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Antimicrobial Resistance Interagency Task Force
2007 Annual Report (Released June 2008)

Action Item 67

Focus Area III: Research

Action Item #67: Additional Research, Including High Risk and High Payoff Research in Nontraditional Fields, Is Needed.

Project Title: AR mechanisms of S. pneumoniae (Alaska)

• Agency: CDC
• Description: PCR methodologies are used to assist ongoing population-based surveillance of invasive disease in Alaska. We rapidly screen S. pneumoniae isolates for genetic determinants of resistance; monitoring the emergence, spread, persistence, and decline of multidrug-resistance organisms by molecular-based typing capabilities to include multilocus sequence typing (MLST).
• Results: Serotype 19A has emerged as the most frequent cause of IPD in Alaska. Sequence type 199 remains the most frequent sequence type among 19A isolates and was present prior to the recent increase in aerotype 19A disease among rural Alaska Native children. Recent identification of a multidrug-resistant Sequence type 320 isolate is of concern as it has emerged as the second most common clonal group in the rest of the U.S.

Project Title: Defense Threat Reduction Agency (DTRA) Biothreat Reduction Program (BTRP)

• Agency: DoD
• Description: The BTRP is a DTRA project to engage former soviet weapons scientists in public health activities and health related scientific research. The program concentrates on the states of the Former Soviet Union (FSU), especially the "stans". DTRA funds projects which develop collaborations between US scientists and FSU scientists to address critical questions about diseases caused specifically by biothreat agents but is expanding to other public health threats.
• Results: DTRA is funding the construction of a human and agriculture research institute in the Republic of Georgia. The plan is for the laboratory to become another DoD overseas facility with funding by GEIS, other DoD agencies and other US government agencies. The GEIS portion of the work will concentrate on disease surveillance including antimicrobial resistance.

Project Title: DNA microarray profiling of antibiotic resistance genes.

• Agency: FDA
• Description: Develop DNA microarray techniques and DNA chips for characterizing antibiotic resistance genes for multiple bacterial pathogens.
• Results: CVM scientists partnered with academic investigators at the North Carolina State University and Ohio State University in using microarray technologies to detect antimicrobial resistance and virulence genes obtained through foodborne bacteria collected via the NARMS program. Scientists from the Center for Food Safety and Applied Nutrition and the Center for Veterinary Medicine collaborated with researchers from the J. Craig Venter Institute in sequencing the genomes of 17 Salmonella serovars of public health importance. The strains chosen for genomic analysis were selected based on an extensive examination of their potential to provide information needed for examining pathogenicity, transmission, origin, ecology, evolution, and dissemination of antimicrobial resistance.

Project Title: Studies on the Mechanism of fluoroquinolone (FQ) resistance and molecular screening for resistance determinants in Campylobacter, E. coli, and Salmonella

• Agency: FDA
• Description: Isolate and characterize FQ resistant Campylobacter, E. coli and Salmonella from chicken and turkey farms.
• Results: 21 FQ resistant campylobacter were isolated from chicken liver samples and characterized by PCR-RFLP and Pulsed field gel electrophoresis (PFGE). Seventy-eight campylobacters were isolated from turkey litter samples and characterized for the presence of galE gene, PCR-RFLP and PFGE. Quinolone resistance determining regions (QRDR) from campylobacters and E. coli were PCR amplified and sequenced for the detection of silent mismatched mutations. The FQ resistant E. coil strains isolated from chicken and turkey litter were typed by ribotyping. Completed in vivo studies examining the development of fluoroquinolone resistance among Campylobacter from chickens administered approved fluoroquinolones. Continue to characterize at the molecular level, resistant Salmonella, Campylobacter and E. coli as part of the NARMS retail program.

Project Title: Fate and degradation of antimicrobials, oxytetracycline (OTC) and sulfadimethoxine-ormetoprim (Romet 30) from aquaculture environmental samples

• Agency: FDA
• Description: To isolate and characterize OTC and Romet 30 resistant Aeromonas spp., Pseudomonas, Citrobacter and E. coli. From aquaculture and catfish tissues.
• Results: 30 OTC resistant Aeromonas spp. have been isolated. These isolates have been characterized by PFGE. These investigations are still in progress.

