Executive Summary

Introduction

The Division of Microbiology at the National Center for Toxicological Research (NCTR) serves a multipurpose function with specialized expertise to perform fundamental and applied research in microbiology in areas of the Food and Drug Administration’s (FDA's) responsibility in toxicology. The Division of Microbiology also responds to microbial surveillance and diagnostic needs for research projects within the NCTR and FDA. Projects are selected based on FDA priorities and programmatic expertise. The research program is divided into six focal areas: 1) Foodborne pathogens, food safety and methods development; 2) Antimicrobial resistance; 3) Gastrointestinal microbiology and host interactions; 4) Environmental biotechnology; 5) The use of microorganisms as models to predict the metabolic pathways by which drugs are metabolized in mammals; and 6) Microbiological surveillance and diagnostic support of research.

FY 2003 Accomplishments

The Division of Microbiology research scientists continue to provide valuable information to FDA for evaluating key regulatory issues in food safety and environmental biotechnology, with special emphasis on antimicrobial resistance in the food animal production environment.

Food Safety and Antimicrobial Resistance

Reports of antimicrobial-resistant bacteria from farms, animal carcasses and aquaculture facilities are raising concerns that antimicrobial use in food-producing animals may play a role in selecting for antibiotic resistance. The research and regulatory issues on antimicrobials used in food-producing animals are of great importance to the FDA. A number of collaborative research projects with other FDA Centers are being conducted in the Division of Microbiology.

In FY 2003, researchers in the Division of Microbiology collected litter, feed, and water samples from farms to isolate Salmonella, Campylobacter, and Escherichia coli to determine if they are fluoroquinolone-resistant. Molecular methods, such as ribotyping, pulsed field gel electrophoresis and polymerase chain reaction, were developed to screen for fluoroquinolone resistance genes in Salmonella spp., Campylobacter spp. and E. coli isolates from chicken and turkey farms. Molecular characterization of the fluoroquinolone-resistant strains was conducted. Romet (oxytetracycline and sulfadimethoxine-ormetoprim) is an antibiotic of choice to control pathogenic bacteria in aquaculture. Intensive use of the antibiotic has resulted in the occurrence of antibiotic resistant bacteria. In addition, several tetracycline-resistant bacteria were isolated from samples obtained in aquaculture facilities. We characterized tetracycline resistance in the fish pathogen Aeromonas spp. by various molecular biology methods such as pulsed field gel electrophoresis (PFGE), polymerase chain reaction (PCR), southern hybridization and plasmid isolation. A multiplex PCR method was developed that will detect the presence of all known tetracycline-resistant genes in Aeromonas spp. We also developed and evaluated an oligonucleotide-microarray method to rapidly detect multiple antibiotic resistant genes from foodborne and clinical pathogens. Over 100 genes related to antimicrobials that are used in animal and poultry farming have been identified and the microarray antibiotic resistance gene chip has been completed. The utility of this method for testing the impact of antimicrobials in the food animal production environment and probiotic products is currently being investigated.

Since there has been concern about the use of antibiotics in agriculture, other approaches are also being evaluated to minimize contamination of animal products with foodborne human pathogens. Reducing colonization of animals by pathogenic bacteria by using competitive exclusion treatments is being considered as an alternative to antimicrobial feed additives. Competitive exclusion products must adhere to FDA regulations that the bacterial mixtures be well defined, pathogen-free, not resistant to antimicrobials and effective. For commercial use, competitive exclusion preparations for poultry must be free from all known human and avian pathogens and from any microorganisms with unusually high resistance to antimicrobials. The FDA has approved a competitive exclusion product designed to prevent the colonization of chicken intestines by pathogenic bacteria, such as Salmonella spp., Campylobacter spp., and E. coli, and also to reduce the use of antimicrobials and the spread of antimicrobial-resistance genes.

These products are reviewed as new animal drugs for safety and efficacy because of claims that they affect the health of the chicken. The composition of these complex mixtures is not simple to define, and in the Division of Microbiology, scientists compared automated conventional techniques, including biochemical analysis and cellular fatty acid analysis, with newer automated molecular methods (16S rRNA sequence analysis) for characterization of the mixtures. Our studies provide the FDA with methods that will help to standardize the identification techniques used to characterize the components of competitive exclusion products. These results will provide guidelines for manufacturers of competitive exclusion products to submit more reliable product information for market approval by regulatory agencies.

In addition, researchers in the Division of Microbiology found that the bacteria in a competitive exclusion product harbor multiple resistance markers against several drugs; including resistance to erythromycin by enterococci and lactobacilli, to fluoroquinolones by E. coli, and to vancomycin in Lactobacillus lactis. The possibility of interspecies transfer of these resistance mechanisms, and transfer eventually to humans, makes this discovery a matter of concern for food safety.

