listening mode, and responds to questions or a need for clarification by the committee as needed through the chairs.

     Thank you very much.

Remarks by Chairpersons

     DR. HEUBI: Good morning.

     We would like to have all of the committee members identify themselves and what their skill set is that they bring to the table that helps them be uniquely qualified to come today.

     I'm Jim Heubi.  I'm a pediatric gastrologist.  I run a clinical research center at the Children's Hospital, and I participate and actively pursue nutrition-related research.

     I think if we'll just go around the table, from Dr. Tompkin.

     DR. TOMPKIN: I'm Bruce Tompkin, and I'm retired now from ConAgra Foods, where I was Vice President of Product Safety.  I am a food-market biologist, and basically that's all I've done throughout the 39 years' experience I've had in the food industry.

     DR. BEUCHAT: I'm Larry Beuchat.  I'm from the Center for Food Safety, University of Georgia.  My area of research is in food-borne pathogens-mostly.  I do also work with yeasts and molds, spoilage problems.  I've worked with a number of pathogens.  I've worked, on occasion, with infant foods, but not with E. sakazakii--rather bacillus serius and, and never hemorrhagic before.

     DR. TARR: I'm Phil Tarr.  I'm from Washington University in St. Louis. I'm a pediatric gastroenterologist, and also have appointment in microbiology.  My interest is in food-borne infections, particularly bacterial infections.

     DR. ACHOLONU: My name is Alex Acholonu.  I'm from Alcorn State University in Mississippi.  I'm a professor of biology.  I'm an epidemiologist in the area of microbiology and parasitology.

     DR. THUREEN: My name is Patti Thureen, and I'm a neonatologist at the University of Colorado, and my particular research interests are in fetal and neonatal nutrition and metabolism.

     DR. FULLER: I'm Marion Fuller.  I'm with the Florida Department of Agriculture and Consumer Services.  I run the Division of Food Safety.  I'm a veterinarian by training, with certification in general toxicology.

     DR. LEE: I am Ken Lee.  I chair the Food Science Department at Ohio State University.  My work area is in the process-induced changes in nutrients.

     DR. MOYER-MILEUR: I'm Laurie Moyer-Mileur.  I'm from the University of Utah.  I'm a research associate professor in pediatrics and a registered dietician with over 20 years of clinical experience in the NICU, monitoring babies with lots of feeding intolerance.

     DR. NEILL: Good morning.  I'm Dr. Peggy Neill.  I'm an adult infectious disease specialist, currently at the Brown University Medical School in Providence.  I have background in public health epidemiology and food safety, with a particular interest in E. coli 0157 infections.

     DR. BAKER: I'm Robert Baker.  I'm professor of pediatrics at University of Buffalo.  I'm a pediatric gastroenterologist at Children's Hospital of Buffalo.  My particular research interest is in gut barrier function.

     DR. BLUMBERG: Good morning.  My name is Henry Blumberg.  I'm in the Division of Infections Diseases at Emory University in Atlanta.  I'm also the hospital epidemiologist at Grady Memorial Hospital, and my interests include hospital and molecular epidemiology and clinical research training.

     DR. BRILEY: I'm Margaret Briley, profession of nutritional sciences at the University of Texas at Austin, and my research is with children and pre-school and nutrition issues.

     DR. FISCHER: I'm Larry Fischer, from Michigan State University.  I direct the Institute for Environmental Toxicology there.  I'm a pharmacologist and a toxicologist.  My research often deals with age-related changes in susceptibility to chemical toxins.

     DR. STALLINGS: I'm Virginia Stallings.  I'm from Children's Hospital in Philadelphia, and professor of pediatrics, and at times have run a nutrition support service--a large part of that service is in neonatal intensive care units.

     Most of my research is on children with chronic disease and in nutrition.

     DR. KUZMINSKI: My name is Larry Kuzminski.  I'm retired from the food processing industry.  I was with Ocean Spray Cranberries, where I was a vice president of research and development and operations.  And prior to that I was with the Kellogg Company, where I was vice president of science and quality in Canada, and director of food and research in the United States.

     My interests are in food safety, and in the application of HACCP principles in manufacturing processes.

     DR. BUSTA: And I'm Frank Busta, and I'm a professor emeritus a the University of Minnesota, food microbiologist and have worked my entire life, mostly, with the bad guys.  And I'm pleased to welcome all of you.  It looks like a tremendous committee, and the entire group is covered.

     DR. HEUBI: Jeanne tells me that I should announce that Dr. Anderson is unable to attend because of family-related problem today.  And I do want to remind you to use the microphones whenever you speak because, obviously, the audience can't hear unless we do, plus we can't hear ourselves.

     And I want to refer to your package that includes materials in this blue folder--that includes the handout slides from the presenters.

     And we're going to make every effort to stay on time today, so that we can give full time and deliberations that they're asking us to make today.

     Yes?

     DR. STALLINGS: Who is our consumer representative?

     MS. LATHAM: Dr. Margaret Briley.

     DR. STALLINGS: Okay.  Good.  I just wanted to know for sure.  Thank you.

PRESENTATIONS BY GUEST SPEAKERS

     DR. BUSTA: So, our first presentation today is going to be on current marketing and use of powdered infant formulas, by Sue Ann Anderson.

     The guidelines state that we're to have 20-minute presentations, with 10 minutes for questions from the committee.

     Current Marketing and Use of Powdered

Infant Formula

     DR. ANDERSON: Good morning.  This morning I'm going to talk about how powdered infant formula is currently marketed and used in the United States.

     If I could have the first slide, please?  Next slide?  There should be one before that.  Well, in any case then, my talk will focus on three topics: the types and uses of formulas currently marketed in the United States; preparation of powdered formula for feeding; and feedings to infants in health-care settings.

     My remarks this morning will be brief and very general in nature.  Greater detail can be found in the White Paper that is part of the briefing materials for the Committee for this meeting.

     In order to identify what the products are and who the consumers are, I should begin by defining "infant formula" and "infants."  Infant formulas are products intended for use solely as a food for infants.  They are simulate human milk and are suitable as a complete or partial substitute for human milk.      Next slide, please.

     [Slide]

     Infants are defined as persons not more than 12 months of age.  This slide set is not my slide set.  We'll go ahead and work with it, though.

     With those definitions in mind, I would like to provide a little information on the introduction of formula for infant feeding.  Powdered infant formulas were introduced well over a hundred years ago, in the late 1800s.  Liquid forms were not introduced until the mid 20th century, with liquid concentrates coming onto the market in the early 1950s, and ready-to-feed products coming onto the market about 1960.

     It should be noted that powdered infant formula are not sterile products, however the liquid products are.  After the liquid forms were introduced, the proportion of infant formula sold in powdered form decreased.  The lowest proportion of sales of powdered infant formula occurred from about 1970 to 1980.  Around 1980, technical advances in the processing of powdered infant formula resulted in products that were more readily dispersed in

water, making it easier to reconstitute the powders for feeding. Since 1980, the proportion of powdered infant formula sales has risen and in 1999, sales of powdered infant formula accounted for about half of the dollar sales of infant formula products in the United States. This estimate was based on data collected from supermarket, drug store, and mass merchandiser scanners by

Information Resources, Inc.  I should also note that the proportion of infant formulas sold in powdered form in the United States differs from that of other countries. In most other countries, almost all infant formula is sold in the powdered form.

I should also note at this point that the proportion of infant formula sold in powdered form in the United States is quite different from that in other countries.  In most other countries, almost all infant formula is sold in powdered form.

     We'll see what the next slide is.

     [Slide]

     There is also a difference in the cost of feeding of the infant formulas, with the powdered infant formula being the least expensive, and concentrate intermediate, and ready-to-feed the most expensive product.

     Most infant formulas are formulated to meet the nutritional needs of infants with generally good health status.

     And, if you will bear with us for just a couple of minutes, we're going to see if we can find the right slides here.

     [Pause.]

     MS. LATHAM: This is Jeanne Latham.  I just want to apologize for our technical difficulties.  This is what happens when you try to perfect a presentation and you provide many, many different versions of it.  So, hopefully, we're going to find the correct one.

     [Pause.]

     DR. ANDERSON: Okay,.why don't you advance and I'll tell you where we are.  One back.

     Most infant formulas are formulated to meet the nutritional needs of infants with generally good health status. Included in this group are term and preterm infants, with term infants being  those born at or later than 37 weeks gestational age, and preterms born before 37 weeks gestational age. These products are available in both powder and liquid form.

     Could I have the next slide, please?

     [Slide]

      Other infant formulas are designed to meet the needs of infants with special medical conditions such as genetic disorders of metabolism (for example, PKU) or other system disorders, for example gastrointestinal diseases. With the exception of protein hydrolysate formulas, this category is available only in powdered form.

     Could I have the next slide, please?

     [Slide]

     Okay.  Because the infant population is at risk for nutrition insult and infection, proper preparation, storage, delivery, and disposal of their feedings are critical for prevention of food-borne infections.  In order to provide consumers with accurate information for preparation and use of infant formulas, there are specific Federal regulations for infant formula labels. Preparation and use instructions required on infant formula labels include 1) product storage, 2) sterilization of water, bottle, and nipples, when necessary, and 3) dilution for powder and liquid concentrates showing major steps for preparation, including a required pictogram for illustration.

     Could I have the next slide?

     [Slide]

     This next slide shows the pictogram from the Code of Federal Regulations, depicting instructions for formula dilution. The wording on the slide is difficult to read, but it does say "Sterilization is recommended. Your physician will decide if it is not required."

     Next slide.

     [Slide]

     Other information is also required on infant formula labels, and it's required  to caution against improper preparation or use. Cautionary statements such as the following are required:"The health of your infant depends on carefully following the directions for preparation and use;" "Use as directed by a physician."  And, also, a "Use by" date required on the infant formula.  The "use by" date  is a date selected by the manufacturer on the basis of tests or other information showing that the formula, until that date, under conditions of handling, storage, preparation, and use prescribed by label directions, will contain not less than the quantity of each nutrient as specified on the label and will otherwise be of acceptable quality.

     Next slide.

     [Slide]

     Each manufacturer designs their product labels to conform to these regulations and the wording and pictograms on labels will vary for each manufacturer. For example, directors for preparation of water for reconstituting infant formulas generally fall into two categories.  On this slide, you see the first category, which  includes some direction to boil the water

and to cool it before adding to the infant formula.

     Next slide.

     [Slide]

     This may be hard to see, too, but this is a sample from an infant formula label where it does say to boil the water.  It says to boil the water for a minute, then cool;  pour the cooled water into a bottle;  add the powder and cap bottle, shake well and feed immediately.

     The second category ---next slide.

     [Slide]

     The second category of instructions for water preparation includes an instruction to consult with the  doctor about the need to boil water.

     Next slide.

     [Slide]

     And a sample pictogram from a label with this sort of information says, "Your baby's health depends on carefully following these easy directions.  Ask your baby's doctor if you need to boil sterilized water for formula and bottle preparations, pour the desired amount of warm water into a bottle, add the powder, cap, shake very well and it does include a statement "After feeding, throw away any formula remaining in the bottle."

     Next slide.

     [Slide]

     Instructions for feeding and storage direct the consumer to feed immediately or cover,   refrigerate, and use, either  within 48 hours--next slide--

     [Slide]

     or, on some formulas, in 24 hours.

      Some labels--next slide--

     [Slide]

     --also include instructions for disposing of the unused formula, such as "Discard unused formula after feeding," or "Throw away prepared formula left in the feeding bottle, or cap within one hour after feeding begins.". Examples of such statements are shown on the next slide.

     [Slide]

     Use of commercially sterile infant formula products is not always feasible in health care settings and, in some situations, formulas must be prepared from nonsterile, powdered products for hospitalized infants. The American Dietetics Association (ADA) has developed guidelines for preparation of infant formulas in health care facilities. These guidelines include detailed recommendations intended to decrease the risk for microbial contamination and growth during infant formula preparation, storage, and use in health care facilities.  The guidelines have been updated recently, and are available on line at the URL shown on the screen.

     I will not go into detail about the recommendations for preparation of formulas, but I should say that the ADA guidelines for delivery and disposal of infant formulas for nipple-fed and tube-fed infants ---there are guidelines for these groups in health care settings.

     Next slide.

     [Slide]

     For nipple-fed infants, the instructions are given to feed within 4 hours of preparation, cover and refrigerated for up to 24 hours.  If the formula is warmed, it should be warmed quickly, in less than 15 minutes; and to discard any product remaining in the bottle one hour after feeding begins.

     Next slide.

     [Slide]

     For tube-fed infants given intermittent feedings, the formula should be, again, fed within four hours of preparation, covered and refrigerated for up to 24 hours if not used immediately.  For intermittent feeding, it should be packaged in amounts for one feeding or for a 4-hour period and for continuous feedings, it should be given in a way so that the hang time would not exceed four hours.

     This has been a brief and very general overview of the current marketing and use of infant formulas. What I would like for you to remember--if I can have the last slide--

     [Slide]

     --what I would like for you to remember as we proceed through today's agenda are the  following points: About half of the market share--the dollar sales in the United States, is for powdered products.  This is based on 1999 dollar sales.  Powders are not sterile products.  There is  a small percentage of infant formulas that are not available in commercially sterile, liquid  forms, and methods given for reconstitution and delivery are variable.

     Thank you.

     DR. BUSTA: Thank you.

     Are there any questions?

     Robert.

     DR. BAKER:   I'm Baker.  I have a clarification point.

     The labeling is required, is that right?  But the exact wording, and sometimes even the content of the label is decided by the manufacturers.

     DR. ANDERSON: There are certain requirements--there are certain elements that are required on the label, and there are certain statements that are required.

     Linda, if you would go back to--about six or seven slides?  Keep going.

     Okay.  This one.

     [Slide]

     The statements that are in quotation marks--the first two statements--those statements are required.

     If you go back one more slide.  One more.  One more.

     [Slide]

     Information related to these topics is required, but there is no wording that is specified or directed by FDA.  The manufacturers is given latitude in that regard.

     DR. HEUBI: I have a question--Jim Heubi.

     Do we know, as an example--first--I have a two-part question.  One, do the powdered formula labels include a statement that they're not sterile?  And then, secondly, do we have any knowledge about how informed neonatologists are, or providers for preterm infants are in terms of understanding the non-sterile nature of powdered formulas that might be used in that venue?

     DR. ANDERSON:   To answer your first question, it's not required that there be a statement on the label that says that the powders are not sterile products--commercially sterile products.  And I'm trying to think quickly--and I don't remember seeing a statement on a powdered product to that effect.

     With regard to knowledge of neonatologists about the nature of these products, I'm not aware of any surveys that have been done to collect that information.

     DR. HEUBI: Is it the general sense that they understand this, or not.  I guess that's a question that I have in the back of my mind right now.

     DR. ANDERSON: My inclination would be to say they have a better understanding of it now than they did before the E. sakazakii incidents occurred, but I don't really have a basis for saying how much they do or do not know.

     DR. HEUBI: Okay.

     DR. THUREEN: I polled our neonatologists at several hospitals in town, and one of about 25 had any clue how to prepare the infant formulas.  It's usually done by a milk lab, and they really--you know, it was totally out of their realm of knowledge.  It just wasn't anything they thought about.

     DR. BEUCHAT: A question concerning the tube feeding--

     DR. HEUBI: Please identify yourself.

     DR. BEUCHAT: Larry Beuchat.

     A question concerning tube feeding and perhaps also relates to some of the other recommendations: are there recommendations with regard to the tubes themselves, or any other vessels or containers into which the actual formula comes in contact before the baby consumes it?  The re-use, the hygiene and so on?

     DR. ANDERSON: Those would be products that would be packaged for delivery of the formula, and those would have to meet guidelines from one of our sister centers.  I'm trying to figure whether it would be drugs or devices--probably devices--that they would have specifications for delivery systems--for those types of products.

     DR. TARR: Is it possible for us to get a list of--

     DR. HEUBI: Please identify yourself.

     DR. TARR: Phil Tarr, from St. Louis.

     Is it possible for us to get a list of the formulas that are not available as liquids?

     DR. ANDERSON: As specific products, or as types?

     DR. TARR: Probably both.

     DR. ANDERSON: We could certainly do it as types, and I could see if we could get the specifics for you.

     DR. MOYER-MILEUR: Moyer-Mileur.

     In regard to delivery systems, as far as tubing for continuous drip feedings, while the products themselves may be sterile when they're initially used, if they are reused then they're no longer that.  And that would all be dependent on each individual NICU and their policies.  So it would be really hard to--for us to answer that or even to control that, I would think.

     Hopefully at a level-three--in a level-three nursery the delivery systems would not be re-used, and that would not be their standard of practice.  The worry would be if you had a level-two, or say even a level-one nursery where they're keeping babies who are 32, 33 weeks and feeding, and maybe not quite as knowledgeable about these products.

     DR. HEUBI: Heubi.

     Just as a point of clarification, do we have a general idea about how long NICUs utilize this equipment that is used for feeding?  Is it 24 hours, 48 hours, 72 hours?  Is there any sense about this?

     DR. ANDERSON: I'm not able to comment on that.  I think--I can say it varies among institutions, but further than that, I couldn't comment.

     DR. MOYER-MILEUR: You know, I can't either, because I think if you're dropping in a continuous drip-feeding tube, you leave it as long as possible.

     DR. HEUBI: I guess I was more thinking about the bag and the tubing that leads to the tube--

     DR. MOYER-MILEUR: They should be changed every four hours.

     DR. HEUBI: The tubing should be as well?

     DR. MOYER-MILEUR:  The connecting tube, not the actual--

     DR. STALLINGS: Stallings.

     I would echo what Laurie was saying.  I think in most level-three nurseries, the feeding system would be treated more like an IV feeding system, but with the--you know, when you change the set you change the whole set.

     For a nasogastric feeding, of course the tube in the infant would not be changed four times a day and, in general, it would be left in I think it's about three days before it would be changed electively.

     So, you really do, in the NICU, when you're talking about these products, you're generally going to be thinking about feeding tubs.  And then as the infants age and approach term, they'll go to nipple-feeding and more to the devices that we think of as usual infant feeding.

     But, you're right, we have to think about the plastic bag--the formula being mixed, storage and all of that, but then the plastic back, the feeding tube, the connection to the feeding tube that's in the infant.  So there are a number of different components.

     DR. HEUBI: Are there any additional questions?  Thank you.

     Oh, I'm sorry--Dr. Beuchat.

     DR. BEUCHAT: Beuchat.

     Just one question--for Virginia.  What is, then--what could be the length of time that the tube would be in the infant without removing it, and still feeding it?

     DR. STALLINGS: The nasogastric?  It probably varies per center, but in the stressed neonate, I think it's about three days that because of the stress of moving a tube in and out of the baby, that you wouldn't be changing it every 24-hours unless it occluded, or there was some other reason.

     I really can't--somehow three days sounds about right, but I can't remember exactly.  But every nursery should have a protocol about electively changing that.  But, again, as Laurie was saying, I think some of our concerns--there's so much movement now to keeping more stable babies in second--the different level nurseries that there's probably more nasogastric feeding that's being done in those settings than used to be done.  So there is a site issue and an education issue here.

     DR. HEUBI: Heubi.

     And you have to realize that some of these babies have naso-duodenal tubes placed and that's the context.  There's not a big move to replace them periodically.  So they might be in for longer periods of time.

     Any additional discussion or questions?

     [No response.]

     All right.  I think we have to move on to our multi-media presentation.   Dr. Kuehnert  is not available to present personally, but he's going to be available by telephone.  He's involved with the investigation for the current respiratory outbreak in Asia, and we're pleased to have him available to present today for us.  And as soon as we make our connection, we'll be ready to go.

Tennessee Investigation

     DR. KUEHNERT: [Via speakerphone]  Can you hear me okay?

     Thanks for inviting me to speak.  I'm sorry that I couldn't be there in person, but an emergent investigation precluded me coming.  But I appreciate the opportunity to present by phone.

     What I wanted to do today is discuss primarily the case in Tennessee in 2001 that resulted in Enterobacter sakazakii meningitis and death in an infant who was hospitalized.

     MS. LATHAM:  Dr. Kuehnert?

     DR. KUEHNERT: Yes.

     MS. LATHAM: This is Jeanne Latham.  You are obviously not able to hear the mikes at the table very well, and I just wanted to remind you that we will be recording the presentation, and we will have to try to figure out how we're going to ask you questions, because Dr. Heubi was trying to get your attention, and we weren't able to, but we will work that out.

     So, go ahead and do your presentation.  Thank you very much.

     DR. KUEHNERT: Okay.  Okay.

     And, to discuss the case, discuss the resulting MMWR and interim recommendations, and also offer some discussion concerning the implications and possible activities going forward.

     Could I have the next slide, please.

     [Slide]

     So this should say "Background."  Enterobacter sakazakii is a gram negative rod that was classified as a yellow-pigmented variant of E. cloacae, until it was designated as separate species in 1980.  And the reservoir is unknown, as far as the original source of the organism.

     Next slide, please.

     [Slide]

     Clinical characteristics.  This is a pathogenic organism with a particular affinity for the nervous system.  Complications are commonly quite serious, including necrotizing enterocolitis, sepsis, meningitis.  And in addition to meningitis, cerebral abscesses, cysts, infarctions, fairly extensive cerebral damage.  And outcome is poor.  In most of these infections, there is an impaired neurological outcome.  In fact, that's expected in the vast majority of cases that occur, and fatality rates has ranged, in the literature, between 40 and 80 percent.

     Next slide, please.

     [Slide.]

     Potential sources of the organism--as I said, the reservoir is unknown, but powdered infant formula has been associated in the past, before the case in 2001, with outbreaks of E. sakazakii infections in neonates.  The organism has bene traced to a number of steps in the powdered formula preparation process.  It's been found in freshly prepared or refrigerated powdered formula, in utensils and equipment that's used in formula preparation, such as blenders and other tools used to make formula, in the unreconstituted product, and also in unopened product.

     Powdered formula products, as I said, have been associated with these outbreaks, including meningitis, sepsis and necrotizing enterocolitis.  Other studies have looked at powdered formulas for contamination by bacteria and there was one particular study that looked at Enterobacteriaceae in general, and found contamination at low levels, including over half of products from a number of different countries, including the United States, and specifically 14 percent of powdered formula samples were contaminated with E. sakazakii, and most of you are probably familiar with this literature.  However, the concentrations of the organisms were quite low.

     Next slide, please.

     [Slide.]

     This is outline of presentation.  Let's skip over this.

     Moving on to case description--the next slide.

     So, on to the 2001 case.  A male patient was admitted to the neonatal intensive care unit in April 2001.  Gestational age was 33-1/2 weeks, delivered by C-section.  APGAR scores were 4 and 7 at one and five minutes, and the birth weight was 1,270 grams.

     Patient was started on enteric feeding with powdered formula and breast milk, and then on day 11 of life, developed sepsis and neurologic symptoms.

     Lumbar puncture was performed which was consistent with meningitis.  White cells and red cells were present, although at actually low numbers, but the protein was high, and the glucose was remarkably low.

     Cerebral spinal fluid culture grew Enterobacter sakazakii.  The patient was treated with ampicillin and Cefoxitime, but did not do well despite full support, and became pulseless and was resuscitated with pressors.

     As the clinical course progressed, it was clear that the neurologic was severe, and that there was no significant brain activity.  And so support was withdrawn, and the patient expired at day 20.

     Next slide.

     [Slide]

     The facility in which this occurred with the University of Tennessee at Knoxville.  It's a regional referral and tertiary care center; fairly large facility--360 beds.  It has a level-three NICU with 55 beds that's composed of two parts: and intensive care nursery with 27 beds, and an intermediate care portion with 28 beds.

     They looked back--because this was an unusual organism, the hospital epidemiologist looked back to see whether they had seen E. sakazakii in specimens in the recent past.  In fact, they looked in the last three years and didn't find anything from 1998 to 2000.  However, they did find two isolates detected in March 2001, in addition to this case.  So this prompted a further investigation.

     Next slide.

     [Slide]

     So the study objectives were to ascertain whether there were additional cases of E. sakazakii infection or colonization that they had missed clinically to determine the source of the organism and to develop measures to prevent further infection.

     Next slide, please, that says "Case Finding."

     [Slide.]

     A cross-sectional prevalence survey was performed, and what this was was basically looking at all patients in the NICU during the case patient with meningitis was ill, and that was defined as April 10th to 20th 2001.  I'll refer to that as the study period.

     They assessed all these patients for stool colonization.  In addition, they looked for clinical reports from the microbiology laboratory for E. sakazakii and did a review to see what symptoms, if any, patients had.  And a "case patient" was defined as any NICU patient with the E. sakazakii-positive culture during the study period, either from the stool or other sources.

     Next slide.

     [Slide]

     Case finding: 49 patients were hospitalized during the study period.  There were nine case patients, and of these nine--this slide shows the site of infection or colonization--six E. sakazakii from the stool, two from tracheal aspirates, one from the urine, and one from cerebrospinal fluid, which was the index case.

     I would want to add here the footnote that you have 10 sites here, and that's because one patient had E. sakazakii in the stool and the urine.

          Next slide, please.

     [Slide.]

     Next, a cohort study was performed.  These are the variables that we looked at, combining the literature concerning infant formula and also just nosocomial infection variables that are commonly looked at.

     Just to high the highlights, we looked at, obviously, modes of nutrition: split formula into powdered versus liquid versus ready-to-feed; also looked at whether the formula was given by continuous feed or by bolus.  And that's pretty much what I wanted to highlight there.

     Next slide, please.

     [Slide.]

     These were the results of the cohort study.  The only variable found to be significant was powdered formula use.  So, just to walk you through this table, of the 30 patients that were exposed during the study period, nine were ill and were case patients.  I'm sorry--of the 49 in the cohort, 30 were exposed to powdered infant formula and, of those, nine had E. sakazakii and were cases.  Of the remaining in the cohort, 19 did not have powdered formula, and none of those patients had been given powdered formula.

     There were some other things that I can't really say approached the statistical significance but were, I think, of some note, is that the continuous had a P-value of .16, and the absence of breast milk use was also--had a P-value of .16.

     Next slide, please.

     [Slide.]

     Next, observational laboratory studies were performed in the facility; looked at formula preparation, storage and administration.  I'll just briefly gloss over these; cultured the environment and materials for formula preparation in patient care, including the preparation area in the NICU, and cultured lots that were in use during the study period, including powdered formula from open containers.

     Next slide, please.

     [Slide.]

     So this should say "Laboratory Studies."  Laboratory studies were performed by CDC and included confirmation of isolates from the cohort study, cultures of open cans of formula.  They were also able to obtain unopened cans of formula of an identical lot number that was supplied by the manufacturer, and we cultured that as well.  Culture method was done according to a modified protocol of inisol, and all study isolates and selected historical isolates were compared by pulse-field gel electrophoresis.

     Next slide, please.

     [Slide.]

     Environmental and formula cultures--these are the results--showed that none of the on-site cultures that were performed, either in a preparation area or in the NICU, grew E. sakazakii.  CDC cultures grew E. sakazakii from a single lot of powdered formula, and this was from both an unopened can and an open can.  The PFGE patterns were indistinguishable between isolates of either the cerebrospinal fluid of the case patient that died from meningitis, and also the opened and unopened containers of powdered formula.

     And the other interesting sort of aspect of this is the PFGE results also suggested pattern diversity among other isolates from the cohort study compared with previously collected strains, and this is shown graphically in the next slide, which says "PFGE Results.

     [Slide]

     And what you see here is a gel, and the PFGE results, and you see lanes two through six showing a CSF isolate, a respiratory isolate, stool isolate, urine isolate and the two formula isolates being identical.  However, there were other patterns that are different, and these include some stool isolates from the cohort study, as well as historical isolates from isolates that were pulled from the freezer, from other previous investigations.

     The implications of this, I guess we can get into in the discussion.

     Next slide.

     [Slide]

     Observational studies.  The hospital detected no breaches in infection control.  The formula was prepared according to manufacturer's instructions on the label.  It was mixed with sterile water, refrigerated for less than 24 hours.  And their guidelines at the time were to use the mixed product within eight hours, and the hang time, although I'm not sure of the exact time, they said it was around six hours.

     Next slide.

     [Slide]

     Intervention.  The facility, at the time, prescribed a fair amount of powdered formula in the facility, to about 50 percent neonates in the NICU.  As a result of this event, they changed the formula preparation site from the NICU to the pharmacy.  The principal formula used was switched to liquid ready-to-feed, although they still had to use some powdered formula selectively for special formulations.  They changed their allowable hang time for mixed feeds from eight to four hours.  And, to date, they haven't seen any further E. sakazakii or clinical isolates.

     So, in conclusion, concerning this case the source of the case of Enterobacter sakazakii infection was traced to receipt of powdered infant formula.  The only significant--it was the only significant risk factor in epidemiologic study, and there were matching isolate patterns on PFGE concerning--from the powdered formula and from the patient.

     So we concluded that powdered formula, which is a non-sterile product can be contaminated with E. sak and can cause fatal meningitis, and that use of powdered formula should be carefully considered in the neonatal health care setting.

     Next slide.

     [Slide]

     After this, Mead Johnson voluntarily recalled Portogen powder.

     Next slide.

     [Slide]

     And an MMWR was published which included summary interim recommendations for the NICU.  This included to select formula products based on nutritional needs; that trained personnel should prepare products using aseptic techniques; to follow manufacturers recommendations for preparation of formula; that the administration of hang time should be less than four hours; and that there should be written hospital guidelines, including notification, reporting and follow-up available in the event of a product recall.  And there are some aspects of that last bullet that I think are difficult for hospitals to follow, and I'll get into that a little bit in the discussion.

     Next slide.

     [Slide]

     We also asked that cases be reported, particularly concerning invasive infections in infants younger than 12 months to the state health departments, CDC and, of course, FDA Medwatch.

     Next slide.

     [Slide]

     So, just to conclude here, the questions I think that sort of come up with this case is when this case first emerged, well, the question was is whether this is an emerging pathogen.  And in trying to answer that, there are a subset of questions that come up.  Again, what is the reservoir of the organism?  Is this--has it changed?  Is there something new as far as the reservoir?  Has this been around all along?  And what is the endemic rate of the E. sakazakii colonization or infection due to powdered infant formula?  Since we don't have surveillance for it, we don't know, you know, how many cases there have been.

     Just as an aside, we have a nosocomial infection surveillance system, and we looked back at isolates--at infections that have been reported due to E. sakazakii, and we found one where there was an isolate from the CSF in 10 years of surveillance.  And this is approximately 300 hospitals.  So this is a very unusual organism for a hospital to find, and I don't think that this Tennessee facility's experience in not seeing an isolate for three years from NICU patients is unusual.

     And, next, what's the role of specific methods for preparation and use to promote growth and reach the threshold in clinical significance.  So if it exists in formula, what are the factors that make it matter in causing clinical disease.  And, as an adjunct to that, what are the predisposing risk factors for infection?

     Next slide.

     [Slide]

     And so I just sort of split sort of the separate issues, which I think encompass the spectrum of issues that cross agencies and different both clinical, laboratory and manufacturing arenas concerning the manufacture of the formula: screening during manufacture, issues about preparation and storage and use; treatment of infection if there--you know, there's very little in the literature about treating E. sakazakii in general; and, finally, issues about case reporting and surveillance of infection, which I've already touched on.

     Next slide.

     [Slide]

     So, concerning manufacturers specifically, I'm sure what's going to be discussed today is going to be concerning processing and implementation of screening, which I understand has already begun to some extent.  Concerning preparation, storage and use, development of guidelines or recommendations.  The American Dietetic Association had had some recommendations previously that had been disseminated but was out of print at the time of this event, and I understand that they're developing revised guidelines.

     And, finally, case reporting and surveillance--this was the issue I touched on before, which was about hospital record-keeping.  They usually don't record whether--they record, of course, in the orders whether formula has been given, but not necessarily whether it's liquid or powdered, and certainly not what lot number has been given.  So, in trying to track--do a trace-back investigation of the case, it's very, very difficult, because oftentimes the hospital doesn't now whether the patient even got powdered formula, or whether liquid ready-to-feed.  And so this provides a particular challenge in the way formula documentation is currently set up.

     Next slide.

     [Slide]

     So, as far as future plans, what we've currently been involved with FDA has been sporadic case investigations, and sort of case finding.  I think a case series description would be very useful, and there are a couple people working on that.  As far as policy on formula preparation, I mentioned the American Dietetics Association.  They also have done surveys of preparation and use.  I think that's important.  And also, obviously, revising the guidelines.

     Concerning laboratory research, we've been involved with some, and I know FDA has been as well, concerning the growth characteristics of E. sakazakii, and also, specifically, the effect of two things: one, competitive microbial flora.  I mean, we've done studies looking at E. sakazakii growth, but one of the questions is how does it grow differently when it's in sort of the milieu with different organisms, as it is with infant formula, and also about the effect of heat inactivation on growth.

