Introduction

Microbiology is an exceptionally broad discipline encompassing research areas as diverse as taxonomy, physiology, biochemistry, molecular biology, pathogenesis, food and industrial microbiology, and ecology. In fact, modern biotechnology rests upon a microbiological foundation. The microbiology research at the NCTR serves a multipurpose function with specialized expertise to perform fundamental and applied microbiology research in areas of the FDA responsibility. The microbiology research also responds to microbial surveillance and diagnostic needs for research projects within the Agency. The major aims of the microbiology research are to raise the general awareness of the importance of microorganisms in public health and to provide data to improve our understanding of the mechanisms by which toxic events occur in humans. The research is organized to handle many aspects of microbial toxicology and continue to train staff to meet the research and regulatory needs of the FDA. The microbiology research at NCTR is divided into five focal areas with strategies and objectives unique to the problem posed. Goals and accomplishments for each focal area are discussed separately below.

FY 97 Goals

1. Determine the role of intestinal microflora in the activation or detoxification of xenobiotics.

   Research on the role of gut microflora in human carcinogenesis is an important FDA need since a high proportion of human cancer is caused by environmental factors, and diet may be particularly important.

   Since the bacterial flora are in a uniquely favorable position to mediate the interaction between the gut contents and the host, it would be surprising if bacteria were not implicated in human carcinogenesis. Therefore, the focus of this research component is: 1) to use existing models for determining the contribution of the gut microflora to foreign compound metabolism in humans and laboratory animals; 2) to relate bacterial metabolism to toxic events occurring in mammals; 3) to consider the interrelationships of bacterial and mammalian metabolic pathways; 4) to determine the effect of dietary components on the composition of the microflora in the human gastrointestinal tract; and, 5) to determine the genes involved in the metabolism and activation of pharmaceutical azo and nitrocompounds in normal populations and in patients with intestinal disorders.

   Research goals for this subprogram are: 1) to delineate the metabolic potential of intestinal microorganisms and the enzyme mechanisms by which they transform drugs, azo dyes and food additives; 2) to develop additional models for assessing the risk to human health posed by exposure to synthetic and naturally occurring chemicals; and, 3) to determine the pharmacological and toxicological effects of the metabolism of chemicals such as food additives, azo compounds used as protective coating for drug delivery and prodrug azo compounds, and antimicrobial compounds on the intestinal microflora.

2. Use microorganisms as models to predict the metabolic pathways by which drugs are metabolized in mammals.

   In recent years, interest has turned to the development of alternative systems for decreasing the use of animals in laboratory studies. Microbial systems are an attractive alternative to mammalian xenobiotic and toxicity studies. The advantages are: 1) ease of experimental manipulation; 2) ease of scale-up for production of metabolites which other investigators could use for structure elucidation, biological evaluation and analytical standards; 3) lower cost; and 4) reduction of the use of laboratory animals. The focus of this research component is to develop alternative methods for toxicity testing of drugs for the FDA. This research will provide more accurate quantitative risk assessments and a better understanding of the mechanisms of toxicity.

3. Develop environmental biotechnology.

   FDA's premarket review considers potential environmental impact during the entire life cycle of a regulated product, including its manufacture, use and disposal. Under the FDA's environmental regulations, the industry sponsor of an application or petition may be required to prepare an environmental assessment of the proposed action. To support the assessment, appropriate testing of the environmental fate and effects of chemicals entering the environment may be required. The need for testing is determined by evaluation of the potential environmental exposure and the toxicity information available for a given chemical.

   Due to the high cost associated with trapping, incinerating or physically removing toxic chemicals from the environment, there has been an increased interest in the use of microorganisms for the biological decontamination and detoxification of hazardous waste sites. Because the environmental risk assessment and management of potentially hazardous chemicals requires information on their occurrence, toxicity, bioavailability and persistence in the environment, we have developed multi-component environmental microcosms. These microcosms are useful for determining the rate and pathways for the environmental biodegradation of xenobiotics. The focus of this research is to isolate microorganisms which can degrade, detoxify, or accumulate hazardous chemicals and to determine the potential for their use in the bioremediation of toxic waste sites. This methodology will be used for several FDA-related research problems.

