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Carl Cerniglia, Ph.D., Director
870-543-7341
carl.cerniglia@fda.hhs.gov
Executive Summary
Introduction
The Division of Microbiology at NCTR serves a multipurpose function with
specialized expertise to perform fundamental and applied research in
microbiology. The Division of Microbiology also responds to microbial
surveillance and diagnostic needs for research projects within NCTR and FDA. The
Division of Microbiology has a multidisciplinary staff including 14 research
scientists and 17 research support staff, postdoctoral fellows, undergraduate
and graduate students, visiting scientists, and program support specialists. The
Microbiology Division has the staff, the know-how, and the facilities to help
address the scientific challenges encountered by FDA and other government
organizations. Some examples of the research projects within the Division and
collaborative research with scientists from other NCTR Divisions, FDA Centers,
academic institutions, and industry are described below. Projects are based on
FDA priorities and programmatic expertise. The research program is divided into
five focal areas: 1) food safety, food biosecurity, and methods development; 2)
antimicrobial resistance; 3) gastrointestinal microbiology and host
interactions; 4) environmental biotechnology; and 5) microbiological
surveillance and diagnostic support of research.
FY 2006 Accomplishments
Division of Microbiology scientists are engaged in research addressing a
variety of critically important issues in food safety and biosecurity relevant
to the missions of FDA and other regulatory agencies. Recent outbreaks of E.
coli O157:H7 and Salmonella underscore the necessity to address the
threat of foodborne pathogens in our food supply. For example, in collaboration
with FDA’s Center for Food Safety and Applied Nutrition (CFSAN) and United
States Department of Agriculture’s Agricultural Research Service (ARS),
microarray biochips are being developed and validated for rapid and accurate
identification of multiple virulence and antimicrobial resistance genes in
Salmonella serovars. In collaboration with CFSAN, the Division has
characterized Salmonella and Vibrio spp. isolated from seafood
samples using the most current state-of-the-art-molecular techniques.
Scientists in the Division of Microbiology collaborate with microbiologists
at the FDA’s Arkansas Regional Laboratory (ARL) to survey imported fish samples
for antibiotic-resistant bacteria. The genetic determinants conferring
antibiotic resistance in these bacteria are also being characterized.
In collaboration with NCTR ARL Bioterrorism Mass Spectrometry (MS) programs,
methods are being developed for the rapid detection of bacterial foodborne
pathogens using pyrolysis and matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-ToF MS) technology with multivariate
statistics and artificial neural-network pattern recognition. Division
scientists have also developed and completed research for an interagency
agreement (IAG) with the USDA and the Department of Homeland Security for a
project on the survivability of Bacillus anthracis in foods.
FDA is increasingly being called upon to make regulatory judgments on safety
questions related to the gastrointestinal microfloras of humans and animals. The
Division of Microbiology research staff has the expertise and long-standing
interest in assessing the risks to the gastrointestinal microflora of humans of
antimicrobial compounds ingested in food residues, probiotics, and dietary
supplements. Division scientists provide guidance and expert advice to FDA,
other national regulatory agencies, and the World Health Organization on the
potential human-health risks associated with the use of antimicrobial agents,
competitive exclusion products, probiotics, and dietary supplements in
veterinary and human clinical medicine.
Microbiology scientists have developed a series of molecular techniques that
can measure perturbations of the intestinal microflora population resulting from
exposure of the consumer to antimicrobial residues, probiotics, and dietary
supplements. Research has also been undertaken to address the role of intestinal
microflora in the metabolism of xenobiotic compounds. In addition, experiments
are being conducted on antimicrobial resistance mechanisms, such as efflux
pumps, found in commensal organisms in the gastrointestinal tract. The Division
is also evaluating the effects of fluoroquinolones on resistance development in
bacteria from the human intestinal tract.
Division scientists have entered into a Cooperative Research and Development
Agreement (CRADA) with Pfizer Animal Health to study the degradation of the
veterinary antimicrobial ceftiofur by the normal bovine intestinal microflora.