     Next slide.

     [Slide]

     I'd like to acknowledge many people who have given input over the last, actually, couple of years and, particularly, the hospital that originally detected the case in Tennessee.

     Next slide.

     [Slide]

     That's the end of my presentation.  I'd be happy to answer questions.

     DR. HEUBI: In order to answer questions, either we'll have to--

     DR. KUEHNERT: Oh, yes, I'm having a lot of trouble hearing you.

     DR. HEUBI: I think what we ought to do is each of us, maybe, should go and pose the question with the microphone to him, and then he can respond.  That way we can all hear what he says.  Do you want to do that?  There's a microphone over there.

     So, if you'll queue up, that would be great.

     Now, please identify yourself.

     DR. STALLINGS: Hi.  It's Virginia Stallings, from Philadelphia.  Thank you for being with us.

     My question is: how hard is this bug to grow and identify in a busy microbiology clinical lab?  Because that will give us some framework to consider the data we have at hand.

     DR. KUEHNERT: Yes.  Yes.  I heard the question.

     There are two aspects to that.  One is whether E. sakazakii can be easily grown in a clinical specimen.  I think that most laboratories are pretty well suited to that.  We have had situations where they weren't sure whether it was run of the mill E. cloacae or E. sakazakii, because sometimes the yellow pigment isn't that strong.  But it appears that from what I understand--I'm not a microbiologist--but from my understanding, is that the methods used in most clinical microbiology laboratory should pick up most of them.

     Now, the issue concerning finding it in infant formula is different.  You know, some hospitals that have suspected that they might have E. sakazakii in their formula have tried, you know, a number of different methods which really don't work, like putting the powdered formula just directly on media, or even trying to do--trying to culture it, just using standard methods.  And I'm sure FDA will speak to this, but there are--I mentioned a modified protocol, and it is a fairly intensive, somewhat complicated protocol that takes a number of days to grow it out of formula.  And the biggest problem--I don't want to belabor this too much, because I'm sure it's going to be discussed later--I mean, the big problem is that you've got a lot of microbial flora in the formula that competes against the E. sakazakii, and so you have to pick--it's sort of a needle in a haystack to pick it out.  And that's some of the difficulty in trying to isolate it from formula.

     So, the short answer to the question is: yes, I think microbiology laboratories should pick it up in clinical specimen, but no, I think they're going to have difficulty from infant formula.

     DR. NEILL: Peggy Neill, from Brown University Medical School.  I have two questions, and they're unrelated.

     The first one is that in your slides, it would probably be approximately "9," for "facility characteristics," at the bottom of the slide it indicates that there are two isolates of E. sakazakii detected in March 2001, but I'm trying to discern from the text here that they appear not to have been either a clinical isolate at all?  Or they were certainly not a clinical isolate from the NICU.

     DR. KUEHNERT: Right.  Yes.  Let me address that and try to remember what the significance of those isolates.

     If I remember right, one of those patients was included in the cohort study--well, let me back up.  The two isolates, I believe, were respiratory isolates.  They were not thought to be causative for infection.  One of the patients was included in the cohort study and, I believe, had a positive stool but did not have a clinical isolate during the study period.

     The other patient, I think, had already been discharged, and their illness was due to another issue, and they did not have the isolate for comparison--they did not have either respiratory isolate for comparison at the time of the study.  They had been discarded at the time that they noted the problem.

     DR. NEILL: Okay.  Thanks.

     The second question pertains to your PFGE profiling?

     DR. KUEHNERT: Yes.

     DR. NEILL: And I suspect you know where I'm going with this.  You alluded to the fact that lanes two through six are clinical isolates that are identical, and they are from this particular outbreak investigation.  But if I understood you correctly, you have other isolates farther to the right in that gel, but I don't know which lane, and that those have different pulsotypes?

     DR. KUEHNERT: Right.  Yes.  Let me try--I was going to discuss this, and in the interest of time I did not.  But I'm glad you brought it up.

     Okay.  There are two groups of isolates here.  Unfortunately, I don't have them labeled, so I can't tell you which is which, but there are some--there are stool isolates there from the cohort study that had different PFGE patterns from the index case and the isolate from formula.  And then two or three of the other ones are from historical isolates from the CDC freezer.

     Now, concerning the stool isolates, I mean there are a couple of possibilities for explaining this.  One is that the patients just happened to be colonized with E. sakazakii from the stool, which is possible--although we don't usually look for it in clinical microbiology labs.  So it might be there, but I think that's probably unlikely, because people do do screening for gram negatives in stool, and have not frequently found this.

     The other explanation--which I think is more likely--is that there were multiple--there may have been multiple--this is speculation on my part--but there may be multiple isolates of E. sakazakii from that--either that implicated lot, or other lots is possible, and that this represents isolates that were below our level of detection for the lot that we sampled.

     But, I mean, there's obviously got to be some explanation as to where these came from, and we don't have an answer for it.

     DR. HEUBI: Hi.  Jim Heubi.

     After this initial case series was identified in the hospital, was there an effort made then to check any of the formulas given to these infants, later during the follow-up or new additional cases to know whether actually those formulas [off mike].

     DR. KUEHNERT: Could you repeat that?  I'm not sure I fully understood that?

     DR. HEUBI: Well, what you reported was that there were no additional cases after this series, including the nine that were colonized with one case.  And then you made the comment about the fact that there's been no additional cases since that time.

     DR. KUEHNERT: Mm-hmm.

     DR. HEUBI: The question I had was: was there any effort made to try to make sure that the formula was or was not contaminated in any of those additional infants might have been exposed to after the initial reports?

     DR. KUEHNERT: Well, my understanding at the hospital is I don't think they've done any further stool colonization studies, nor have they done any cultures of the formula.  I think that they've done--I mean, I don't know--a somewhat active surveillance, certainly in looking for E. sakazakii on clinical specimens.  But as far as stool colonization, or directly culturing formula, I don't think that they've done that.

     Again, I don't think they had the resources to do, you know, the modified protocol in the first place to culture formula.  And so I don't think they've been doing that on an ongoing basis, either.

     Does that answer your question?

     DR. HEUBI: Yes.  I guess as the devil's advocate what I'm saying is that the changes they implemented may or may not have had any impact on whether they have any future cases.

     DR. KUEHNERT: Yes.  I think that's a valid statement.

     DR. HEUBI: Thank you.

     DR. BEUCHAT: Larry Beuchat, University of Georgia.

     I have two questions.  One is a follow-up to Peggy's.  Were the two March 2001 isolates, you know the PFGE patterns determined in this film--were they similar or not from isolates that are shown on the gel?

     DR. KUEHNERT: Sorry, this is the March 2001 isolates?

     DR. BEUCHAT: Yes.

     DR. KUEHNERT: Yes, those were discarded before they knew there was a problem.

     DR. BEUCHAT: My second question is dealing with the four-hour hang time.  What criteria--what science-based information was used to determine and recommend that a four-hour hang time should be used?

     DR. KUEHNERT: That's a very good question.  At the time that we made these recommendations, I think it was an educated guess.  We weren't sure even that the hang time had a role in this.  I think the previous question, you know, spoke to that, that they made these changes and didn't see any more isolates, and that doesn't mean that the changes they made resulted in fixing the problem.  But also the flip side of that is we don't know whether the hang-time played role in it being a problem.  But we knew that it made sense, you know, concerning microbial growth to shorten the time to as short as feasible.  And I think four hours got to the limit of what we thought was feasible in a NICU.

     Since that time--I'm sorry I'm not there, because I would have brought some extra slides, which I'm looking at here--our lab has done some studies on E. sakazakii growth in the implicated formula, Portogen with Iron.  And what you see is a curve that shows very little growth until you get to about--beyond six to eight hours, in which case, between eight hours and 24 hours you see a jump in growth from a log of 4 to about a log of 9.  And also we've done studies looking at not only that formula, but also other manufacturers formulas that have shown almost--well, actually, identical, statistically significant differences in that growth.

     DR. BEUCHAT: On that growth curve that you were just describing, what was the initial number of E. sakazakii in the formula?

     DR. KUEHNERT: At t-0 hours, 3 logs--so, 1,000 organisms were inoculated.   And then you see about a log of 3 at t-2, t-4--it goes to about a log of 4 at t-6 and t-8, and then it goes up to--actually, it's log 8 at about 24 hours.

     I want to explain how this done.  It was done under clinical conditions, so the formula was mixed with sterile water, and E. sakazakii was put in at that time.  Then it was refrigerated for 24 hours, and then hung.  And that's t-0 was when it was hung.  And so what I just described was under those conditions.

     DR. BEUCHAT: So the temperature would have been, perhaps, 70 Fahrenheit?  The room temperature?

     DR. KUEHNERT: Yes--I don't have the breakdown to the exact temperature, but it was ambient temperature, which I'd have to look at what actually she did.  I think it was a little warmer than that, I think.

     DR. ACHOLONU: Doctor, my name is Alex Acholonu.  I'm from Alcorn State University in Mississippi.

     During your delivery, talking about the case findings, you talked about examining stool samples, tracheal aspirates, urine and CSF.  May I know why blood is not checked as one of the fluids in the body?

     Number two question: we have been told that this disease is most prevalent in neonates, which mean children less than one month old.  Did you, in order to rule out cases of prenatal infection, check the mothers of the babies?

     DR. KUEHNERT: That's a good question.

     We did not do cultures of the mothers or of siblings, or household contact at that time.  Subsequent to this, there was another investigation in another hospital in Tennessee, where we did do those cultures--vaginal cultures and other--rectal cultures--of the mother, and did not find it.  But that's a very good point, and that was not done in this investigation--to my knowledge.

     The first question, I didn't quite get.  It was something about checking some site?  What site was it?

     DR. ACHOLONU: Yes--checking the blood.  We are told that one of the effects of the disease is sepsis.

     DR. KUEHNERT: Right.

     DR. ACHOLONU: And I take it to be septicemia.  May I know why blood samples were not checked.

     DR. KUEHNERT: Yes.  Okay.

     That's sort of a complicated issue.  I guess--I mean, at the time it was sort of dealing with, certainly, an infection-control issue and an investigation.  I'm not sure--I wasn't the hospital epidemiologist who had to make this decision. Inga Himmelreit was.  But I mean, if I were her, I might be uncomfortable doing blood cultures where it's not clinically indicated.  So I think that, you know, all these patients were clinically stable, and so I think that probably was the decision why they didn't do blood cultures.  Although blood culture is obviously a low-risk procedure, it's not a no-risk procedure.  And so I think that, you know, that may have been why it wasn't done.  Also, the yield probably would have been pretty low.

     DR. ACHOLONU: My last question: since the infection is more in neonates in the intensive care unit, is it possible that some of the infections are nosocomial?

     DR. KUEHNERT: Do you mean that some of this--

     DR. ACHOLONU: Picked up from the hospital, rather than coming from the baby formula?

     DR. KUEHNERT: Right.  Right.

     Yes, I think that's a good point.  I mean, as far as the stool--I mean, we don't know--okay, just looking at the cohort study, we can't tell whether these stool cultures represent transmission between the patients or that health care workers were a vector between patients.  But the epidemiologic study, I think, clinches it, because though, again, looking back at that, those that were exposed to powdered formula were the only ones that had the stool cultures or cultures at other sites.  Of all the patients who did not get powdered formula, none of those had E. sakazakii.  And I think that sort of--although there might have been something confounded exactly with powdered formula, I think that really leans against nosocomial infection.  So that's what this investigation--overall, I would say that it isn't a common nosocomial pathogen.  I mean, I mentioned that NIS surveillance, and we just don't see it.  And we've also done some case finding, which I didn't have time to present, both clinical microbiology laboratories, ClinMicroNet, and also infectious disease consultants, and over a three-year period, there were very, very few isolates from NICUs.  So, overall, it's probably pretty unusual, and I think the epi really points to the powdered formula rather than nosocomial transmission.

     DR. ACHOLONU: Thank you.

     DR. LEE: Hi.  This is Ken Lee, from Ohio State.

     There was almost an offhanded reference to competing flora, and I'm just wondering, is the competing flora a significant factor in E. sak infection, or is it even present?  What's your best guess about other microorganisms that might be present?

     DR. KUEHNERT: Right.  Well, "guess" is a good word as far as what I'm going to say on that, as far as what the significance of it is.  I mean, like the lab studies that I mentioned, I mean that was inoculating E. sakazakii into sterilized formula.  We have not done studies yet, but I think it's important that we do, looking at sort of the in vivo environment of powdered formula, and how E. sakazakii behaves in that environment.  We don't know if it's going to make it grow more slowly, grow faster.  We don't know if other organisms are deleterious or actually whether it augments growth.

     It obviously is a complicated situation when you have, you know, multiple bacteria in the formula.  And this also gets back to the difficulty in growing E. sakazakii out.  I mean, again to just emphasize the obvious here, I mean formula is not sterile.  When you try to grow--when you try to isolate E. sakazakii you get all kinds of organisms growing from it--you know, from bacillus, to other enterobacters, to other gram-negatives.  And so the difficulty, actually, is in picking the E. sakazakii out.  And what we don't know is whether the bacterial flora--what role the other bacterial flora play in the growth of E. sakazakii.

     DR. TARR: Phil Tarr, Washington University.

     Is there any chance that these culture-positive cases are the tip of the iceberg?  Was there any evidence of clustering of sterile CFS with the presence of cliacytosis that might be represented in partially treated cases of E. sakazakii?

     DR. KUEHNERT: Yes, that's a good question.  We actually went back in the hospital and looked over a multi-month period at nosocomial infections of any kind; those due to enterobacter cloacae, because we thought, well, the point you bring up or touch on is that if formula has multiple organisms, could there be other organisms causing infection that maybe we don't notice because they're not as striking as a sentinel organism as E. sakazakii.

     We didn't see any association of nosocomial infections in general, or nosocomial meningitis associated with E. cloacae or with--sorry, let me back up.  We didn't see any association of powdered formula with either nosocomial infections or specifically nosocomial meningitis over the time period that we looked at at this hospital.

     I think it would be an interesting idea to look at that in a multi-center fashion.  Again, it gets back to the difficulty and challenge of how hospitals record powdered formula.  But if there were a way to get over that hurdle, I think this would be a very good idea to look at.  Because we just don't know.

     DR. TARR: What particularly--I'm thinking of missed E. sakazakii, if it was partially treated prior to obtaining the CSF.  Have sera been obtained from the survivors?

     DR. KUEHNERT: Ahh--well, we have--when we confirm organisms, we get the CSF and I'm not sure if we have sera.  We have the CSF stored, but I'm not sure if we have sera.

     DR. TARR: Thank you.

     DR. BLUMBERG: This is Hank Blumberg from Emory University.

     There have been previous reports of E. sakazakii causing necrotizing enterocolitis, and I was wondering, in the outbreak that you investigated in Tennessee, were there any cases of necrotizing enterocolitis in the patients--in the neonates--who had positive stool cultures?

     DR. KUEHNERT: Yes.  Thanks for that question, because it reminded me that--no, the answer is no, we didn't see any NEC.  And also, when we looked at that association between nosocomial infection and powdered formula, we also looked at NEC over the previous few years, and did not find an association.

     DR. BLUMBERG: Thank you.

     DR. TOMPKIN: Good morning.  This is Bruce Tompkin, and I'm the industry representative t this meeting.

     You mentioned a statistic that I hadn't heard of, where you'd gone back to 300 hospitals over the past 10 years.  Would you please repeat that, because I'm not sure I heard it correctly.

     DR. KUEHNERT: Oh, yes.  Sure.

     Umm--this was not published anywhere.  We did this case finding after this case, so this may be the first time you've heard it.  Let me just go through it again.

     We have a national nosocomial infection surveillance system, and this is a voluntary participation of hospitals in the United States.  We looked back at--and this is only nosocomial infections, you know, acquired in the health care setting--and we looked back for E. sakazakii infection.  This was during a 12-year period of surveillance.  And we found one case of E. sakazakii meningitis in that time period.

     DR. TOMPKIN: And did you say it was 300 hospitals--approximately?

     DR. KUEHNERT: Yes, it's about 300 hospitals.

     DR. TOMPKIN: Thanks.

     DR. KUEHNERT: Are there any other questions?

     DR. HEUBI: One comment.  Jim Heubi.

     We would actually--Dr. Busta and I were discussing the fac that we would actually appreciate it if you could provide us with the slide of the log growth for us to look at, if you have that.

     DR. KUEHNERT: Oh, sure.

     DR. HEUBI: I think that would be very valuable for us.  And just to clarify--this was E. sakazakii that was placed in sterile formula, and then hung and then cultured repetitively thereafter--is that correct?

     DR. KUEHNERT: That's right.  I'm sorry--so you were asking for me to e-mail it now, or--

     DR. HEUBI: Right--we'll actually--someone from the FDA will contact you and we'll make arrangements to get this up here so we can have it for our books.

     DR. KUEHNERT: Okay.

     DR. HEUBI: Thank you for your presentation.

     [Recess.]

     DR. HEUBI: I think we'll reconvene.

     Dr. Karl Klontz is going to present other relevant investigations.  Where's Dr. Klontz.

Other Relevant Investigations

     DR. KLONTZ: Good morning.  Karl Klontz, here.

     I want to spend the next 20 minutes or so--oh, by the way, I'm with the Epidemiology Team at the Center for Food Safety and Applied Nutrition.

     The title of my talk today is "Enterobacter Sakazakii Case Reports and Outbreaks Involving Infants, As Reported in the Peer Reviewed Medical Literature."

     Next slide, please.

     [Slide]

     Now, before I go any further, you have probably--you on the panel have a more extensive slide set than the one I'm covering here. But I will, in the interests of time, just cover the most salient ones.  And if there are questions on the others you have, I'll be happy to entertain those.

     Okay.  For the purposes of summarizing the literature, we defined a case of Enterobacter sakazakii infection as an infant meeting at least one of the following criteria.   First, E. sakazakii was recovered from one or more of the following normally sterile sites: blood, cerebrospinal fluid, brain tissue or urine.

     Second, an infant could have been classified or defined as a case of E. sakazakii if that infant was involved in an outbreak of necrotizing enterocolitis, and E. sakazakii was recovered from blood, stool or stomach aspirate of more than one infant in that outbreak.

     A third possible criterion was that the infant had bloody diarrhea and E. sakazakii was recovered from a stool specimen in pure culture; that is to say, no other pathogen was in the stool that could have accounted for the bloody diarrhea.

     So any infant that met one or more of these definitions, we called a "case" of Enterobacter sakazakii.

     Next slide, please.

     [Slide.]

     This slide summarizes the initial reports of Enterobacter sakazakii in the literature in chronological fashion.  The first thing I'd like to point out is that the first published report occurred in 1961, by Ermenye, et al.  This was a report that described two fatal cases of neonatal meningitis.  And, by the way, in some of these early reports, the name Enterobacter sakazakii, of course, wasn't used.  That term was introduced--proposed--in 1980 by Dr. Farmer, et al.  Some of the terms used earlier in these reports were "yellow-pigmented Enterobacter cloacae" and some other terms.  But, nevertheless, for our purposes today, some of these following publications that came after the  Ermenye, et al., study, described also infections in neonates or very young infants, and it really wasn't until 1982 that Jimenez, et al., reported the first case of Enterobacter sakazakii infection in an adult--a 76 year old man with a history of rectal adenocarcinoma who had urosepsis and recovered after antibiotic therapy.

     Now the first case series was by Muytjens--and, by the way, I'm pronouncing this "Moyt-yens," with a little bit of counseling from Dr. Muytjens himself.  We were fortunate enough to reach him by e-mail in the last couple of weeks, and one of our questions, among other technical issues--

     [Laughter.]

     --was how to pronounce his name.  And he was very kind to tell us "Moyt-yens," although he did say, curiously, that his colleagues in the Netherlands, even in his area, have trouble pronouncing this name.  So I'm sure I'm not doing a very good job here.

     But nevertheless, he published the first case series, and this was the first study to propose a possible link to powdered formula.  And we'll go over this study in more detail, but--let's go to the next slide.

     [Slide]

     I would like to stand back a bit now, and look at the literature as a whole, and point out that there were 27 references in the literature that described Enterobacter sakazakii infections, encompassing a total of 58 cases.  As you can see, the majority of the cases were in infants; 17 percent in children over the age of one, or in adults.  And in the bottom half of the slide, we've broken down these cases by age group; the number of cases, and then the percent.  And, as you can see, the majority, again, were in the first year of life--83 percent.  But even in that group, the majority were in the first month of life, underscoring--at least from the literature standpoint--that this appears to be largely a neonatal infection.  That's not to say there aren't older cases.  For example, there were six cases in individuals over the age of four.  And if you look at their median age, they were actually at the other end of the spectrum--median age 74 years of age.

     Okay.  Let's go to the next slide, please.

     [Slide]

     Now this slide summarizes the 48 cases of Enterobacter sakazakii infection in infants, and selected clinical features of these cases.

     About half the cases were in males; half in females.  Meningitis, however, was the predominant syndrome, accounting for 58 percent of the cases.  Sepsis and bacteremia, 17 percent; and necrotizing enterocolitis, 29 percent.

     Overall, the case fatality rate was 33 percent.  And, again, in the bottom half of this slide we've broken down for you the birth weight, where reported, for these infants, and the number that were in each category, and then the case fatality weight.  And, as you can see, there was really no increasing or decreasing trend of case fatality by birth weight.  It's notable here that one of the stratum had a higher case fatality rate, but I think that's largely because a number of the cases in this group came from one paper--and that's the Muytjens article that we'll discuss in just a minute--which was a retrospective survey in the Netherlands, and probably more likely to have identified the most severe cases.

     Let's go to the next slide, please.

     [Slide]

     Now, this is the Muytjens article of 1983, the case series I mentioned.  And it was an analysis of eight cases of neonatal meningitis and sepsis due to Enterobacter sakazakii.  What they did here is they re-analyzed 20 Enterobacter strains that had been isolated from cerebrospinal fluid, going back six years; and 25 strains from blood, going back two years--all in the Netherlands.

     Now, their hypothesis was that Enterobacter sakazakii may not have been originally in this group of organisms because this organism produces its characteristic yellow pigment at temperatures generally lower than 36 degrees centigrade, which was the temperature that these strains were identified.  So what they did is they used various biochemical and growth parameter systems, including a lower temperature, and they were able to identify eight of these organisms as actually being Enterobacter sakazakii.  These were confirmed as being E. sakazakii by the Centers for Disease Control subsequently.

     And so the investigators then went back to medical records to obtain clinical data, retrospectively.

     Next slide, please.

     [Slide.]

     This slide now summarizes the clinical features of these eight cases of neonatal meningitis and sepsis.  The first thing to point out is that three of the eight cases were delivered by Cesarean section, and this is notable, because in the literature, until this point, there had been some discussion about the possibility that Enterobacter sakazakii was acquired from the vaginal canal during the normal birthing process.  But here we have three of the eight cases that had been delivered by Cesarean section, and one of which was delivered to a woman who gave birth less than 45 minutes after the membranes were ruptured.

     Now, as a rule, these infants progressed well in the first few days of life.  The first signs of illness occurred between days four and eight.  Two of  the cases actually had necrotizing enterocolitis along with meningitis.  The case fatality rate was 75 percent, and even the two survivors had significant neurologic sequelae.

     When death occurred, it did so within hours to several days after onset of illness.  And the investigators actually used the term "hemorrhagic encephalitis" to describe the illnesses, because on autopsy, a number of these infants had large, swollen brains that were soft and showed signs of intra cerebral hemorrhage.

     Next slide, please.

     [Slide.]

     Same study--now let's look at some of the epidemiologic features of these eight cases.  Six out of eight had birth weights of less than 2,500 grams, with the lowest being 850 grams.  Five of the eight cases were premature; that is, their gestations were less than or equal to 36 weeks.

     There was geographic clustering in this case series, in that five of eight cases occurred at the same general hospital in the Netherlands, whereas the other three cases were born at three other facilities.  But there was also temporal clustering in this series, in that three of the five cases at the general hospital became ill within three months of each other, and even at the other hospitals, two of the three cases became ill within two months of each other.

     Next slide, please.

     [Slide.]

     Because of this geographic clustering, Muytjens and his colleagues did environmental sampling at the pediatric ward of the general hospital where five of the eight cases occurred, and they were able to recover Enterobacter sakazakii from prepared formula, from a dish brush, and from a stirring spoon--but not from powdered formula itself, nor the water that was used to prepare that formula.

     Now, let me just say a word about the methods they used to test the powdered formula, because one of the questions we asked Dr. Muytjens in the last couple weeks--because it's not stated in the article--how much powdered formula did you look at?  And he responded by saying it was somewhere between four and 10 grams of powdered formula--which, in terms of today's methodologies is a relatively small amount.

     They did do plasmid profile analysis, and they concluded that "three or four of the five isolates from the patients at the general hospital were probably the same strain."

     Now, it's important to note, though, that these plasmid: profiles differed between the isolates from the cases, versus the isolates from the prepared formula, but that may not be surprising, because keep in mind that this was a retrospective study.  At the time of testing the prepared formula, some of the cases had become ill months, if not years, earlier

     Now, evidence that the formula, however, may have served as a vehicle for transmitting the infection was found in a 1990 letter to the editor by Muytjens, et al., to an infectious disease journal, in which he stated: "No cases of Enterobacter sakazakii were observed at the general hospital since the powdered formula was replaced by liquid formula eight years ago."  Again, liquid, of course, being a sterile product.

     Next slide, please.

     [Slide.]

     All right.  Before I turn to three outbreak investigations, I would like to summarize, in this slide, some of the key developments that took place regarding Enterobacter sakazakii in powdered infant formula for the period 1961--that is, the date of the first publication--through the '80s.

     The first thing to note is in 1980 Farmer, et al., in studying 57 isolates of Enterobacter sakazakii, note in one of their tables that one of these isolates came from a "pan of dried milk."  This is a sample that came from a central public health laboratory in London, but there's no other details about it.  We've seen from the 1983 Muytjens article that Enterobacter sakazakii was recovered from prepared formula, and that prompted the investigators to recommend, in fact, that sampling of "milk" take place as part of future investigations of Enterobacter sakazakii infections.

     And then in 1988 Muytjens and colleagues obtained 141 powdered infant formula products from 35 countries, tested them for the presence of Enterobacteriaceae and found 52 percent of the samples were positive; 14 percent of 20 powders were also positive for Enterobacter sakazakii.

     Important, thought, is to underscore that the concentration of  Enterobacteriaceae in these powdered formulas was uniformly less than or equal to one colony-forming unit per gram.

     Next slide, please.

     [Slide.]

     Okay.  What I'd like to do now is use the second half of this talk to go over three particular outbreak articles that I think are salient to today's discussion.  The first one is by Biering, et al., 1989.  It was entitled, "Three cases of Neonatal Meningitis Cause by E. Sakazakii in Powdered Formula."

     These illnesses occurred in 1986 and 1987 in Iceland--two within a month of each other.  Two of the infants were normal at birth.  Their gestations are listed there.  One baby was a Downs syndrome baby.  All three did well until day five, when they became ill.  The Downs baby died.  The two other infants recovered, but with several neurologic sequelae.  The investigators found that all three had been fed powdered infant formula, and this was the first study to show, at least in an illness setting, that Enterobacter sakazakii could be recovered--albeit in low numbers--from freshly prepared formula from a previously unopened can.

     Now, the investigators did not recover, or were not able to recover, the organism from the environment; specifically not from the milk kitchen, nor the utensils, nor the ward.

     Next slide, please.

     [Slide.]

     Staying with this same article by Biering, et al., they note that in addition to three ill cases, there was an infant with colonization, and that these four Enterobacter sakazakii strains that were isolated from the neonates had the same plasmid profile as 22 strains recovered from the formula.  They also point out, however, that there was anecdotal evidence that the formula bottles had occasionally been kept at fairly warm temperatures--35 to 37 degrees centigrade--for extended periods in bottle heaters.  However, they also note that in one instance they were able to recover the organism via direct plating from a bottle that had been refrigerated for an unknown amount of time.

     From this outbreak investigation, the investigators concluded: "Milk powder can be the mode of transmission for Enterobacter sakazakii meningitis or sepsis in neonates."

     Next slide, please.

     [Slide.]

     The final two studies I'd like to discuss with you I bring up because I think, importantly, can be discussed in the context of what we call "historical cohort studies" in epidemiology, which are very useful because they allow one to calculate rates of infection by exposure status--whether infants ate the formula or did not eat the formula.

     And the two in particular I want to discuss with you are the Simmons, et al., article in '89, and the Van Acker in 2001.  But keep in mind--we heard this morning from Dr. Kuehnert, the Tennessee outbreak of 2001 published in the MMWR, that too was handled as a historical cohort study.

     Okay, Next slide, please.

     [Slide.]

     The first study is the one by Simmons, et al., 1989, entitled, "Enterobacter Sakazakii Infections in Neonates Associated with Intrinsic Contamination of Powdered Infant Formula."  These illnesses occurred in February and March of 1988.  There were two cases of bacteremia, one urinary tract infection, and one case of blood diarrhea.  Enterobacter sakazakii was recovered from the stool of all four infants, which prompted the investigators to go back and look at the feeding practices in that neonatal intensive care unit.  And they found that all four had been fed the same powdered formula.

     This formula had been prepared in a blender which was then rinsed with tap water between uses.  On culturing, the blender yielded a heavy growth of Enterobacter sakazakii, but once a policy of sterilizing the blender was instituted, no further clinical isolates were obtained.

     They then conducted a historical cohort study in the neonatal intensive care unit to assess risk factors for infection and colonization.  And, as you can see, this historical cohort study took place--or covered a period of about five weeks in February and March of '88.

     Next slide, please.

     [Slide.]

     All right.  Here, then, are the results of the Simmons, et al., historical cohort study.  And it's just the standard setup for a two-by-two table, where we've got "infection and colonization" up on the top--"yes/no;" whether the infants ate the implicated formula, "yes/no" on the left.

     And, as we can see from the slide, in this outbreak setting, four of the five infants who consumed the implicated product developed infection or colonization, as opposed to zero of 40 infants who did not eat the implicated product.  That yielded a highly statistically significant association between the implicated formula and infection/colonization.

     Next slide, please.

     [Slide.]

     Staying with the same outbreaks, they note that the implicated elemental formula had been given primarily to the most premature infants, largely because it required relatively little digestion.  They obtained samples from an opened yet un-mixed can of powdered formula, and they cultured this powdered formula using the 1988 methods of Muytjens, et al., which called for at least 100 grams of powder to be sampled in each sampling frame, and then repeated three times for that matter.

     They were able to recover Enterobacter sakazakii from the powdered formula at a concentration of approximately eight colony-forming units per 100 grams.  In addition, they cultured--they recovered Enterobacter cloacae at a concentration of 48 colony-forming units per 100 grams.  And, with respect to the Enterobacter sakazakii, they had the same plasmid and multi-locus enzyme profile--both the isolates from the formula as well as the isolates from the patients.

     Next slide, please.

     [Slide.]

     All right, the final outbreak I would like to discuss with you is the 2001 Van Acker paper, entitled "Necrotizing Enterocolitis Associated with E. Sakazakii in Powdered Formula."  In this study there were 12 cases of necrotizing enterocolitis that had been diagnosed in June and July of 1998 in Belgian neonatal intensive care unit.  In this period--June and July--a total of 50 neonates were admitted to that neonatal intensive care unit, but with respect to the 12 cases of necrotizing enterocolitis, all 12 had birth weights of less than 2,000 grams, and had been fed powdered infant formula.  Six of the 12 neonates with necrotizing enterocolitis had positive cultures for Enterobacter sakazakii--and the specimens are listed up here--versus zero of the 38 without necrotizing enterocolitis, and that was a statistically significant difference.

     In addition, 10 of the 12 neonates with necrotizing enterocolitis had been fed the same powdered formula, and the article states that this was a product called Alfare, a semi-elemental formula with a low osmolarity.

     Next slide, please.

     [Slide.]

     Here, using the same setup that we used before, are the results of the historical cohort study in this outbreak.  And, as we can see, six of 14 infants who were fed the implicated formula developed Enterobacter sakazakii-positive necrotizing enterocolitis, versus zero of 36 infants who did not--were not fed that implicated formula.  Again, a statistically significant association between the implicated formula and the development of necrotizing enterocolitis.

     Next slide, please.

     [Slide.]

     Van Acker and his colleagues noted that Enterobacter sakazakii was recovered from the prepared formula, as well as from unopened cans.  They did molecular typing, using arbitrarily primed polymerase chain reaction, and they confirmed a partial strain similarity between the powdered formula and the patient isolates.