As part of the NCTR Counterterrorism Initiative, scientists in the Division of Microbiology have collaborated with investigators in the Division of Chemistry on the rapid identification of bacteria by mass spectrometry. They have used this method to fingerprint Vibrio parahaemolyticus isolates from seafood and environmental samples and antibiotic resistant Salmonella spp. from poultry facilities.

Gastrointestinal Microbiology and Host Interactions

Intestinal microflora play significant roles in human health because they aid in the digestion of food, metabolize drugs and foreign compounds, mediate hormonal activities of phytoestrogens, and help prevent pathogens from colonizing the gastrointestinal tract. Because these bacteria play critical roles in human physiology, scientists have devised many methods for identifying them in fecal samples. Investigators in the Division of Microbiology have studied the variation of intestinal microflora from different individuals in modification of hormonal activities of phytoestrogens. Since identification of GI tract bacteria by traditional methods is time consuming and many molecular methods are limited in their ability to identify bacterial species, the Division of Microbiology, in collaboration with scientists from the University of Arkansas for Medical Sciences, has shown that microarray technology can identify the 40 most common bacterial species in the human gastrointestinal tract. These results demonstrate that microarray methods can reliably and rapidly identify intestinal flora and in most cases are more sensitive than culture methods. The microarray method has many more potential applications, for example, examining bacterial species in various patients clinically treated for intestinal diseases, and for experimental animal studies to determine the effect of food additives, antimicrobial residues, phytoestrogens, and xenobiotics on the intestinal microflora.

In response to FDA’s need for assessing the microbiological safety of animal drug residues in food, the Division of Microbiology and CVM have been investigating an in vitro culture system that examines the effect of low-level antibiotic residues on the human intestinal microflora by using a continuous culture to model the human intestinal tract. In FY 2003, fed batch culture systems were tested, and molecular methods were used to identify changes in the bacterial populations in response to antimicrobial residues. Recommendations on the methods and protocols for determining the effect of residual levels of antimicrobials on the human intestinal microflora were presented at several meetings of the Microbial Safety Task Force of the Veterinary International Cooperation and Harmonization Safety Working Group. Guidance documents have been drafted on determining the effect of residual levels of antimicrobials on the human intestinal microflora.

Another essential study in the Division of Microbiology is the elucidation of the mechanism of resistance to antimicrobial agents among bacteria from the human gastrointestinal tract. The resistant bacteria are of particular concern, because not only do they act as a reservoir for antimicrobial resistance genes, but also if they establish themselves in other parts of the body, they can cause diseases that cannot be treated. The Division of Microbiology research scientists have detected anaerobic bacteria from the human intestinal tract that are resistant to high concentrations of various fluoroquinolones. They also determined that one of the mechanisms of fluoroquinolone-resistance is due to the bacteria having an active efflux pump, which effectively reduces the intracellular concentration of the antimicrobial and drug efficacy. The antimicrobial agent metronidazole is the drug of choice for prevention and treatment of many anaerobic infections. Metronidazole is a product that requires conversion by nitroreductases to demonstrate its bactericidal activity. Resistance to the drug is commonly associated with decreased nitroreductase activity in the target bacteria. Scientists in the Division of Microbiology tested Enterococcus species bacteria from the feces of an individual who had been treated repeatedly with metronidazole for nitroreductase activity. The drug did not kill the bacteria even though they contained full nitroreductase activity. Further analysis showed that these metronidazole-resistant bacteria likely inactivated the drug by metabolism.

Environmental Biotechnology

The environmental fate of veterinary drugs and factors that influence the persistence and biodegradation of antibiotics used in farm animals and aquaculture have been investigated. Both fundamental and applied studies on the biodegradation pathways of erythromycin and the fluoroquinolones ciprofloxacin, norfloxacin, and sarafloxacin have been conducted. These studies indicate that microorganisms may play an important role in the detoxification and removal of antimicrobials from animal wastes and aquaculture sites. Scientists in the Division of Microbiology and the University of Mississippi determined that the common poultry litter, rice hulls and ground corncobs, supplemented with the nonpathogenic fungus Pestalotiopsis guepini degraded the norfloxacin. By comparison, pine shavings did not support the growth of

P. guepini or degradation of the antimicrobial. These experiments suggest a technique to reduce or eliminate contamination of the environment in agricultural uses of clinically important drugs.