Results from the investigation will allow FDA to evaluate the long-term use of
this antibiotic on the development of antimicrobial resistance and may give
guidance for understanding the drug characteristics that minimize resistance
development. An FDA Office of Women’s Health project has been initiated to
investigate the protective effect of Lactobacillus against
Staphylococcus aureus-mediated toxic shock syndrome. This represents a new
area of research for the Division in potential probiotic issues in women’s
health. Another new field for investigation in the Division of Microbiology is
determining the critical role that the normal microflora of the human skin has
in the metabolism of tattoo pigments and topically applied colorants. A
comprehensive study has been completed on the enzymatic mechanisms of azo dye
degradation by skin microflora. Microbiology scientists are developing proteomic
approaches to identify in Staphylococcus aureus the extracellular
proteins responsible for staphylococcal pneumonia.
The environmental biotechnology research program in the Division of
Microbiology supports FDA in its assessment of potential environmental impacts
of regulated chemicals during their lifetime in the environment. To this end,
the research program is incorporating the new omics technologies to elucidate
the microbial degradation of polycyclic aromatic hydrocarbons (PAHs) found in
industrial, agricultural, and municipal effluents and in contaminated sediments.
The use of systems-biology approaches for elucidating the metabolic pathways of
PAH degradation will further help FDA understand the fate of these biohazardous
compounds in the environment and in the human body. Research on the degradation
of antimicrobial agents, such as fluoroquinolones, to biologically inactive
products continues to be a major emphasis in the Division of Microbiology. This
investigation has increased our understanding of the ability of microorganisms
to degrade residues of antimicrobial drugs, thereby reducing the pressure for
the selection of drug-resistant strains of bacteria.
The continuing primary mission of the Surveillance and Diagnostic Program is
to provide the assurance that NCTR research data is not compromised by the use
of infected or unhealthy experimental animals. During FY 2006, the
Surveillance/Diagnostic Program prevented pathogen introduction by quarantined
animals by 1) detecting bacterial pathogens in the cage water from commercially
purchased mice; 2) monitoring the health, environment, and food of the animals
in the established NCTR breeder colonies; 3) working closely with the Division
of Veterinary Services to monitor the success of a bacterial pathogen
eradication program in the NCTR breeder mouse population; and 4) expanding the
use of molecular techniques to detect the presence of difficult-to-culture
pathogenic bacteria. The Division provided microbial cultures to researchers in
the United States and foreign countries as well as to researchers at NCTR.
Microbiology scientists provided technical support to NCTR researchers by 1)
supplying and maintaining microbial cultures used for research; 2) supplying
culture media and reagents; and 3) providing culture identification and
antibiotic sensitivity testing.
FY 2007 Plans
Food Safety and Biosecurity, Food Biodefense, and Methods Development
- In collaboration with CFSAN and USDA, two Salmonella biochips are
being developed and validated for rapid and accurate identification of
multiple virulence and antimicrobial resistance genes in Salmonella
serovars. The joint effort between the FDA Centers and USDA will be useful in
transferring microarray technology from the research stage at NCTR to the FDA
field laboratories and law enforcement mobile laboratories.
- In collaboration with the ORA ARL Bioterrorism Mass Spectrometry project,
researchers in the Division of Microbiology are continuing to develop and
validate methods for rapid detection of multiple bacterial pathogens using
pyrolysis and MALDI-ToF MS technology with multivariate statistical and
artificial neural-network pattern recognition.
- Salmonella and Vibrio spp. isolated from seafood samples
will be characterized by pulsed-field gel electrophoresis (PFGE), multiplex
PCR (polymerase chain reaction) ribotyping, ERIC (enterobacterial repetitive
intergenic consensus)-PCR, multilocus seqencing, and RAPD (randomly amplified
polymorphic DNA) methods. Multidrug-resistant bacteria, including sulfonamide
and ciprofloxacin-resistant Salmonella spp., will be characterized for
integrons and plasmids. The amplified integrons will be sequenced to determine
any unique characteristics.
- The Division will continue working on a project in collaboration with USDA
on the survivability of Bacillus anthracis.