     There were no further cases of necrotizing enterocolitis observed after the implicated powdered formula was stopped in that neonatal intensive care unit.  But it's interesting to note that during the outbreak period there was an inadvertent challenge test that took place as follows.

     The implicated formula was stopped on July 10th of 1998, as soon as the investigators had a suspicion that there was an association between illness and that formula.  But then the formula was released again 10 days later, when all cultures to that date had been negative.  However, three days after releasing that formula, there was a new case of necrotizing enterocolitis linked to the same formula, and it was on that same day that the cultures came back showing intrinsic formula contamination.

     Next slide, please.

     [Slide.]

     In the same article, they also provide information regarding the manufacturers quality control data for the implicated formula, and that is as follows.  Of five samples analyzed, one yielded 20 coliforms per gram; four yielded less than one coliform per gram.  And these results fulfilled the requirements of Codex Alimentarius, which call for a minimum of four of five control samples to have less than three coliforms per gram, and a maximum of one in five samples that may have more than three, but less than or equal to 20 coliforms per gram.

     However, the implicated formula did not meet Belgian law, which called for less than one coliform per gram in all samples, and therefore the product was recalled.

     Next slide, please.

     [Slide.]

     I think this is my final slide, and I want to end it, again, on the Van Acker publication, because they note in the article that the facility that produced the implicated formula in the Netherlands was upgraded.  More stringent standards were adopted for products; specifically, calling for less than .3 coliforms per gram, and zero Enterobacter sakazakii isolates per 10 grams.

     After these changes were instituted at the production facility, the implicated powdered formula was reintroduced to the very neonatal intensive care unit where the outbreak had taken place.  Reintroduction took place in 1999, and Van Acker, et al., note that as of the publication date--2001--no further cases of necrotizing enterocolitis associated with E. sakazakii had taken place--or had been diagnosed--in that neonatal intensive care unit where the outbreak occurred.

     All right.  That's my last slide, and I'll stop at this point.

     Do we want to take questions now?  I saw by the schedule Dr. Alexander is coming up, but I defer to you as to what you'd like to do.

     DR. HEUBI: I think we're going to take some questions now.

     DR. KUZMINSKI: Dr. Klontz--Kuzminski, here--just a question on your very last slide, please, if you could replace that on the screen.  Thank you.

     Do you have information on the nature of the upgrades at the production facility?

     DR. KLONTZ: No.  The article doesn't get into the specific upgrades that were made.  The reference to this is in the discussion section, and it's fairly limited.  No, I don't know exactly what took place there.

     DR. BEUCHAT: Larry Beuchat.

     Out of these cases that you've summarized here, or other information that you might have, what is the minimum infectious dose of E. sakazakii, in terms of eliciting illness?

     DR. KLONTZ: The literature--I don't think the literature answers that question.  Certainly, not in the articles I read could one ascertain a definitive infectious dose.  There are a lot of complicating factors here, as I'm sure you're aware--how much is in the powdered formula that leaves the plant, versus what sort of treatment that product received in the hospital setting, in terms of hang time and so on.  So those are a lot of complicating factors.  I don't think the literature tells us what the infectious dose is here.

     DR. ACHOLONU: You said that--Alex Acholonu, from Alcorn State University in Mississippi.

     You said that you recovered the bacterial organism in unopened cans.  My question is: what is the longevity of the bacterial organism; how long was it in the can when it was checked?  And when we consider the fact that you're dealing with a powder in an unfavorable environment for the bacteria, is growth sustained while the organism is in the can?

     DR. KLONTZ: Again, the articles don't go into those details.  That information is just not there.  I saw no discussion whatsoever that would answer any of those questions--how long the organization was in the can, or whether it reproduced in the can, how the environment could have affected its numbers and viability--none of that discussion is in these articles.  So I just can't answer that question.

     DR. ACHOLONU: But you are aware of the fact that they don't grow very well in dry conditions.

     DR. KLONTZ: That's what I hear.  Right.  That's what I hear.

     DR. ACHOLONU: So it is necessary to find out what is keeping them alive.  They are not spore-forming bacterial organisms, as far as I know.  So how do they stay in the can--indefinitely--until it is opened?  Maybe this is food for thought; something we may have to think about.

     DR. KLONTZ: Yes.  And I think as the day goes on we're going to have some more microbiologically-oriented discussions, and this would be a very relevant question to arise then.

     DR. NEILL: Peggy Neill.

     I was just trying to quickly go back through this, but it's probably faster just to ask you.  In your literature review, then, are all the cases--are any of the cases reported associated with non-powdered formula preparations?

     DR. KLONTZ: Let me start to answer that by saying--no, I didn't see any cases that were linked to non-powdered formula preparations.  However, a number of these articles don't even comment on source at all.  There's just a question mark, when one finishes reading the article, as to the source.

     The ones I've presented, I picked out largely because they focused, they commented on, or at least took effort to check the source.  And, of those, they were all powdered produces that were implicated.

     DR. FISCHER: [off mike]

     DR. HEUBI: Please identify yourself.

     DR. FISCHER: Larry Fischer.

     You mentioned that several of the publications indicated that they fixed the problem by either using a sterile liquid, or they upgraded production facility.  But did any of them say that they altered the procedures used to prepare the formula in the hospital and fix the problem?

     DR. HEUBI: No.  From my reading of the literature, I didn't see any comment about that specific aspect of changing the way that they prepared it.  There was actually relatively little discussion in these articles regarding--after the outbreak, so to speak.  And this is one area that I didn't see any comment, really, in terms of what they did in-house, in terms of their mechanism of dealing with powdered formula.

     DR. TARR: Tarr--Seattle--ah, St. Louis.

     [Laughter.]

     DR. TARR: Did the incidence of NEC total go down after these outbreaks, or was there inadequate data in the paper?  And, also, you talked about powdered formula versus no powdered formula.  Among the "no powdered formula," was that heterogeneous or homogeneous, with respect to breast milk, or not eating anything at all?

     DR. KLONTZ:  Regarding the first question, did necrotizing enterocolitis decrease?  Do you mean in this particular outbreak setting, or just as a literature--sort of an aggregate view?

     DR. TARR: Total cases of NEC in these facilities.

     DR. KLONTZ:  Yes.  The only article that commented, to my knowledge, on that was Van Acker, who said following the changes made at the firm, product reintroduced in 1999, and then until 2001, no further cases of necrotizing enterocolitis diagnosed in the facility.

     There's relatively--there's very little sort of  post-outbreak surveillance that's discussed in these articles.

     DR. TARR: [off mike] I'm wondering about NEC not associated with--

     DR. KLONTZ:  They do not discuss that.  I can't answer that.

     DR. TARR: And the second question is: the no powdered milk group, that was supposedly protected from the disorder, do we know--is that NPO--children no eating anything?  Versus breast milk?

     DR. KLONTZ:   They don't--again, unfortunately, there's a lack of details on sort of other feeding practice attributes of the unexposed group.  So I can't be too specific about that.  Nevertheless, they shared the same environment.  They were in the same place, over the same period of time that the exposed infants became infected.  That's about as far as one can say.

     DR. KUZMINSKI: Thank you.  Larry Kuzminski.

     Dr. Klontz, in one of your earlier slides of the Muytjens paper from 1983, there was a bullet that says, "E. Sakazakii recovered from prepared formula but not from powdered formula and water."  And you commented on that four to 10 grams of the powdered formula--which you indicated was a small amount--relatively small amount--was used for the sampling here.

     And I relate that observation to what you have in the last slide here, on the second bullet: "More stringent standards adopted for products.  Zero E. sakazakii per 10 grams of product."--a relatively small amount when you relate it back to your observation on the Muytjens sampling--environmental sampling paper published in '83.

     So I'm just wondering: is, indeed, 10 grams, the more stringent sample--quantity for a more stringent standard, when historically the literature would indicate it wasn't found with that amount sampled in the past, and perhaps not being found now?  Just your comment.

     DR. KLONTZ: Yes, that's an interesting point.  That's an interesting point.  And, in fact, today, as some of the talks that we get into shortly on the microbiological aspect will underscore is that generally 100 grams of powder are taken, or 111 in three different sub-samples, and repeated three times.  So much larger amounts than 10 grams are used today , you know, in the process of looking for this organism.

     So one could argue that 10 grams is--you're right--a relatively small amount.  And certainly today all I can say is larger amounts are used, in part because this organism may be uniformly distributed in dried powder.  And if one is going to detect it, if it's indeed present there, one needs to look at larger amounts. It's a good point.

     DR. TOMPKIN: This is Bruce Tompkin.

     During the public open comments, there will be some information relative to the specific plant that was involved in the outbreak as Muytjens has reported.  Okay.   And could you clarify again, then, you're essentially summarizing all the data of cases reported internally, from across the world--you mentioned 48--but within the United States, there's a little confusion as to the number of cases, or clusters, that have occurred in the U.S.  Could you--

     DR. KLONTZ: In the--I don't have the actual count.  I believe you have a line list in your--or, if you don't--in your packet, that actually goes through each of the outbreaks and each of the single cases, as well, as to what country they were from and so on.  So, if that's not in there we can get it to you.

     A quick count?  I know that in terms of outbreaks, there was the Tennessee 2001 Dr. Kuehnert described.  Simmons outbreak was four neonates.  That was also the United States outbreak there.  And then there have been some sporadic individual cases reported in the United States.

     But I think the bottom line is that this is truly an international phenomenon.  And the Muytjens culture study of 1988 showed that, you know, formula from 35 different countries he tested and found a 14 percent positivity for E. sakazakii.  So I think international is really sort of the bottom line here.

     DR. HEUBI: Any additional questions?

     Thank you.

     Now, Dr. John Alexander is going to discuss clinical consequences of E. sakazakii infections.

Clinical Consequences of E. Sakazakii Infections.

     DR. ALEXANDER:  : Good morning.  My name is John Alexander.  I'm an ID trained  pediatrician who works in the Division of Anti-Infective Drug Products, in the Center for Drugs at FDA.  And I was asked to give a presentation on what the clinical consequence of Enterobacter sakazakii infection in infants is.

     Next slide.

     [Slide.]

     So what I'm going to do briefly is give a presentation talking about the three major manifestations of infection that have been identified in infants: neonatal meningitis, necrotizing enterocolitis and bacteremia and sepsis.  And I'm going to spend most of the time talking about neonatal meningitis, because that's--we have the clearest information about it, in terms of the consequences of the disease and the infection.

     Next.

     [Slide]

     Now, overall, neonatal meningitis is a disease that occurs with an incidence of about .25 to 1 per thousand live births.  But most of this is from the usual pathogens: Group B streptococci and Escherichia coli, with a small proportion cause by listeria monocytogenes, as the three major pathogens.

      There are, of course, a lot of other bacteria, other gram negative organisms, other types of bacteria that can cause neonatal meningitis.  In one survey that was done in Dallas, Enterobacter species--and I use "species" because of the fact that this was done before sakazakii was even separated from other Enterobacters--accounted for less than 4 percent of the organisms.  So this is talking about a rare organism in what is a rare disease.

     Next slide.

     [Slide]

     Now, the clinical manifestations of neonatal meningitis are often difficult, just as it is often difficult in neonates, to separate any type of infectious disease one from the other.  There are a lot of findings that are non-specific and may represent multiple different infections, including fever or temperature instability, lethargy or just poor feeding, and some infants, as they become more ill, develop respiratory distress.

     The more specific findings of neonatal meningitis are infrequent, including a bulging fontanelle--so the soft spot on the top of the head starts to raise up.  Stiff neck, or epistotonus--some type of posturing, or convulsions that develop from the infection.

     Next slide.

     [Slide]

     Now, the information that we have on E. sakazakii itself causing neonatal meningitis is based on the collective literature.  And there is a predisposition for this occurring in neonates that are less than 2,500 grams, as a symbol of prematurity.  So that it occurs more often in premature infants.  And about half of the pediatric cases that have been reported, that were summarized by Lai, et al., in 2001, occurred in children that were less than one week of age, and almost 75 percent of all cases occurred in less than one month of age.

     But I would point out that this just represents what is collected in the literature as case series, and this predisposition may be just the fact that we are identifying through epidemiologic outbreaks in NICUs that might sort of push you towards thinking that this is much more of a disease in neonates and isolated to the neonatal intensive care unit.  But there were a couple of cases that were reported in otherwise health term newborns, including a case that was reported of a five-week old who was healthy, left the hospital on time, who at five weeks of age developed neonatal meningitis.  So that is important to keep in mind, that we aren't just talking about a NICU disease.

     Next slide.

     [Slide]

     One of the important manifestations that needs to be kept in mind with Enterobacter sakazakii infection is a predisposition towards developing these large cystic lesions that are believed to be brain abscesses in the brain.  And this is important because in--this is much more likely to represent severe disease, as opposed to other causes of meningitis.  Other causes of neonatal meningitis that were listed--the Group B, strep, E. coli and such--actually cause brain abscess in a minority of cases, but from the cases that we have identified so far in the literature, approximately 75 percent or so are cases that involve brain abscess.  And this is something that's recognized.  There are other bacteria, including one called Citrobacter diversus, that's recognized to cause--to have a predisposition to causing brain abscess, so that when it's there in the brain you have to look at CT scans to see whether these brain abscesses are present.

     Next slide.

     [Slide]

     Now, for neonatal meningitis, the outcome that we're concerned most about is certainly fatality.  And for gram negative meningitis--so, gram negative organisms of any variety that cause meningitis there's approximately a 17 percent case fatality rate.  For E. sakazakii meningitis, from the reports, Lai, et al., had given about 45 percent, and the different reports that looked at the collective literature ranged from 40 to 80 percent.

     Now, it's a little difficult to take this as an indicator of what the overall case fatality rate is, because what you're doing is collecting the literature reports.  But I do believe that we are talking about something that's at least 17 percent, and probably greater.

     Therapy for infants who have neonatal meningitis is usually at least three weeks of IV antibiotics, and would be longer for persistent positive cultures, which would be expected in those cases where you have brain abscesses.  So you are talking about prolonged IV antibiotic treatment.

     Next slide.

     [Slide]

     Now, this study by Onhanand, et al.--and I didn't check beforehand about how to pronounce that name--

     [Laughter.]

     --gives the sequelae for gram-negative meningitis overall in a his institution.  And they had sequelae in approximately 58 percent of those children who survived.  You have children with developmental delays, seizure disorders, cerebral palsy, hydrocephalus, and hearing loss as the main sequelae that are recognized from gram-negative meningitis.  And, again, these are from a variety of other organisms, and I think it is appropriate to show these numbers because, if anything, they would probably be an underestimate of what Enterobacter sakazakii that causes these brain abscesses could do.

     Next slide.

     [Slide]

     So now we move on to necrotizing enterocolitis.  And as an ID-trained pediatrician, I never thought I'd be speaking to a bunch of gastroenterologists and nutritionists about this disease, but here goes.

     It's a disease of the GI tract that is seen mostly in premature infants.  Up to 10 percent of NICU admissions, and approximately 10 percent of affected infants, though, are term infants.  So this is, again, mostly a disease that is seen and recognized in premature infants.  It does, though, occur in some term infants, as well.

     And this is a multifactorial disease, so that there are other factors besides just the presence of organisms that are believed to play a role in this disease.  But the reason that infection in some cases is considered important is because of the fact that there are the outbreaks that occur in association with infections.

     Next slide.

     [Slide]

     Now, it's important to remember that Enterobacter sakazakii is only one of a number of organisms that have been associated with necrotizing enterocolitis.  These are lists from a pediatric infectious diseases textbook of the different bacteria and viruses that have been associated with outbreaks of necrotizing enterocolitis.  So, in response to some of the questions that had been raised earlier about the effect on the incidence of NEC, I don't think it's that surprising that there may not be a change in the incidence of NEC overall from changing whether the exposure to E. sakazakii occurs.

     Next slide.

     [Slide]

     Now, the clinical manifestations of this disease--again, there's a wide spectrum of disease, ranging from a sudden or to an insidious onset.  Again, there are nonspecific findings, as you expect with neonates, of a bunch of different symptoms of infection or disease: feeding intolerance, temperature instability, lethargy.   Some develop apnea and respiratory distress; evidence of metabolic acidosis, unable to maintain their blood glucose, and then a bunch of more specific GI findings, where you have infants who develop abdominal distension, tenderness and erythema, bilious emesis, blood in stools that's either grossly visible or microscopically tested for; and then a bunch of radiologic findings--pneumatosis intestinalis being sort of a specific finding of air within the wall of the intestine that indicates that necrotizing enterocolitis is occurring.  And then when you get to portal venal gas or pneumo turg, in the end you're talking about a--usually a rupture of the intestinal wall and leakage of the gas into the systemic circulation or into the peritoneum.

     Next slide.

     [Slide]

     Briefly, therapy includes discontinuing feedings and nasogastric decompressions following serial radiographic examinations; blood cultures and antibiotics are usually given.  IV fluid and supportive care when there are systemic manifestations of disease, so when the infant starts to appear shocky; and then for advanced disease, surgical intervention to remove part of the bowel that's infected.

     Next slide.

     [Slide]

     Now, the outcomes from both textbooks of neonatal medicine and a recent surgical article give overall mortality rates to NEC of around 9 to 28 percent.  The survival is around 98 percent for medical management--and medical management, again, is the less severe cases.  It's the more severe cases that usually end up going to surgery.

     The surgical mortality rate was approximately 45 percent in the article by Grosfeld, et al., and ranged up to 60 percent from various sources.  And, again, surgical mortality is inversely related to gestational age and size, so it's the younger and smaller infants that are going to have more problems overall and a higher risk of mortality.

     These are other complications of necrotizing enterocolitis: GI strictures occurring in 25 to 35 percent, whether after medical or surgical therapy; other gastrointestinal dysfunction; neuro-developmental sequelae---whether that's directly related to the NEC or related to prematurity and NEC is difficult to tease out; and then short-gut syndrome, which is a syndrome where children have problems and require other forms of alimentation because of the fact that they don't absorb quite enough food from the feedings that they get.

     Next slide.

     [Slide]

     Now, for bacteremia and sepsis, just to give you a separation here, bacteremia is talking about bacteria in the blood, and it can occur with many different infections.  A lot of the bacteremia that is reported with Enterobacter sakazakii is actually in association with the meningitis, which isn't surprising because you have to get the organism into the blood to get it to the brain; but also in cases of NEC.

     Sepsis is actually a clinical syndrome where you're talking about fever, systemic illness progressing to shock, and it is associated with morbidity and mortality.  And from bacteremia to sepsis, there's actually a spectrum of disease, and different organisms can cause different things.   Some organisms--for instance pseudomonas--are more likely to present with sepsis, even in normal hosts.  Other organisms, like Enterobacter sakazakii, are more likely to be organisms that would be identified as bacteremia and in populations that are populations that are predisposed to developing the infection.

     Next slide.

     [Slide]

     So, what we're talking about here is basically an opportunistic pathogen; an organism that isn't usually something that's identified in children who are otherwise normal and healthy.  The predisposing factors that have been recognized include the neonate and premature infants, where meningitis and NEC cases have been seen.  And one of the--what I'm going through here is basically the reports that we have from the literature of infants who were septic, so there were the many reported cases with meningitis.  There was also a separate report of a seven-day-old infant who developed fever and sepsis, but not meningitis, that was hospitalized and, I believe, recovered with antibiotic treatment.

     There are also other--two other reports of children with bacteremia.  One was a three-year-old with a rhabdomyosarcoma who recovered with antibiotics and removal of the lung from which the organism was identified, and what his exposure is is unknown.

     There's also the issues of altered host defense.  The other case report of bacteremia is in a six-month-old who had had an intestinal resection.  So that's probably a potential site of entry for the organism from the intestine.  And this patient also recovered with antibiotics.

     So it's overall, in terms of talking about bacteremia, we have to recognize that it's difficult to just take the incidence of sepsis from other organisms and apply it to this disease.  We are talking about, here, bacteremia with an opportunistic infection, so that you're talking about infants that are sickened in some way, have altered host defenses, or altered immunity that sort of predisposes them to developing infection with this organism.

     In conclusion, Enterobacter sakazakii is a cause of meningitis, NEC and bacteremia--all diseases that are associated with serious morbidity and mortality, and it's found especially in neonatal disease but not exclusive to that population.

     DR. HEUBI: Questions?

     Heubi.  My first questions would be: the last two--the last slide you showed, the patient with rhabdomyosarcoma, and one with short-gut, were either of those patients on powdered formula during the course of their therapy; specifically, the short-gut patient?

     DR. ALEXANDER: I don't know.

     Actually, for the--you're talking about the six-month-old who had the surgery?

     DR. HEUBI: Right.  Right.

     DR. ALEXANDER: She had the surgery, for a jejunal atresia, actually.  And I believe that she was receiving powdered formula.  I'd have to go back and check that.

     DR. HEUBI: Thank you.

     Virginia?

     DR. STALLINGS: Stallings.

     We haven't discussed antibiotic coverage.  Is this a particularly difficult infection to treat once you've identified it?  Because the mortality rate seems to be high, in addition to the brain injury.  What is it usually sensitive to, and why are the kids dying so fast?

     DR. ALEXANDER: Well, part of it is that meningitis, and especially meningitis that causes brain abscesses in premature neonates is more likely to be something that's going to be associated with mortality.

     Part of it, again, is that collecting this information from the case reports, we're more likely to be getting a snapshot of those patients with severe disease.  We don't necessarily have a case series for the experience at one or a bunch of hospitals with this disease where we could feel more comfortable that we are sort of collecting all of the cases.  So, there may be some of the--there may be some bias towards seeing reports of higher mortality, and that's why we're dealing with that.

     In terms of the susceptibility of the organisms, it is susceptible to most of the immunoglycosides, which are typical treatment for neonates.  So, I think that I saw one report for Enterobacter where gentamicin susceptibility for the genus as a whole is approximately 94 to 95 percent.  And from the case reports, I didn't see anything that really spoke to anybody having a problem with treatment, other than a very recent report, about a month or two ago, of an adult who had an Enterobacter sakazakii infection that was resistant to immunoglycoside.

     DR. HEUBI: Margaret?  Oh.  I thought you were going to ask a question.

     DR. BAKER: Baker.

     I wanted to just think a little bit about the susceptible populations.  The original--the FDA "Dear Doctor" letter was specifically for NICUs, and we've seen that several of these outbreaks have been in NICUs, so our sort of--the feeling was that this is a neonatal and maybe specifically for premature kids.  On the other hand, we had--there's been two things that have sort of contradicted that, and that is--there was a slide earlier this morning which didn't show any greater susceptibility with decreasing birth weight.  And then you talked about a five-day-old who was supposedly full term and was infected with E. sakazakii.

     DR. ALEXANDER: It was actually a five-week-old.

     DR. BAKER: Five-week-old--so--and then there have been some of these sporadic cases of older children and adults with E. sakazakii.  So can we identify a susceptible population, and are there other susceptible populations outside the NICU?  That's my question.

     DR. ALEXANDER: Well, I guess that for neonatal meningitis, and for the meningitis manifestations of Enterobacter sakazakii it really does seem to be something that is isolated to those infants that are within the first months or two of age, because for the reports that we've received--for the reports that I've seen, at least, of Enterobacter sakazakii in anybody that's older than a month or two of age--which is around the cutoff where you start transitioning from what would be considered the normal neonatal meningitis pathogens to community-acquired meningitis pathogens.

     After that transition period, you don't really see any reports of people who have meningitis, other than one report where I think there was an infection of the central nervous system in somebody who had some type of congenital malformation of the central nervous system.  So, neonatal meningitis itself is something that is more likely to be seen early on, and more likely to be seen in more premature infants, and that may be why what we're seeing is mostly reports that are associated with the NICU.

     For the bacteremia, I think that we are talking about a population that has some altered host defense, so that you're not really talking about children who are otherwise normal and healthy, but it can be as simple as children who have had previous surgery, as well as children who have immunosuppression, or some other altered host defense system.

     DR. HEUBI: Phil?

     DR. TARR: Tarr.

     I'd like to probe again about the issue of potentially missed cases.  Are there data in NICUs, on a population basis, of culture-negative meningitis that could conceivably have been caused by E. sakazakii, or another organization?  And are there cases of brain abscess caused by this organism in which the CSF was sterile?

     DR. ALEXANDER:   I think, actually--I wouldn't necessarily know from the literature whether there are going to be cases of culture-negative meningitis, or culture-negative brain abscess that could be related, because you wouldn't figure that out unless you got a culture that identified this organism.  I do think that in most cases where somebody has a culture-negative infection, whether the spinal tap is negative or whether the source of an abscess doesn't grow our any bacteria, you do start looking for other potential causes, including tuberculosis, parasitic infections, different fungal infections that can manifest in that manner.  So it's difficult to say how much of these other culture-negative things would be related to that.

     The other difficulty that I have is, again, we're dealing with the case reports.  And we don't necessarily know whether that represents the tip of a large iceberg, or whether what we're seeing as a report is a lot, or a majority, of the cases that have been identified.  I would think that it's more likely to be the tip of the iceberg, because I don't think that the literature reports represent all of the sporadic cases, and we certainly don't get investigations of individual sporadic cases to start looking at things like powdered formula, and what the source of infection would be.

     DR. STALLINGS: Stallings.

     Would you concur with the previous speaker that if you have properly obtained biological specimens--the blood, the CSF, pathological tissue--that we should be able to grow this bug and identify it, you know, in a U.S./Canadian setting?

     DR. ALEXANDER: I mean, I think that the cases where you're talking about isolation of the organism from CSF, or isolation of the organism from blood should be possible.  I don't think that this Enterobacter sakazakii as a subspecies is going to be any more fastidious than normal Enterobacter, and our hospitals have plenty of experience with isolating those.

     The only caveat may be whether the testing is specific enough to separate Enterobacter sakazakii from other Enterobacter cloacae.  So whether you're getting some cases that are reported out as Enterobacter cloacae that are actually this organism.

     DR. STALLINGS:   But the issue of how useful--if we wanted to go back and look at cultured-negative CSF, I mean in a neonatal setting, if you're working up a baby who's become ill, you're going to get, in the U.S. anyway, you're going to get blood and urine and CSF, and those will be managed in a way we ought to be able to identify that it was bacterial, at least, and at least to the species.

     DR. ALEXANDER: And certainly the other things that you look at in the CSF--the white count, and glucose and things like that--should be pointing you in the direction as to whether this is a likely bacterial infection or not.

     DR. BEUCHAT: Beuchat.

     The information that you and other speakers have given this morning on surveys of the dried powder for the presence of E. sakazakii indicate, at least in one instance, up to 52 percent of the samples were positive.

     Given that number of servings per annum is probably a billion or more, the question is why aren't we seeing more cases?  What do we know about the level of virulence of these strains?  Are some avirulent under any conditions, while others are highly virulent under all conditions?  What do we know about the virulence and pathogenicity of E. sakazakii?

     DR. ALEXANDER: Well, I'd say that overall what we can tell is that the virulence of the organism is likely low, until you get to a particular patient that may be predisposed.  And I'm not sure that there's any way, really, to identify all of those patients ahead of time.  And then when you have an isolate that in neonates and young infants, specifically--so children that we're probably talking about less than four to six weeks of age--that they may be predisposed to developing meningitis.  And I think that has something to do with the mechanism of neonatal meningitis overall, which we still have a lot of--we still really aren't very clear about.

     DR. BEUCHAT: Are then all the strains equally virulent?  Do we know that?

     DR. ALEXANDER:   I don't know.  I mean, so far all we've done is sort of identify this organism as a particular species.  If you're comparing the species of E. sakazakii to other species of Enterobacter cloacae, there are certainly indications that Enterobacter cloacae and other Enterobacter species may be more likely to cause bacteremia and sepsis in an older age population because of the fact that it's just isolated so infrequently.  But, again, it's a question--is it that it's isolated infrequently because of the fact that you need a certain exposure and you're only getting it from powdered formula?  Or is it because of the fact that the organism is less pathogenic?  Can't tell.

     DR. HEUBI: Larry?

     DR. KUZMINSKI: Larry Kuzminski.

     I think you've answered partially, in your answer to the previous question, but I look back to part of our charge here--based upon your survey--and I apologize if I'm asking a question that's been at least partially ask before--but, in your opinion, what do you think are the populations of infants at risk to this?

     DR. ALEXANDER: Well, again, I certainly think that the infants who are in neonatal intensive care units, who are premature, are probably a population that's at the greatest risk.  But I do think that there's also a somewhat lesser risk, but a risk of a fairly serious fatal disease--neonatal meningitis--that goes to term infants and probably out to a month to six weeks of age, which is the period that we see neonatal meningitis in.  And then, again, for the older children and te bacteremia, it's certain children with altered host defense, immunosuppression, that are likely to be the ones who are at risk of developing bacteremia from the organism.

     So you have a population of infants that are susceptible because of cancer, because they have central lines for some other reason, that are fed powdered formula and then could develop a bacteremia due to the organism.

     DR. HEUBI: I think we're going to bring the rest of the presenters up, and we could actually pose these kind of questions to them collectively--although if you have a question--so if the remainder of the presenters from the morning, except for Dr. Kuehnert, could please come forward, we'll organize this, and have a little question-and-answer period.

     DR. BLUMBERG: Henry Blumberg.

     One question I had for you: do you know if FDA or CDC, or other groups are trying to do any surveillance to look at rates of necrotizing enterocolitis, specifically, you know, since the CDC report came out in the MMWR, at least at my institution, and I think probably others, there's been a shift away from using--in the neonatal intensive care unit-from used powdered milk, and I think there's a lot more use of the liquid, sterilized product.

     And I was just curious if anybody has any data, is that impacting rates of NEC?

     DR. ALEXANDER: I'm not aware of any.

     DR. HEUBI: So I think at this point--thank you.  I think at this point the committee members can pose questions to any of the presenters from this morning.  And I think Larry's question is pretty relevant, because I think we're trying to define what populations may be at risk, because ultimately we're going to be asked to provide some counsel to FDA about which populations should be protected.

     DR. STALLINGS: Stallings.  So, actually, thinking about that, and thinking about the clinical practice setting, I was sitting here going--we've talked about the pre-term infant in the NICU.  And just to be sure that all of the people at the table may not be aware of how many term infants are NICUs, because of the different kinds of presentation.  So not everybody there is a premature infant with all of the things that go with that, including less well-developed immune systems.

     So then I was thinking, well, the other couple of groups--and I would just like for your response--there's a whole subset of infants and lung children with poor gut function.  And some of these are the ones that have got motility problems we don't understand.  Some of them are post surgical.  Some of them may have been the babies who had NEC and now have short-bowel syndrome--and, again, a group that we've often used special formulas in.

     And then the third group are children who we would recognize as immunosuppressed.  So, discounting the premies, children who might have HIV, or cancer, or even of the new biological agents that are coming out to treat all sorts of diseases we're not sure.  So there's a whole spectrum of children who might be at risk, as we open the umbrella from the case presentation, which was--it was sort of a classic premie presentation.

     So, for the committee, just as we start to think about that, what do you think about those different scenarios, or other clinical ones you might think of from your experience, as well?

     DR. ALEXANDER: Well, again, I think it's difficult to try and define well a population that might be at risk.  Now, it's easier to define the populations that are at risk for neonatal meningitis or for NEC, because what you are usually talking about are more often premature infants, but can occur in term infants.  And then there's a defined window of time that we're usually looking at, something along the line of four to six weeks of age, which is when the neonatal meningitis would usually manifest--whether early-onset, which is defined as just less than seven days, versus later-onset.

     And so for those two diseases--and certainly neonatal meningitis being the most serious manifestation that we've seen of Enterobacter sakazakii--you have what would be an easily defined group that's based on what their chronological age is: about six weeks of age, or exposure within NICUs and prematurity.  So it's potentially the case that you're talking about an infant who is very premature, who might go beyond that four to six week age period and then develop NEC at some point later on because of exposure.

     For bacteremia, when you get beyond that four to six week age population, you are really talking about populations that may have some form of immunosuppression, whether it's due to cancer treatment, whether it's due to an unrecognized immunologic deficiency--and there are several different forms that it could take.  And then altered host defenses.  So if what's happening is that this organism is entering through the intestine, then potentially anybody who'd had prior GI surgery or resection of either  congenital anomalies or for treatment of NEC could be a population that's potentially exposed.  And then also other populations that, for whatever reason, require a central line for treatment.

     DR. KLONTZ: Karl Klontz here.  I'd just like to comment briefly on that issue.

     In looking at the literature again, one of the things I did was to go through the line list of all the cases that were reported--in infants--n=48 cases.--just to look at gestations.  And of those 48, there were five reports that don't comment on how old the baby was, in terms of gestation.  Then I think there were 13 cases that were described as either--quote-unquote--"term," or gestations greater than 36 weeks, or--some of the older reports--"normal baby" at birth.  So there were 13 of those, leaving 30 who fell into the category of prematurity.