Polycyclic aromatic hydrocarbons (PAHs) constitute a class of organic compounds whose environmental fate is of concern because some PAHs have mutagenic, ecotoxic and carcinogenic potential. Scientists in the Division of Microbiology have elucidated the biodegradative pathways of benzo[a]pyrene, benz[a]anthracene and 7,12-dimethylbenz[a]anthracene and the enzymes involved in PAH metabolism. Proteomic and genomic techniques have been developed to characterize protein expression and the genes involved in the bacterial metabolism of PAHs. This research increases our understanding of the environmental fate of PAHs and in developing practical PAH bioremediation strategies in the future.

Microbial Models of Mammalian Metabolism

Another ongoing research initiative within the Division of Microbiology is to exploit the use of microorganisms as models of mammalian drug metabolism. Studies were completed which determined that the fungus Cunninghamella elegans mimics mammalian metabolism of several tricyclic antidepressant drugs. Microbial metabolites of a wide range of drugs can be produced more cost-effectively and in less time than those produced by experimental animals, cell cultures or mammalian enzyme systems for structural elucidation and toxicity evaluation.

Microbiological Surveillance

The primary mission of the Surveillance/Diagnostic Program in the Division of Microbiology is to assure that the experimental animals at NCTR are healthy and free from infections that could compromise research data. The staff also provides researchers critical support in microbial culture identification, contamination investigation, stock culture maintenance, media preparation, and technical assistance. A major initiative in FY 2004 will be to develop molecular biology detection procedures for each of the microorganisms on our potential animal pathogen list and incorporate these methods into our surveillance screening.

FY 2004 Plans

Work will continue on a number of ongoing projects, including:

1. The Importance of Human Intestinal Microflora in Conversion of Phytoestrogens to Estrogenic Compounds – The phytoestrogen metabolites produced by colonic bacteria of different individuals will be characterized and the bacteria involved in the metabolic process will be identified;
    
2. Studies on Mechanism of Fluoroquinolone Resistance in Salmonella spp. Isolated from Animal Feeds (Poultry), Animal Production and the Development of Molecular Methods for Screening the Drug Resistance Genes – We will characterize E. coli, Salmonella spp. and Campylobactersp. strains from turkey and chicken samples using a variety of molecular biology methods and study the effects of environmental enteric pathogens on intracellular signaling of the host;
    
3. Studies on the Fluoroquinolone Resistance in Campylobacter sp. Isolated from Poultry - Characterization of campylobacters from turkey litter will be continued and data on the correlation of environmental factors on the occurrence of campylobacters in turkey and chicken farms will be conducted;
    
4. Characterization of Tetracycline and Sulfadimethoxine-Ormetoprim Resistant Pathogenic Bacteria from Catfish Tissues – Molecular probes will be developed and used to localize the DNA fragments that harbor these tetracycline resistance genes. Similar molecular methods will be used to determine the epidemiology of the occurrence of tetracycline-resistant genes in E. coli, Vibrio spp., Salmonella spp., and Citrobacter in catfish tissues;
    
5. Develop gene array chips for rapid microbial pathogen detection of Salmonella spp. and Vibrio spp. in ocean-derived products – The method developed will be used as a template for development of a diagnostic array that is capable of simultaneous detection of multiple foodborne pathogens;
    
6. Microbial Models for Biotransformation of Fluoroquinolones – We will identify the transformation products that are produced by fungi and bacteria from flumequine, norfloxacin, and ofloxacin;
    
7. In Vitro Assay for Perturbation of Colonization Resistance by Antibiotic Residues – We will test the effects of antimicrobial agents on the ability of the new model human intestinal microflora to protect against Salmonella spp. and Campylobacter sp. invasion of Caco-2 cells and test a new human intestinal cell line in the assay;
    
8. Determining the Effect of Low Levels of Antibiotic Residues on the Human Intestinal Microflora using an In Vitro Continuous Culture System – We will continue to develop and refine methods for detecting specific intestinal bacterial in complex mixtures such as feces or fecal cultures. In vitro cultures of the human intestinal microflora will be exposed to different concentrations of a variety of antimicrobial compounds, and the changes in bacterial populations will be monitored using both qualitative and quantitative methods;
    
9. Elucidation of the Mechanism of Resistance Development in Anaerobic Bacteria from the Human Intestinal Tract – We will evaluate the roles of alteration of target genes and efflux pumps in the mechanisms of resistance to antimicrobial agents;
    
10. Probiotic Effects on Host Defense Against Enteric Pathogens – We will acquire germ-free mice, colonize them with the model intestinal microflora and evaluate the colonization of the mice by the intestinal microflora;
     
11. Proteomic Approaches to Elucidate Biodegradative Pathways – We will identify differentially expressed proteins by N-terminal sequencing and mass spectrometry and determine condition-specific marker proteins, which are part of the response of the bacteria under different conditions. Furthermore, we will elucidate the metabolic pathways for high molecular weight PAHs in Mycobacterium vanbaalenii PYR-1 and characterize the PAH degradative genes using molecular techniques;
     