Antimicrobial Resistance
- The van operons from 17 human vancomycin-resistant enterococcal
isolates were amplified and tested for differences by a PCR-RFLP (restriction
fragment length polymorphism) method. These isolates had differences in the
operons. The van operons from these isolates will be cloned and
sequenced to determine differences at the sequence level.
- Aeromonas spp. isolates obtained from FDA’s Center for Veterinary
Medicine (CVM) will be analyzed by PFGE, PCR, and other molecular
biology-based typing methods.
- The Division plans to study host-pathogen interactions, especially the
expression of host and pathogen genes, by microarray analysis. Human
intestinal epithelial cells and foodborne pathogens will be used as model
systems.
- The Division will conduct conjugation experiments to determine the rate of
tet gene transferability from tet-resistant Aeromonas
spp. to tet-sensitive E. coli. Similar studies will be done with
tet-resistant Citrobacter isolates. In addition, tet-resistant
Citrobacter, E. coli and Pseudomonas isolates will be
characterized from aquaculture samples.
- The Division will continue evaluation of the effect of fluoroquinolones on
resistance development in bacteria from the human intestinal tract. Analysis
of the fluoroquinolone-resistance mechanisms in anaerobic bacteria from the
human intestinal tract and other sources will also be determined.
- Our study of efflux-mediated drug resistance will address the
physiological role of multidrug efflux pumps in the Escherichia coli
model system using microarrays to monitor changes in the transcriptome upon
their knockout (deletion), inhibition, or substrate challenge. This will be
done through collaboration with the NCTR Center for Functional Genomics.
- In light of the grave potential outcome of an influenza pandemic, research
efforts in FY 2006 included collaboration with St. Jude Children’s Research
Hospital in Memphis, Tennessee. Preliminary data resulted in the submission of
a proposal titled “Interaction of influenza and Staphylococcus aureus
in the mouse and ferret models of severe pneumonia” to the National Institutes
of Health. While not funded on its initial submission, the animal models of
influenza and bacterial superinfection that have been established in mice and
ferrets will be used to identify virulence factors in the virus and bacteria
that contribute to the synergism. Genes encoding suspect virulence factors
will be altered or deleted and the effects on disease will be examined.
Gastrointestinal Microbiology and Host Interactions
- Vancomycin-resistant enterococci (VRE) have become a major food safety
issue. We recently discovered that a vancomycin-resistant Lactococcus
lactis that we isolated from a competitive exclusion product used in
poultry could transfer the vanA gene to Staphylococcus aureus,
which is often found in humans. The Division of Microbiology will continue to
explore the potential for bacteria in competitive exclusion and other
probiotic products to transfer potentially hazardous genes to human
gastrointestinal (GI) tract-associated bacteria.
- Lactobacillus serves as an important indicator of GI and vaginal
tract health and is heavily used by consumers, either intentionally as
probiotic supplements or unintentionally in microbially fortified foods. Less
complicated studies, such as assessing growth fitness or identifying
metabolites of endogenous steroid molecules (hormones and bile acids), will be
conducted in Lactobacillus, as will studies assessing the effects of
microbicides and spermicides. In vaginal health, Lactobacillus
microarrays will be used to address questions on efflux pump function.
- The Division will analyze degradation products of azo dyes by skin and
intestinal microorganisms, using specific enzymatic treatments,
high-performance liquid chromatography (HPLC), and LC-MS/GC-MS methods. Since
the three-dimensional structure of the azoreductase from Enterococcus
faecalis has been solved, site-directed mutagenesis will be employed to
study the enzyme activity center and flavin mononucleotide (FMN) binding motif
and to characterize properties of the mutant proteins. Microbiology scientists
will continue to use DNA probes and antibodies from E. faecalis and
S. aureus azoreductases for screening similar genes in skin and intestinal
microflora to determine the distribution of the azoreductase genes among
predominant bacteria and the enzyme expression levels in these microorganisms.
The Division of Microbiology will also continue to study structure and
function of the azoreductases from S. aureus and environmental
microorganisms.