     But beyond that, the articles really don't get into the sort of the molecular biology, so to speak, of immune systems.  It's just insufficiently detailed to--in my opinion--to decipher clearly, from an immune status, who exactly is at risk.  I don't think the literature does that for us.

     DR. ALEXANDER: Right.  I think my comments, in terms of the bacteremia and sepsis are meant to be just an extrapolation of what you would think of as the populations at risk for an opportunistic pathogen to cause infection.

     DR. BEUCHAT: Beuchat.

     I tried to find it in the handouts, but can't.  I'll ask--I think it was Dr. Klontz--you had indicated that four cases, median age 74 years--what was the

     DR. KLONTZ: Six cases, I believe it was.

     DR. BEUCHAT: What was the vehicle--do we know what the food of the vehicle was, that may have been associated with those cases?

     DR. KLONTZ: No, I don't think any of the articles described what the vehicle was.  So, no, I don't know the answer to that.

     DR. BEUCHAT: Presumably it wasn't infant food, but--we don't know that.

     DR. KLONTZ: We don't know that.  I mean, the gentleman, 76-year-old, with a history of rectal adenocarcinoma, presumably he was doing reasonably well, but then suddenly came down with, you know, urosepsis.  No comment on dietary factors in that article nor, to my knowledge, the other five.  I don't recall.  I'll check during the break, but I don't recall any distinct dietary information given in those cases.

     DR. BEUCHAT: I guess what I'm really asking: are there other foods that also may be vehicles of E. sakazakii, other than the powdered infant formula.

     DR. KLONTZ: I don't have enough information to answer that question.  I don't know.  I don't know the answer.

     DR. HEUBI: Dr. Fischer?

     DR. FISCHER: Fischer.

     I'm not a physician, so I can ask this question.  Why would you fed the powdered formula in the hospital instead of using the liquid formula? Under what circumstances would you want to take the risk of feeding the

powdered formula?

     DR. ANDERSON: There are certain formulas that are only available in powdered form.  In particular, the formulas for infants with metabolic disorders, the amino acid-based formulas, and some formulas for infants with special medical conditions are only available in powdered form.

     In addition, human milk fortifiers, which are added to human milk for premature infants are available in powder form.  There is also one product on the market that's available in fluid form.  The advantage to added the powder in this situation is that there is a minimal increment in volume that the infant must consume, and that's an important consideration for pre-term infants.

     DR. FISCHER: Well, let me follow up by asking what is the percentage of times that you need to use these special formulas, as opposed to the non-special powdered formula?  You're saying the reason you're doing it is because it's a special case and special diet.  What's the percentage of time that has to be used?

     DR. ANDERSON: I don't have specific information.  It would be small, but I can't say further than that.

     DR. THUREEN: I can tell you that having talked to many of my colleagues across the country, that the use of powdered formulas has evolved over the last five to 10 years.  Many infants can take the regular prepared formulas for preterm infants, but there's a perceived notion that better nutrition can be supplied if you supplement the existing liquid formulas with powdered formulas.  Many times it's given to increase calories or proteins, calcium--whatever--but many units, individual, without any protocols, concoct a variety of different formulations using powdered formulas added to liquid formulas.  And they don't just use more liquid formula, because many of these infants have fluid volume restrictions.  So it's a widespread practice.  And I contacted a number of units around the country when this first occurred, and about half of them stopped using powdered formulas, or had never really used them much and always used ready-to-feed formulas.

     But many other units have, and are continuing, to make their own concoctions, and it's highly variable as to what exactly they're making up, but they individualize it to different infants.  So it's a very common practice.

     And why would they do it?  Well, it's just the belief that if you strategically want a certain type of preparation for infant, you can make it using what's available on the market, and just mixing up what you think makes sense.  It's a widespread practice.

     DR. HEUBI: Margaret?

     DR. BRILEY: [Off mike.]

     DR. HEUBI: Please identify yourself.

     DR. BRILEY: [Off mike.]  Margaret Briley.

     DR. HEUBI: Speak up, please.

     DR. BRILEY: Margaret Briley.

     In regard the population that might be involved with this organism, there's been some part of our American adult group, as well as children, that are still trying to find raw milk products to consume, even though we do not advocate that, and we have rules against that.  But there are rules in Texas where people can produce raw goat milk, for example, and sell it on their place.  And we've had some real serious situations about that.

     Does anybody here know if this organism is present in the raw state?  Is that not where it's, maybe possibly, coming from?

     DR. ALEXANDER: I'm not sure that we have clear idea about the source.  I know that one of the reports--I think it was Muytjens, et al., that tried to look at a bunch of environmental sources; so, looking at soil, looking at a bunch of sources, didn't isolate it from anywhere.

     I think there was something else that I had also seen about rice paddies as an environmental source of where this organism is able to grow.  But I'm not sure how that would be involved, and whether that would be the source at all, of the infant formula exposures that we're talking about.

     I don't know--I can't speak to whether it's something that's identified from cow milk or not.  But I don't think that that was considered to be the source, ultimately, of the organism.

     There was another interesting report, I think, for one of the adults, where it was actually found within the hospital environment, but it wasn't clear as to exactly where.  So that there's the possibility that with the older adults, you may be talking about some sort of exposure through environmental--through an organism that was present in the environment, but it's not clear how that happened, and there are certainly other reports with the different NICU outbreaks that didn't find the organism in the environments.

     DR. HEUBI: Dr. Stallings?

     DR. STALLINGS: [Off mike.]  Stallings.

     I was just going to add [inaudible] so that just so--there are some children with some diagnoses that would have to be on these things almost for their whole life.  If you looked at something like PKU, or MSUD.  But there are many children with GI diseases outside of infancy where we use all of these products, as you were saying, in whatever creative way that the group decides.  So I think there is a lot of use.  There's a lot of use beyond sort of the original intent.  And, you know, again, the case, if I recall correctly, was a product that wasn't available in a liquid or ready-to-feed product.

     The other thing--it has been my impression, although I don't know the data and maybe the manufacturing people will help us with this--is there was an element of the dried products are cheaper and easier to store.  It just takes up shelf space.  So if you have a big setting that you're willing to have a formula room and that sort of thing to mix some of these things up.

     So there are a lot of different issues going on about who's exposed.  But even if we trained all the neonatologists to do this perfectly, there are a lot of other people in pediatric practice who are using all these formulas to put together whatever they think they need for the patient.

     DR. ACHOLONU: Alex Acholonu.

     Has any survey been done on neonates outside the hospital environment, who are breast fed?  And the reason why I ask this question is that they may be getting colostrum from their mother, and that is believe to have agglutinates that may be able to control the disease.

     DR. ALEXANDER: Again, I think that what we're dealing with in terms of what we can report about Enterobacter sakazakii is only the information that we have on these sporadic cases from the literature.  I think it would be very difficult to try and identify and tease out specific factors as to whether children who are breast fed are less likely to have this organism, or more likely to have this organism--and how do you necessarily separate that out from the fact that those children who are breast fed are less likely to be receiving powder or any type of formula at all.

     So, I don't think that there's anything out there that's going to be able to help use in terms of a survey, because I think we are still talking about a fairly rare organism.

     DR. BUSTA: Frank Busta.

     When I look at the Tennessee cohort study, which was given to us indirectly, seven of the nine were on continuous feeding, which I assume is tube feeding.  Is there ever any discontinuous tube feeding?

     VOICE:   Uh-huh.

     DR. BUSTA: So that the other two could have been a discontinuous tube feeding.  Also, that same--seven of the nine never received any breast milk.

     Is there a possibility that the infants that are infected are not exposed to the normal inoculum flora, like the bifidabacter, or lactobacilli, that normally-fed infants are exposed to, and colonize their digestive tract?  Or is there any similar type of data in the other studies that you did internationally that would show that it's tube feeding, and maybe a lack of good competitive flora?

     DR. KLONTZ: No.  Karl Klontz here.

     From the published literature there is really not that depth of discussion--even in terms of how the formula was fed.  I'm thinking back to the Van Acker study, some of the major--large outbreak publications, and I don't recall details on mechanism, or mode of delivering the prepared formula, nor were was there--certainly, there were no features on sort of the microbiology of the intestines, to my knowledge, in those outbreaks.

     DR. ALEXANDER:   I think part of the problem that you're going to get in trying to look at the idea of whether it might be associated with something like the tube feedings, or the plastic tubing, or things like that is when it comes to the disease of NEC, the infants that you're talking about that are more likely to develop that disease are also the infants who are going to be receiving this almost exclusively through some type of gastric tube feeding, and they're not the infants who are going to be sucking on a bottle.

     So it's not going to be possible for the NEC cases that were identified to see whether it's--to try and tease out the differences between whether it may be related to the plastic tubing, versus the powdered formula itself.

     With the meningitis cases, I'm certain that, for instance, that the five-week-old infant that I was talking about was most likely a child who was normal and healthy and wasn't receiving any kind of gavage feedings, so that some of those cases of meningitis, if we could find more information from--you know, from the cases themselves, would probably indicate that those were children who were on otherwise normal feedings, and not related to gavage.

     But it's not something that's clear, that we clearly know at this point.

     DR. HEUBI: Dr. Fuller.

     DR. FULLER: Changing topics a little bit, could you refresh my memory or give us a little--go back over--what information do we have in E. sak in the ingredients used to formulate the powdered formula?  Or do we have that information?

     DR. ANDERSON: I believe information on that topic may be part of the talks this afternoon.

     DR. HEUBI: Rob?

     DR. BAKER: Baker.

     I just had two comments.  One was about breast feeding.  And just sort of a priori, you would sort of expect breast feeding or breast milk to be protective, in that the information did sort of suggest.  So I would think that would be something to look at.

     The other thing I wanted to mention is that there is another population that we haven't really talked about that may be at risk, and that's the graduate of the NICU.  And babies are graduating from the NICU at earlier and earlier stages, so there are some quite premature infants that are being cared for in other parts of the hospital or even at home, and they may be at risk for this.

     DR. HEUBI: Last question

     DR. LEE:  Yes, going back to the earlier question--

     DR. HEUBI: Please identify yourself..

     DR. LEE: This is Ken Lee.  And going back to Dr. Busta's question about feeding by gavage and establishment of favorable flora, is there any attempts in a neonate to try to establish a helpful flora.  Obviously the line of questioning is that if friendly flora is established, then there's less opportunity for an opportunistic pathogen like E. sak to establish itself.  And that kind of thinking leads you to the idea that well, maybe, that ought to be put in the powder itself to help the patient.

     DR. THUREEN: I'd like to just make a comment on that.  This is Thureen.

     I would have to say that most of these infants that are preterm have very altered flora.  They've gotten antibiotics staring at birth--often many courses.  They've gotten medications that have altered the pH of their GI systems.  They've undergone many insults.  So their GI flora is anything but normal.

     I think there are a lot of studies that are now underway, looking at probiotics as a means of establishing a more normal flora.  But I think this population is so vulnerable, just because they're set up, for many, many reasons, for infection from this organism.

     DR. LEE: We had a little conversation over here--the idea, of course, is--that's a very good point, in that perhaps there are things that--any growth condition that would lead to an outbreak of the pathogen E. sak could also lead to the growth of something that's non-pathogenic.  And if one could find a non-pathogen that would inhibit E. sak, then that would also be a helpful thing to be able to do.

     DR. THUREEN: Thureen--one more comment.

     [Laughter.]

     DR. THUREEN: You know, I wonder if the E. sakazakii's not just sort of a marker that's given us an interesting epidemiologic chance to study these infants, because also my understanding is in any infant formula there's very low levels of contamination with other coliforms, staph aureus, and other organisms, and those are very common pathogenic organism for NEC, sepsis, etcetera--and that we are just focusing on E. sak because it's such an unusual organism.  But, in fact, this could be a contributor to a lot of the other infections that we see going on.

     DR. ALEXANDER: Certainly, it seems like there is--it's easier to focus on E. sakazakii because of the fact that there is what appears to be a more clearly epidemiologic association between its presence in the infant formula and lack of identification in other settings.  I mean, for these other organisms that you're talking about--the coliforms, E. coli, klebsiella, the other enterobacter species--the problems that you run into are those are part of normal human GI flora, and so how do you tease out the fact that formula would have been what introduced the organism that caused the disease, as opposed to just passing through the birth canal, or some other source, whether related to what's on the health care worker's hands, or what would be normally the process of sort of GI colonization that, for whatever reason in this infant, leads to a more serious infection?

     DR. HEUBI: I'd like to thank the presenters and the committee members for the lively discussion.  The committee members and the speakers are invited to lunch, behind us.   The guests are on their own.

     [Laughter.]

     DR. HEUBI: And, I forgot--if you haven't found this out already, the restrooms are out here.  If you didn't know already, I think you're probably in trouble.

     [Laughter.]

     [Luncheon recess.]

A F T E R N O O N  P R O C E E D I N G S

     DR. BUSTA: I f you will take a seat we will get started with this afternoon's presentation.

     The committee has received the information requested from Atlanta.  You see the two graphs that we were sent.  This is just for the committee, not for public distribution.  It's unpublished data, not for further dissemination . I don't know how much more that can be emphasized.  It's right across the top of the slide, but it's to help us understand the discussion this morning.  So, please bear that in mind--not for public distribution.

     We also have a table to supplement the presentation this morning, that you received.

     Thank you for the prompt return to the committee.  Brief luncheon period, but we will get on our way.  We'll do our best to stay on time this afternoon.  It's a little tighter.  And we had the good fortune of having a little extra time this morning.  We'll have to be tighter this afternoon.

     The first presentation is on the general microbiology of Enterobacter sakazakii.  This is from Dr. Maria Nazarowec-White.

     DR. HEUBI: As your co-chair, I'd like to remind everyone to speak--into---the--microphone.

     [Laughter.]

General Microbiology--Ecology, Pathogenicity, Subtyping, Etc

     DR. NAZAROWEC-WHITE: How to speak into the microphone.  Can everybody hear me?

     First of all, I'd like to thank you for inviting me to speak about Enterobacter sakazakii.  This is an organism that I spent a number of years studying during my doctoral research.  And at that time--and I'm talking about the early '90s, because I went back to school quite late in life--there was very little information available.  And it would have been wonderful to have had a meeting like this where different people were presenting different bits of information that they have.

     I do notice, though, that in the last couple of years, you do hear people talking about Enterobacter sakazakii.  I work for the Canadian Food Inspection Agency in Canada, and we are already in work planning meetings talking about maybe putting in a monitoring program.  Health Canada is planning to include a method for isolation of Enterobacter sakazakii in its official analytical methods.  CODEX is also talking about this organism, through its committees on food hygiene, as well as the Committee on Nutrition and Foods for Special Dietary Uses.

     Next slide, please.

     [Slide.]

     Now, I'm just going to briefly touch on a number of things, give you a little bit about the history of this organism, and what we actually don't know bout the ecology or the environment that this organism can be found in.  I did a bit of a study on the incidence on the Canadian retail market.  I want to describe some growth studies, looking at generation time and lag time of this organism in reconstituted infant formula.

     I also did a little bit of work on the phenotypic and genotypic characterization of the strains that I worked with.  And, finally, a number of people have asked the question--a number of people have asked a lot of questions this morning--a little bit about the initial work on pathogenicity and virulence factors. The paper is actually just out in the Journal of Food Protection, on the pathogenicity.

     Next slide.

     [Slide]

     Okay.  As we know, until 1980, Enterobacter sakazakii was called "yellow-pigmented cloacae."  However, there was a proposed name change in 1980 based on differences between Enterobacter sakazakii and Enterobacter cloacae, using DNA hybridization studies, biochemical reactions, and pigment production.

     Enterobacter sakazakii has a biochemical profile very similar to Enterobacter cloacae, but unlike Enterobacter cloacae, it's always sorbitol negative, and positive for deoxyribonuclease.

     Next.

     [Slide]

     It think it's time we saw a picture of this organism that we've been talking about all morning.  And someone this morning has said that the yellow-pigmented colonies were difficult to identify.  Well, the yellow pigment has a stronger hue when the organism is grown at 15 degrees Celsius, rather than at 36, and often growth or isolation is done at 36.

     The other thing that we found--and other researchers, as well--that there are two morphologically different colony types.  This slide illustrates a colony that is mucoid, or dried, and it has a slightly--well, it doesn't really show the scalloped edges, but it's a three-dimensional thing.  And when you are trying to touch it with the loop on the actual plate, it's very rubbery, and it's sort of almost difficult to get off the plate.

     It looks sort of like a raspberry upside down, sitting on the plate.

     Next slide, please.

     [Slide.]

     Now, the second colony type is a typical smooth and soft colony.  It is easily removed with a wire loop.  And what also we found is the colonies--the rubbery, scalloped kind--revert sometimes to this type of colony on sub-culturing.  At one point in time, when we first saw this, we were wondering whether differences in virulence or any other phenotypic characteristics would be different between these two morphology types.

     Next slide, please.

     [Slide.]

     Here's a picture--an electron micrograph--of an individual bacterium.  The blue color, of course, has been added.  And, as you know, it is a motile, gram-negative rod.  It grows very easily on laboratory medium.  And another interesting observation was that after 24 hours of growth, all the strains that we had produced a large amount of sediment.  It appeared that--it was really quite interesting--and it contained sort of clumped cells and masses that were sort of stuck together.  I have no idea why that was.  But that's something that could be explored a little further.

     Next slide, please.

     [Slide.]

     Ahh--the environment.  Little is known about the environment.  There was this one study--or it was mentioned in a letter to one of the journals--and it was--now I call him "Mutagens," but I guess it's "Moyt-yens?"  Am I pronouncing it correctly?  He was the one who showed that it could not be isolated from surface water, mud rotting wood, bird dung, rodents, domestic animals--I'm not quite sure which ones they mean--and cows milk.

     Now, aside from the infant formula that we have seen the association there, there is a paper where sakazakii was isolated from a UHT tetrapack box of milk.  I have heard that bottled water--this is certainly not a scientific study--someone mentioned, "Oh, yes, by the way, we did find it there"--and raw ground beef.  Then there were the studies where they were looking at the actual illnesses and meningitis, I guess, cases, where they talked about the dish brush and the blender that the infant formula was reconstituted in.

     So this is certainly an area where research is needed.  When we're looking at the infant formula, I certainly did not look at any of the specific ingredients that go into the formula.  We know that the formula is not sterile, but is the milk?  Does it go through--sometimes the milk will go through a pasteurization process prior to being put into the formula, and sometimes not.  And they are--because it's going for further processing--meaning that it has to go through the powder--to be made into a powder, through the drying, so we don't know whether it's coming from the milk or other ingredients.  Are they sterile?

     Next slide, please.

     [Slide.]

     At the beginning of my research, I contacted children's hospitals and general hospitals across the country to see if their microbiology departments maintained Enterobacter sakazakii strains in their culture collections.  I managed to obtain nine clinical strains.  There was a St. Joseph's Health Center, affiliated with the University of Western Ontario, they sent me two isolates.  These isolates are from a one-month-old child that had had meningitis with cerebral abscess formation.   I received another strain from the Montreal Children's Hospital, and then the Toronto Hospital for Sick Children sent three specimens.  Two isolates were isolated from cerebrospinal fluid, and one strain from a blood culture.

     Each of these strains are from different patients, isolated in different years.  The Laboratory Center for Disease Control in Ottawa kindly provided three clinical Enterobacter sakazakii isolates, but they did not provide any history as to where these strains came from.  So these were my clinical strains that I worked with.

     There are a number of dried infant formula available on the Canadian retail market.  We don't have very many actual manufacturers in Canada.  And although there were no incidents reported in the literature of E. sakazakii meningitis related to infant formula in Canada, in 1990, two incidents of infection were reported to Health Canada.  In one incident, analysis of two cans of formula obtained from the home of the one-month-old infant showed no microbiological contamination.  However, the original can was discarded, and therefore could not be evaluated.

     The second incident involved a neonate in a hospital from a different city.  It appears that in this case, there was a misconception that all powdered formula are sterile.  And, again, we don't know whether the less developed intestinal flora or, as someone this morning described, the totally different flora in these children that are ill.  Even--it sort of emphasizes that we should be very careful in all the procedures we use to ensure as low a microbial load as possible.

     Samples of dried infant formula were obtained from the manufacturers or from retail.   And strains of E. sakazakii were isolated.  Five cans, a retail unit, from five different lots manufactured on five different days were used in my study.

     Next slide, please.

     [Slide.]

     This slide shows the prevalence of E. sakazakii in the formula we evaluated.  The incidence varied from zero percent to 12 percent for Company A.  And we already know that there was the study that was in the literature talking about the 141 cans from 35 countries, and they found sakazakii in 14 percent of them.

     The levels of Enterobacter sakazakii were low, at 0.36 coliform-forming units per can.  However, there was no information on the pathogenicity of this organism, and we wanted to see--learn more about the growth of sakazakii.

     Next slide, please.

     [Slide.]

     Farmer et al., in his initial paper, tested 57 strains for growth.  He found that all 57 grew at 25, 36 and 45 degrees Celsius; 50 strains grew at 47, but none grew at 4 or 50.  Ten Canadian strains--five clinical and five food isolates, were selected, and growth was observed over a temperature range of 4 to 50 degrees.

     Temperature gradient incubator was used.  Laboratory media was spike with a thousand cells, and growth was observed over a 15 to 20-day period.

     Next.

     [Slide]

     This slide shows the minimum growth temperature for both the clinical and food isolates.  None of the tested strains grew below 5.5 degrees Celsius. Now this would indicate that Enterobacter sakazakii would not grow at refrigerator temperatures, which we consider 4 degrees Celsius.  However, in looking at studies of in-home refrigerators, they are well above that 4 degrees Celsius.  They have shown that the temperature range between 7 to 10 is probably what you're going to find in most in-home refrigerators.  And this, in turn, could help Enterobacter sakazakii to grow.

     And this morning we were hearing how there was a study that was done which made up the formula, spiked it, refrigerated overnight, and then put it to hang for four hours.  Well, depending on what temperature that refrigerator was at, if it's in there for 24 hours, would allow the sakazakii to grow.

     In order to evaluate growth in reconstituted dried infant formula, three formulae were selected.  These three had the highest market share on the retail market in Canada at the time.  A cocktail of five clinical strains and five food isolates were evaluated in three different formulae.  The formulae were spiked with 103 CFUs, and incubated at three different temperatures: 4 degrees, 10 degrees and 23 degrees.  Four was considered the refrigeration temperature; 10 was considered a slightly abusive temperature; and 23, room temperature.

     Sampling was done at timed intervals, using direct plating, which provided colony counts for determination of generation time and lag time.  After calculating generation time and lag time, results were subjected to analysis of variants to calculate and determine if there were any significant differences.

     [Slide]

     This graph shows the generation time.  The dark blue is 10 degrees, and the light blue is 23 degrees.  And there was no statistically significant differences found, either among the tested formulae--in other words, formula 1, formula 2--or between the clinical isolates and the food isolates.

     At 10 degrees, the generation time varied from 4.18 hours to 5.52 hours.  Food isolates have a slightly lower generation time--although, again, it's not statistically significant.

     At 23 degrees, the generation time was around 0.67 hours, which translates into 40 minutes.

     The data for 4 degrees Celsius is not shown here.  It was found that it remained at the initial levels that were--that the formula was spiked with, or declined over time.  And, again, this certainly confirms the importance of storing reconstituted infant formula at the correct temperature.

     When prepared bottles are stored overnight at room temperatures, we looked at the data and you could get at least 105 CFU per mil, and this is easily obtained.

     Next slide, please.

     [Slide.]

     And this is the lag time.  Again, there was no statistically significant differences among the three formula for both clinical and food strains.  The lag time varied for from 19 hours--is it backwards?--19 hours for formula in the food, to 47 hours for formula in the clinical.  Again, no significant difference.

     And, again, you can see the generally shorter lag times for the food isolates when compared to the clinical strains. And this is really just sort of initial work, and so only using a very few strains.

     Next slide, please.

     [Slide.]

     Okay--typing systems, of course, are based on the premise that clonally related isolates share characteristics by which they can be differentiated from unrelated isolates.  Methods for identification and discrimination of bacterial isolates have always been divided into two broad categories: phenotype and genotype.  The traditional micro-techniques, phenotypic typing based on the secondary characteristics of bacteria, including biochemical reactions, antibiograms, serotyping, bacteriophage typing.

     We used the API 20 miniaturized biochemical test strip to identify Enterobacter sakazakii. as we isolated it from the infant formula.  And the 18 strains in our studies were grouped into three biotypes.

     Biotype I, included nine strains, and that includes the type strain.  Six strains out of the 18 were inositol-negative, and three strains were VP-positive.  Farmer, et al., also found that 98 percent of his 57 strains tested to be VP-positive, and 25 percent were inositol-negative.

     Next one.

     [Slide]

     Somebody this morning was talking about antibiotics and antibiotic resistence.  Again, this is very preliminary work.  And, as you know, all clinical laboratories do antibiotic susceptibility testing routinely. |We used just the disk diffusion method, using commercial antibiotic disks to determine antibiotic resistence in the 18 strains.  Now, these 11 antibiotics are commonly administered in gram-negative infections, and were evaluated using the Enterobacter sakazakii isolates that we had.

     Included in this list are the gentamicin and ampicillin which, if you look through those literature reports that many people were describing this morning, if you look at the actual antibiotics that were given to some of these infants, the ampicillin/gentamicin combination is often considered the gold standard.  I think it was Robinson that said that.

     Next slide, please.

     [Slide.]

     Now, what I'm showing here is the resistence to the certain antibiotics.  We found that there was one--it was a food strain--that was resistant to ampicillin.  The other interesting thing is that there were 17 strains that were resistant to cephalothin, and there was only one clinical strain that was susceptible to it.  The sulfisoxazole--all 18 strains showed resistence to this antibiotic.

     So when we looked at the antibiotics, 14 strains fell into Biotype I, and this was including the type strain.  One strain in Biotype II--antibiogram II--and two strains that fell into the third category, and one strain was antibiogram IV.

     Now, antibiograms are not a very discriminatory test, and there have been reports in the literature, by Clark, et al., in 1990, that found this same thing; that the strains that she tested were categorized into--there were many strains in certain categories.

     The other interesting thing was if you took colonies that were supposedly clonal on one plate, if you picked five colonies, sometimes you would get two or three different antibiotic patterns.  So--don't know what's going on with that, where we're assuming they're the same organism, where the same antibiogram should come up, it doesn't.

     Next slide, please.

     [Slide.]

     I'm not going to talk about the actual methodology here, but I do want to mention that we did use ribotyping, pulse field gel electrophoresis, and random amplification of polymorphic DNA.  It's come a long way since the early '90s as to some of the work that I did and what is being done now.

     And the next slide shows the diversity among those 18 strains.  It you look at the ribotyping, it was grouped into 10 different ribotypes--those 18 strains.  In both pulse field electrophoresis, and the RAPD, it depending on the restriction enzyme that was used in the pulse field, because you can see with XB-A1, we had 18 different pulsovars, and with SP-E1, we had 17.  And the same thing happened with the random application for polymorphic DNA.  It depending on which primer was used.

     Now, the interesting thing--if you look at the ribotypes--see these three here?  All Number III--these are the three isolates from the one hospital--Toronto sick kids hospital--but they were isolated in three different years.  So, with ribotype, they fell into the same profile.

     And here we have MNW III, IV and V--these are food isolates.  These are all the ones from one company.  Interestingly, they also fell into one category.  However, if you go to where we have only 17 different profiles, again, these two strains--MNW III and MNW IV, both in the pulse field as well as the RAPD, fell into the same category.  So, it was interesting.  And whereas the third isolate from that particular company did not fall into the same category as it did in the ribotyping.  So, really, what I'm showing here is the heterogeneity among the strains we looked at.  There were only 18 strains, but they all appear to be genetically heterogeneous.

     Okay--the next slide, please.

     [Slide.]

     Virulence factors.  Now, I could find nothing that reported anything with respect to Enterobacter sakazakii.  There was one study, where it was a retrospective in a Danish hospital, that looked at enterobacters--not particularly Enterobacter sakazakii.  And it stated in that paper that a portal for entry for enterobacter species, infection can be the gastrointestinal portal.

     So we took that statement and thought, well, we can also find Enterobacter sakazakii in infant formula and, of course, it goes through the GI tract, and in that paper, again, it said that they did find that Enterobacter cloacae produced exotoxins, and E. sakazakii and Enterobacter cloacae are kind of related, so it was decided to explore a little bit on the pathogenesis or virulence factors of this organism.

     We used a suckling-mice assay to test for enterotoxin production, and then three cell culture lines to look and see what the impact was on the morphology of those cells.

     Pathogenicity was assessed with the use of suckling mice challenged intraperitoneally and orally, with both the clinical and the food isolates.

     Next slide, please.

     [Slide.]

     For the enterotoxin production, we found of the 18 strains, four produced and enterotoxin.  Three were clinical, and they were all three from three different hospitals, and in three different geographic regions in Canada.  One food isolate also produced an enterotoxin.  And the type strain was negative.

     Next slide, please.

     [Slide.]

     As far as the tissue culture, we only looked at three isolates.  There was the type strain, and two clinical strains were used.  We used three cell lines: Y-1, CHO, and vero cells.  Once clinical strain was toxic to all three cell lines.  On the Y-1 and vero cells, approximately 75 percent of the cells died.  On the CHO cells, there was cell shrinking and vacuation.

     We also boiled the cells to see if there were any differences there, and the boiling produced the same results on the Y-1 and the Chinese--the CHO cells, however the boiling decreased the impact on vero cells.

     The other clinical strains--the type strain and one from one of the hospitals in Montreal--had no effect on all three cell lines.  Again, these are very preliminary and initial work.  But we wanted to see whether we could, you know, sort of at least see what was going on.

     Next slide, please.

     [Slide.]

     I'm going to talk just a little bit about the infectivity.  IP injections were lethal at 108 CFU per mouse for all strains tested.  Death usually occurred within three days of dosing, and the lowest level causing death by IP injection was two strains: one food and one clinical.  The clinical one was at 105--both of them were actually at  105.  And the clinical strain also produced an enterotoxin.

     By the oral route, only two strains caused death: one clinical and one food.  These results are the first to be reported with respect to any virulence factors in Enterobacter sakazakii.  As you can see, much work remains to be done.

     The paper on the infectivity has just been published in this month's issue of Journal of Food Protection.  So details and tables are in that particular paper.

     Next slide, please.

     [Slide.]

     Just in summary, incidence in dried infant formula in the Canadian market was found to be about 6.7 percent.  We've seen values of 14 percent in the one Dutch study.  Minimum growth temperature is between 5.5 and 8 degrees, and emphasizes the importance of refrigeration.

     Generation time at room at that temperature of 40 minutes.   If we thinking of putting that bottle at the bedside table so you don't have to get up in the middle of the night, or taking bottles with you when you're going shopping and keeping it in the stroller--looking at that time/temperature relationship.

     Four of 18 strains produced enterotoxin.  The enterotoxin was not characterized.  Infectious dose--we have some initial results that maybe we could use in further work--certainly needs to be done in this area.

     And when we're looking at the molecular level, we see that very heterogeneous strains.

     Next slide, please.

     [Slide.]

     So it was very interesting working on this organism, because anything I did was new at the time.  I'm glad to see that there is more interesting in it on the one hand; on the other hand much remains to be learned about this organism.

     And I look forward to the discussion over the next couple of days.  Thank you.

     DR. BUSTA: Thank you very much

     This afternoon, being that we have a fairly tight schedule, I would like to restrict this to questions of clarification, and then we'll have all the speakers up at 3:50 for questions in general.

     Are there questions for clarification.

     VOICE: [Off mike.]

     DR. BUSTA: The question is: a definition of lag time?

     DR. NAZAROWEC-WHITE: Oh, lag time is the time once we spiked the formula--and, as you know, bacteria grows sort of in that S-curve way, well, it's that beginning of that S, before it begins to double, and the doubling is the generation time.

     DR. BUSTA: For my clarification--this is Frank Busta--for my clarification, what was the shortest lag time?  I couldn't tell that from the--

     DR. NAZAROWEC-WHITE: Oh, I know, because it was very--there was no significant difference.

     DR. BUSTA: It looked like one of the food ones was very small column, and I was wondering what the shortest lag time is--for an idea of--now, you were using cocktails in each case, so that you in fact, were measuring both generation time and lag time, you're measuring the fastest strain of the five strains.

     DR. NAZAROWEC-WHITE: That's right.

     DR. BUSTA: While you're looking for that, can you handle another question and look at the same time?