12. Novel Molecular Approaches for the Detection and Analysis of the Predominant Bacterial Species in the Human Gastrointestinal Tract – We will evaluate microarray-slides to detect 40 intestinal bacterial species from human fecal samples and determine its applicability to clinical samples;
     
13. Development of a microarray chip for the detection of multiple antibiotic resistance markers - We will correlate microarray results with disk diffusion, broth dilution, and PCR assays to analyze the signal intensity of microarrays with quantification software, and to characterize a vancomycin-resistant mechanism employing wild type, intermediate and high mutator strains of Enterococcus faecium ATCC 51559;
     
14. Characterization of Antimicrobial Drug Resistance Genes from Lactococcus lactos P1-79 – We will identify and isolate the genes for multiple antibiotic drug resistances in a Lactococcus lactis isolate from a competitive exclusion product; and
     
15. We will also continue to collaborate with investigators in the Division of Chemistry on the rapid identification of bacteria by mass spectrometry.

We hired four research microbiologists last year and they have developed protocols that will be initiated in FY 2004. Three of the projects will compliment existing studies on antimicrobial resistance. They are on the contributions of membrane-associated efflux systems to antibiotic resistance in Lactobacillus, molecular epidemiology and characterization of multiple antibiotic resistant Salmonella spp. isolated from the turkey production environment, and development of proteomic approaches to identify Staphylococcus aureus extracellular proteins responsible for causing pneumonia. The fourth project is molecular cloning and characterization of genes coding for tattoo degrading and permanent cosmetic pigment degrading enzymes from human skin microflora.

Public Health Significance

The FDA, various national and international committees, and the general public are concerned about the increased multiple antimicrobial resistance that has recently been found among pathogenic microorganisms. This may be due in part to the veterinary use of antimicrobials, which will potentially bring about a general increase in the numbers of antimicrobial-resistant bacteria in food animals and the environment and increased amounts of antimicrobials and their biotransformation products in meat, milk or egg products that could affect consumers via the intestinal microflora. The issue of microbial drug resistance has significance both to health and regulatory agencies. The FDA has expressed an interest in research that would determine whether antimicrobial resistance occurs in bacteria isolated from animal feeds containing antibiotics, the pattern of resistance development in bacteria found in animals fed antibiotics, and differences in survival rates of drug-resistant pathogens compared to nonresistant pathogens. Various antimicrobial drugs are currently approved for growth promotion in food animals by Canada, Mexico, Australia, New Zealand, and the European Union as well as the United States. Furthermore, the Division of Microbiology is involved in basic research for the advancement of biochemical and molecular technology for further understanding of the role of microbial communities in human health. It has taken a multidisciplinary approach to provide fundamental information to the FDA on antimicrobial resistance, environmental biotechnology, and food safety issues.

Research Projects

PI: Cerniglia, Carl

Title: Proteomic Approaches to Elucidate Biodegradative Pathways
Project Number: E0711801
Strategic Research Goal: Method Driven Research

Objective(s):

Use proteomic approach to isolate putative catabolic proteins that are over-expressed when microorganisms are grown in the presence of polycyclic aromatic hydrocarbons; and
Develop software to analyze 2-D gels.

Status: Started/Ongoing

PI: Chen, Huizhong

Title: Molecular Cloning and Characterization of Genes Coding for Enzymatic Degradation of Tattoo and Permanent Cosmetic Pigments from Human Skin Microflora
Project Number: E0717901
Strategic Research Goal: Predictivie Toxicology

Objective(s):

1. The research will be focused predominately on human skin and intestinal microflora of genera Staphylococci, Propionibacteria, Clostridia, and Enterococci. The objectives of the projects are:
2. Biodegradation and bioconversion of the tattoo, topically applied colorants, and permanent make-up pigments in the selected bacteria;
3. Clone and over-express genes encoding for azoreductases and nitroreductases, which are able to decolorize the pigments, from the bacteria;
4. Determine physicochemical properties of the purified native enzyme from the bacteria and the expressed recombinant enzymes cloned in E. coli;
5. Elucidate the role of the microflora with potential genotoxic effects of tattoo and permanent make-up pigments; and
6. Study effects of sunlight and tanning lights on skin microfloral populations.