- Entering into the second year of the CRADA with Pfizer Animal Health, we
will continue our study of the metabolism of the third-generation
cephalosporin ceftiofur by the bovine intestinal microflora.
- In FY 2006, our proposal “Protective effect of vaginal Lactobacillus
species against Staphylococcus aureus-mediated toxic shock
syndrome” was funded by the FDA Office of Women’s Health. In FY 2007, the
Division will carry out this research to investigate whether Lactobacillus,
when supplemented in tampons, has the capacity to exert probiotic benefits by
decreasing either S. aureus proliferation and/or TSST-1 exotoxin
production.
Environmental Biotechnology
- The Division will conduct transcriptomic and proteomic analyses to
understand PAH metabolism in M. vanbaalenii PYR-1. The results will be
integrated with metabolomic and genomic data, which will give us a deeper
understanding of how the bacterium responds to its environment and degrades
PAHs at a systems-biology level.
- To identify microorganisms that degrade fluoroquinolones, samples from
soils and wastewater treatment plants to screen for the presence of
fluoroquinolone resistance genes and for bacteria that degrade or transform
N-phenylpiperazine and fluoroquinolones will be obtained. The Division
will prepare DNA and sequence the 16S ribosomal RNA genes from any bacteria
that appear to produce metabolites from either N-phenylpiperazine or
fluoroquinolones, and will submit the 16S rRNA sequences of bacteria to the
GenBank.
Microbiological Surveillance and Diagnostic Support of Research
The goal for FY 2007 is the development and implementation of new techniques
for the detection and identification of pathogenic microorganisms.
Contribution to FDA’s Strategic Goals
The Division of Microbiology uses an interdisciplinary approach to be
responsive to FDA-regulatory needs. The Microbiology Division staff has a number
of projects in conjunction with other FDA Centers to provide critical research
to address FDA’s strategic goals. Division of Microbiology scientists provide
research, guidance, and expert advice to other FDA Centers, national regulatory
agencies, and the World Health Organization for emerging public health issues.
In addition, Division scientists respond to FDA-regulatory research needs in
food safety, antimicrobial resistance, gastrointestinal microflora, and host
interactions and environmental biotechnology.
A significant portion of the research in each focal area in the Division of
Microbiology is focused on addressing specific FDA high-priority issues.
In food safety, researchers in the Division of Microbiology have developed
diagnostic microarray assays and other rapid molecular techniques for
high-throughput screening of virulence and antibiotic resistance genes in
foodborne pathogens to assess the threat of bacteria in foodborne outbreak
investigations.
The Division of Microbiology developed an interagency agreement with USDA to
study the growth and inactivation of a surrogate strain of Bacillus anthracis
in liquefied egg products at storage, permissive, and nonpermissive
temperatures. The research in Food Biosecurity and Bioterrorism in the Division
of Microbiology not only relates directly to the FDA’s mission but also shows
the adaptability of the scientists to develop liaisons with other federal
agencies to deal with perceived threats of bioterrorism through the food chain.
The intestinal bacterial population plays an important role in human health
acting as a barrier to infection as well as contributing to the digestion of
dietary components and metabolism of drugs. Scientists have developed the
methods to predict and monitor changes in this complex bacterial population. In
addition, research is being conducted on the importance of human intestinal
microflora in the metabolism and conversion of food additives, food supplements,
and antimicrobial agents which have altered biological activities from those of
the parent compounds resulting in activation or detoxification. These approaches
will allow FDA to gain a clearer understanding of how drug residues, probiotic
products, dietary supplements, and xenobiotic substances affect the intestinal
microflora and how changes in this population may affect human health. This
research supports the FDA’s Strategic Goal 3 (Increase access to new medical and
food products) and increase access to safe and effective veterinary products and
to safe and nutritious food products, including products for unmet animal and
human needs).