4. Develop methods for detection of contaminants

   Foodborne bacterial pathogens have been detected in contaminated foods using molecular genetic methods. Effective and sensitive methods are needed to detect contamination in foods to determine if the levels of contamination pose a public health risk. Polymerase Chain Reaction (PCR)-based methods have the potential for revealing the presence of pathogenic microorganisms in foods in a few hours while current methods require two days or longer. Rapid detection and identification of bacteria are important not only for food safety, but also for the study of the significance of the species on both in vitro and in vivo metabolic activation and detoxification of chemical toxicants and drugs and for the diagnosis of the diseases caused by these species. Development of better in vitro methods for rapid detection of bacterial pathogens and toxins will provide the FDA with analyses critically needed for assurance of food safety and enforcement of regulatory compliance.

5. Continue microbiological surveillance and diagnostic support of research

   Laboratory animals are susceptible to a wide variety of bacterial, viral and parasitic infections, resulting in an altered animal model that consequently affects research and testing by introducing variables that confound results. Routine screening for various infectious diseases assures reliable animal models and prevents costly, time consuming delays of research which could affect FDA regulatory decisions. Studies utilizing animals are dependent on healthy test animals; therefore, it is NCTR's responsibility to maintain the best microbiological diagnostic laboratory possible. The investigators and the FDA should be able to depend upon NCTR to support their efforts. Research goals for this subprogram are: 1) establishing and maintaining pathogen-free animals; 2) developing bacteriological assays for determining chemicals, such as folate in culture fluid, for research projects within NCTR; 3) culturing and identifying microbial contaminants for other projects and programs within the NCTR and other FDA centers; and 4) developing and testing new methods in diagnostic microbiology for other FDA centers.

FY 96 Accomplishments and FY 97 Plans

In FY96, microbiology-related research issues were discussed with microbiologists from other FDA centers and field laboratories. The scientific exchange led to the initiation of new projects and exchange of scientists between laboratories. A short summary of some resulting collaborative research projects are listed below:

I: Development of quantitative assays for measuring the tuberculocidal activity of chemical disinfectants.

Tuberculosis, once considered a disease brought under control through use of antibiotics, has re-emerged as a serious health concern in the United States. The percentage of tuberculosis cases caused by strains of Mycobacterium tuberculosis that are resistant to one or more of the antibiotics used in therapy is increasing. While tuberculosis is not readily transmissible by casual contact, it can be spread where individuals live or work in very crowded conditions, and perhaps by certain medical procedures as well.

Numerous chemical agents are used to disinfect and sterilize medical instruments, such as endoscopes, that cannot be autoclaved. Endoscopes contain crevices and channels that are difficult to clean and can harbor bacteria. Many of the liquid chemical germicides on the market claim the ability to kill Mycobacterium tuberculosis, yet improperly washed and disinfected endoscopes have been linked to the transfer of this organism from tuberculosis patients to previously uninfected individuals. This has raised the concern that some of the disinfectants may not be fully effective under the prescribed conditions.

The FDA is preparing to evaluate the tuberculocidal activity of a large number of liquid chemical germicides. The Division of Microbiology at the NCTR has been instrumental in the preparation for this evaluation by developing the expertise required to perform the Association of Official Analytical Chemists (AOAC) tuberculocidal assay, clarifying and expanding the protocol for this assay, and training ORA personnel to conduct this assay at their own facilities.

The current methods for determining the tuberculocidal activity of disinfectants are difficult to perform, poorly reproducible, and require up to 90 days to obtain a result. Scientists in the Division of Microbiology are attempting to implement molecular methods to both improve the sensitivity and accuracy of the test, and shorten the time required for a definitive answer. One such approach is the use of a mycobacteriophage carrying the firefly luciferase gene to quantitatively determine if the mycobacteria survive exposure to the disinfectants (E-6965.01). This phage should promote light production from surviving bacteria, allowing quantitation of the disinfectant activity. By developing and validating such an assay, we hope to be able to rapidly assess the effectiveness of both currently available disinfectants and future products.

Three FDA analysts from Denver, Minneapolis, and Winchester Engineering and Analytical Center (WEAC) were trained in the AOAC tuberculocidal assay by scientists at the NCTR. The topics covered were biosafety and facility requirements, contamination control, growth and standardization of the test organism, media preparation, carrier preparation, disinfectant preparation and neutralization, phenol standardization, test performance, and interpretation of results. Discussions also included how their current facilities could be modified to provide appropriate conditions for the test.