Status: Project Under Review

PI: Elkins, Christopher

Title: Assessment of Membrane-Associated Antibiotic Resistance Mechanisms in Lactobacilli

Project Number: E0718001

Strategic Research Goal: Predictivie Toxicology

Objective(s):

1. Obtain Lactobacillus cultures from available commercial or private culture collections and test such cultures for multiple drug resistance;
2. Compare these “intrinsic resistances” in species used routinely by the food industry to GI tract commensuals;
3. Search current sequence databases and determine putative membrane efflux transporters in lactobacilli based on sequence similarity to functionally identified MDR proteins;
4. Close and test such genes for MDR phenotype in a MDR-sensitive E. coli;
5. Create genomic libraries of lactobacilli to determine the extent of “genetic dedication” to these activities by identifying genes associated with drug efflux; and
6. Develop a microarray, if feasible, with genes identified in this study and test it with the culture collection to determine the contribution of efflux to the observed resistances in objective 1.

Status: Project Under Review

PI: Erickson, Bruce

Title: Determining the Effect of Low Levels of Antibiotic Residues on the Human Intestinal Microflora Using an In Vitro Continuous Culture System

Project Number: E0709201

Strategic Research Goal: Method Driven Research

Objective(s):

Determine the concentration of selected fluoroquinolones that produce no adverse effect on the human intestinal microflora. Hypothesize that an in vitro chemostat culture system that mimics the human intestinal tract can be used to detect and characterize the effect of low-level antibiotic residues in food on the human intestinal microflora.

Status: Started/Ongoing

PI: Hart, Mark

Title: Development of Proteomic Approaches to Identify Staphylococcal Aureus Extracellular Proteins Responsible for Staphylococcal Pneumonia

Project Number: E0717501

Strategic Research Goal: Knowledge Bases

Objective(s):

1. Develop a new, more effective approach to prevent and treat staphylococcal pneumonia;
2. Develop a proteomic approach of identifying proteins by first fractionating proteins in spent media using isoelectric focusing followed by nonporous, reverse phase HPLC. Proteins isolated in this manner will be submitted to protease degradation and peptide profiles will be generated using LC/MS/MS. Peptide profiles will be searched against the public NCBI protein database to identify the proteins; and
3. Generate a proteomic profile for S. aureus RN6390 and its AGR and SAR isogenic mutants. These profiles will be compared to identify differences between strains and thus, preliminarily identify potential proteins responsible for the lethality observed in the mouse model of pneumonia.

Status: Project Under Review

PI: Khan, Ashraf

Title: Studies on Mechanism of Fluoroquinolone-Resistant Salmonella spp. Isolated from Animal Feeds (Poultry), Animal Production Environment and the Development of Molecular Methods for Screening the Drug Resistance Genes

Project Number: E0704801

Strategic Research Goal: Method Driven Research

Objective(s):

1. Isolation, identification and characterization of nalidixic acid and fluoroquinolone-resistant Salmonella spp. from chicken farms (animal feed, feces, manure, litters and animals) by biochemical and Polymerase Chain Reaction;
2. Determination of minimum inhibitory concentration for environmental isolates, development of molecular techniques and its comparison with clinical strains;
3. Determination of drug-resistance mechanisms in the environmental isolates and their characterization by molecular techniques; and
4. Determination of influence of seasons and the frequency of isolation of fluoroquinolone-resistant Salmonella spp.

Status: Started/Ongoing

PI: Khan, Saeed

Title: Molecular Screening Methods for the Determination of Vancomycin-Resistance in Selective Competitive Exclusion Product CF3 (PREEMPT) Bacteria

Project Number: E0705301

Strategic Research Goal: Method Driven Research

Objective(s):

1. Isolation, identification and biochemical characterization of vancomycin-resistant bacteria present in a commercially available competitive exclusion product CF3;
2. Development of a rapid PCR method of the detection of vancomycin-resistance determinant genes, namely, the Van A0, Van B, Van C and D-ala-D-lac ligase gene Ddl.;
3. Characterization of plasmid DNA Profile and plasmid-mediated drug resistance transfer;
4. Genetic fingerprinting of the vancomycin-resistant microorganisms present in PREEMPT culture; and
5. Nucleotide sequence analysis of the PCR products of vancomycin-resistant determinant genes showing interesting restriction profiles.

Status: Started/Ongoing

Title: Development of a Microarray Chip for the Detection of Multiple Antibiotic-Resistance Markers

Project Number: E0715101

Strategic Research Goal: Method Driven Research

Objective(s):

Develop a microarray-based method for the detection of 150 genes associated with 22 antibiotics; some of which are used to promote growth in poultry and animal farming while others are used to treat infections in both humans and animals. The data generated by the use of the chip in monitoring and tracking the spread of resistance markers may be helpful for the FDA in making regulatory decisions that require banning and/or approving the use of certain antibiotics in poultry and farm animals.