Before a veterinary drug can be granted approval for marketing, FDA requires
that the drug be subjected to environment risk assessment. Research being
conducted in the Division of Microbiology on the environmental fate of
FDA-regulated drugs is highly relevant to the prevention of microbial antibiotic
resistance. Drug residues that persist in the environment may select for
resistance strains of bacteria. If the drugs can be metabolized to inactive
products, they will no longer select for bacterial resistance. The research
conducted by the Microbiology Division is increasing our understanding of the
metabolism of these compounds and will further help FDA understand the fate and
environment effects of antimicrobial compounds.
Microbiology Ongoing Research Projects
NCTR's Strategic Goal 1 — Advance the scientific approaches and tools to
attain personalized nutrition and medicine for the American public
PI: Chen, Huizhong, Ph.D.
Genomic Approaches to Determine the Role of Skin Microflora in the
Metabolism of Tattoo Dyes (E0717901)
Collaborating Division(s):
Biochemical Toxicology
Objective(s): To focus on metabolic capacity and enzyme expression in human
skin microflora: 1) Biodegradation and bioconversion of pigments used for
tattooing and permanent makeup pigments; 2) To determine the effects of the skin
microflora on tattoo and topically applied dyes that reside in the dermis; 3) To
isolate, clone, and overexpress genes encoding for azoreductases and
nitroreductases, which are able to decolorize the pigments; 4) To determine
physicochemical properties of the purified native enzymes from the bacteria
and/or the expressed recombinant enzymes cloned in E. coli; and 5) To
elucidate the role of the microflora with potential genotoxic effects of tattoo
and permanent makeup pigments.
Novel Molecular Approaches for the Detection and Analysis of the Predominant
Bacterial Species in the Human Gastrointestinal Tract (E0711901)
Objective(s): 1) To develop a rapid method for quantification of intestinal
bacteria; 2) To perform qualitative analysis of the communities for several
major genera and discovering the species which are noncultivated; 3) To
isolation and identify the bacterial species from probiotics used for human or
animal health; and 4) To develop a microarray method for the detection of
intestinal bacteria.
PI: Elkins, Christopher, Ph.D.
Assessment of Membrane-Associated Antibiotic Resistance Mechanisms in
Lactobacilli (E0718001)
Collaborating Division(s):
Systems Toxicology
Objective(s): 1) To evaluate of intrinsic drug resistance of Lactobacillus
isolates from various sources; 2) To achieve functional identification of
MDR genes from currently sequenced Lactobacillus genomes using genomic
and proteomic approaches; and 3) To achieve epidemiological profiling via
pulsed-field gel electrophoresis (PFGE) and microarray technology for resistance
determining factors (RDFs).
Protective Effect of Vaginal Lactobacillus Species Against
Staphylococcus Aureus-Mediated Toxic Shock Syndrome (E0725501)
Objective(s): To determine whether probiotic administration of
Lactobacillus can thwart S. aureus TSST-1 production if supplemented
in women’s tampons. Alternatively, bacteriophage therapy may be investigated as
a multifaceted approach to strengthening probiotic introduction for such
conditions.
PI: Erickson, Bruce D., Ph.D.
Evaluation of the Mechanisms of Inactivation and Degradation of Third
Generation Cephalosporins by the Bovine Intestinal Microflora (E0721901)
Objective(s): 1) To evaluate the ability of the bovine intestinal microflora
to inactivate ceftiofur using pure culture isolates and mixed fecal cultures; 2)
To identify primary metabolites of ceftiofur degradation; 3) To isolate
ceftiofur-resistant bacteria and determine the primary mechanisms of drug
inactivation; 4) To investigate the metabolic potential of anaerobic fungi
isolated from bovine fecal samples to degrade ceftiofur; and 5) To compare the
metabolism of ceftiofur with the human third-generation cephalosporin,
ceftriaxone.
PI: Hart, Mark E., Ph.D.
Development of Proteomic Approaches to Identify Staphylococcal aureus
Extracellular Proteins Responsible for Staphylococcal Pneumonia (E0717501)
Collaborating Division(s):
Systems Toxicology
Objective(s): 1) To develop a proteomic approach of identifying proteins by
first fractionating proteins in spent media using isoelectric focusing followed
by nonporous, reverse phase HPLC; and 2) To generate a proteomic profile for
S. aureus RN6390 and its agr and sar isogenic mutants.
PI: Khan, Saeed A., Ph.D.