     DR. NAZAROWEC-WHITE: No--lag time--let's see.  I don't have the numbers, but I will be able to supply them for you.  They're in the paper--the actual numbers.  I don't have them.

     DR. BUSTA: All right.  Dr. Fischer?

     DR. FISCHER: What was the dose used to kill animals when given orally?  The oral dose?

     DR. NAZAROWEC-WHITE: Oh, the oral dose? We did--se started off with, you know, 103, 104, 107.

     DR. FISCHER: What dose killed the animals.  You said it killed two strains--I mean--killed the animals

     DR. NAZAROWEC-WHITE: Oh, I'm sorry--107.  Yes.  Orally.  And it was the two strains.

     DR. BUSTA: Any other questions for clarification?

     [No response.]

     DR. BUSTA: All right.  Thank you very much.

     The next presentation will be by Dr. Burr; Microbial Detection--Clinical and Food--from the FDA.

 Microbial Detection--Clinical and Food

     DR. BURR: I want to thank you for inviting me here today.  One of the nice things about going at this time of the afternoon is that essentially everyone's covered just about everything I'm about to say.  So it makes it kind of nice.  But you're not going to learn very many new things; hopefully a few pictures will come in.  But most of the stuff that I'll talk about has been briefly touched upon already.

     I've been given the assignment of discussing the microbial detection of Enterobacter sakazakii, both in food and clinical.  And--

     Can I have the next slide?

     [Slide]

     --what I'd like to do is provide a summary of the methods that are available for isolating and quantifying levels of E. sakazakii in food and clinical samples.  But I am not planning on covering everything that is in the White Papers.  I'll try to go over the high points of the methods.  And as you'll see in your papers, there's a bit more information that's provided.

     Next slide.

     [Slide]

     In terms of presentation, I'd like to divide it into two aspects: one, talk a little bit about the initial isolation reports of E. sakazakii, and then turn our attention more to the development of the quantitative methods for E. sak.

     Initially, the first isolation was in 1980.  And, again, you've heard about all this from previous speakers.  1983--I think we'll go now to just Dr. M--at this point--

     [Laughter.]

     --and in 1984, we had Dr. Postupa and Aldova.

     Next slide.

     [Slide]

     The initial isolation, or the initial description of E. sakazakii being associated with a can of dried milk came in Farmer's initial description of E. sak as a new species, and that was in 1980.  But like the other papers that will talk about the initial ones, there's no real isolation detailed.  Okay?  They talk about it's just being an isolate, but they don't describe how they got it, where it really came from, or what it took to get it.

     The next slide, again, is Dr. Muytjens in 1983, and this is the study that Karl and Maria talked about, where eight cases of neonatal meningitis associated with E. sakazakii.  They isolated the organism several times, as has been pointed out, from the prepared formula, but never from either the powdered formula itself, or the water used in preparing the formula.  Again, no information was reported on the quantity of powdered formula analyzed.  However, as Karl has pointed out this morning, we have had several conversations or e-mails, and we have been informed that the quantity analyzed in this 1983 paper was the 10 gram sample.

     Next slide.

     [Slide]

     In 1984, Postupa and Aldova--again, I apologize if I've done something to the names here--described four strains of E. sakazakii from powdered milk, two strains from powdered milk infant formula.  The only details provided was that it was isolated on deoxycholate-citrate agar, incubated at 37 degrees for 48 hours, but no details on anything in terms of the quantity analyzed, again.

     Okay, and now I'd like to turn attention to the development of the quantitative methods, and essentially we'll deal with three methods, bringing us up to the current one that the FDA is using.  And sort of for ease of discussion, we'll call the first one the "European," then we'll move to the "Canadian" and then we'll move to the FDA method.

     Each one of these--the '97 and the FDA method--is essentially a minor modification of the 1988 paper.  Sample size, sensitivity remains the same and, for the most part, the change has been in the ease of being able to do the method.

     As we spoke about, this method was first described in 1988.  And in referring back to their 1983 paper, they commented that although it was not cultured from the formula powder itself, this might have been due to an unequal distribution in the powder, or it was present in such a low concentration that it escaped detection by conventional methods.  And for a long time we were sort of hindered by, you know, what exactly does "conventional methods" mean?  And that sort of put us on trying to get in touch with Dr. Muytjens as quickly as we could, and that's when we got the information about the 10 gram sample.

     So, based on their early results, in the 1988 work they decided to culture a large quantity of breast milk substitutes for the presence of all Enterobacteriaceae, including E. sakazakii.

     Next slide.

     [Slide]

     So essentially what their method is--and a method paper is probably not the best thing after lunch--but hopefully we'll go through it.  In triplicate, they mixed 100, 10 and 1 gram samples with 900, 90 and 9 mils, respectively, of buffered peptone water, 45 degrees, until completely dissolved, and this gets incubated overnight at 36 degrees.  From these overnight cultures, you innoculate 10 mils from each of the flasks into 90 mils of Enterobacteriaceae enrichment broth--EE broth--and, again, this is incubated overnight at 36 degrees.

     This first incubation is non-selective, and this is the first area where you get an enrichment broth which has some selectivity to it.  In duplicate, innoculate from these cultures in duplicate; innoculate 1 mil from each of the enrichment broths into 20 mils of fluid, violet-red bioglucose agar.  And again incubate this overnight at 36 degrees C.

     Suspect colonies sub-cultured to sheep blood and eosin-methylene blue agar, and the strains were identified via the API 20-E system.  And, as I said, this initial paper was for all of the Enterobacteriaceae, so they did additional testing for E. sakazakii by looking at the production of yellow colonies on nutrient agar after 48 hours at 25 degrees, production of extracellular DN-ase. And a positive alkaglucocytase reaction.

     Next slide.

     [Slide]

     For those of you that are not familiar with what an API system is--and I know Maria talked a little bit about it--API system is a rapid identification process, where essentially you have plastic strips that have 20 small wells consisting of dehydrated media.  You then add your bacteria suspended in saline to each of the wells, and then incubate for 16 to 24 hours, and then look at the color reaction.  So, essentially, you have each one of these wells is an individual biochemical reaction.  And so after incubation, you then end up with--and this is a series of four different bacteria--four different examples--of what you see following 24, or 16 to 24 hour incubation.

     Next slide.

     [Slide]

     What you then do is, based on the color of each of the reactions, it will be scored as either a plus or a minus and so on.  So you very easily just score.

     Next slide.

     [Slide]

     This is just a typical reaction of what E. sakazakii would look like in an API strip.

     Next slide.

     [Slide]

     So then what happens is that you take your score here, and you transform the biochemical reaction; so you had a plus here, a plus reaction, a negative reaction.  You then transform that into a numerical profile.  And that numerical profile you then just take to either a computer program that they provide, or a book that they provide, and that then comes up with an identification of E. sakazakii.  And you have different levels, based on the variation; you have good identification, you have excellent identification; good identification, acceptable.

     Now, sometimes this numerical profile is not considered sufficient enough to be able to identify the organism, so API then recommends additional testing.  And for E. sakazakii, generally the additional test would be testing for the yellow pigmentation, which API has, and I think the Journal of Clinical Micro has listed as 98 percent of the isolates are yellow--produce yellow pigmentation.

     So this is basically what you end up with after doing an API.

     Next slide.

     [Slide]

     Now, in terms of the levels of E. sakazakii in the sample, in this method and, again, in all the later ones that we're going to talk about, the actual level is determined by the most-probable-number procedure.

     Next slide.

     [Slide]

     And I don't want to go into a lot of detail, because I'll confuse myself quicker than I'll confuse you, but it's a statistical method, and it assumes that the bacteria are separate, and the conditions of incubation such that every inoculum that contains even one viable organism will produce detectible growth.  And it's based on the number of positive samples from each of the series of triplicate cultures of the three inoculation levels.  So essentially what you're doing is you're scoring the number of positives in the three 100 gram samples, in the three 10 gram, and in the three 1 gram.  And the tables that you go--the standardized table--and I'll show that in a minute--provides you an MPN number--most probable number--plus a 95 percent confidence interval.

     Next slide.

     [Slide]

     This is a typical MPN table.  And I think this is actually taken out of the BAM manual, and this is for a three-tube at an inoculation of .1, .01 and .001 gram.  And what it give you--as I said, it gives you an MPN per gram in the 95 confidence level.  So, essentially, you look here and you're saying how may tubes of the three were inoculated with the .1 gram are positive?  So here you have one positive, zero positive, and this gives you an MPN number of 3.6 MPNs per gram, and then here is your 95 confidence level.

     Now, down here it describes how we will go from this standardized table to go into an E. sak of an MPN per hundred gram.  And essentially what you do is you take the MPN per gram from this table, you divide by 1,000 to adjust for the larger sample size.  So that gets you down to these sort of values here.  And that would be giving you an MPN per gram.  And then you simply multiply 100 to obtain per 100 gram.

     So, in this case, if you had 1.00, you would have .36 MPNs per hundred grams.

     Next slide.

     [Slide]

     The results of the European survey--and these, I think, have been talked about I think twice already, maybe three times.  But essentially, from the 35 countries, 141 powdered formula samples were analyzed. E. sak was isolated from 20 samples, from 13 of the countries.  And the levels recovered using the MPN was .36 to 66 colony forming units--or CFUs--per 100 gram.

     And the lowest level of detection reported in this method was 0.36 CFU per 100 grams.

     Now, on the next slide I want to show a little bit about where that comes from here, and I want to distinguish sort of a 0.36 per 100 gram detection limit, versus a 0.31 per 100 gram--actually, the lowest possible limit.

     [Slide]

     If you take this chart, in order to get this .36 per 100, that's essentially you have one tube out of the--of the largest sample.  So one of the 100-gram sample is positive, and then the 10 gram and the 1 gram are both negative.

     It's theoretically possible that you could have zero out of three in the 100 gram, zero out of three in the 10 gram, and one out of three in the 1 gram, giving you the lowest possible level of a .3 per 100 grams.  But most of the papers that reported, this is the number--where they're getting that one particular number that it's coming from.  Okay?

     Next slide.

     [Slide]

     In 1997, Maria--as she discussed--and this we'll call the Canadian method--made modifications of this initial method.  And, essentially, they just--the dried infant formula was suspended in sterile water.  Suspect colonies from the VRBG plate sub-cultured to TSBYE agar.  And, again, they're just very minor modifications.  And as in the previous study, the API 20 was used for confirmation, and no additional biochemicals were used.  The levels were again determined by MPN, and again you're left with the same sensitivity: .36 per 100 grams.

     The results of this study: E. sak was isolated from eight of 120 cans; this representing five different manufacturers.  And, again, the level reported is the .36

     Next slide.

     [Slide]

     Now, in 2002, the FDA was charged with developing its method.  And rather than reinventing anything took from the Canadian method and, again, just made some minor modifications.  And what we did was went to direct spreading or streaking over the overnight EE broth, rather than the pour plates.  The pour plates are a bit laborious in order to set up, so this was a much easier method of getting the isolated colonies.

     Five presumptive colonies are  sub-cultured to tryptikase soy agar, and incubated at 25 degrees C for 48 and 72 hours.  And here, again, at the lower temperature where we're looking for only yellow-pigmented colonies from the TSA plates are then further worked up in the API 20 system.  Like the Canadian method, we do not do any additional biochemical tests, and we just rely on the results from the API 20.

     The level of detection by MPN, again we report as the most likely one as the .36 per 100 gram.  And we can detect levels of E. sak much lower than recommended--by the recommended FAO level of 3 CFU per gram of powdered infant formula.

     These are just some pictures that have been provided from Sharon Mallow who was c--author in producing this method.  This just shows the mixture of the powdered milk in the flask.

     Next slide.

     [Slide]

     These are an example of the typical colonies of E. sak on the VBGP.  And, again, you're looking for the purest purple colony, surrounded by a purple halo of precipitated bio-acids.

     Next slide.

     [Slide]

     And this is just the description of the yellow pigmentation that you get at room temperature.

     Next slide.

     [Slide]

     Just briefly want to touch on clinical isolation, and this was one of the questions was brought up earlier this morning.  E. sakazakii is isolated from clinical samples using standard methods for the isolation of Enterobacteriaceae.  There's no special media has been developed for E. sak, and it grows well on all the standard media that is used in clinical laboratories.  And for the most part, when you read the papers, they don't even describe the method.  They just say "cultured from blood," "cultured from spinal fluid."  And in talking to several friends who do clinical microbiology, again their comment is you just follow the normal routine for blood cultures, and that there's nothing different, nothing unique.

     You can confirm with either API 20, as we've done, or there's an enterotube 2 system.  But, again, it just follows the standard methods for identification.  And essentially because you're dealing for the most part with supposedly sterile sites, again much easier identification process.

     So then, finally, and just in conclusion then, the procedures E. sakazakii from powdered formula and clinical samples: follow the standard microbiological methods for the isolation of other members of the family Enterobacteriaceae.  Normally, sterile clinical samples pose no major problems for isolating E. sak.

     Because of the very low levels of E. sakazakii in powdered formula samples and its non-random distribution in the powder, larger quantities and sub-samples should be cultured for isolation.  And in both clinical and food microbiology laboratories, appropriate incubation times and temperatures should be applied if the diagnostic tests were precise.  And, again, this appropriate temperature would be going down to the lower temperature for pigment production.

     I think that's the last slide.

     So, hopefully that--as I said, a lot of the information was covered in some of the earlier talks, but hopefully this gives it a bit more of a clear picture of what exactly is done to get it out of these food samples.

     Thank you.

     DR. BUSTA: Questions--preferably for clarification.

     Dr. Blumberg.

     DR. BLUMBERG: Yes--Henry Blumberg.

     Do you know--generally in the clinical micro lab they're not using an API strip to identify gram-negative bacteria, and most of the identification of susceptibility is done by automated methods like Vitec or Microscan--things like that.

     And do you know how well those identify Enterobacter sakazakii, versus, you know, other enterobacter species, like cloacae or things like that?

     DR. BURR: I don't, actually, because the only thing that I got from the two friends that I had was just that, "We just do the standard way."  So I'm assuming that it's easy, and that the automated ones do pick it up, but I don't have the experience with those.

     DR. BUSTA: Other questions?

     Dr. Baker.

     DR. BAKER: Baker.  This morning people talked about difficulty picking out these organisms among a whole lot of other organisms.  Your slide certainly didn't show that.  Is that a problem or not?

     DR. BURR: Well, in this only, hopefully it's the combination of the EE broth plus the selective plate that actually is cutting down on whatever else is in there.  So we're really trying to do this--the selectivity of the method removes a lot of the other bacteria.

     DR. BUSTA: Other questions?

     Dr. Fuller.

     DR. FULLER: I apologize for what may be a very stupid question.  When you started with the European method, I think it said triplicate runs using 100, 10 and 1 gram.  Again--pardon what may be a pretty stupid question.

     Are we talking about--

     DR. BURR: If I can't answer it, you're really setting me up here, now.

     [Laughter.]

     DR. FULLER: Well, I'm just asking--is this from a single can, or are these nine different--you know, is this for a lot?  What are we sampling?

     DR. BURR: It's for--you take--out of one can you would take--

     DR. FULLER: Each of those.

     DR. BURR:  --for each of those.

     DR. FULLER: Okay.

     DR. BURR: So you would take a total of 333 grams out of one can--

     DR. FULLER: One can.  Okay.

     DR. BURR:  --and do it in the separate things.  Oh, that was a good one.

     [Laughter.]

     DR. BUSTA: Dr. Acholonu.

     DR. ACHOLONU: You said that there is no special medium or media for isolating E. sak.  Is there any special need for developing one, since you have indicated that it grows in different kinds of media?

     DR. BURR: It doesn't appear that there is a need for one.  As I said, for the clinical, it will grow on any of the media that's used for enterics.  And so there's been no need to actually get one that's just specific for E. sakazakii.

     DR. BUSTA: Dr. Tompkin.

     DR. TOMPKIN: Yes--Tompkin.

     Due to  the low prevalence of this organism in the product--you make a statement--or a statement is made that it is "non-randomly distributed."  How can you differentiate low prevalence and just failure to detect it, versus whether, in fact, it is non-randomly distributed.  Does this have to do--did you actually go into a large quantity of a given lot and try to determine random distribution, or is this just an assumption.

     DR. BURR: It's just a generic way of--

     DR. TOMPKIN: It's an assumption.

     DR. BURR: Right.

     DR. TOMPKIN: Okay.

     DR. BUSTA: I have a question--this is Busta.

     When you're doing a normal Enterobacteriaceae enrichment, and then you go on to an Enterobacteriaceae agar, what portion of the colonies that you're selecting would be sakazakii?  What portion of the colonies?  Do you run all colonies from that enrichment onto the API?

     DR. BURR: No, as you--you're just taking--when you go to that second plate--okay--when you're going from the broth to the VRBG plate, then you're taking--you're looking for the purple colonies with the haze around them.  So, again, you're picking what you think are going to be those colonies.  Okay?  So you're looking for typical colonies of E. sak on the selective plate.

     DR. BUSTA: Dr. Tarr.

     DR. TARR: I have two questions that straddle the question and clarification border.

     First, when you state your sensitivity is 3 or 3.6 colony-forming units per gram--

     DR. BURR: 100 grams.

     DR. TARR:  --per 100 gram--do you ever normalize that to the ambient flora?  I can imagine one E. sakazakii in a kilogram being detected if there's nothing else there when you amplify it as if it were coming our of a blood culture.  And I can imagine many E. saks being swamped by ambient flora in the grow-up stage.

     That's my first question.

     DR. BURR: I'm not sure if I--again, I'm not sure if I get that question.

     DR. TARR: Because, again, you're growing it up--

     DR. BURR: Remember, you're looking for just growth in that one particular tube.  So you're not looking for any particular level within that tube when you're doing the MPN procedure.

     DR. TARR: But you have a subsequent step when you have a sample--the sampling comes out on your plates, looking for the yellow colonies.  If you have many additional organisms there, you might miss the low frequency E. saks.  If you have few other organisms there, you might get them.

     DR. BURR: Right.  But again--

     DR. TARR: It's a variable that's not--is that taken into account?

     DR. BURR: Right.  Remember you're doing that--the hundred grams is first going in to a non-selective, and then you've got selective.  So you've got two, really, grow-outs--

     DR. TARR: Right.

     DR. BURR:  --that are occurring before you're going to any plates.  So that's supposedly going to bring up the levels of any low levels of bacteria, to bring it up to where you've got lots in those cultures.  So, essentially, you've got 48 hours' growth is what you're actually putting on plates.

     DR. TARR: But that will be limited by the other organisms in there, won't it?

     DR. BURR: And everything should grow.  In at least the first one, you're essentially allowing everything to grow.  That initial one is non-selective.  Everything is going to grow out there.  And then when you take the smaller portion of that to the enrichment, then you're more selective for the E. sak colony.

     DR. TARR: Okay.  And that laterals into my second question.

     Have you or anyone else explored the thermal tolerance of this organism, as an initial selective step, where the first incubation is perhaps done in 45 degrees--

 

     DR. BURR: We have not net--

     DR. TARR:  --to give competing flora a penalty/

     DR. BURR: Yes--we have not yet.

     DR. BUSTA: Thank you very much.

     To continue on, with Dr. Buchanan, talking on resistence--thermal and otherwise.

 Resistence--Thermal and Other

     DR. BUCHANAN: Thank you.   Okay.  I've got a microphone and I'm armed.

     [Laughter.]

     What I'd like to do today is talk mostly about thermal resistence of Enterobacter sakazakii.  I have a few additional items that I want to mention in passing; one that may help address one of the questions that came up earlier today.  And in talking about resistence, I'm talking now about resistence to the type of things that this organism might encounter during its manufacture or distribution.  I'm not going to be talking about antibiotic resistence, which was covered earlier.

     Next slide.

     [Slide.]

     And I'm going to break this up into the--a quick look at a number of studies that have been done, first at a laboratory level, and then at a pilot plant level.  I am going to be focusing a great deal on the latter study, that has been completed.  It has been submitted for publication, but will be relatively new data to the members of the committee.

     We have had two studies done--laboratory trials.  They were done by Maria, who talked to you earlier.  And then Sharon and I did one using a somewhat different technique.  And just so you have an idea of what are covered in these, Maria's used a stainless steel tube and a constant water bath as the heating system.  It used five pooled food strains and five pooled clinical strains, in separate trials.  And in that kind of a study, the most resistant organism is what comes out at the end.

     We used a submerged coil apparatus .and 12 individual strains.  A submerged coil apparatus is a fairly sophisticated instrument for determining thermal resistence, allowing holding times in factions of a second.

     Next slide.

     [Slide.]

     Before going on, I'm going to be mentioning a couple of terms.  And, knowing that this is a mixed group, for the food microbiologists, I'm sorry to bore you--the others, two terms that I will be using: "d-value," is the time at any given temperature that would be needed to reduce a microbial population by 90 percent.  And it's a common term that's used for measuring the relative thermal resistance of an organism.

     A second term that's used commonly is referred to as the "z-value."  This is a value that--what change in temperature, what increase in temperature, you would have to have in the processing in order the decrease the d-value by 90 percent.

     So there are two ways of describing the time-temperature relationship of the organism's resistence.

     Next slide.

     [Slide.]

     And, typically, when we do a thermal resistance curve what we do is we do a survival curve.  These are two examples.  They cover the extremes that we saw with 12 individual strains.  The organism on that has a d of 591 is the most resistant strain that we ran into.  And the one on the left, with a d of 30.5, is the least resistant strain.  And the d-value is simply the slope of those inactivation curves.  That's what we're really measuring in this case.

     So those are what I'll be referring to as I go along.

     Next slide.

     [Slide.]

     We looked at the 12 individual strains and they fell into two distinct groups as we examined them.  They fell--half of them fell in the group there on the far left.  Those six strains had d-values actually less than 35 seconds.  So they were very sensitive.  The group on the right all had d-values greater than 300.  They range from 320 up to about 590.  And so we found two distinct populations--in itself is very interesting.

     Next slide.

     [Slide.]

     Trying to put this in perspective so that you can sort of judge this against other pathogens of a similar nature, we did have some exact data that was generated under the same exact conditions, and so I plotted these so you can get a relative idea.  The Enterobacter sakazakii 607 was the most resistant strain we ran into.  And, like I said, it had a d-value of around 590.  So it bracketed at one end.  And the Enterobacter sakazakii 51329 was very heat sensitive and it bracketed the others.  The other strains that are of note here: this is E. coli 0157, Klebsiella pneumoniae, Salmonella Hartford, which is a relatively heat resistant salmonella; a biotype-1 E. coli, and Enterobacter aerogenes.  And then this, here, is the pooled strains from Maria's study.  So, again, it covers a wide range and sort of behaves like other Enterobacteriaceae do.  They, themselves, often have a distribution of thermal resistences.

     Next slide.

     [Slide.]

     Now, this is the--when you do a thermal resistence determination, and you do it at different temperatures, and then you plot the d-values you calculate on a log scale.  Typically, if it follows the rules that everyone says it's supposed to follow, it winds up producing a straight line.

     Well, Enterobacter sakazakii--this is strain 607--did a very nice job of following the rules.  It's just--I've never--

     [Laughter.]

     --I couldn't draw a better line if I did it on purpose.  Each of these points represent at least three independent trials.  I mean, it just fell out beautifully.  So this is--its d-value's over a--almost a 15-degree range.

     If you then--well, you're not supposed to--

     Can I get the next one?  Next slide?  Oh, that's this one.  Okay.

     Just to show you that the gods do shine down on you sometimes when you're doing experimental work, when Maria did her pooled food isolates, and pooled clinical isolates, She found that there were no significant differences between the two groups, and that they had an overall d-value of 5.8 degrees Celsius.  And when we did it by an entirely different method four years later we came out with 5.6.  I mean, you can't get much closer than that.

     Next slide.

     [Slide.]

     Going back to the z-value--and while you're not supposed to extrapolate z-values past your experimental ends, we did anyway as a way of predicting how the organism should behave when you go up to even higher temperatures.  And I might note going above higher temperatures in the apparatus we used is virtually impossible, because you're down at fractions of a second.  So we did just simply extrapolate this starting at the 70, which was our upper end, and it basically suggested that when you get above the temperature of 70, inactivation becomes almost instantaneous.

     But following the linear relationship, for example, to get a 5-d inactivation--which is something we typically would shoot for with these kinds of treatments--a pasteurization-type treatment--you can see that by the time you're up to 80 degrees, to get a 5-d kill you should be, at this point, at about .7 seconds.  And if you go up even higher, you see it becomes literally a small fraction of a second to get that kind of inactivation.

     So one of the things we wanted to check out was whether or not this actually occurs in reality, when you're dealing with an infant formula, and so we designed a simple experiment where what we did is we took a commercial dried infant formula.  We inoculated it with a concentrated culture of Enterobacter sakazakii 607, which was, again, our most thermally resistant isolate.  We did it under a condition where we maintained the dry nature of the product.  We pelleted a colony--basically, we pelleted about 1015 cells and then distributed it throughout a large volume of formula, so we had a dry formula with approximately a million cells per gram--or would produce a million cells per gram when it was rehydrated.

     We then distributed that inoculated formula into standard plastic baby bottles, following the manufacturer's instruction on how to weigh it out, with the appropriate volume.  We then added preheated water to those bottles; capped it, agitated the bottles--the formula--as one would normally do, shaking it periodically; and we allowed it to stand for 10 minutes, and then we sampled the bottle and determined the remaining cells to see how much inactivation we got.

     Next slide.

     [Slide.]

     Just to show you what the heating looks like, we did a series of temperatures in approximately 10 degree Celsius ranges, starting at boiling water and then going down to 50 degrees Celsius.  And then our control was just simply room temperature water that was added to it.

     Next slide.

     [Slide.]

     So, basically, we have the control is on the far left.  This is room temperature water added to the formula.  You can see that when we added them back we had approximately a million cells per gram.  And at 50 we got a very small degree of inactivation.  At 60 we got approximately a little over one log cycle inactivation.  And then, as predicted by our original studies in model systems, at temperatures at 70 above, we got greater than a four log kill.  We basically got below the limit of detection that we could use.  We didn't try to go into an NPN at this point, but certainly we got a very substantial inactivation.

     Next slide.

     [Slide.]

     We also wanted to have some information on what would be the impact of this type of heating on nutrient content.  And so we did have the same procedure done by our nutrition analysis center in Atlanta.  They basically did the same procedure s we did, except they only used boiling water as the single temperature they examined.  They did that because that would have been the worst case in the scenario we set up.  We did the analysis in triplicate, using four different commercial infant formulas.  And I might note that--I'm going to have to explain how the results are expressed, but they were all normalized against units per hundred calories, depending on what nutrient we're analyzing.

     Next slide.

     [Slide.]

     Now, this is a very busy series of slides, but I do want to point out what you should be looking for.  This is the nutrient being analyzed. This was the four commercial formulas being analyzed.  Within each of the boxes there are four values.  This is the value on the label.  This is the value that was found when the product that was rehydrated with room temperature water was analyzed.  This is the value that was found when it was rehydrated with boiling water.  And this is the change between this and this.

     So you're basically looking for two things when you're looking; you're looking for the differential between these two and then you're looking for whether or not what was remaining, versus what was on the label.  So, in this case, the addition of boiling water produced a 3.7 percent decline in the nutrient, but it's still well in excess of the reported value on the label.

     We've done this now for a series of them, and basically here very little change in vitamin A, very little change in vitamin D, E, K, thiamin.

     Next slide.

     [Slide.]

     Riboflavin--again, you know, we see some fluctuation plus or minus between the two groups, but basically little change; little change would be 12.  Niacin--again, little change; folic acid, little change.

     Next slide.

     [Slide.]

     Pantothenic acid--actually, a small increase pretty much across the board.  Biotin--little change.  The only nutrient we found that was a significant decrease, in terms of the concentration across the board was with vitamin C.  This would be expected.  This is the most thermally sensitive of the nutrients.  In some cases it did fall below the label value.  In other cases, even though there was a decrease, it was still above what was indicated on the label.

     So, overall, with the exception of vitamin C, the addition of boiling water had really not much impact on nutrient content.

     Next slide.

     [Slide.]

     There have been only, as far as we can tell, only one study that was done at a pilot plant level on Enterobacter sakazakii.  This was done by Maria also.  It used pooled strains, we think--and, Maria, if this is a question you can help answer that.  It was unclear.  And it was done with an HTST pasteurizer.

     Next slide.

     [Slide.]

     It was done in a manner that bracketed the different times and temperatures associated with pasteurization.  And I've tried to take what was a great deal of information and reduce it down to a single table.  But basically, this value you see here is the percent surviving population of the initial inoculum.  So you can see here with a three-second heating at 63 degrees C you got about a 60 percent reduction, down to, you know, several log cycles when you get down here--and that it follows pretty closely what we would expect in normal time-temperature relationships.  That is, it's not a very heat sensitive organism, and normal pasteurization would certainly knock it out.

     Next slide.

     [Slide.]

     Okay.  Let's switch to other treatments or resistences.  And when we're talking about Enterobacteriaceae as general--and it's important to keep in the back of your mind that Enterobacter sakazakii is a normal Enterobacteriaceae.  It is one of the fecal coliforms that we have a great deal of experience with in general terms.

     Next slide.

     [Slide.]

     and we know, in general Enterobacteriaceae are not heat resistant.  They can be moderately acid resistant, particularly if they have been pre-adapted.  Conversely, they can become moderately alkaline resistant if pre-adapted.  They have low to moderate chlorine resistence; low to moderate irradiation resistence.  They will remain viable in refrigerated and frozen products for extended periods, particularly if the pH is neutral.  And then, finally, they have moderate to good resistence to drying.

     Next slide.

     [Slide.]

     Now, when you get specifically to Enterobacter sakazakii there is virtually no information available in the literature specifically related, and there's very little when you start looking at the distributions of resistences the way we did with the 12 individual strains.

     Just summarizing very quickly what we found, the isolation of it from a variety of dried foods indicates that it's probably resistant to drying.  And I'll come back to that.  We did find one paper where it tends to be found after treating seeds that are going to be used for sprouting with chlorine.  This is one of the organisms that remained on the dried seeds, and it would be indicative of at least having moderate resistence to chlorine, though this would have to be confirmed.  And, just an odd thing, it seems to be pretty sensitive to kitazans, which is used as an inhibitor.

     Next slide.

     [Slide.]

     The one additional piece of experimental information that we do have and can share with you is this slide--when we prepped up the samples--the

dried infant formula samples that we used for the baby bottle experiment I showed you--we did keep the leftover formula, and we have been periodically sampling it over the course of almost a year now.  And this is the survival in that infant formula.

     Again, we started with about a million per gram upon rehydration, and then we've been following it over the course of nine months.  There was an initial die-off in the first four months of about two log cycles--two-and-a-half log cycles.  Since that time, it has basically maintained itself as a steady-state level.  We anticipate if its behavior is like most other Enterobacteriaceae that this value will change little over the next year or two.  And it's worth noting for any of you that follow archeology, they have opened up tombs in Egypt that have been basically sealed for 10,000 years, and they have found fecal coliforms.  So, in a very arid and dry environment, don't expect that it's going to disappear.  It will probably hang around for at least the shelf life of an infant formula.

     And I think that's the last slide.

     Summary--this is not a particularly heat resistant organism.  There seems to be a substantial diversity in the thermal resistence among the strains, and this seems to be in keeping with the substantial genetic diversity that you've heard discussed in other attributes today.

     There's a good agreement among the studies that have been done.  There's been a limited number of studies but, I mean, they've matched up, despite the fact that they were done in different ways, the agreement is great.

     Inactivation at temperatures above 70 degrees occurs almost instantaneously, and certainly within a few seconds.  And then specific information on the organism's resistence to other factors or stresses it might see in a food-processing type environment, or food preparation environment are generally lacking, though I would say that every indication is that it will survive dehydration for extended periods.

     Thank you.

     DR. BUSTA: Thank you, Dr. Buchanan.

     Are there questions for clarification?

     DR. FULLER:  We asked it here, and I just want to make sure. On your resistence to dehydration slide, that was stored at room temperature?

     DR. BUCHANAN: Yes.

     DR. FULLER:  Is that a correct assumption?

     DR. BUCHANAN: Yes, that's sitting on a lab bench in the lab.  It's sitting at room temperature.

     DR. FULLER:  Right.

     DR. BUCHANAN: It would probably show it down if we refrigerated it, but this is--we tried to mimic conditions that would occur in the home.

     DR. BUSTA: Dr. Moyer-Mileur.

     DR. MOYER-MILEUR: Moyer-Mileur.

     I have a question concerning your results and how they relate to the USDA letter that went out to health professionals where it advised not using boiling water to reconstitute powdered formula, one, because of loss of heat sensitive nutrients, which your results would say is not the case, except for vitamin C, as well as the inability to assure adequate destruction of E. sakazakii.

