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 20^th 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 10^th to 20^th 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 10^th 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.