Status: Started/Ongoing

PI: Kurniasih, Dedeh

Title: Characterization of Antimicrobial Drug-Resistance Genes from Lactococcus lactis P1-79

Project Number: E0716201

Strategic Research Goal: Knowledge Bases

Objective(s):

1. Determine whether the antimicrobial-resistance genes are encoded on the bacterial chromosome or on episomes;
2. Screen for the presence of common resistance genes;
3. Clone the resistance genes in E. coli and evaluate their DNA sequence; and
4. Evaluate the potential for L. lactis P1-79 to transfer antimicrobial-resistance genes to Enterococcus faecium or Staphylococcus aureus.

Status: Started/Ongoing

PI: Nawaz, Mohamed

Title: Studies on the Fluoroquinolone-Resistance in Campylobacter sp. Isolated from Poultry

Project Number: E0705001

Strategic Research Goal: Method Driven Research

Objective(s):

1. Isolation and identification of fluoroquinolone-resistant Campylobacter jejuni and C. coli from water, feed and litter samples in poultry houses;
2. Determination of the optimum concentration of nalidixic acid and fluoroquinolone resistance in C. jejuni and C. coli;
3. Determination of the influence of various seasons and the frequency of isolation of fluoroquinolone-resistant C. jejuni and C. coli; and
4. Molecular characterization of fluoroquinolone-resistance by polymerase chain reaction (PCR), nucleotide sequencing and single-strand conformation polymorphism (SSCP).

Status: Started/Ongoing

Title: The Fate and Degradation of Antimicrobials, Oxytetracycline (OTC) and Sulfadimethoxine-Ormetoprim (Romet-30) from Aquaculture Environmental Samples

Project Number: E0707501

Strategic Research Goal: Method Driven Research

Objective(s):

1. Determine the biodegradation rates and metabolic fate of antimicrobials, oxytetracycline and sulfadimethoxine-ormetrorpim (Romet-30) (SDO), used in fish farming systems;
2. Isolate, characterize and identify OTC- and SDO-resistant organisms from aquaculture sediment and natural environment samples and conduct molecular characterization of the genes that regulate resistance to the drugs.

Status: Started/Ongoing

PI: Nayak, Rajesh

Title: Molecular Epidemiology and Characterization of Multiple Antibiotic-Resistant Salmonella Isolated from Turkey Production Environment

Project Number: E0717301

Strategic Research Goal: Knowledge Bases

Objective(s):

1. Determine the preharvest sources and/or vectors of horizontal transmission Salmonella in turkey flocks;
2. Evaluate the intrinsic resistances of Salmonella isolates to multiple antibiotics;
3. Assess the genetic diversity and epidemiological profiles of Salmonella strains isolated in a turkey production environment; and
4. Develop DNA-based and microarray assays to detect genes in Salmonella isolates that are involved in antibiotic-resistance and pathogenicity.

Status: Project Under Review

PI: Paine, Don

Title: Animal Husbandry Breeding Support

Project Number: E0002200

Strategic Research Goal: Center Support (Research)

Objective(s):

Microbiological evaluation of animals and non-animal samples not specifically designated to an ongoing experiment.

Status: Started/Ongoing

Title: Conventional Mice Breeding

Project Number: E0010900

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine health status of mice breeding colonies maintained under conventional conditions.

Status: Started/Ongoing

Title: SPF Rat Breeding Colony

Project Number: E0011000

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine health status of rats breeding colonies maintained under specified pathogen free conditions.

Status: Started/Ongoing

Title: Conventional Rat Breeding Colony

Project Number: E0011100

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine health status of rats breeding colonies maintained under conventional conditions.

Status: Started/Ongoing

Title: Conventional Guinea Pigs Breeding Colony

Project Number: E0011200

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine health status of guinea pigs colonies maintained under conventional conditions.

Status: Started/Ongoing

Title: Quarantine Animals

Project Number: E0011300

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine health status of animals received at NCTR and held under quarantine conditions.

Status: Started/Ongoing

Title: Diet Prep General Support

Project Number: E0014500

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine the microbial contamination level in dosed- or control-feed and water lots prepared for animal use but not designated to a specific ongoing experiment.

Status: Started/Ongoing

Title: Primate Colony Surveillance

Project Number: E0023500

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine the health status of the primate colonies maintained at NCTR.

Status: Started/Ongoing

Title: Microbiological Diagnostic Methods: Development, Testing, & Evaluation

Project Number: E0026200

Strategic Research Goal: Method Driven Research

Objective(s):

Improve diagnostic and epidemiological capabilities in bacteriology, parasitology, mycology, virology and serology as applicable to NCTR programs and projects.

Status: Started/Ongoing

Title: General Microbiological Support - Bacteriology, Parasitology, Mycology & Virology

Project Number: S00006

Strategic Research Goal: Center Support (Research)

Objective(s):

Determine health status of animal colony and their environment.