Development of a Microarray Chip for the Detection of Multiple Antibiotic
Resistance Markers (E0715101)
Objective(s): To 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 FDA in making
regulatory decisions that require banning and/or approving the use of certain
antibiotics in poultry and farm animals.
PI: Paine, Donald D.
Microbiological Diagnostic Methods: Development, Testing, & Evaluation
(E0026200)
Objective(s): To improve diagnostic and epidemiological capabilities in
bacteriology, parasitology, mycology, virology, and serology as applicable to
NCTR programs and projects.
PI: Rafii, Fatemeh, Ph.D.
Elucidation of the Mechanism of Resistance Development in Anaerobic Bacteria
from the Human Intestinal Tract (E0709301)
Objective(s): To evaluate the effects of fluoroquinolones on resistance
development in bacteria from the human intestinal tract and analysis of the
fluoroquinolone-resistance mechanisms in anaerobic bacteria from the human
intestinal tract.
PI: Wagner, Robert D., Ph.D.
Characterization of Antimicrobial Drug Resistance Genes from Lactococcus
lactis P1-79 (E0716201)
Objective(s): 1) To determine whether the antimicrobial resistance genes are
encoded on the bacterial chromosome or on episomes; 2) To screen for the
presence of common resistance genes; 3) To clone the resistance genes in E.
coli and evaluate their DNA sequence; and 4) To evaluate the potential for
L. lactis P1-79 to transfer antimicrobial resistance genes to
Enterococcus faecium or Staphylococcus aureus.
Measurement of Antimicrobial Drug Concentrations that Inhibit Colonization
Resistance (E0708601)
Objective(s): To adapt an enterocyte culture model of colonization resistance
by enteric microbial flora against Salmonella sp. colonization/invasion
to measure concentrations of antimicrobial drugs as food residues that would
inhibit the barrier effect of the consumer’s intestinal flora.
Probiotic Effects on Host Defense Against Enteric Pathogens (E0709701)
Objective(s): 1) To establish a model intestinal bacterial population in mice
that consists of human intestine-derived bacteria; 2) To observe the fate of
members of the model bacterial population when probiotic bacteria are fed to the
mice; 3) To observe the fate of the probiotic bacteria fed to the human
flora-associated mice; 4) To observe the effects of the human-derived flora on
the host protective systems of immunodeficient and immunocompetent mice; 5) To
observe effects of adding probiotic bacteria to the human flora-associated (HFA)
mouse on immunodeficient and immunocompetent host-protective systems; and 6) To
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.
NCTR's Strategic Goal 2 — Develop science-based best practice standards and
tools to incorporate translational and applied toxicological advancements into
the regulatory science process to create a seamless bench-to-bedside continuum
PI: Cerniglia, Carl E., Ph.D.
Proteomic Approaches to Elucidate Biodegradative Pathways (E0711801)
Collaborating Division(s):
Biochemical Toxicology
Systems Toxicology
Objective(s): 1) To use a proteomic approach to isolate putative catabolic
proteins that are overexpressed when microorganisms are grown in the presence of
polycyclic aromatic hydrocarbons; and 2) To use omics approaches to understand
the environmental fate of PAHs.
PI: Sutherland, John B., Ph.D.
Microbial Degradation of Fluoroquinolone Antimicrobial Agents (E0722701)
Collaborating Division(s):
Biochemical Toxicology
Objective(s): To identify microorganisms that either completely degrade
fluoroquinolones or modify the fluoroquinolone molecule so as to reduce its
toxicity to bacteria and levels in the environment.
NCTR's Strategic Goal 3 — Develop and apply rapid detection technologies and
testing platforms to assure food safety, biosecurity, food biodefense, and to
combat bioterrorism
PI: Khan, Ashraf A., Ph.D.
Molecular Characterization of Salmonella spp. and Vibrio spp.