II: Develop molecular and mass spectrometry methods for the detection of foodborne pathogens.

Despite the fact that the United States food supply is the safest in the world, tens of millions of cases of foodborne illnesses occur in the United States every year with a cost to the economy of an estimated 1 to 10 billion dollars. Therefore, the microbiological safety of food has become an important concern of consumers, industry and regulatory agencies. The U.S. Food and Drug Administration gives a high priority to protecting the public from microbial contamination of the food supply. The research program in the Division of Microbiology in FY96 has a project (E-6988) to develop molecular methods to detect and identify foodborne bacterial pathogens. In addition, scientists in the Division of Microbiology are collaborating with scientists in the Division of Chemistry to use mass spectrometry methods for the rapid identification of bacteria (E-6785 and E-6931).

A protocol (E-6988.01) for the detection of 13 species of foodborne pathogens in foods using the polymerase chain reaction (PCR) technique was developed in FY96. The method used a universal enrichment medium and the same PCR conditions with 13 sets of specific primers for the detection of foodborne pathogens. The foodborne pathogens examined were Escherichia coli, Shigella, Salmonella, Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. No interferences were observed using the PCR assay for food samples artificially inoculated with each single bacterial species.

In addition, a 16S rDNA-based PCR method was developed for the specific detection of Aeromonas caviae and Aeromonas trota. The detection limit was between 50 and 100 cells per gram of crab meat. This method is very rapid, obviates the need for DNA isolation from complex food matrices, and is specific for screening two pathogenic species of Aeromonas.

Mass spectrometric methods for the identification of bacteria were also evaluated in FY96. Scientists from the Division of Chemistry and the Division of Microbiology used pyrolysis mass spectrometry for the rapid identification of whole bacterial cells. Suspensions of five strains of bacteria (Escherichia coli, Bacillus sp., Pseudomonas aeruginosa, P. mendocina and P. putida) were placed in wells in a Teflon cell culture plate. The cells were electrically deposited on the sample wires for pyrolysis; the mass fragments were scanned over the m/z range of 30 to 300. Pattern recognition software was used to analyze the various factors that characterized the pyrolysis mass spectra. The mass spectra obtained from 30 out of 31 different cultures of the five bacterial strains were identified correctly to the species they represented.

Another mass spectrometry method was evaluated to identify bacteria. Scientists from the Divisions of Chemistry and Microbiology used matrix-assisted laser-desorption-ionization time-of-flight (MALDI/TOF) mass spectrometry to identify intact cells of eight strains of bacteria that may be found in foods. The bacteria (Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Serratia marcescens, Shigella flexneri, Pseudomonas aeruginosa, P. mendocina, and P. putida) were grown on tryptic soy agar plates, suspended in matrix solution containing -cyano-4-hydroxycinnamic acid, and used to obtain reference MALDI/TOF mass spectra over the m/z range of 4,500 to 14,500. The mass spectra of these and other bacterial strains were analyzed for diagnostic ions that might characterize the different species. Two approaches for identification were successful: 1) comparison of new mass spectra with archived spectra from known species, and 2) co-analysis of unknown bacteria with cultures of known bacteria. Although some diagnostic ions were observed with more than one strain, enough unique ions were observed to allow all of the strains to be unambiguously distinguished from each other.

III. Assessing the effects of food additives and drugs in food on the human intestinal microflora. Determining the role of intestinal microflora in the metabolism of therapeutic drugs, food additives and cosmetics.

In recent years, questions have been raised concerning the consumption of low levels of food additives and antimicrobial residues in foods and the effect of these residues on the indigenous human intestinal microflora. Intestinal microflora are an essential component of human physiology because they act as a barrier against colonization of the gastrointestinal tract by pathogenic bacteria. They also play important roles in the digestion of food and the metabolism of drugs, xenobiotics and nutrients. Repeated exposure to antimicrobial residues and food additives may perturb the normal population density of intestinal microflora, altering enzyme activity for the metabolism of endogenous and exogenous substances, and impairing colonization resistance, which may increase susceptibility to infection by enteric pathogens such as Salmonella, Shigella and Escherichia coli.