Status: Started/Ongoing

Title: Microbiology Division - Media Preparation

Project Number: S00064

Strategic Research Goal: Center Support (Research)

Objective(s):

Provide media and reagent preparation to both research and surveillance/diagnostic needs.

Status: Started/Ongoing

Title: Special Epidemiology Investigations of Potential Microbiological Contamination Problems

Project Number: S00185

Strategic Research Goal: Center Support (Research)

Objective(s):

1. Investigate potential microbiological contamination problems; and
2. Report nonroutine sample time which is not recorded on Sample Collection Report (SCR).

Status: Started/Ongoing

PI: Rafii, Fatemeh

Title: Importance of Human Intestinal Microflora in Conversion of Phytoestrogens to Estrogenic Compounds

Project Number: E0700701

Strategic Research Goal: Concept Driven Research

Objective(s):

1. Detect various metabolites of phytoestrogens, produced by the metabolism of these compounds by pure culture of bacteria typical of that isolated from human microflora, and elucidation of the metabolic pathways of phytoestrogens by human intestinal bacteria;
2. Assess the estrogenic effect of each phytoestrogen metabolite produced by intestinal bacteria;
3. Determine the bacterial species producing estrogenic metabolites from phytoestrogens and elucidation of enzymes involved in various steps of these metabolic processes; and
4. Evaluate the effects of phytoestrogens and their metabolites on the population, composition, metabolic activity and enzyme production of bacteria from the human gastrointestinal tract.

Status: Started/Ongoing

Title: Elucidation of the Mechanism of Resistance Development in Anaerobic Bacteria from the Human Intestinal Tract

Project Number: E0709301

Strategic Research Goal: Knowledge Bases

Objective(s):

The aim of this study is the evaluation of the effect of fluoroquinolones on the resistance development in the bacteria from the human intestinal tract and analysis of the fluoroquinolone resistance mechanism in anaerobic bacteria from the human intestinal tract.

Status: Started/Ongoing

PI: Sutherland, John

Title: Biotransformation of Fluoroquinolones by Fungi

Project Number: E0705201

Strategic Research Goal: Method Driven Research

Objective(s):

1. Measure the kinetics of biodegradation of veterinary fluoroquinolone drugs in natural matrices;
2. Identify the potential metabolites produced by fungi from fluoroquinolones; and
3. Assess the residual antibacterial activity and potential risks of the metabolites formed from these drugs.

Status: Started/Ongoing

PI: Wagner, Robert

Title: In Vitro Model and Molecular Analysis of Competitive Exclusion Products

Project Number: E0704901

Strategic Research Goal: Method Driven Research

Objective(s):

1. Evaluate individual component bacteria in a defined competitive exclusion (CE) product for exclusion of enteric pathogens from Caco-2 and CRL-2117 cell monolayers;
2. Define the antimicrobial susceptibility patterns of the component bacteria using Minimal Inhibitory concentration measurements;
3. Sequence analysis of 16s rRNA Polymerase Chain Reaction (PCR) products from defined culture component bacteria and development of a database containing the sequences for use in subsequent identification of the organisms in undefined CE products; and
4. Application of the 16s rRNA sequence analysis procedure to detect and identify effective CE component bacteria in undefined CE products.

Status: Started/Ongoing

Title: Measurement of Antimicrobial Drug Concentrations that Inhibit Colonization Resistance

Project Number: E0708601

Strategic Research Goal: Method Driven Research

Objective(s):

An enterocyte culture model of colonization resistance by enteric microbial flora against Salmonella spp. colonization/invasion will be adapted to measure concentrations of antimicrobial drugs as food residues that would inhibit the barrier effect of the consumer's intestinal flora.

Status: Started/Ongoing

Title: Probiotic Effects on Host Defense Against Enteric Pathogens

Project Number: E0709701

Strategic Research Goal: Knowledge Bases

Objective(s):

1. Establish a model intestinal bacteria population in mice that consists of human intestine-derived bacteria;
2. Observe the fate of members of the model bacterial population when probiotic bacteria are fed to the mice;
3. Observe the fate of the probiotic bacteria fed to the human flora-associated mice;
4. Observe the effects of the human-derived flora on the host protective systems of immunodeficient and immunocompetent mice;
5. Observe effects of adding probiotic bacteria to HFA mouse on immunodeficient and immunocompetent host protective systems; and
6. Observe the roles of model host flora and probiotic bacteria to modulate host protective systems of immunodeficient and immunocompetent mice from Salmonella typhimurium and Campylobacter jejuni.