     From your findings, would you say that those are no longer concerns?

     DR. BUCHANAN: What I would suggest is when that original was put out, there was a lack of data upon which we could evaluate the efficacy of that treatment.  Based on that, these experiments were done.  I would say that the results of these suggests that one, nutrient loss does not appear to be an issue--or not a substantial issue, and that at temperatures above 70, it is a viable means of reducing the level that may occur within a dried infant formula.

     DR. MOYER-MILEUR: Okay.  Thank you.

     DR. BUCHANAN: The only issue that was not there is that we still have not completed studies on the extent of clumping that might occur.  Those are being evaluated.  Our problem right now is we don't have a standard for what "clumping" means for actually measuring clumping.  We have empirical observations at this point that it does not appear to be a major factor, at least in most formulations, but we don't have an objective measure of the extent of clumping.

     DR. BUSTA: Dr. Acholonu.

     DR. ACHOLONU: Just for curiosity--Alex Acholonu--I saw a slide--I think it was an electromicroscope slide by one of the previous presenters.  And then I've seen a slide that you showed of E. sak.  Some look like they have--

     DR. BUCHANAN: By the way, that wasn't E sak.  Actually, that was E. coli.

     DR. ACHOLONU: Oh, I see.  Okay.

     DR. BUCHANAN: Okay?  That was just a general Enterobacteriaceae.

     DR. ACHOLONU: Okay.  Well, anyway, my question still is: do the E. sak organisms have flagella?  Are they flagellated?

     DR. BUCHANAN: Mm-hmm.  Yes.

     DR. ACHOLONU: Okay.  That's what I wanted to find out.

     Also, are they supposed to be facultative anaerobes, or just anaerobes?

     DR. BUCHANAN: They're facultative anaerobes.  They grow best in the presence of oxygen; can grow quite nicely in the absence of it, though.

     DR. ACHOLONU: Thank you.

     DR. BUCHANAN: They have all the normal characteristics of an enterobacter.

     DR. BUSTA: I have a question, Bob.

     How did you inoculate the infant formula with that 106?

     DR. BUCHANAN: How did we--okay.  What we did is we grew up a very large culture of Enterobacter sakazakii.  We spun it down into the centrifuge.  We took it up into a small volume of water.  We then added it, dropwise, to the infant formula that was in a--I think it was a four-liter flask, as I remember.  After adding a drop, we would then shake the material to get it transferred as beset we could.  We then continued that process until we added the entire inoculum to make sure that we had a reasonably distributed material.  We had added a small amount of a blue dye to the inoculum so that we could see how the color was distributed throughout.  And while I can't say that it was homogeneously distributed, it had to be pretty close, because we got a nice flat color across the entire lot.

     DR. BUSTA: Still could have been clumps, though.

     DR. BUCHANAN: There could have been very small clumps, but we got it distributed as beset we could.  And it's interesting to note that as we pulled samples out over time, the amount of fluctuation or differences between the levels we saw was very, very small.

     DR. NEILL: Dr. Fischer?

     DR. FISCHER: You did this experiment where you inoculated infant formula and then looked at the rate of killing?

     DR. BUCHANAN: Mm-hmm.

     DR. FISCHER: The strain that you used, was that the most resistant--thermally resistant strain--or-

     DR. BUCHANAN: Yes.

     DR. FISCHER: It was.

     DR. BUCHANAN: Yes--607 has been consistently the most resistant strain we have in our collection now.  And that was the strain we used for all subsequent experimentation.

     DR. BUSTA: Dr. Tompkin.

     DR. TOMPKIN: Yes.  Tompkin.

     Do you have any idea as to the code date and the freshness of the four formulas that were used for the vitamin studies?

     DR. BUCHANAN: No, but I can get that information, if it would be helpful to the committee.  We have the details of all of that.  I just didn't try to put it all on one slide.

     DR. BUSTA: Dr. Kuzminski?

     DR. KUZMINSKI: Kuzminski.

     If you'd had clumps, though, wouldn't your death-time curves have reflected the presence of clumps by the changing slopes?

     DR. BUCHANAN: The question was posed to me.  We tried to get as homogeneous of a distribution as possible in adding it to it.  All indications, by the methods that we used, both using the dye to see that it was evenly distributed, by the linear nature of the death-time curve, and the fact that we didn't get substantial shoulders, and by the fact that we were able to take samples throughout this large lot and they all pretty much came out within .2 log cycles, are indicative of the fact that we did a pretty good job.

     DR. KUZMINSKI: I agree.

     DR. BUSTA: Other questions?

     We have a point in the agenda, now, where I'd like your feeling.  I arbitrarily postponed the preliminary subcommittee discussion on clinical presentations because I felt these last two had some implications on some of the clinical questions this morning.  We can have that discussion now and then go on to the item on the agenda for the production situation, by Dr. Zink, or we can have Dr. Zink's presentation, take a break and come back for an overall preliminary discussion, and then have the public comment, and then have another discussion.

     What is your preference?  The other alternative is you really don't care.

     [Laughter.]

     Next speaker.  As close a consensus as we need.

     Is Dr. Zink ready?  He's ready.  Here he comes.   And Dr. Zink will give us the FDA field survey of powdered formula manufacturing.

FDA Field Survey of Powdered Formula Manufacturing

     DR. ZINK: Can you hear me?  I figure, if nothing else, I'll try to speak up.

     What I want to do is accomplish two things with this presentation.  I want to take what you've learned this morning and put it into an industrial scale, using the information we gained from our field survey to talk to you about manufacturing practices; give you a real good idea of how formula is made; start you thinking about potential opportunities of how the organism could get into formula; and then talk to you in some detail about our findings.

     Could I have the next slide, please?

     [Slide]

     The objective of our field survey was to describe manufacturing processes that were used--excuse me.  This is presentation objections--talk about the manufacturing practices used, summary micro-criteria used in various jurisdictions and by various governing bodies, and to review the results of our survey.

     Next slide.

     [Slide.]

     Powdered infant formula manufacturing , we can roughly talk about two kinds of processes.  One is dry blending, and the other is where all the ingredients are mixed together into a homogeneous liquid, and then spray dried to produce a dry powder.  Each of these processes has different challenges and benefits.  I wouldn't characterize one as being preferable or inferior to the other.

     Among dry blending, certainly it minimizes the use of water in the processing area.  Bacteria need water to grow.  Anything that minimizes the use of water aids your ability to control the growth of bacteria in the environment of a manufacturing plant.  However you're vulnerable to the contamination of ingredients.  If your buying dry ingredients from various suppliers, and you're not, you know, hearing those ingredients in any way, pretty much what you get in the finished product is whatever you start with in these dry ingredients.  Also, if you're going to use microbiological testing of the incoming raw materials to qualify them, that's a difficult thing to do because of the limitations of lot testing, the potential for non-random distribution of organisms, and the difficulty of getting a microbiological sampling plan that truly detects a positive organism.

     Wet mixing, on the other hand, you have the advantage that you're hydrating all your ingredients and you can pasteurize them to destroy all harmful bacteria, including organisms like  sakazakii, but this requires frequent wet cleaning of very large equipment items.  And also this use of water in the plant promotes the growth of bacteria in the plant and makes it a little harder to control.  So you see some pros and cons with these.

     Next slide.

     [Slide.]

     This is a typical wet mixing, spray drying type process.  This does not show any one manufacturer's process.  It's sort of a stylized, simplified summary of the types of processes used by manufacturers in the survey.

     In general, you begin with the protein, mineral oil and carbohydrate--often corn syrup--components.  These all go into a large tank.  It can be three to five thousand gallons in this tank.  They're mixed to homogeneity, and then they go through the pasteurizer.  I should add that in some cases, manufacturers homogenize before they pasteurize, and some pasteurize before they homogenize.  But, at any rate, knowing from Bob's presentation, you're dealing with pasteurization temperatures that could be anywhere from 170 to 260, this is a process that effectively destroys, completely, Enterobacter sakazakii.  The holding time on this can range from, oh, 15 seconds to more than a minute.

     Then the product is usually cooled down to something like 170 degrees Fahrenheit--maybe a little bit less.  Some heat-sensitive ingredients, such as vitamins and perhaps some mineral component, fatty acid component, could be added here.  Then it's homogenized.  This is generally done at about 170.  Again, remember Bob's presentation; 170 is pretty close to 70 degrees centigrade, so you're still doing some killing here, even though you're not documenting it.

     Then it goes to a holding tank--if it goes to a holding tank.  This is optional.  If it goes to a holding tank, it's usually cooled down to about 40 degrees centigrade, and may be held for some period of hours at that temperature.  And then it's preheated before it goes into a spray dryer.

     This spray dryer--and I'll show you some pictures of them later--can be a very large structure; seven, eight, nine stories tall.  And it's sprayed into that structure at the top as a fine mist, very small droplets.  It can have a temperature anywhere between 160, 200 degrees Fahrenheit.  When the liquid goes in here the air temperature of the spray dryer can run anywhere from maybe 260 Fahrenheit to over 400 degrees Fahrenheit.

     So, up to this point the chances that Enterobacter sakazakii would have been in this material, survived this process and be alive and be contaminating product at this point--not very great.  The lethality steps are considerable up to this point.

     However, after this point, that is where, should the organism be present in the manufacturing environment it could certainly contaminate the powder.  As this powder falls through the spray dryer, the swirling hot air, it very rapidly dries the droplets into particles of dry powder.  By the time the powder falls to the bottom of this funnel-shaped spray dryer, you have a temperature of maybe 170, 180 degrees Fahrenheit; maybe as high as 200 degrees Fahrenheit in the powder.  And it goes into what's called a "fluidized bed."  And you can think of a fluidized bed as a kind of a churning conveyer of powder, if you will, which has air flowing through it.  And usually these fluidized beds are two stages: one that will pump hot air in there to do some additional drying, and then maybe several stages of cold air to cool it down.

     When the powder comes out of the fluidized bed it goes through a sifter.  And at this point the powder is about room temperature.  The sifter is to remove large clumps.  And from the sifter it can go directly to a can-filling line, where it's filled, purged with nitrogen and hermetically sealed; or, it can be stored temporarily in totes or large bags for, say, quality assurance analysis, and then scheduled for filling.

     This is fairly typical.

     There's more than one kind of spray dryer.  I've diagramed a kind of a vertical funnel-shaped dryer here.  You can also have box type dryers which are more room-shaped.  But the principle of operation is the same.

     Next slide.

     [Slide.]

     In a dry blending type of process, actually most--the formula manufacturers that use dry blending more heavily, it really should be thought of as a combination of wet mixing-dry blending.  They'll take some dry materials, particularly the protein fraction, and the oil components, and they will wet mix those, again in a perhaps several thousand gallon tank, pasteurize them, cool them, homogenize them as before.  Homogenization, if you're not familiar with it, is simply a step where the product is raised to several thousand pounds of pressure and forced through a small orifice to make a uniform distribution of the components, particularly the oils.  The oils are reduced to very small micro-droplets so that they stay uniformly in suspension and don't settle out at the top of the product.

     Then, again, the holding tank, the pre-heater, and this protein and fat fraction is then spray dried.  Now, this is--percentage-wise, this is a smaller amount of the formula, and this process is advantageous to manufacturers, because they're not having to put water in and take water out by expenditure of energy into such a large quantity of powder.

     They then take this dry protein and oil fraction.  It goes through the same sort of fluidized bed and sifter process, and into drums or totes or bags for storage.  And then what happens is additional raw materials, particularly the carbohydrate fraction, but also potentially other kinds of fractions, are added to this pre-mix, if you will, in a ribbon blender.  Again, it gets sifted and it goes to the can-filling line.  The purpose of the ribbon blender here is to blend all of the materials to homogeneity.

     These are basically the two kinds of processes that are used.

     Next slide, please.

     [Slide.]

     This is a diagram, a little bit more detail about spray dryers and fluidized beds.  First, I want to point out to you: this is the bottom of a funnel-type vertical spray dryer. That little figure right there is a man.  Okay?  It's a large piece of equipment.  And this piece of equipment has doors--access doors into it.  They have gaskets.  It's--we're not talking about a smooth tube here.  So periodically these things have to be cleaned.  And they have to be wet cleaned.  And sometimes you have to open an access door, and people have to go into them, or people dangle in a harness in them, trying to clean it.  This is not what we would call a clean-in-place type operations, where you just flush some water through there and away you go.  It's a challenging thing to clean, with lots of little nooks and crannies, when you think about it.

     So what you see here is this portion of a diagram.  So here you have a large spray dryer.  Hot air is generated here.  It passes through high efficiency filters that filter out any microorganisms, and it goes in the top of the spray dryer, along with the heated liquid formula.  The whole mixture swirls around and dries and falls to the bottom, goes into the fluidized bed.  This is a two-stage fluidized bed shown here, with a hot air stage and cold air state.  Again, this air is filtered when it goes in there to prevent microbial contamination--and comes out of the fluidized bed through the sifter, as I showed you in the diagram.

     Things are always more complicated than you typically can draw them and show them.  And, in fact, you get some very fine particles of powder which don't readily settle out to the bottom.  These can be taken out of the spray dryer, and they go through what are called "cyclones," which is, in essence, a centrifugal type of centrifuge.  And these very fine particles can be separately collected.  And those may be used for infant formula, or they may be--excuse me.  Those may go to waste as animal feed, or they may be recycled back into formula through a wet-mixing process.  It varies.

     Next slide.

     [Slide.]

     This is the inside of a box dryer.  And a box dryer is just a square spray dryer.  It's hard to see here--it's dimly lit--but these are--there's nozzles and hot air inlet up here.  Imagine this room was a box dryer.  You have hot air and sprayed liquid coming in at this end, swirling around and falling to the floor, where it's collected.  This picture showed liquid being sprayed in a very fine mist, and it dries.  This detail here shows a chain drive.  And how do you get the powder off the floor?  Well, you just have a bar that's pulled by a chain that scrapes it over to one side, and then you have an auger--if you go to the--yes, you can see the auger here, I think, in this slide--and then you have an auger in the corner there against the wall that carries the powder away.  So that's the other type of spray dryer used.

     Next slide, please.

     [Slide.]

     This is a diagram of a fluidized bed--not a small piece of equipment, either.  If a person were standing here, they would be about so tall--okay?  So if you're standing on the floor, certainly the apparatus is taller than you are.  It gives you some idea of the scale.  If you look inside of it--and, of course, this is clean and not operating right now--you see this kind of a porous bed here that vibrates and that air can come up through, and the powder just moves along like it's flowing in a turbulent stream over the bottom of the fluidized bed.

     And you can see, there's some nooks and crannies in here, too, that pose cleaning challenges.  And it is--it's a large piece of equipment.  So there's opportunity for contamination to occur here if the equipment is not perfectly clean.

     Next slide.

     [Slide.]

     On to microbiological standards.  Before talking about our findings in formula, we thought it would be good if we could review with you what we knew about the various microbiological standards currently being applied to powdered infant formula.

     This is the CODEX standards. You heard it referred to earlier in one of the presentations, in relation to an outbreak that occurred in Belgium.  It's permissible--they take five samples of a lot.  It's permissible to have one of those samples with as many as 20 coliforms in it, but you should have fewer than 3--and "fewer than 3" in this case means no positive tubes in a three-tube MPN--but you should have fewer than 3 in the remaining four samples; n, number of samples, c maximum number of samples that can have a number in excess of m, and M is the "no single sample shall exceed" limit.  Okay?

     Next slide.

     [Slide.]

     This is the Canadian standards, and I'm going to focus in here.  They don't specifically have one for coliforms or Enterobacteriaceae, but Escherichia coli comes close.  Here they're taking 10 samples.  One of those would be permitted to have as many 10 organisms in it, but the remaining nine would have to be less than 1.8--essentially, probably a negative results.

     Next slide.

     [Slide.]

     This is a standard currently being used in China: aerobic plate count less than 30,000 per gram.  They have a coliform standard of less than 40 per 100 grams.

     Next slide.

     [Slide.]

     This is a standards that was proposed--or, guidelines, really, that were proposed I guess in our--I don't know the exact term we would use for it--but it was proposed as criteria that we would use in judging the acceptability of a lot of infant formula.  And it was less than 3.05 MPN per gram coliforms.  There were similar separate standards for fecal coliforms.

     Next slide.

     [Slide.]

     In our survey of infant formula plants we surveyed all of the plants that produce powdered formula in the United States.  There were 15 such plants.  Some of these plants produced concentrates and ready-to-feed only.  Some produced--I think seven of the plans produced powdered only.  Some plants produce ready-to-feed and powder, and I think a total of 10 plants each produced powdered formula and all were included in this assignment.  So this is a complete survey, if you will, of the U.S. powdered formula market.

     Next slide.

     [Slide.]

     What we wanted to do with this study was to get an official sample of powdered infant formula and raw materials to assess the prevalence and the number of E. sakazakii in the samples.  And the sampling protocol that we devised was based on prior literature reporting numbers as few as .36 per 100 grams, and we wanted to be certain to have a sampling protocol that could quantitate down to that limit; also to conduct an inspection at each major domestic infant formula producer in order to understand any possible relationship between manufacturing practices and the prevalence of E. sakazakii.

     Next slide.

     [Slide.]

     Here you can see the 10 plants that were studied. The total number of samples collected was 92.  Of this 92, you'll probably be most interested in the finished product samples, of which there were 22.  We also collected separately samples of carbohydrate fraction, fat, if it was present as a dry powder, and protein samples.

     These samples were collected regardless of the process used.  In other words, in some cases this carbohydrate was directly dry blended into the finished product without further heat treating.  And, in some cases, this carbohydrate was part of a wet mixing-spray drying operation.  It went in into a wet mix and then it got pasteurized before it was spray dried.  But we wanted to look at prevalence in raw materials as well.

     Next slide.

     [Slide.]

     Looking at finished products by the types of products--again, there were 22 finished product samples.  Fourteen of these were formulas for full-term infants; four were formulas intended for pre-term infants; and two were metabolic formulas, and two were hydrolysates.

     Next.

     [Slide.]

     I want to talk to you a little bit about our procedure.  If you've gotten the impression everybody does their own thing, sort of, when they develop these that's kind of the nature of microbiology.  We have essentially, as we said, used the procedure of  Nazarowec-White, with very little modification.  Okay?  However, it's important to note that we started out with 20 cans here.  In the study Don talked to you about earlier, they were actually looking at 330 grams in a given lot or can of sample.  We're looking at 1,332 grams, and here's how we come to it.

     Twenty cans were collected, and each of these cans had at least eight ounces of powder in it, and a composite was constructed by sorting these cans into four groups of five; 100 grams came out of each can in the group, so we now had four groups of 500 grams.  And those four groups of 500 grams were each treated as four separate three-tube MPNs.  So you had 4 times 3 times 100, times 10, times 1, for a total--out of any given lot sampled--of 1,332 grams.   That's important to note when you consider the prevalence of the organism reported by other investigators looking at smaller quantities of powder versus our results.

     The theoretical limit of detection here would be one in 1,332 grams.  That could occur, provided you had optimal conditions; you didn't have competing microorganisms, etcetera.  We don't know what the actual limit of detection is, of course, because we don't know what the true contamination was.  The minimum limit of quantitation--as Don Burr explained to you--is 0.31 most probable number in 100 grams.

     I want tot diverge just a minute so you don't get to where you misunderstand what's meant by "most probable number method."  It has limitations, and you need to think of those limitations, and you need to think about the statistics associated with it.  First thing, the most probable number, to be a valid method of quantitating something, the main assumption you have to meet is that whatever it is you're measuring has to be uniformly distributed in what you're measuring.  And that is almost certainly not the case here.

     If you remember the paper reporting the outbreak in the Belgian hospitals, they referred to the fact that they analyzed five samples; four of them were negative and one of them had 20 per gram.  Well, that shows you what I think a lot of microbiologists--food microbiologists--know, is that this organism is not uniformly distributed in the powder.  Contamination theoretically could occur from sloughing off of a equipment.  That could happen sporadically.  So, in our MPN analysis, we're already violating one of the assumptions of the MPN, simply because we had no other alternative way to do it that was do-able.  And you should keep that in mind.  The confidence interval for this result--say you get a result of 0.31 MPN in 100 grams, the confidence interval for that--95 percent confidence interval--is anywhere from 0.15 per 100 grams, all the way up to 1 per 100 grams.

     Now, the only way to really understand--I think it's important to think about what might actually go into a bottle of formula.  On average, I guess an eight ounce bottle of formula has something like--what?--34 grams of powder, something like that, in it.  And the only way we could really find out what might actually be going into a serving would be to do an experiment like this where we culture everything in little 28 or 34 gram aliquots.  But when we report this result of 0.31, or 0.36 per 100 grams, you could very well have a gram of that powder with 8, 10 or a dozen organisms in it, and then many aliquots of that powder with nothing in it. Okay?   So this is a statistical result, if you will.

     Next slide.

     [Slide.]

     What we found, overall, in finished product, we found five positive samples, for a total of 22.7 percent of the sample--finished product sampled--were positive.  In every case, when you analyze this by the three-tube MPN tables, we found 0.36 per 100 grams--at or very close to the limit of detection--22 samples tested.

     In the carbohydrate fraction, we found only one positive result.  Now, I should say we only did quantitative MPN test on finished product, not the components of it.  And we found one protein fraction that was positive.

     Next slide.

     [Slide.]

     If you break this down by type of formula, four of 14 were full-term formulas; one of four was a pre-term formula.  The metabolic and hydrolysates weren't positive.  There's no significant difference in that result.

     Next slide.

     [Slide.]

     All positive finished product samples were at the lowest limit of quantitation.   So we're talking about low numbers here.  There was no correlation between the test results and manufacturing practices.  In other words, you know, we got positives from people that wet-mixed, and we got positives from operations that incorporated dry blending, without apparent difference.

     And there was no relationship between the results and product type.   We had soy products that were positive; we had milk-based products that were positive .  So there's no reason to believe that this has got any particular type of association.

     I believe that's it.  Thank you.

     DR. BUSTA: Are there questions for clarification for Don?

     Dr. Fuller.  Oh, I'm sorry.  Dr. Moyer-Mileur.

     DR. FULLER:

.    DR. MOYER-MILEUR: Moyer-Mileur.

     Could you just clarify--for the pre-term formulas, did that include your transitional feeding formulas for pre-term babies, or just the in-hospital 24 calorie-per-ounce products.

     DR. ZINK: I believe that the pre-term did include what you would call transitional.

     DR. BUSTA: Dr. Briley.

     DR. BRILEY: Margaret Briley.

     Could you clarify for me the differences--you said there were no differences in cleaning the vats when they were dry or wet milled.  Would you say that possibly it would be more difficult in one, or that one would be a slight bit more toward the possibility of contamination, or--

     DR. ZINK: Okay--talk about equipment cleanability?

     DR. BRILEY: Yes.

     DR. ZINK: Dry versus wet blending?  All right.

     In the wet blending and spray drying, since everything has to be spray dried, you're generally dealing with some very large capacity spray drying systems.  And those spray dryers, on average, probably have to be cleaned once a week by wet cleaning.  And that can take a day or two, you know.  It's a difficult process.  The fluidized bed has to be cleaned, and all of that associated equipment with it has to be thoroughly dried out.  That's a challenge as well.

     In a dry blending operation--like I said, most people still have some wet blending and spray drying going on.  But they're generally not having to clean as large a piece of equipment.  And on the dry side of it, you can go quite a long time between cleaning, or you can use strictly dry cleaning methods for your brushing, vacuuming--that sort of thing.  So, on a purely dry side of the process, oh you can go weeks, perhaps months without using much water.  And keeping it dry is a huge advantage in controlling these organisms.  You can't deny them food, but you can certainly deny them water, and they need that water to grow and without it they don't grow.

     DR. BUSTA: Dr. Fuller?

     DR. FULLER: Given the--you mentioned the potential for clumping in equipment to release chunks of bacteria, if you will.  Did you all do any looking at--do any environmental sampling?  And, if not, was there any reason for not doing that at the same time?

     DR. ZINK: We've had limited ability to go back and do those kinds of investigative samples.  I believe it was done in one case.  I believe that organisms were recovered from the environment.  Perhaps some of these investigations will still be ongoing.  I don't know what the status of that is.

     But by the time we get a sample, analyze the sample, and interpret the results--this wasn't done to be in real time.  So a sample simply went in and they were enqueued with the labs other work.  Oftentimes, by the time we got that result, it might be a month or more from the time the sample was collected, and by that time the equipment has gone through many cleaning cycles, and the manufacturers themselves were monitoring equipment and environment.  So I think going back in and doing a detailed equipment survey in the timeframe we were working in here, that was clearly a secondary or tertiary objective.

     So I don't think we sought anything meaningful out of that or got anything meaningful out of it.

     DR. MOYER-MILEUR: So let me just--let me ask my question a little bit differently.

     The assumption, then, is that the primary source of the E. sak is coming from the ingredients, not from equipment contamination.  You feel that there's enough cleaning going on to prevent that?  Or--I mean, I was just trying to get a better understanding.

     DR. ZINK: I think if I had to offer you my opinion, I would say the organism is probably coming from the equipment, post spray drying.  I think that the organism, by whatever means unknown, gets into the plant and can contaminate that equipment.  And the difficulties of cleaning this equipment, you can never make that equipment completely sterile.  And so I think that what low levels of contamination do occur are probably coming from that handling equipment.

     DR. MOYER-MILEUR: Okay.  So we've got that assumption, but no data to back that up yet.

     DR. ZINK: Nope.

     DR. MOYER-MILEUR: Okay.

     DR. ZINK: I'm--that's going to have to be the subject for further study.

     DR. BUSTA: Dr. Blumberg.

     DR. BLUMBERG: Henry Blumberg.

     Did you look at other organisms besides Enterobacter sakazakii?  Or did these studies that you were doing just focus on that?

     DR. ZINK: We were specifically looking for Enterobacter sakazakii.  They were specifically picking yellow colonies.

     When they first started this study--and going over the work sheets of the labs, I think they were more liberal in picking colonies as they were trying to become familiar with this organism.  So I think they cast a wider net at first.   And, certainly, we did see, in some of those early samples, some things like Serratia, Panatea--other organisms cropped up.  But, for the most part, after the first few positive samples, they were zeroing right in and just picking sakazakii.

     DR. BUSTA: Dr. Tompkin.

     DR. TOMPKIN: This is Tompkin.

     You didn't do MPNs on the carbohydrate and protein fractions, the ones that tested positive.  What was the amount of product, or ingredient?

     DR. ZINK: We were looking--I'm trying to remember.  You would ask me that.  I believe we looked at 500 grams as one sample.  Is that right?  Don, do you remember?  Is Don Burr back there?

     DR. BURR: No, I don't remember.

     DR. ZINK: I'm pretty close to that.  I'll check.  But I believe it was 500 grams.

     DR. BUSTA: Dr. Neill?

     DR. NEILL: Peggy Neill.

     Don, do you recall what month or season of the year the sampling was done?

     DR. ZINK: I think we began collecting in August and continued through January, or maybe even February.

     DR. BUSTA: Dr. Tarr.

     DR. TARR: Tarr.

     What governs the interval between cleanings?  Is it tradition, or is it driven by colony counts or other data?

     DR. ZINK: It's driven by need.  You begin to see accumulation of material on the walls of the spray dryer.  Or else they need to switch from product type to some other product type.  That's another thing that would drive it--say, going from milk to soy, or soy to milk.

     DR. BUSTA: Dr. Briley?

     DR. BRILEY: Margaret Briley.

     Did you, in your survey, by any chance make record of any of these plants that had HACCP procedures in place?

     DR. ZINK: The question was did we, in our, surveys make a record of any of the plants that had HACCP procedures in place.  No, I don't recall that we were specifically looking "Did you or did you not have a HACCP plan?"

     The nature of how these plants operate is it's HACCP by any other name.  In other words, all of these manufacturers had detailed procedures.  Every one of them had SOPs for cleaning the equipment.  They all identified critical control points in their process.  It was--you know, there was a remarkable sameness as you read through all the reports about the methodology of controlling the process.

     DR. BUSTA: Dr. Stallings.

     DR. STALLINGS: Stallings.

     We've really been waiting to see some of these data.  So, to clarify the one the FDA field survey results--you did have starting product--carbohydrate and protein--that had positive cultures.

     DR. ZINK: Yes.

     DR. STALLINGS: And then the finished product.  And so you were saying, though, that you really felt like the source of the organisms was during the manufacture.  So the protein and carbohydrate--I mean, just explain that.

     DR. ZINK: I can talk about those in a little bit more detail.

     DR. STALLINGS: Okay.

     DR. ZINK: In the case of the protein fraction, that was used in a product which went through a traditional wet mixing-spray drying process.  So all of the protein was hydrated into water with the other ingredients, and then it went through pasteurization.  I remember doing a calculation on the lethality, taking that processor's exact time and temperature conditions, and then only at the pasteurizer--okay?--not considering other heating contributions.  And I think I came up with a theoretical lethality of around 200 log cycle reduction.  Very substantial.  No way that organism could possibly have survived that process.  So, I do not believe that that protein contributed--you know, although it was contaminated, it would not have been a factor in contaminating the other product.

     You always have to consider, when raw materials come into a plant and they're contaminated, even though you're going to mix them wet and pasteurize it, if they get opened and tracked around the plant, or--you know, that could be a problem.  But in the case of the contaminated carbohydrate, it did go into a product through a dry blending process.  So, most certainly, what was in it would have been expected to also show up in the formula.

     DR. BUSTA: Dr. Kuzminski.

     DR. KUZMINSKI: Thank you.  Larry Kuzminski.

     A number of questions.  In your survey, did you get a chance to review--and it sounds like you did--the QA practices---the standard quality assurance practices--during the manufacturing process?

     DR. ZINK: Yes.  All of those procedures were documented and recorded, and there was nothing remarkable or deficient showing in any of that documentation.

     Bear in mind, you know, when all of that field survey material comes back to us, they were following specific instructions to collect certain kinds of information, and collect certain kinds of documentation.  I think one of the things you have to ask is: do these traditional kinds of critical control point documentation systems and SOPs--do they adequately address risk of contamination with an organism like this?  I mean, certainly, in looking at the data as it came in, you know, it seemed to meet, you know, all of the requirements one would expect of a manufacturer of that product--as documented on those reports.

     If you were standing there in those plants, I think you have the ability to assess things, and consider things, you know, that might not be part of formal control programs that might be contributing factors.

     DR. KUZMINSKI: One of the points that you made was the potential for contamination post--in the process flow, past the spray dryer, once heat's stopped being applied to components of other process, back to ribbon blending and to addition of the minor ingredients; the vitamin and mineral pre-mixes.

     I noted that--you perhaps omitted this, but did you get a chance to take a look at the vitamin components in the pre-mix for microbiological quality?

     DR. ZINK: They did not sample any of the vitamin components in this survey.

     DR. KUZMINSKI: There must--also from the data on the distribution of the nutrients that, I think, Dr. Buchanan showed, it looks like the efficiency of the ribbon blender is a pretty efficient blender, in terms of getting distribution pretty homogeneously throughout a batch, even up into a 5,000 pound batch--quite a size batch.

     Would you think, though, that if something if something is sloughed off from the equipment--a chunk of dried material, caked on material, from something that's been running for awhile, a number of shifts, a number of days, especially in the dry process, that the efficiency of the ribbon blender would be there to get that distributed through the batch also?

     DR. ZINK: Well, to the extent that--I mean, not all formulas go through that kind of ribbon blender dry blending process.  Some are strictly the wet mix-spray dry, more of a continuous stream type of thing.

     Yes, I would think the ribbon blender would tend to take non-random contamination and randomize it to a degree.  I'm not familiar with any kinds of studies--and I don't have any experience to say how effective in doing that it would be.  But I would expect that the ribbon blender, on those dry blended products, would tend to randomize the contamination a bit--yes.

     But in the other process, you really don't have that at work.  So you would maybe expect more non-randomness in the wet mix-spray dry type of process.

     DR. KUZMINSKI: And, lastly, there have been a couple of recalls--voluntary recalls--in the industry due to E. sak presence.  Very responsible companies are involved.  And in the follow-up investigation to these recalls, in terms of process analysis, the efficacy presence perhaps and the line flow of ingredients, right back to the raw materials--individual raw material supplier--to the discharge end of the packing line--can you share with us any information that has been gained from those recall incidents, as to the occurrence?

     DR. ZINK: I can say--you know, did someone--did an investigator or a company come forward with the smoking gun in each of those cases?  No.  Okay.  I mean, I am familiar--some manufacturers shared the fact that, indeed, they were finding the organism in the food processing environment, and that controlling the organism in the food processing environment is a real challenge.  And perhaps we'll hear more about that later.