Isolated from Seafood and Development of Microarray Detection Method (E0720801)
Collaborating FDA Center(s):
ORA
Objective(s): To characterize representative isolates of Salmonella
and Vibrio spp. by molecular techniques, such as pulsed-field gel
electrophoresis (PFGE), multilocus sequencing, ERIC (Enterobacterial repetitive
intergenic consensus), and REP-PCR (Repetitive Extragenic Palindromic) methods.
The results of this study will be used as a template for development of a
diagnostic gene chip capable of simultaneous detection of multiple foodborne
pathogens.
PI: Khan, Saeed A., Ph.D.
The Survival of Bacillus Anthracis in Processed Liquid Eggs
(E0725101)
Objective(s): 1) To determine of the lag phase duration (LPD), growth rate
(GR), and maximum population density (MPD) of B. anthracis Sterne strain
at different temperatures used for storing and cooking liquid eggs; and 2)
Inactivation kinetics of spores of Sterne strain at different temperatures.
PI: Nawaz, Mohamed S., Ph.D.
The Fate and Degradation of Antimicrobials, Oxytetracycline (OTC), and
Sulfadimethoxine-Ormetoprim (Romet-30) from Aquaculture Environmental Samples
(E0707501)
Collaborating FDA Center(s):
CVM
Objective(s): 1) To determine the biodegradation rates and metabolic fate of
antimicrobials, oxytetracycline (OTC), and sulfadimethoxine-Ormetoprim
(Romet-30®) (SDO) To used in fish farming systems; and 2) To 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.
PI: Nayak, Rajesh R., Ph.D.
Molecular Epidemiology and Characterization of Multiple Antibiotic-Resistant
Salmonella Isolated from Turkey Production Environment (E0717301)
Collaborating Division(s):
Systems Toxicology
Objective(s): 1) To determine the preharvest sources and/or vectors of
horizontal transmission Salmonella in turkey flocks: 2) To evaluate the
intrinsic resistances of Salmonella isolates to multiple antibiotics; 3)
To assess the genetic diversity and epidemiological profiles of Salmonella
strains isolated in a turkey production environment; and 4) To develop DNA-based
and microarray assays to detect genes in Salmonella isolates that are
involved in antibiotic resistance and pathogenicity.
PI: Wagner, Robert D., Ph.D.
Measurement of Antimicrobial Drug Concentrations that Inhibit Colonization
Resistance (E0708601)
Objective(s): To adapt an enterocyte culture model of colonization resistance
by enteric microbial flora against Salmonella sp. colonization/invasion
to measure concentrations of antimicrobial drugs as food residues that would
inhibit the barrier effect of the consumer’s intestinal flora.
Microbiology Research Projects Completed in FY 2006
PI: Wagner, Robert D., Ph.D.
In Vitro Model and Molecular Analysis of Competitive Exclusion Products
(E0704901)
Objective(s): 1) To 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) To define the antimicrobial
susceptibility patterns of the component bacteria using Minimal Inhibitory
concentration measurements; 3) To perform 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) To apply the
16s rRNA sequence analysis procedure to detect and identify effective CE
component bacteria in undefined CE products.
Results: 1) The in vitro assay measured the amount of a defined mixture of 29
human bacterial isolates (7.83 log10 CFU) and of the Preempt competitive
exclusion product (4.05 log10 CFU) that significantly reduced Salmonella
invasion of 6.41 log10 Caco-2 human intestinal-like cells and 6.89 log10 of
CRL-2117 chicken intestinal cells; 2) Most of the bacteria isolated from the
Preempt competitive exclusion product were susceptible to antibiotics; however,
there were tetracycline-resistant Bacteroides spp.,
erythromycin-resistant enterococci, and vancomycin-resistant Lactococus
lactis isolates that should be studied further for abilities to transfer
resistance genes to other bacteria; and 3) The composition of the Preempt
competitive exclusion product was confirmed for 48% of the strains reported to
be present in the product. Of the 20 strains isolated, 48% were not strains
reported to be present in the product. The 16S rRNA sequence comparisons with
GenBank entries provided more reliable identifications of anaerobic bacteria
than fatty acid methyl ester or biochemical profiling techniques.
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