Status: Started/Ongoing

PI: Wang, Rongfu

Title: Novel Molecular Approaches for the Detection and Analysis of the Predominant Bacterial Species in the Human Gastrointestinal Tract

Project Number: E0711901

Strategic Research Goal: Method Driven Research

Objective(s):

1. Develop a rapid method for quantification of intestinal bacteria;
2. Qualitative analysis of the communities for several major genera and discovering the species which are noncultivated;
3. Isolation and identification of the bacterial species from probiotics used for human or animal health; and
4. Develop microarray method for the detection of intestinal bacteria.

Status: Started/Ongoing

Publications

Cho, B.P., Yang, T., Blankenship, L.R., Moody, J.D., Churchwell, M., Beland, F.A., and Culp, S.J. 2003. Synthesis and Characterization of N-Demethylated Metabolites Of Malachite Green and Leucomalachite Green, Chemical Research in Toxicology. 16:285-294. Accepted 1/2/2003

Elkins, C., Savage, D.C., Cbst2 From Lactobacillus Johnsonii 100-100 is a Transport Protein of the Major Facilitator Superfamily that Facilitates Bile Acid Antipot., Journal of Molecular Microbiology and Biotechnology. Accepted: 9/5/2003 (E0718001)

Kim, Y., Kim, S., Cerniglia, C.E. and Heinze, T.M., Adsorption And Clay-Catalyzed Degradation of Erythromycin A on Homoionic Clays, Journal of Environmental Quality. Accepted: 6/23/2003 (E0690101)

Kim, Y.-H., Engesser, K-H., and Cerniglia, C.E. 2003. Two Polycyclic Aromatic Hydrocarbon O-Quinone Reductases from a Pyrene-Degrading Mycobacterium. Archives of Biochemistry and Biophysics 416:209-217. Accepted: 08/06/2003.

Kim, Y., Pak, K., Pothuluri, J.V. and Cerniglia, C.E., Desorption and Degradation of Erythromycin a in Aquaculture Sediments, Aquaculture. Accepted: 6/1/2003 (E0690101)

Mattia, A., Cerniglia, C.E., Safety Evaluation of Certain Food Additives and Contaminants: Natamycin, WHO Food Additives, Geneva, Series 48:49-76. Accepted: 11/15/2002 (N/A)

Moody, J.D., Fu, P.P., Freeman, J.P. and Cerniglia, C.E., Regio- and Stereoselective Metabolism of 7,12-Dimethylbenz[A]Anthracene By Mycobacterium Vanbaalenii PYR-1, Applied and Environmental Microbiology, 69:3924-3931. Accepted: 4/1/2003 (E0707501)

Nawaz, M.S., Wang, R., Khan, S.A. and Khan, A.A., Detection of Gale Gene by Polymerase Chain Reaction in Campylobacters, Molecular Cellular Probes. Accepted: 8/16/2003 (E0705101)

Rafii, F., Heinze, T.M., Beger, R., Park, M. and Davis, C.L., Variation in Metabolism of the Soy Isoflavonoid Daidzein by Human Intestinal Microflora from Different Individuals, Archives of Microbiology, 180:11-16. Accepted: 4/14/2003 (E0700701)

Rafii, F., Wynne, R.A., Heinze, T.M. and Paine, D.D., Mechanism of Metronidazole-Resistance in Isolates of Nitroreductase-Producing Enterococcus Gallinarum and E. Casseliflavus from the Human Intestinal Tract, FEMS Microbiology Letters, 225:195-200. Accepted: 6/1/2003 (E0709301)

Roberts, G., Cerniglia, C.E., Toxicological Evaluation of Certain Veterinary Drug Residues in Food, WHO Food Additives, Series 49, 49:29-62. Accepted: 11/1/2002 (N/A)

Shahverdi, A.R., and Rafii, F., Inhibition of Nitrofuratoin Reduction by Menthol Leads to Enhanced Antimicrobial Activity, Journal of Chemotherapy. Accepted: 5/5/2003 (E0709301)

Wagner, R.D., Paine, D.D., and Cerniglia, C.E., Phenotypic and Genotypic Characterization of Competitive Exclusion Products, J. Applied Microbiology. 94:1098-1107. Accepted 02/21/2003 (E0704901)

Concept Papers

PI: Wang, Rongfu

Title: Development of Molecular Methods Including Oligo-Microarray Methods for the Detection and Monitoring of Foodborne Pathogenic Bacteria

Project Number: E0715401

Strategic Research Goal: Method Driven Research

Objective(s):

1. Development of an oligo-microarray method for the detection of foodborne pathogens based on 16S rDNA sequences;
2. Development of oligo-microarray methods with multiplexed PCR based on many genes for the detection and genotyping of specific pathogenic bacterial strains; and
3. Development and modification of PCR methods for the detection of all above pathogens.

Status: Approved Concept Paper