     DR. BUSTA:  Dr. Neill

     DR. NEILL: Don, can you tell me--I have two very different question, one of which is how analogous is this process for the production of the powdered formula to the production of powdered milk?  And then, kind of a--

     DR. ZINK: Virtually .identical.

     DR. NEILL: All right.  And then perhaps somewhere in the later batch of questions, one of the individuals who spoke can tell us whether any similar work has been done trying to analyze, microbiology speaking, any powdered milk for this.

     DR. ZINK: Her question was how similar is the infant formula manufacturing process to, say, what would be used to produce spray-dried milk.  And my answer is it's virtually identical; same types of equipment, same types of processes, probably even similar temperatures involved.

     We certainly haven't done a survey of spray-dried milk plants for this organism.

     DR. NEILL: And then the second question has to do with the derivation of the sampling plan.  Since, in looking at that I notice that there's quite a difference in the distribution of the sample types--finished product versus component ingredients, etcetera--across the 10 plans, and was this derived on the basis of volume of product, or what?

     DR. ZINK: I think probably others could talk about that sampling plan more, but--correct me if I'm wrong--we wanted to get a good sampling of the various different kinds of products that were out there.  I don't believe there was aan attempt to bias it in any way.  It was really what we could get at within the timeframe allocated for the study, really.  And we wanted to look at some of each thing, but--you know, some of these formulas are produced infrequently--once or twice a year.  And that had a lot to do with it, particularly some of the speciality formulas and stuff--our ability to get those samples.

     DR. BUSTA: I'll take three more questions.

     Dr. Acholonu.

     DR. ACHOLONU: Doctor, you discussed the microbiological standards in Canada, in China and as used by FDA.  And each of them has aerobic plate count.  Since we have been told that E.sak is a facultative anaerobe, can that method be used?

     DR. ZINK: It will certainly grow up as a colony on the aerobic plate count.  A lot of other things will too, of course.  You would have to, at that point, pick the colonies and identify them to differentiate it and detect it.

     To a microbiologist--the ideal method to a microbiologist is that you take a whole lot of the sample, throw it in some magic broth and only what you are interested in grows, and it's not the least bit inhibited by anything in there, but everything else is inhibited--and out comes the bug.  We don't have that method for this organism, unfortunately.

     Infant formula is very clean, microbiologically.  Although you saw aerobic plate count limits up there of 10,000, 30,000--things like that, most manufacturers are producing more in a norm of about 500 per gram.

     DR. BUSTA: Dr. Beuchat.

     DR. BEUCHAT: Beuchat.

     My question concerns the physical structure, or the layout, of a typical wet mixing-spray drying process.  Is the area in which the raw ingredients handled separated physically and/or by air flow, ventilation and so on, from the post-spray dry area?

     DR. ZINK: In the plants I have been in--I'll answer that question by saying yes.  In some plants, it's outstandingly well separated.  And in somse plants, there's room to improve--okay?  But I think all manufacturers strive to achieve segregation, and all of the manufacturers I know of have very strict physical environmental isolation once they're dealing with a finished powder.  You just can't stroll from one area of the plan to the other.

     DR. BUSTA: Thank you, Don.

     We will now take a 20 minute break.  Is that long enough for you?  A 20 minute break, so we'll aim at 3:25 reconvening, and we'll ask any questions of the three speakers so far this afternoon first.

     [Break.]

     DR. BUSTA: Will all of you that are asking questions--now that's how to make a point--right?  You're asked to talk loudly, as well as into the microphone.  But even with the microphone, talk loudly, because all of this is being recorded and someone down the line has to try and transcribe it all.  And that's--we're trying to make their job not as terribly difficult as it might be.

     At this point we have about, maybe, 20 minutes if we need that to ask questions for any of the four speakers from this afternoon.  And we're open to questions.

     Dr. Tompkin.

     DR. TOMPKIN: Bruce Tompkin.  This is for Maria.

     I don't recall that you mentioned it in your presentation, but in the manuscript that you had published, at 4 degrees centigrade, which is almost 40 Fahrenheit, you found that E. sakazakii actually died?  But I don't recall that it stated how rapidly.  Could you comment on the rate of death at refrigerated temperature?

     DR. NAZAROWEC-WHITE: At refrigerated temperatures we took samples, I think it was every two days.  And there was nothing--in some of them it would be 103 and 103, and then after about--I don't know--two weeks, it was down.

     DR. BUSTA: Dr. Thureen.

     DR. THUREEN: Thureen.

     If this committee--this is a hypothetical question--if this committee can agree that there should be a standard above which there should be no higher level of E. sakazakii contamination, and we decide that this is an index marker, because it has such virulence, and because it's easy to detect, and we establish that standard as being the limit above--or at which we feel comfortable of having to be a slight contamination rate, do you think then that we will also eliminate risk for serious infection from other organisms that are found within the powdered infant formulas?  Would that be a good benchmark to set to provide overall safety, not just against his particular organism.  And this is for any of you.

     DR. BUCHANAN: You're asking a really big question.  You've posed a hypothetical question that--the best that I could give you is a hypothetical answer--with all the disclaimers, caveats, etcetera.

     The suggestion is that if you could have a standard that controlled this organism, would you control others?  And the real question here is: is their mode of action the same?  Is their mechanism of pathogenicity the same?  Is the impact of the interventions or standards that you would use the same?

     If you could fulfill all those criteria--probably.  And I hesitate to say anything more than probability.  If their method of control was different, then I would say that you'd have to actually go through and make the case.  And, gee, if you'd ask me, you know, are there infections due to Enterobacter cloacae, and would controlling Enterobacter sakazakii control Enterobacter cloacae?  Yes, I think you'd probably have a good chance.  Salmonella?  I'd be less sure of.

     DR. THUREEN: And so, along that line, do you think it should be E. sakazakii, or do you think it should be enterobacter in general that should be sort of the target organism for eradication?  Or do you think it makes a difference.

     DR. ZINK: I think I'd be more comfortable answering if I knew a little more than I know right now.  I agree with Bob.  If you choose to pick an organism as the focus of your control, and it happens to be part of a family of organisms that have a similar mode of contaminating the product, and similar resistance or susceptibility to control measures, then I think picking the most onerous of the family probably would be a good way to go.

     Talking about Enterobacteriaceae, as you've seen, they're all pretty similar in their susceptibilities and resistences to control measures.  So that may be an approach you could use.  The trouble is that I'm not--I don't think any of us have a lot of data on the diversity of organisms in formula and how they get there, you know.

     DR. NAZAROWEC-WHITE:  That's what--I agree.  I think that we don't know what's in there yet.  We do know that there are some salmonella, maybe, in the dried formula.  So you have one option of sort of setting that standard is a possibility.  On the other hand, are there any intervention strategies--can we do something with that final product to inactivate organisms?

     DR. BUSTA: Dr. Tompkin.

     DR. TOMPKIN: I didn't look it up, Don, but maybe you might remember.  In the '96 ANPR for FDA for listeria monocytogenes, do you recall what the amount of product it's--your slide says it's negative, but is it negative in a 25 gram, or 50 grams?  Do you remember?

     DR. ZINK: I don't remember.

     DR. TOMPKIN: Or was it specified?

     DR. ZINK: It would be specified, if by reference to a method.  Okay?  I don't remember whether it was actually spelled out in the ANPR.  If it wasn't, it would have been referred back to the method we used.  I don't know--Ada, do you know?  220?  250?  250 grams?

     You know, you hardly ever see listeria in these dry powder plants.  It just doesn't like dry powder plants.  I don't think I've ever recalled it being an issue.

     DR. BUSTA: Dr. Fuller.

     DR. FULLER: This is Fuller.

     Not being particularly knowledgeable of manufacturing practices, etcetera, is it possible--or maybe it's cost prohibitive--but to apply a heat step at the--you know, just prior to filling the cans?  Is there--I mean, does it make any sense?  Can you use dry heat?  Do you have to--you know, is there anything that would--could be done at that step?

     DR. BUCHANAN:  I'll jump in here for a minute, because I looked at dry heating for a number of issues.

     The big thing about dry heating is whether or not your product is a mixture of protein and carbohydrate, which infant formula are.  In some instances where you have a single entity, and you don't have a browning reaction taking place, you can actually get away with dry heating--long enough and hot enough that you would inactivate microorganisms.  A classic example is salmonella control in egg white.  But egg white is interesting because they have to de-sugar the egg white before they can dry it because if not, it turns brown.

     The same thing would be expected in infant formula.  You'd get a lot of browning reaction products.

     DR. ZINK: One of the problems is these bacteria get a lot more heat resistant when they dry.  I guess this is best studied with salmonella.  And there are garden variety salmonella strains that can resist 200 degrees Fahrenheit for hours.  And that's a real problem--when they're dry, when you dehydrate them.

     So, I think that applying heat to the dry powder would be--it would be challenging to get significant log reductions.

     DR. BUSTA: Dr. Tarr.

     DR. TARR: Since looking for low counts of specific pathogens can be frustrating and quite variable, let's go to the more robust total colony counts, or Enterobacteriaceae counts.  Are there trends over time that you are observing?  Are they generally going down year by year in industry, and are they associated with any new practices that could be either accelerated or amplified in your production line?

     You're clearly exceeding the CODEX and the FDA guidelines, but you're still getting some counts coming through.  Are there trends in those that would be informative for making a safer product?

     DR. ZINK: That might be a better question for industry.  I don't know that we have--at least not in a readily analyzable form--data that would say, "In 2003 we're better than we were in 1985"--in terms of counts.

     I know there are specific instances within industry where--going back to paper on the Belgian outbreak, citing the plant that was the source of that powder.  I do know there are specific instances where improvements in manufacturing process were introduced that had dramatic improvements in the microbiological quality of the product.

     DR. BUCHANAN: Phil, I'd also caution you to be a little careful about the use of indicator and index organisms.  Because there's no guarantee that the reduction that you see in total aerobic plate counts are equivalent reductions taking place in the pathogens of concern in the product.  In fact, you could, for example, eliminate large numbers of vegetative cells from a product, and if the organism of concern was a spore former, it would have no meaning in terms of reducing the level of risk.

     DR. TARR: I agree with that, but at least with Enterobacteriaceae, if we got to zero Enterobacteriaceae we would get to zero E. sakazakii.  And that should be a target.  Maybe not this year.

     DR. BUSTA: Dr. Stallings.

     DR. STALLINGS: Stallings.

     One of the overriding principles we're still dealing with is that this is a rare disease.  You know, that if--that it's a rare bacteria in the product, and that it's rarely pathogenic, even in the setting we're using.

     And I go back to the concern that someone brought up earlier about the changes in clinical microbiological methods, in that many things have become more automated.  And I don't know if any of you know, or if it's something that we can just get for the committee--just some reassurances that the systems that are now being used, where basically they're put in banks, and heated, and no human eyes see them until, you know, there's something that says something's growing--that for this fairly rare bug, that we would still be picking it up.  Because if--we know the neonates are getting smaller and smaller, and sicker and sicker, and there would be some reassurance if we really knew that we were doing very good clinical microbiological surveillance, and not picking this up, and that it was being identified properly when it happened, and that sort of thing.

     Do any of you have any information in really what's going on in clinical hospital settings?

     DR. BUCHANAN: I think we can try to find that out.  But the only comment I would offer is that the most important thing for getting a clinical sample done is having the physician ask for it.

     DR. STALLINGS: Well, to address that, I think any baby--if we're talking about the high risk nursery setting, the signs and symptoms you're going to see were described.  It's temperature instability, it's food intolerance if they've been eating, and--it's very soft signs.  But in a level 3 neonatal nursery, at the drop of a hat you do blood, urine and spinal fluid.  That's just the way we manage the highest risk babies.  So, in this one rare setting, I think we probably do--we don't do stools, but stools would not be the specimen of choice for this.

     So--I'll defer to my neonatologist, but it's usually a pretty--if you are  at all worried about these babies, you do the drill and you do it comprehensively.

     DR. THUREEN: One of the problems is that, in theory, you'd like to do lumbar punctures on all these infants, but they're usually too unstable to have that done.  Anda I think in many nurseries, just signs of NEC would not be enough to trigger spinal sampling being done.  And it varies from nursery to nursery.

     While I have a microphone, I have a question.

     DR. BUSTA: Dr. Thureen.

     DR. THUREEN: Thureen--for the record.  And I'm sure there's a good reason, but why isn't irradiation a solution to this problem?

     DR. BUCHANAN: I'll take that one on.  And, again, it's the characteristics of these organisms in a dry environment.

     Just as the thermal resistence of a normally sensitive organism goes up drastically when you put it in a dry state, so does the irradiation resistence.  So you would probably exceed the level of irradiation that's allowed at this point.

     Now, I'd have to say that I don't know of any specific information on the irradiation tolerance of this organism in an infant formula and, for that matter, in just about anything.  It certainly is an area that could be looked at.  But, traditionally, as you decrease the water activity of a product its resistence to radiation goes up dramatically.  Because the way that you kill organisms by irradiation--there's two; there's direct and indirect effects.  The indirect effects are due to the formation of free radicals because of, actually, the irradiation of the water.  And if you eliminate the water, you eliminate all of the secondary effects, which is the greatest one for these organisms, and you go back to single hit targets on DNA.  And it gets to be much more difficult.

     And I think if I remember--your Chair knows a lot about that.

     [Laughter.]

     DR. BUSTA:  Don, are you just holding the phone?

     [Laughter.]

     Dr. Kuzminski.

     DR. KUZMINSKI: Thank you.  Larry Kuzminski.  Just a brief question to Dr. Nazarowec-White.

     You mentioned in your introductory comments to your presentation that Health Canada was considering a monitoring program.  Can you share any more details about that, please?

     DR. NAZAROWEC-WHITE: I guess we've talked about it for the past couple of years, and it's the Canadian Food Inspection Agency.  At this point in time, we are set up very similar to what you have here in the U.S.  And we do not have any infant formula companies producing powdered formula at this point in time.  But monitoring the, you know, prevalence of E. sakazakii on the formula that we do import, for instance, is something that they are considering doing, but looking at other programs that right now have a higher priority.

     So I think that depending on how the meetings go, and what we learn, as far as that importance, we'll see what happens with next year's programming.

     DR. BUSTA: Dr. Beuchat.

     DR. BEUCHAT: Beuchat.  This question is directed to Maria.

     You made a comment, almost in passing, about the observation of some of your strains clumping or sedimenting.  There's some evidence to make us think that some enterics that are capable of producing capsules--exopolysaccharide-are more heat resistant, are protected from the heat assault, are perhaps more resistant to harsh environments--as simple as acid conditions that would be occurring if the cells were taken into a body.  And perhaps also virulence.

     Do you have any indication of those strains that you did observe that did have this trait of sedimenting, of being in the category--or among those strains that you've reported on in your paper on infectivity and pathogenicity?

     DR. NAZAROWEC-WHITE: We found that all of the strains actually produced this sediment.  And there were only the infectivity--you know was not that--you know, the strains that cause infectivity also produce sediment.  But strains that did not cause infectivity also produced sediment.  So we don't know at this point in time.  Further research, I guess, to look at how many other strains that are in various culture collections--what's happening with them, what they're doing.

     DR. BEUCHAT: And, along that line, do we know anything about biofilm formation by E. sakazakii?  And also, these strains, if you transfer ad infinitum, do they lose this capability?  This is something we need to be aware of if  research is conducted in this area.

     We've observed some things along that line with listeria.

     DR. NAZAROWEC-WHITE: We haven't done anything.  And, at this point, I'm not sure what the plans are for the work with this organism.

     DR. BUSTA: Dr. Heubi:

     DR. HEUBI: Heubi.

     I had a side conversation with Dr. Buchanan during the break, and I actually wanted to raise this for the group as a whole, because I was a little confused about this myself.

     According to the proposed standards of the FDA for microbiological standards, it doesn't include any comment about E. sakazakii.  And are there new standards that are not printed here that specifically relate to E. sakazakii?  And can you specifically comment on that for the group?

     DR. BUCHANAN: Yes, there seems to be a little confusion about microbiological standards, or the way we work.

     For the standards that you've been talking about--the CACFH, etcetera, those are largely good manufacturing, or good hygienic standards; this is what we would expect of an industry that's producing a product under, you know, what would be good conditions.

     Our standard for a ready-to-eat food for a pathogen is the absence, usually specified by some sampling protocol, some standardized method.  But here, sakazakii was considered--this was considered a ready-to-eat food.  Sakazakii was considered a pathogen, and the level of sensitivity of the test determined whether or not we were finding it or not.

     This is basically the same standard that we have for, essentially, all ready-to-eat food: absence based on the detection using a certain method.

     Does that help clarify?

     DR. BUSTA: Okay.  Chris.

     DR. TAYLOR: Thank you.

     Just to add a little bit to what Dr. Buchanan has said, in the 1996 proposal, there was an effort to update some of the microbial standards and reopening the comment period on this rule in order to make it a final rule, there will be the opportunity again to talk about specific microbial standards for infant formula specifically.

     I just wanted to add that.

     DR. BUSTA: Thank you.

     Dr. Briley.

     DR. BRILEY: Margaret Briley.  I wanted to clear up something.

     It seems that this pathogen is suspected--

     DR. BUSTA: Dr. Briley, could you speak louder?  It's on, it's on, but you just have to speak a little louder.

     DR. BRILEY: This pathogen, from what I understand--maybe you need to correct me--is suspected to be in the equipment, possibly, in the manufacturing stage.  Did I understand that to be a possibility?

     Can you make some comment about any improvement in the cleaning of the equipment, or in the process of moving it through the equipment that industry might take that would allow this to be corrected?

     I don't know enough about what's inside the big funnel.  I can see a little man with a bonnet and a robe and covered shoes climbing up and scrubbing the walls.  But I just would like to know if there's some comment that you could give, based on your observations?

     DR. ZINK: I think maybe some from industry could comment on it better than I could.  In my capacity at FDA, you know--that was another life, a prior life for me.  But, you know, you can always design something a little better, or clean something a little better than you have in the past.  Things come along, and we're required to raise the bar.  I mean, were it not for the Titanic, would we have life preservers on cruise ships.

     I'm sure that the discovery of this organism and its significance has caused a reexamination of cleaning methods and procedures.  I think the question becomes to what extent, by procedures and equipment-tweaking alone can you get to some other level, and what is that other level.  And that I don't know.

     DR. BRILEY: Hopefully, zero tolerance maybe.

     DR. BUSTA: Dr. Thureen?  Okay, Dr. Tompkin.

     DR. TOMPKIN: I sensed a perception that the frequency of cleaning could be an issue with these facilities.  And I'd like to have some comment, probably from Don, on this.

     But, in operating a dry operation it's better to run it, I think, as long as you can.  And actually, from my own experience, I've seen situations where your problems occur as a result of cleaning, and that this non-random distribution of certain bacteria can occur in the first product through the system.  So sampling the first product through the system may, in fact, pick up a contamination that may have occurred in the process of cleaning, or while the equipment was sitting and still wet, allowing for some growth.

     So, in terms of recommendations or strategies, one that the group may come up with is clean more frequently, but I don't feel comfortable with that, and I would like to know what your view is.

     DR. ZINK: I think most manufacturers would agree with you, that they'd rather not break and clean very often; that if they could run longer and drier, they would.  The fact is that if you've got one spray dryer, and you make four different kinds of formula, you know, and you have to do these changeovers, that will dictate some need to clean.

     Also, these formulas have a fair bit of carbohydrate or sugar in them, and that can tend to make them form a cake or adhere to the walls of the spray dryer.  There's other operational things that you have to go in there, such as, maybe, the accumulation of some small number--you know, burnt particles and things like that might dictate a need to get in and clean.

     But for the spray dryers, I seem to remember that somewhere between once every five to 12 days seems to be the usual frequency at which they're cleaned.  And I think cleaning more often--I agree with you, it might cause more trouble, you know, by the additional water it puts in the system.

     DR. BUCHANAN: Also, I'd like to also respond or expand your question out a little bit, because I have some concern about misinterpreting what is meant by "low levels of contamination," versus an nonhomogeneous distribution of a sample.  And while I have not seen a rigorous evaluation--I haven't seen a chi-square test yet--on looking across a large number of cans out of a single lot that's known to be contaminated, I do have to reflect that we have examined numerous cans from several of the recalled lots.  And I think we're more in an issue of the level of contamination, at least with those lots, than we are with "It's in the first three cans and it's not in the next 400 cans, and then it's in the next three."  Because we have found it.

     You also have to keep in perspective, when we're talking about a classic one-pound can, and you're down in an MPN of .3 per 100 grams, that basically means that you're dealing with one-and-a-half in every can.  No, it's not even that.  It's one per every three cans.  So you're already in the type of distribution that you're--it's a rare event.  You're not going to find it in every can.  But that doesn't mean that it's not homogeneously distributed within the lot.

     DR. BUSTA: Dr. Lee.

     DR. LEE: Perhaps a generic question--as I understand it, E. sak wouldn't be a problem if we are rehydrating with boiling water.  And certainly, one can formulate, in anticipation of that, by just putting in more vitamin C to offset any potential losses.

     So I'd like to know what the contraindications are to formulating this product with boiling water?

     Well, as far as I can tell, there are two contraindications--or two factors that haven't been taken into account.  And first I want to emphasize--you don't have to use boiling water.  That's 30 degrees too high.  It really only has to be at 70.  It needs to be hot water.

     Second, is we still don't have sufficient data on clumping.  And while we don't think it is a large problem, it's material that has to get through enteral feeding tubes.  And so clumping is something that certainly needs to be examined.

     And then--and probably the most significant thing--when you do heat, the formula retains its temperature for quite a long time.  I showed you a cooling curve of the first ten minutes following the heat.  And there is--realistically, you have to be careful.  You can't feed 100 degree formula to an infant.  They'll get burned.

     So it has to be balanced by the fact that if you use hot water to rehydrate the formula, there does have to be adequate cooling.

     DR. LEE: Just to follow up on clumping--if clumping does occur, is this formula normally turbid?  Is it clear if it's fully dissolved?  No?

     DR. ZINK: No.  I mean, it looks kind of like, you know, milk.  And you can get coagulation of protein that's fairly significant there without it's being obvious.  You know, I can see it going into an enteral feeding tube and clogging that tube up, and outwardly not looking like it had a precipitate or clumps in it.

     DR. LEE: But it will pass through cheesecloth or some kind of filter, if that's needed?

     DR. ZINK: Well, I think it would--you know, when it clumps it clumps.  You would get some of it retained on cheesecloth, probably.

     DR. BUSTA: Dr. Fischer.

     DR. FISCHER: Fischer.

     I was surprised to hear that--no, I'm not surprised to hear that powdered--formulation of powdered milk, or the production of powdered milk was very similar to the production of infant formula.

     If that's the case, have you looked in powdered milk for the organism?  And if it is there--and if it isn't there--mightn't this not be clue as to where it might be coming from, in terms of the raw ingredients involved?

     First of al, have you looked in DR. TARR: ?

     DR. ZINK: We haven't specifically run a survey of powdered milk.  And having just recently joined the agency from industry, let me say that in this job your capacity to cause mayhem by your actions far exceeds that of what it was when I was in industry.

     [Laughter.]

     You know.  No, we haven't gone out and looked at, you know, producers of dry milk for this organism.  And while that might seem like a good idea at the moment, I think I would want to think about that long and hard.  You know, what am I going to do when I find it, and what does that mean?

     I really would expect, if I did do that, to find it.

     DR. BURR: Actually, from the 1984, the Postupa and Aldova paper, they have four strains coming from powdered milk.

     DR. BUCHANAN: Most normal analysis of powdered milk for gram-negative Enterobacteriaceae is to determine whether or not there's E. coli in it.  And so you have E. coli, and then you have everything else, and you forget about everything else.  No one tries to differentiate all the--enterobacter serratia, klebsiella that may show up as fecal coliforms.  They're worried primarily about E. coli, as an indicator organism.

     So I'm sure that it's been isolated from powdered milk.  I just don't know of anyone that takes the time to try and differentiate all those species.

     DR. FISCHER: Yes, it seems to me that's helpful--that information is helpful, because then it would tell you it's not some substance you've added, particularly after the drying process, like the vitamins, that is the source of the organism.

     I'm just surprised that the source hasn't been stumbled on as yet.  And it seems to me that is a key issue, that would help everybody out, if the source could be found.

     DR. BUCHANAN: Can I answer this?  And I hesitate to do it because this is really a question that needs to be addressed to the industry.  But I know from past experience of looking for the sources of listeria--and there's been a lot of work on that, and people have literally torn their plants apart to find out where a small micro-colony of listeria is harbored and causing problems.

     These plants are really complex places, and all you need is one little small niche somewhere that's feeding into it.  So, it's not as simple a job as one might think to track down where in a plant environment you actually have that organism residing.

     DR. BUSTA: Could I take the chair's prerogative and expand on that just a second?

     Did I hear correctly, in two presentations, that it's not in the environment?

     DR. ZINK:  Oh no, it's in the environment.

     DR. BUSTA: Marie?

     DR. NAZAROWEC-WHITE: [Off mike.].

     DR. BUSTA: Marie,.you said in that list-

     DR. NAZAROWEC-WHITE: Right.

     DR. BUSTA:  --that this was not the source.

     DR. NAZAROWEC-WHITE: Correct.  That's right.

     DR. BUSTA: And that as quite an extensive list of all kinds of environment.

     DR. NAZAROWEC-WHITE: That's correct.  And it was not found in that particular--in those particular areas.  But we have no idea whether they took five samples, or whether they actually--you know, there has been no systematic scientific approach to look at that environment.

     DR. BUSTA: So chances are where Enterobacteriaceae are, it is also.

     DR. NAZAROWEC-WHITE: Well, I would think so.

     DR. ZINK: I think this is a question for industry.  I believe, as Bob alluded to, is what went on with the meats industry and listeria, I think this industry has disassembled their plants and done extensive testing and has, you know, probably isolated the organism from many places in their plant.  And I suspect that, you know, while as an agency we're not in possession of this kind of data and information, I suspect that it's known, the types of plant environment where the organism can be found.

     DR. BUSTA: Two more questions, then.

     Dr. Acholonu.

     DR. ACHOLONU: Alex Acholonu.

     In one of the background materials given to us, the title is "Powdered Infant Formula: An Overview of Manufacturing Processes."  It has a statement which I'm going to read--this is for Dr. Zink.

     "Typically, finished product is held until it undergoes a final check for conformance to specifications--"--and the next statement is  "--including testing for microbiological contaminants."

     My question is what method would you use to check for microbiological contaminants in a finished product?

     DR. ZINK: Well, what manufacturers do right now--what you want to do is you don't want to produce value-added garbage.  Okay.  So if you can run your test for quality conformance before you add value by putting it in a can and labeling it and casing it, only to find out you have to throw it out, if they can they'll get these tests done before they put it in the can.  And they're typically testing to two kinds of standards.  Each manufacturers has its own internal standards; typically much more stringent than what a regulator would require.  So they're going to test for conformance to their own internal quality assurance standards.  And that could include things like aerobic plate count, salmonella, listeria, coliforms, E. coli.  They take the lead, it's been my experience, from what regulators require.  And they'll be also testing for, you know, vitamin content, homogeneity--all those sorts of things.

     Right now, I think most manufacturers test for the kinds of things that a regulator would, and they apply the same or more stringent criteria.

     DR. ACHOLONU: My understanding of a finished product is one that has been prepared and may be canned and ready to go.  Do you understand--

     DR. ZINK: Well, you get into semantics.  Right.  From a standpoint of microorganisms, if you have that powder sitting in a tote or a big bag, it's true that there is some potential for further contamination by the time it goes through a can-filling line and into the can.

     I'm familiar with several manufacturers that will test product while it's in bulk.  Then they'll go ahead and put it in the cans, and then they'll test it again.

     DR. ACHOLONU: They open the can and test it again?

     DR. ZINK: Yes.

     DR. ACHOLONU: Doesn't that give--make it get contaminated--

     DR. ZINK: Well, they open the can--usually, they'll have a biological cabinet to open the can in.  It's very common in these plants to see laminar flow hoods, and--you know, they're able to open those cans in a manner that precludes recontamination.

     DR. BUSTA: Thank you all very much for an extended question and answer session.  I appreciate this.

     And now we will go on to the public comment.  We have three presenters from the International Formula Council.

Public Comment

     DR. BUSTA: The first one that I have on the list is Dr. John Vanderhoff.  He's vice president, Global Medical Affairs, Mead Johnson Nutritionals, a Bristol-Meyers Squibb Company, speaking on Overview and Risk Characterization.

     [Pause.]

     Will there be a copy of the slides available, that you are using?

     DR. VANDERHOFF: If you'd like.  I don't know what we've decided to do about that.  I'm speaking for the entire group, so I'll have to defer that to you, whether we want to leave these slides here or not.  But they're--

     VOICE: [Off mike.]

     DR. BUSTA: All right.  Thank you.

     Proceed.

     DR. VANDERHOFF: Okay.  I think we're waiting on a little warmth from the projector here.

     I very much appreciate the opportunity to kick this off and try to give you what I would like to call an industry overview of Enterobacter sakazakii, and how we see it.  And I think it's probably going to be a rehash of all the different things that we've hear today.  Hopefully we'll be up here in a jiffy.

     I think, as I recall, the first thing that we wanted to do was just to cover what is Enterobacter sakazakii.  And we know that it's a gram-negative rod.  We know that it is--whoops.  Now do we have to wait for it to warm up again?

     Okay.  Tom, do you have the--that way, if it does come up we won't be too far off track here.

     But this organism we've all said is an opportunist, which it's been probably in the environment for some time, and has a predilection to cause central nervous system infections.  I think that's important.  Because of that, these are not infections that we usually miss when we treat children with infectious diseases.  And so I guess the question is: are we dealing with the tip of an iceberg?  And I think probably not.  These things are the sort of things that one usually sees and diagnoses.

     The epidemiology of this organism: we know that it's found in very low concentrations in powdered infant formulas.  This seems to be a worldwide phenomenon.  It's also been shown to be found in hospital environments.  I know I reviewed one paper where numerous swabs had been taken around hospital rooms, and several of the swabs demonstrated the presence of this organism.  It's been found in numerous food products.  It's even been found in the gastrointestinal tract of a number of fruit flies.

     So it's probably--we don't know how common this thing is in the environment, but we know that it's there.  And we also know that it's been--that it can occasionally be found in the stools of infants.

     Yeah--go ahead and bring us up to--next one.

     [Slide]

     And we know that it's been cultured out of the stools of some infants who apparently had no symptoms at all, as part of the epidemiological evaluation of outbreaks.

     Slide, please.

     [Slide.]

     Who's at risk for the infection?  Well, the primary risk appears to be in premature infants, especially small premies that are being treated in neonatal intensive care units.  And these children are, of course, with some degree of impairment of immunological function because of their developmental age.  We also know that occasional term infants have been reported to have become infected with this agent, but if you look at the case reports, a fairly high percentage of these--and there were very few of these--have some underlying condition, either immune or barrier function, or some congenital anomalies or chromosomal anomalies and so forth.

     And we also know that immunocompromised children and immunocompromised adults appear to be able to be infected with this organism.  So it preys on, basically, immunocompromised people, like most opportunistic organisms.

     Slide, please.

     [Slide.]

     What does it do?  Primarily it causes meningitis, ventriculitis, and maybe sepsis.  We know it has a predilection for the central nervous system.  And we know that it can be present in the gastrointestinal tract in asymptomatic children.

     And as a pediatric gastroenterologist, I would discourage you from drawing too close of a connection between the reports of necrotizing enterocolitis and this organism.  Necrotizing enterocolitis seems to be a condition that results when you aggressively feed a premature baby, causing some degree of ischemia, and the bowel breaks down, and you get an invasion with whatever organism happens to be there.  And there's a long list of these.  And everyone's thought they found the cause of NEC, and that's never panned out before, and it probably won't with this organism either.  It can certainly cause a problem if it's there and you have an ischemic bowel, but considering it an etiologic agent for necrotizing enterocolitis is probably not correct.

     Slide, please.

     [Slide.]

     What's the risk involved in this particular infection?  There's 50 r 60 cases reported worldwide.  And this is what we're basing our information on.  Since we're dealing with case reports, there are probably more cases out there than we know about, but it still suggests that it's a relatively rare condition.  Most of these occur in premies.  It appears to be an extremely rare condition in health term infants, despite the fact that there are billions of feedings of term infants with powdered infant formula, we seem to have only a handful of cases occurring in this age group.

     Next slide.

     [Slide.]