The Director of the Division of Microbiology has provided guidance to scientists at the Center for Veterinary Medicine (CVM) and reviewed research protocols for the CVM on the effects of low levels of antimicrobial residues in food on the human intestinal microflora. In addition, he, wrote a guidance document for the World Health Organization on "Assessing the effects of antimicrobial residues in food on the human intestinal microflora." This document will be used by regulatory agencies, industry drug sponsors and the international scientific community as a guideline for making an assessment of the potential risk of dietary intake of residues of antimicrobial animal drugs.

The research program in the Division of Microbiology at the NCTR in FY96 has developed molecular methods for the detection of predominant anaerobic bacteria in human and animal fecal samples. PCR procedures based on 16S rRNA gene sequences were developed and used for quantitative detection of intestinal microflora in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey and rabbit) feces. This method, including the fecal sample preparation method, is rapid and eliminates the DNA isolation steps. The method is being used in research at the NCTR for assessing the effects of food additives, antimicrobial residues and caloric restriction on the human intestinal microflora. In addition, the Division of Microbiology has been contacted by scientists from universities, pharmaceutical industry and regulatory agencies for advice and training concerning the method.

Studies have continued in the division on the determination of the role of intestinal microflora in xenobiotic metabolism. Various enzymes from the human intestinal tract play a role in the activation and/or detoxification of food additives, therapeutics, azo compounds, and nitro compounds. Some azo dyes are reduced to mutagenic compounds following reduction by bacteria from the human intestinal tract. Scientists in the division are investigating the effects of bacteria from the human intestinal tract on seven different azo dyes currently used in the food, pharmaceutical, and cosmetics industries. All of these dyes were reduced by the bacteria isolated from the human intestinal tract. Mutagenicity assays, using two strains of Salmonella typhimurium, showed that none of these dyes or their reduction products were mutagenic. The azoreductase genes from the various anaerobic bacteria involved in the reduction of these dyes were analyzed, and variations were found among the structures of the azoreductase genes from the different bacteria.

Azoreductase and nitroreductase convert some therapeutic azo and nitro compounds to their activated forms. These drugs are used not only for the treatment of bacterial infections but also for the treatment of inflammatory bowel diseases with unknown etiology. The reductase activities in fecal samples from pouchitis patients during the onset of the disease and following recovery was evaluated. Higher levels of azoreductase and nitroreductase were found in all of the patients following recovery. In addition, the role of anaerobic bacteria from the human intestinal microbial flora in the metabolism of nitro-substituted benzodiazepines, which are used extensively for the treatment of anxiety, was studied. These compounds have been shown to be teratogenic in experimental animals, and nitroreduction by anaerobic intestinal bacteria is considered to be involved in the mechanism of toxicity. The bacteria isolated from the human intestinal tract that had nitroreductase activity were shown to reduce the nitrazepam to 7-aminonitrazepam.

IV. Conduct biodegradation assessments of antibiotics used in aquaculture and other priority pollutants.

Bioremediation principles, i.e., the use of microorganisms to degrade pollutants under controlled conditions to an innocuous state or to levels below concentration limits established by regulatory authorities, offers great promise for accelerated removal of chemical pollutants in the environment. A drug registration package must contain data that demonstrates that the proposed substance is efficacious against target pathogen, safe for human use and safe for the environment.

A project (E0690101) was developed in the Division of Microbiology in collaboration with the regulatory scientists of the Center for Veterinary Medicine (CVM) to evaluate the environmental impact of antibiotics and feed additives used in fish farming systems. Aquaculture industries around the world extensively use antibiotic erythromycin for control of bacterial kidney disease in salmon and trout. It is currently under review for approval in the United States. Since aquaculture waste water and sediment are discharged into the environment, there is concern over the potential detrimental effects on the environment and public health. CVM needs environmental impact and biological activity data on erythromycin for its approval.

Upon reviewing the literature, scientists in the division found that very limited studies have reported on the environmental fate of erythromycin used in aquaculture. The extensive review of antibiotics used in fish farming systems also led us to write a chapter on the environmental fate of antibiotics for an upcoming book on Bioremediation: Principles and Practice (in press). Considering the lack of available information on the environmental impact of erythromycin, the first and foremost challenge was to develop a sensitive bioassay procedure. NCTR was successful in developing a sensitive bioassay procedure to determine biological activity of erythromycin in aquaculture and environmental samples. This technique is suitable for testing water from marine and aquaculture environments, as well as extracts of a variety of environmental sediments (posters presented at the World Aquaculture meeting in Dallas and the American Society of Microbiology in New Orleans). Since then the organism Xanthomonas has been identified as the indicator organism (abstract submitted to the American Society of Soil Science). A manuscript describing this bioassay is presently in review for publication in an Aquaculture Journal.