     Well, then why is it so uncommon?  The organism itself appears to be a minimally pathogenic opportunist.  The infective dose is probably high.  It's probably much higher than we normally see in fresh reconstituted infant formulas.  In fact, if you look at the various case reports, almost all of them suggest that there has been some mishandling--I would call it mishandling--of the infant formula from the standpoint of hang times, or preparation or use, that has let the formula sit around and proliferate, so that there's an increase over the background number that's quite significant in a number of these cases in which infection has occurred.

     And most of these have also included something like environmental contamination or breakdowns, or contamination of blenders and so forth.

     Slide, please.

     [Slide.]

     What, then, could the infant formula industry do to deal with this condition?  Well, I think one of the things we can certainly do is work with the FDA, the CDC and other organizations to do everything we can to help educate users and health care providers.  And this would be to do things like minimize hang time with enteral feedings, to minimize the use of refrigeration to 24 hours, to use liquid formulas in hospital setting whenever possible--especially in neonatal intensive care units.

     We've hear a little bit about boiling, and the risks associated with that.  And we an industry have sort of discouraged that because we're very concerned about mishaps occurring when people might give a formula that's too hot to a baby and cause burning in the esophagus or even an injury to the preparer of the formula.  And there's also now been case reports--at least one--of vitamin C deficiency from a mishandled infant formula.

     So these things can happen and I think probably the major efforts we can utilize are on this slide.  And then I think we have to do everything we possibly can to maximize the capabilities to reduce the levels to as low as possible--which our next speaker will discuss.

     Slide, please.

     [Slide.]

     So, in summary, I think Enterobacter sakazakii poses a limited threat--again, because it's a rare condition--to preterm infants and other immunocompromised individuals in a hospital setting.  And because it's occasionally spread through mishandled reconstituted powdered infant formula, the infant formula industry wants to take an active role in supporting the FDA and the CDC efforts to educate consumers and health care professionals, and to work on new policies and procedures to minimize the exposure of these high risk patients to this organism.

     So, now I think Les Smoot is going to tell us a little bit more about the manufacturing side.

     DR. BUSTA: Dr. Les Smoot is Director of Food Safety Quality Management at Nestle USA, Nutrition Division.

     DR. SMOOT: Good afternoon.  Is this on?  Can you hear me all right?

     Okay.  I'm going to spend just a few moments to, hopefully, follow on and answer a fair amount of the questions--or at least provide some further insight on some of the questions that have been asked most of the afternoon.

     Next slide, please.

     [Slide.]

     First of all, some comments on what is not known about this organism.  We know genetic diversity is still something we're debating and learning as we go along.  It would be important for us to understand the incidence and behavior of the organism throughout the entire food chain.  Still we're gripping, trying to get a hold of where this particular organism--the origin of this; how does it get into the food chain?  So the specific environmental niches is something that we don't have a real clear handle on at this point.  And then, finally, optimum methods for control, detection and recovery.  I think we're still in the very early stages of the science involved with, particularly, the detection and recovery phase.  And, as an industry, we're also continuous ly improving the methods for control.

     Next slide.

     [Slide.]

     What is known about the organism--and this is very similar to what you've seen for most of the day--just recently recognized in 1980 as Enterobacter sakazakii.  As we saw this afternoon, it's not unusually heat resistant.  I think it's very fair to say it survives well in warm, dry environments.  We'll talk a little bit more about that in a minute.

     We also know, in industry, based on our internal studies, that it is not overly resistant to typical food sanitizers; and also knowledge that it does appear to have a narrower host range of salmonella, one of the common food pathogens that we worked against in this particular food product category.

     What is known about ES ecology?  This is getting to some of the questions that have bene asked around the table, relative to where we find this in our food supply.  A recent report by Cordier, et al., has shown we've found it in skim milk powder, a lactose, starch, powdered banana flakes and powdered orange flakes--all of these being typical ingredients for infant cereals and infant formulas.

     Now, Castano, et al., found it in raw pork for a dry-cure bacon.  This was a Spanish study.  Soriano, et al.--I think this was a Spanish study, as well--found it in raw lettuce.

     From and environmental standpoint, Kandhai, et al., in 2003--this is a paper that's being submitted to Lancet and should be published this year--out of the University of Wagenen in the Netherlands, they found this organism isolated from the environment--well, there were actually five milk powder factories, one chocolate factory, one infant cereal factory, a potato flour factory, pasta factory and numerous households.  So we're starting to see that this is not necessarily an unusual organism to be found in the environment where food preparation or food ingredients might be prepared.

     As mentioned earlier, there have been reports of its isolation from insects--this one on a Japanese dairy farm, another one from the gut of the Mexican fruit fly, by Kuzina, et al., in 2001.  Two isolations have been reported in hospital settings; one by Masaki, et al., in 2001 in Japanese geriatric hospital environment, and then most recently the Block, et a., a pediatric hospital kitchen in Israel. So this gives us a little bit of the flavor of the type of environmental ecology, as well as food ingredient and food material ecology, hopefully answering some of the questions that have been raised this afternoon relative to the industrial process.

     What I, hopefully, have here for you is a representation of the partial steps within a food supply chain for the preparation of infant formula.  This is kind of a general step-wise progression.  What we're looking at here is potential sources for E. sakazakii, and also roots of recontamination of the product.  And, as mentioned earlier, raw materials--there needs to be a better understanding of the raw that raw materials play as a potential vector, bringing the organism into the environment of the factory.  I believe Don Zink had mentioned this earlier this afternoon. Also, we have been just discussing, and throughout the day, the concept of handling practices during reconstitution and consumption.

     Ut we want to spend a little bit of item, obviously, where there's some interest is, what type of intervention strategies are applied in the manufacturing process, when we look at these various steps throughout the process as were detailed by Don Zink in the wet and dry blending operations.  What types of activities is industry currently applying?  What kind of good hygienic practices are we currently using to reduce the incidence of this particular organism from the factory environment.

     Next slide.

     [Slide.]

     Well, it's not rocket science, really, but it is something that we have to do and continue to do, and we are currently doing.  Key to this is the raw material supply.  And, as mentioned earlier, we adhere very much to the good manufacturing practices and the HACCP principles.  You see here the term "prerequisite programs."  These are programs that are conducted and embraced within the manufacturing arena that are prerequisite for a successful hazard analysis, critical control point system to be in place, assuring the quality and, most importantly, the safety of the food.

     So, as a prerequisite program relative to HACCP on raw materials, it's very important that you have a very rigorous supplier quality assurance program; auditing, working with the supplier, improving their industrial process to ensure a very clean supply of raw materials.  Secondly, though you want to verify how effective they are in their industrial process.  So it's very typical a very rigorous raw material monitoring program.

     All of this supports the use of the HACCP--the hazard analysis critical control point--principles, particularly for raw materials, hazard analysis on the raw material.  Is E. sakazakii, a significant hazard, likely to occur in the various raw materials that we would use within our manufacturing base?  And I believe Don Zink had answered earlier that from the perspective of the FDA, they have evidence that all manufacturers are adhering to these principles in detail.

     Secondly, the processing environment.  This seems to be a little bit of--there's kind of an open question, in terms of understanding our ability to control recontamination of the product stream.  Prerequisite programs here that are very important; obviously they're good manufacturing practices; the standard sanitation operating practices--this is your cleaning practices that were questioned earlier.  The microbial surveillance programs.  These are the programs that are in place to verify the efficacy of those cleaning programs; in other words, are we using hygienic indicators, whether it be Enterobacteriaceae, whether it be coliforms, so on and so forth.

     And very critical to the processing environment, and I think this goes a ways towards answering some of the questions--this "hygienic zoning" concept.  This is something that's embraced in great detail in many, if not all of the infant formula manufacturers, where we have very strict separation of incoming raw material to finished product handling and filling; also very strict separation between the dry aspects of the factory operations as well as the wet processing areas, because we know the Enterobacteriaceae, as well as the salmonella and listeria all need water to grow.  So, as mentioned earlier, the drier we can keep our environments, in and where we have finished product handling, it's most important to minimize potential regrowth and then recontamination of the product stream.

     So these are the cornerstones that we are continuously using to combat the potential for recontamination.  In the process--and additionally a prerequisite program very critical to the ability to exclude organisms like E. sakazakii out of the process--is the hygienic design of equipment.  And I believe this is where, over the recent years, there has been significant improvement.  And this is one of the areas that the manufacturers themselves, in terms of the design--as Don was showing you the various diagrams, the various pictures of the spray dryers, the drying tunnels and so on and so forth.  This is where technology has started to make a difference in terms of the hygienic design.

     In terms of the HACCP aspect on the process, as mentioned very clearly, the application of thermal related critical control points.  And, finally, how effective are these intervention strategies?  Finished product testing is not an intervention strategy.  It's primarily a HACCP verification program, to assure that these particular activities and raw materials, process environment control and process control, have been effective in assuring the highest quality and safety in the finished product.  So the finished product monitoring that we've been discussing about, in terms of the surveillance data, is really there to verify the effectiveness of the HACCP program and these prerequisite programs.  And as mentioned in the White paper on manufacturing, finished product monitoring is used before release of products.

     Everyone has been talking quite at length today about the Van Acker 2001 report.  This is a set of unpublished data.  This is data from that particular factory in the Netherlands.  Shortly after the even occurred in the summer of 1998, there's mentioned in the CODEX risk profile and numerous speakers about the enhanced manufacturing practices were put in place at this particular facility to assure improved safety and quality of the product.

     Here we have four consecutive years of monitoring the finished product.  These were 100 gram samples coming from that particular facility.  This was a targeted study, above and beyond the ongoing monitoring criteria that were utilized--cited in the publications.  But we have here the percent of these samples that were positive for EB, and then the percent of those samples that were positive for E. sakazakii.  And through the years, through continuous improvement in areas, particularly in hygienic design of the process, as well as environmental control, really learning how to manage the use of water in these dry processing environments, through a relatively level number of samples over those four years we can see a dramatic decrease in the percentage of EB-positive finished product and, correspondingly, see a dramatic--or a reasonably significant decrease in the presence of E. sakazakii in those finished products.

     So this coming from a well-celebrated problematic factory can show how enhanced environmental and process controls can make a difference, can drive this number very low, but not necessarily to the point of eradication.

     Next slide.

     [Slide.]

     So, the frequency in finished products has been reduced through environmental and process control.  And historically we know that CODEX standards have been used to assure the safety of infant formula for the intended population.  And the industry is proposing, at this point, a more stringent standard than the CODEX standard that's currently based on coliforms.  Now we've seen those standards cited earlier.  I think they were shown in Don Zink's presentation.

     So this next slide is the industry proposed sampling and testing program for powdered infant formula.  You'll not that we have expanded--as has been mentioned in some discussion--this particular standard to the entire Enterobacteriaceae family.  It's a sampling program on per lot basis, where the sample size would be 5 grams--the analytical unit would be 5 grams.  The number of units tested would be five, and an acceptable marginal quality would equal 1, where if there were no samples--basically, what we have here is a qualitative test, where you have five 5-gram samples; a total of 25 grams being tested.  If all samples are negative, then you would accept the lot unconditionally.  If there were one sample that were to show positive, then you would only accept the lot if that particular positive sample was confirmed to be negative for fecal coliform.  If it was found to be positive for fecal coliform presence, the lot would be rejected.  And then if there was a chance where there were two of the lots found positive for Enterobacteriaceae, then there would be a conduct of an intensive investigation looking for root cause, as well as confirmation of presence or absence of fecal coliforms, and this could lead to either acceptance or rejection of the finished product lot.

     Next slide.

     [Slide.]

     So, in summary, really, the industry's ability to control this organism in finished product really comes to the implementation of a phased effort through raw material management, and working with the suppliers, continually improving our ability to control the organism in the environment; going after, looking at niches in the environment.  We do know that this organism has the ability to survive in dry, warm environments, and in that particular study, performed on the Netherlands factory, we did see a higher prevalence, typically at the bottom of the spray dryer, in the warmest--one of the warmer parts of the building.

     Process control--really, the key there is the hygienic design for us, in terms of improving how the equipment is designed to minimize the possibility for micro-niches to be development; and then, ultimately, then finished product verification testing.

     Thank you.

     DR. BUSTA: Thank you, Les.

     And the third presenter is Dr. Russell Merritt, Director Medical Affairs, Nutritionals, Ross Products Division of Abbot Laboratories, who will be speaking on industry's response to FDA charge.

     DR. MERRITT: Thank you, Dr. Busta, Dr. Heubi.  And Thank you for the time to present hereto the committee today.

     I'm speaking to you from the perspective of a clinician who worked in nutritional support, including neonatal support, for many years, and more recently have been very involved in industry and our concerns with safety.

     You have heard this afternoon a clinical summary of the issues from the industry perspective, and also a summary of the manufacturing issues and some of the steps that we are proposing to move forward with.  It's my goal to bring this industry perspective into the context of the specific charges that FDA gave to this committee.  So I will comment specifically on the FDA questions, and this commentary is available to you as well in written form.

     Next slide, please.

     [Slide.]

     The first charge was: given available information on E. sak and powdered infant formula, is there a risk?  Are there sub-populations at risk?  And what about immune status, general health, etcetera, in regards to the susceptibility to this infection.

     Next slide, please.

     [Slide.]

     For preterm and immunocompromised infants, we've heard considerable material today, and it indicates that there is a rare risk to such infant.  Somewhere in the neighborhood of 75 percent of the identified cases have a birth weight less than 2,500 grams, or the infants were premature.  There's some suggestion--particularly in those cases where there were issues with formula preparation, storage and delivery, that there may be a dose effect.  It appears that it requires more than simply the presence of an organism--one organism in two or three cans of formula--but rather, the presence, a susceptible host, and then presumably some defect in the environment, by which the formula gets from the can to the baby.

     The situation appears to be quite different in regards to healthy, term infants.  The literature does not--particularly in North America--support the evidence of a clear association between consumption of a formula known to possess this organism, and the development of serious clinical infection.  If any risk exists, it needs to be put in the context of the number of feedings of infant formula that are used per year, and the extraordinary rarity of this infection.  If you think about it, a baby probably gets about 2,000 feedings over the course of a year, and maybe close to half of those in the U.S. come from infant formula.  Then crudely, that gives you something on the order of four billion feedings--or two billion feedings a year of the four million babies who are born.  And yet one rarely sees this organism.  We heard earlier about the CDC screening program in 300 hospitals, where a case was identified, to my knowledge, not associated with infant formula.

     So part of the critical task of the committee is to differentiate the possible presence of E. sakazakii on occasion in infant formula from the risk of infection in infants.

     Next slide.

     [Slide.]

     If there is a risk, what intervention strategies can be used in infant formula manufacturing processes and plants?

     Well, you just heard from Dr. Smoot that there is an opportunity in almost any situation to enhance environmental monitoring and controls, using the HACCP principles.  And you have just seen an excellent example of where not only E. sakazakii, but also enterobacter, presence in infant formula was substantially reduced by adherence to these principles.

     Industry is also proposing confirmatory final product testing for enterobacter.  Again, this is a confirmation of the effectiveness of the environmental and the incoming ingredient controls that are placed on the manufacturing process, more than the method of controlling the product.  The testing is not the method, it's the indication that those methods are working.

     Next slide.

     [Slide.]

     Are there other intervention strategies?  And I think, clearly, we've heard that there are.  We can minimize the use of powder in at-risk infant populations to the extent possible.  We've heard from Dr. Thureen's experience a little bit about how difficult this would be to eliminate completely.  And I believe Dr. Bhatia may address this issue as well in his comments.

     We can promote the published guidelines from the CDC, from the American Dietetic Association that emphasize appropriate hygienic practices within the hospital.  And, again, picking up on Dr. Thureen's comment, I was impressed that perhaps if neonatologists understood more readily that powdered formula is, in fact, not sterile, then the motivation no their part to control the use of these products, both in terms of quantity and in terms of practice, might in fact represent an opportunity for us.

     The hot water reconstitution process has been described--also some of its limitations, including possible incomplete bacteriocidal kill, vitamin C loss, protein denaturation and resultant clumping, and injury that may result to personnel preparing the formula, if we have boiling or hot water in the nursery environment.

     In addition, there is opportunity, I think, to limit hang time.  And based on what we've heard today, the four hours seems to make some sense, and more consistent labeling and directions for preparation and use is another opportunity.

     Next slide.

     [Slide.]

     Continuing the second charge: is it possible to identify lower levels of E. sakazakii in powdered formula and, in effect, assign allowable levels?

     Next slide.

     [Slide.]

     For premature or immunocompromised infants, the data are insufficient to really specify an allowable level, given that there's at least some degree of risk there.  For healthy term infants a low, non-zero level can be established, based on the historical exposure data--for example, 5 to 15 percent of infant formula, based on a number of surveys.  The extraordinary rarity of this infection in term infants, and the data we have heard today about the numbers of organisms that appear to be required in neonatal animal inoculation studies.

     Next slide.

     [Slide.]

     What are the critical knowledge gaps and research opportunities?

     Next slide.

     [Slide.]

     Well, certainly we need to understand more about the circumstances that need to opportunistic pathogenicity; whether it's susceptibility factors in the host, or the virulence factors in the organism itself.  We need to understand of the incidence and behavior of the organism in the food chain.  I think Dr. Smoot's comments were helpful in giving us some direction in that regard.  The probability and circumstances under which pathogenic levels--that is, levels capable of causing infection in susceptible populations--occur.  And then, lastly, we need to focus attention to the methods that may potentially give us environmental control, whether that's in ingredients a the level of plant manufacture, or at the site of use.

     So, again, I thank you for the opportunity to provide these industry provisional answers to the questions at hand.

     DR. BUSTA: Thank you, Dr. Merritt.

     The fourth presentation this afternoon is by Dr. Jatinder Bhatia, Medical College of Georgia.  The title is, "Use of Powdered Additives in Neonatal NICUs."

     DR. BHATIA: Dr. Busta, Dr. Heubi, Ms. Latham, distinguished members and guests.  Thank you for the opportunity to share my thoughts with you.

     I come from the perspective of being trained as a neonatologist and a nutritionist who's been practicing his trade for a while.

     Next slide, please.

     [Slide.]

     The needs have been highlighted because we truly don't have a gold standard for pre-term babies.  And the gold standard the AP has put forth is that post-natal growth that approximates the in utero growth of a normal fetus.  Well, the question that can be raised is: should you achieve maximal achievable gain without adverse metabolic and other costs?

     We know from a whole body of literature that growth of most of these infants remains lower that in utero, and that the nutrient intakes remain lower than the fetal counterpart during much of the in-hospital stay, and possibly much beyond.

     Now, what are the consequences?  And this is has been known in the animal literature and the human literature for a long time.  Under-nutrition, especially during these vulnerable periods of brain growth, can have permanent CNS development, cognition, behavior and somatic growth effects.  And these effects may be present, even if there's sub-optimal growth, as shown in animal studies.  For example, an animal doesn't grow, he may have these side effects, but if the animal grows suboptimally, he may still have the same effects.  Therefore premature infants are at high risk.

     And I don't need to remind this body that the extremely low birth weight infant has additional needs for protein, calcium, sodium that far exceed those for the term infant.  And these, perhaps, may not be completely met with the armamentarium that we have at present.

     All this information has ben reiterated by the NICHD-sponsored trials published recently, which has heightened the awareness of practitioners of nutrient intakes and growth curves.  These were something that we knew, but we did not have.  And I want to share with you the data on these three cohorts of very small premature babies--24, 25, 26, 27 and 28, 29 weeks.  In general, birth weight was regained by about two-and-a-half weeks, with the larger babies gaining them a little sooner.  But the more important point is the next one.  By 32 weeks post-conceptional age, growth curves of all cohorts fell below the 10th percentile--and I'll show that to you in the next slide graphically.

     The three cohorts that you have here--the first one being the 24, 25--the largest one--all three of them, even if you look at 36 weeks post conceptional age, all fall below the 10th percentile, and the smaller babies may not catch until almost the first year of life.  This is despite the maximal feeding that we can do currently.  So we are taking appropriate-for--age babies, and now making them small for gestational babies by the time they leave the hospital.

     So, at 32 weeks the average weight of a 24, 25 infant was 50 grams below the 50th percentile; 26 to 27, by 780, and the larger cohort by 715 grams.  And the postulated reasons, at least to practicing neonatologists, is the expected post natal weight loss, and our inability to feed babies to the maximum potential, and therefore under nutrition.

     So what are the strategies that we use?  Early initiation of parenteral nutrition, starting on day one or day two; early initiation of enteral feedings, also limited by fear of necrotizing enterocolitis--although, as Dr. Vanderhoff has pointed out, and other people have pointed out, that the starting or not staring of enteral nutrition early has no association with it.  There are other associations, which I'll come back to later; increasing nutrient density while maintaining relative fluid restriction, because we can only go so far as far as fluid volume goes, and therefore the need for supplements.

     What do we have available?  Liquid concentrate, powdered formula--either the premature or transitional formulas--or single nutrients.  Or we have MCT, vegetable or lipid emulsions.

     Here's an example of what you would do if you want to increase caloric density by either using a single constituent or a balanced powdered formula.  For example, on the left hand slide, a 24 cal premie has 81 calories, and draw your attention to the completely far right hand corner, that if you add a proportionate amount of powder to the premature infant formula, you can achieve 98 calories per 100 ml; you can increase the protein intake and, more importantly, you can also increase calcium intake, which is shown on the bottom right-hand corner; whereas, if you use corn oil, as an example of vegetable oil, or polycose, all you do is increase calories, you do not increase protein, and you don't affect calcium.  So a balanced addition of nutrient is far better than using single product.

     Now, I'm going to share briefly some disease-specific concerns, and why these are important.  For example, bronchopulmonary dysplasia, a chronic lung disease, a fairly common entity known to neonatologists.  Anywhere between 30 to 80 percent, depending on the cohort you study, will have continuing needs.  And there is need for additional nutrients to compensate for the increased work of breathing, and side effects of common drugs.  And, more importantly, these babies also need the same nutritional intervention after they go home, because--several studies have been done.  For example, one study has clearly demonstrated that afterwards 73 percent of babies, after they leave the hospital, now deviate in their growth.  And this can be obviated, to a certain extent, by using nutrient-rich formulas, which then come from supplementation.  We also have an issue of fluid restriction.  And since they are also born prematurely, they also have high needs of calcium, phosphorous and electrolytes.

     Necrotizing enterocolitis, which we've been hearing all day--the only two factors that I know that have held the test of time have been a baby has to be premature, and maybe the feedings have been advanced very rapidly, and this has been reevaluated again as of this month.  More importantly, in the clinical arena, timing of NEC does not always--or usually does not have--a temporal relation to the addition of powdered nutrients.  And there's a wide range of onset of NEC, with less than 40 percent of them having concomitant positive blood or other cultures.  In other words, you heard this morning as well, that there are whole host of organisms that are associated with NEC, but there is also a high incidence of NEC without any organism identified at all.

     So what are the long term consequences?  We know that very little catch up growth occurs.  I've already shown you the growth curves up to post-conception age of 36 weeks.   We have been creating, and continue to create, shorter, lighter infants, with a smaller head circumference compared to the fetal counterpart, and now data as long as eight years of age, because of the early growth restriction that occurs in the NICU.

     And the neurological outcome of these babies is not always explained by disease process alone.  So I believe that nutrition, or under nutrition, has to have played a role as well.

     And here's a good example of that.  These data were published in 2001, and what they're showing, the left hand, at birth, that babies who were above the 10th percentile, and by the time they're discharged, the number of babies who are above the 10th percentile.  And you can see, between 70 and 85 percent were appropriate or above 10th percentile at birth, and barely above zero--way above 10th percentile, by the time they reached 36 weeks post-conceptual age.  And this has been shown, not only in England, it's been shown in other places in the United States as well.

     So, post-discharge, which is now becoming a more and more important thing--evidence demonstrates better growth with enriched formulas, i.e., the transitional formulas, or using premature formulas at home.  These infants are predominantly fed powdered infant formulas due to cost.  And, with earlier discharge from the hospital, calcium and phosphorous, and other nutrient needs cannot or may not be met by term-infant formulas, and this statement is supported by the American Academy of Pediatrics as well.

     So in-hospital practices--given the variety of scenarios in the NICU--and this is my own anecdotal data, and I believe, if you surveyed, you'll find similar data.  And Dr. Thureen can confirm that for me--up to 50 percent of infants in a large NICU will have some form of feeding modification--either addition, thickening due to feeding intolerance or concentrating.  However concentrating inherently has a problem with changing osmolarity, which then limits the feeding problems as well.

     And rather than asks the question of how have the in-hospital guidelines suggested by CDC affect the current practices, what we're now seeing is while trying to adhere to the CDC, ADA and FDA guidelines, that neonatologists and nutritionists are reluctant to use appropriate measures to try and achieve the maximal achievable gain that I said before.  So we've gone backwards, at least in ability to provide the nutrition we believe we need to.

     So how do neonatologists weigh the risks?  We need to establish enteral feedings.  We all agree with that.  We need to establish adequate weight gain, because mortality, morbidity and long-term outcomes are clearly related to weight and gestational age.  And the risk of E. sak--20 cases per 10 years, or however you want to count it--should be put in a perspective that during the same period of time, there were more than four million premature infants who were fed and delivered during that period of time.

     Thank you.

     DR. BUSTA: Thank you.  We have about 10 minutes to ask questions of these last four presentations, if there are any questions.

     Dr. Stallings.

     If the four of you would come up, we'd appreciate it.

     [Pause.]

     You just have a comment, Dr. Tompkin?  All right.  Go ahead.

     DR. TOMPKIN: Yes.  This is Tompkin.

     The white paper that was on the table in front of each of us is the white paper that the industry has prepared and shared with us.  And, likewise, the information that Dr. Merritt presented, in terms of the industry's perspective on the answers that may be the outcome--that also was prepared by industry.  So each of you should have those documents.

     DR. BUSTA: Dr. Stallings.

     DR. STALLINGS: Stallings.

     I have almost a question--short question--for each of us.  So, starting with John--if we were starting at the beginning of this question--and in your slide you summarized that you felt that the pathogen, it was a minimally pathogenic organism, and that the infective dose was high.

     And I guess I'm not convinced of that.  And what kind of animal models, neonatal animal models--I mean, there should be some pretty straightforward ways of testing that.  Do you, or you guys, have any ideas?  Because I would feel a lot more comfortable if I knew that from something other than we've given, you know, four billion feedings and 10 babies have gotten sick.

     DR. VANDERHOFF: I think at this point in time that is an assumption that we're making.  And it would be nice if we could verify it with some animal models.  And I'm probably not the ideal person to tell you which ones would be most valid, but I know there are some.  But this, I think comes from the epidemiological knowledge that there's a huge amount of feedings--Jatinder just told us--of powdered infant formula to pre-term babies; half the premies get added powdered.  And, of course, all the term infants being exposed to low levels of this stuff, 14 percent of the formulas appear to have low-level contamination, according to the data yet, you know, I've never seen one of these.  I asked Jatinder if he'd ever seen one?  I don't know if you've seen one.

     But this is a very uncommon infection.  And it would certainly suggest to us that there must be some other factors, such as mishandling or host defense problems or so forth, involved in doing this.  I agree, I think investigating it with an animal model would be ideal.  I'm not sure we can a hundred percent draw the connection between the two, but it will certainly give us an idea.

     DR. MERRITT: If you think about it, you're never going to have the dose response in the human, because the event is going to occur sufficiently long after the event, that the actual concentration in that formula will never be done.  So that forces you to a dependence, presumably, on an animal model to try and get that answer.  And Dr. Nazarowec-White--I mean, we were desperately searching for some of that kind of information.  And the paper that came out this month I think is the first clue in that regard.  And, at least, I believe, suckling animal model, it indicates that the number of organisms is quite high, and if the practices we are suggesting that we all adhere to in conjunction with the levels of detection that were described were used, we shouldn't see those levels.

     Now, I don't think we have any way of knowing that the mouse is the same as the 800 gram baby, but that's the best information we have, I believe.

     DR. STALLINGS: But, really, it's not the 800 gram baby that's getting the formulas.  It's usually a little bit later.  But--okay.

     So, the next question--for Les.  How big is a lot?  You know, when you were describing what's been proposed, it was, I think, 5-gram samples times five samples.  And I'm just not familiar enough to know what is a lot, and, you know, really what kind of surveillance might that represent?

     DR. SMOOT: Right.  I can only speak from our experience with Nestle.  Typically, a lot for us might range in about a 20 ton--

     DR. STALLINGS: How many?

     DR. SMOOT: 20 tons.

     DR. STALLINGS: Okay.  That's a lot of cans of baby food.

     DR. SMOOT: True.  Yes.

     DR. STALLINGS: 20 tons, and we would be sampling about 25 grams--5-grams times five.

     DR. SMOOT: For the family Enterobacteriaceae--correct.

     DR. STALLINGS: Okay.

     And then my last question is, are the HACCP regulations--are they regulations?  Are they required, or are they voluntary at this point?  Because there was a lot of reference to using that kind of evaluation of manufacturing and risk analysis.  And I'm--

     DR. SMOOT: I don't believe it's mandatory on FDA.  It might be infant formula regulations, the GMPs--we see this more of a mandated effort.  But in our manufacturing base, typically HACCP right now, the use of these principles I would say to be voluntary in these types of processes.

     DR. STALLINGS: So I just wanted to be clear that that might be required in the future, but right now it remains voluntary.  Thank you.

     DR. BUSTA: Dr. Moyer?

     DR. MOYER-MILEUR: Moyer Mileur.

     I think I want to direct my comment to Dr. Bhatia, but then get response from all of you.

     I think your comments--I appreciate your comments, and I think you're preaching to the choir when it comes to Dr. Thureen and myself, as far as the extremely small or extremely premature infants not being served well by--in that growth is minimized during their hospitalization, and continues to be minimized.

     The concern I have is that I don't see this as an--the issue of powdered formula in these babies as an issue until later on in their neonatal period.  And my personal experience with these babies is that we really can't even get up to a full volume of enteral feeding until at least 21 days of age, at which time we would either be using mother's milk or a liquid premature infant formula and then go to 24-calorie, and then if not able to get them to grow, then start to consider adding additives to those products.

     So my bigger concern is that I think the message you had is that you're worried that neonatologists will stop--will not provide sufficient nutrition because of the worry about contamination from E. sakazakii.  And I don't know--and so I think maybe directing this critical need for education to neonatologists, but not only neonatologists, but other care providers who do see preterm infants or other infants who are immunocompromised, and that includes pediatricians, that includes nurse practitioners, both neonatal and pediatric nurse practitioners, as well as registered nurses.

     I don't know if you--

     DR. BHATIA: Well, I would have changed my slides if I'd known you two were going to be here.  Anyway--the point's well taken.  The approach we take is exactly the same.  But the point I think I was trying to get across is that without these additives available, we will not be able to even feed a human-milk fed baby or give the additional needs of specific concerns.  I can go on and on.

     But these are not additives you add in the first two weeks of life.  These are much later.  This is not to the period of time when you see this organism affecting babies, so the temporal relationships seem different.  My issue was: these are the only things we have left now to try and do better with what we have.  The current formulas for premature babies were made when survivors were above 750 to 800 grams.  They were not designed for 500 to 800 gram babies.  So the best we can do is use a balanced additive, and that's the point I was trying to make--including the human-milk fed preterm baby.

     DR. BUSTA: Dr. Heubi.

     DR. HEUBI: Heubi.

     My question is one that I think both John and Russ commented about--immunocompromised and pre-term infants being potentially at risk.

     What are your thoughts about, number one, the issue of kids that have motility disturbances, short gut, other kinds of problems, who are exposed to potential "specialty formulas" that tend to be non-liquid in composition and be powder instead, and whether you would consider them to be a potential risk from exposure because of their underlying condition?  And then I have another question after you answer that for me.

     DR. VANDERHOFF: I think I've seen one case report of a short-gut getting infected with E. sakazakii, despite the fact that there are a lot of these, and they've been fed with this stuff for a long period of time.  And I've seen them get infected with almost every other enteric organism over the years.  So I think it's relatively low risk.

     The motility disturbance kids--probably same argument.  Anything you feed to these kids, because they have mucosal breakdown, anything that grows in their gut can get into their blood stream.  But I don't think it's a big risk for kids with GI tract disease.

     Russ?

     DR. MERRITT: I think, clearly, there's a literature that indicates that short-gut is a special situation immunologically, and these children are often not well nourished as well.  So it's hard to deny some potential there.  They become somewhat more like the premature baby, in terms of having impaired gastrointestinal function and barrier.

     DR. HEUBI: So, my follow-up question is this: what position will industry take in terms of trying to educate physicians and health care providers--just like what Laurie was pointing out--about the fact that these formulae that are powders, are not absolutely sterile.  Because I think, in general, most clinicians don't understand this.  Now, I don't know that we should scare people.  But I think, on the other hand, it should be clear that these preparations are not sterile, and that there are some potential minimal risks, or risks associated with their use.