The division studied the behavior of erythromycin under a variety of physicochemical and environmental conditions and found that a variety of microorganisms native to the aquaculture environment were responsible for biodegradation of erythromycin and a host of metabolites produced lack antimicrobial activity (abstract submitted to the American Society of Soil Science). A manuscript is in preparation on the fate and degradation of erythromycin.

One of the most important aspects of this project is to develop a standard analytical method to detect erythromycin in aquaculture and environmental samples. This method once standardized, will be incorporated into CVM Environmental Assessments - Technical Methods Handbook - and used as a guide for the evaluation of drugs and feed additives requiring FDA's approval. An analytical method was developed which uses HPLC and electrochemical detector to detect and quantify trace-levels of erythromycin (manuscript in review for publication in the Journal of Food and Agriculture).

The erythromycin resistance by bacteria isolated from antibiotic-impacted sediments was evaluated and found that it is inducible and concentration dependent. Studies are in progress to purify, characterize, and identify some of the isolates. The distribution of ErmC gene in these isolates is being determined by DNA-DNA hybridization (abstract will be submitted to the World Aquaculture meeting in Seattle, WA).

V. Develop alternative methods for toxicity testing of drugs using microorganisms.

Because of the high costs of animal maintenance and the need to reduce animal use, alternatives or supplementary systems for animal drug metabolism are in high demand. The advantages of a microbial system as a complementary in vitro model for drug metabolism are low cost, ease of handling, scale-up capability and a potential to reduce use of animals. Filamentous fungi have shown the ability to metabolize drugs in a manner similar to that in mammals and are therefore potential models for mammalian drug metabolism. The goal in FY96 was to investigate further the potential of the fungal model system to produce a broad spectrum of mammalian drug metabolites and to predict mammalian drug metabolic pathways.

The research program in the Division of Microbiology in FY96 had a protocol approved (E0694200) on microbial models of mammalian metabolism. In FY96, a group of tricyclic antidepressants and antihistamines were chosen as probes to investigate their metabolic pathways by C. elegans. This fungus exhibits a wide range of metabolic reactions similar to mammalian livers. Fungal metabolism of cyproheptadine, amoxapine, azatadine, amitriptyline, methadilazine, chlorpromazine, cyclobenzaprine and doxepin were investigated. From the comparisons of the structure-reactivity relation of these drugs, the fungal metabolic pathways were quite similar to those found in mammalian systems including aliphatic and aromatic hydroxylation, heteroatom-demethylation, heteroatom oxidation (S,N), epoxidation, formation of dihydrodiols, as well as conjugate formation. The results of mechanistic studies for both phase I and II metabolism indicated that these reactions were catalyzed by cytochrome P450 monooxygenases similar to those found in mammals, which provided a basis for this fungal system to be used as a predictive model for drug metabolism. These experiments also demonstrated that large quantities of metabolites, which are difficult to obtain by other means, can be easily isolated from this fungal biotransformation system.

Significance to FDA

The Division of Microbiology seeks to continue and expand its scientific exchange and collaborative studies with colleagues at other FDA centers and field laboratories to anticipate their research needs and provide data to support regulatory activities of the Agency.

These studies include: 1) metabolism and toxicological effects of food additives, antimicrobials and macronutrients on the intestinal microflora; 2) microbial production of metabolites of mycotoxins; 3) environmental fate and effects of aquaculture chemicals; 4) tuberculocidal disinfectant testing; 5) detection of foodborne biological hazards; 6) rapid and accurate detection methods for pathogens and toxins; and 7) sensitive methods for the detection of genetically modified microorganisms.

Many of the techniques currently in use within the microbiology research area are of value to other FDA centers and field laboratories. As communication and discussion of mutual research interests between NCTR staff and other FDA scientists increases, many new projects at the forefront of applied microbiology research will be developed.

The Division of Microbiology's vision is to strive for scientific excellence and to strengthen the relevance of its research with the mission of the Food and Drug Administration. It will continue to maintain a world-class research program to solve current issues that face the FDA in the next millennium, so the Agency can make sound, science-based regulatory decisions on microbiology.