Project Title: Blood borne pathogens

• Agency: FDA
• Description: Develop rapid assays to identify blood borne pathogens using nucleic acid based tests (NAT) and a TaqMan assay to detect bacterial contamination in whole blood and platelets. The sequences used in these primer sets are conserved in 19 bacterial species.
• Results: Ongoing research to develop a DNA microarray based pathogen chip that could detect all pathogenic bacteria that contaminate blood and blood products. Project Mgr (PI) filed a TQR on 20, July 2007 for work April -June, and co-PI responded to FDA reviewer comments on 27 August 2007. Progress reported in optimizing techniques to eliminate contamination bacterial DNA from reagents using psoralen/UV treatment and reduce PRC-inhibiting substances, as well as developing a basic PCR-based "universal" method and more specific methods for detecting Gram-positive and Gram-negative bacteria. Investigators have prepared nine working standard preparations of bacteria and fungi clinically import in septicemia, to serve as calibrators for developing real-time PCR detection with increased sensitivity. Cp-PI committed to develop a bacterial concentration step that should also increase sensitivity of detection by PCR and to assemble a collection of representative blood samples from patients with various forms of endocarditis and normal subjects for eventual clinical trials.

Project Title: Small Business Innovation Research and Technology Transfer Research Program (SBIR/STTR)

• Agency: NIH
• Description: SBIR/STTR program is an omnibus solicitation established under federal law that seeks to use small business to stimulate technological innovation, increase the participation of small business in federal R&D, and to increase private sector commercialization of technology development through Federal R&D.  The annual set-aside for agencies with extramural research budgets over $100M is 2.5%.  
• Results: Ongoing.  Examples of recent SBIR/STTR awards include:  Mucin-degrading Microflora for Prophylactic Antibiotics, Quinoline-Based Inhibitors of Botulinum Neurotoxin A, Novel Inhibitors of Staphylococcal Biofilm Formation, Stable Cationic Bacteriochlorins for Antimicrobial Photodynamic Therapy, and Designed Antimalarial Agents Overcoming Chloroquine-Resistance.    

Project Title: NIH CRISP Database

• Agency: NIH
• Description: CRISP <http://crisp.cit.nih.gov/> (Computer Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other research institutions. The database, maintained by the Office of Extramural Research at the National Institutes of Health (NIH), includes projects funded by NIH, Substance Abuse and Mental Health Services Administration (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDC), Agency for Healthcare Research and Quality (AHRQ), and Office of the Assistant Secretary of Health (OASH). Users, including the public, can use the CRISP interface to search for scientific concepts, emerging trends and techniques, or identify specific projects and/or investigators.
• Results: Ongoing.

Project Title: Pharmacological Approaches to Combating Antimicrobial Resistance (R01) Request for Applications (RFA-AI-07-025)

• Agency: NIH
• Description: This solicitation invited Research Project Grant (R01) applications from institutions/organizations that propose to apply pharmacokinetic and pharmacodynamic principles to studies on the prevention of emergence of antimicrobial drug resistance. This initiative is also intended to stimulate and strengthen collaborations between antimicrobial pharmacologists and infectious disease researchers to provide a synergistic, integrated approach that will form the basis for future clinical management of antimicrobial drug resistance.
• Results: RFA was released in April 2007; awards to be made in 2008.

Project Title: Investigator-initiated small research grant award program announcement (R03)

• Agency: NIH
• Description: The R03 award supports small research projects that can be carried out in a short period of time, with limited resources. This solicitation extends its use to unsolicited applications in addition to its use in individual Requests for Applications (RFA) and Program Announcements (PA). This is an important mechanism for attracting new investigators to a field of study and providing sufficient support to allow development of preliminary data that will enable successful long-term funding.
• Results: Program Announcement PA-06-180 was released on March 2, 2006 with expiration date May 2, 2009. Examples of awards made in FY2007 include: Antiretroviral Treatment Strategies with Optional Switching Times, Antibiotics Discovery from the Great Lakes, and Susceptibility of Mycobacterium tuberculosis Clinical Isolates to Moxifloxacin.

Project Title: Clinical Trial for Community-Acquired Methicillin-Resistant Staphylococcus aureus (CA-MRSA) Infections

• Agency: NIH
• Description: These studies are to define the effective outpatient treatment with skin and soft tissue infection in areas where prevalence of CA-MRSA is high. The efficacy of off-patent antimicrobials such as clindamycin and trimethoprim/sulfamethoxazole will be evaluated.
• Results: Two contracts were awarded in 2007, one to University of California San Francisco and one to Olive-View University of California Los Angeles.

Project Title: Food and Waterborne Diseases Integrated Research Network (FWDIRN)

• Agency: NIH
• Description: NIAID's FWDIRN network includes multidisciplinary research on all food and waterborne pathogens (bacteria, viruses, and protozoa), as well as toxins, to facilitate the development and evaluation of products to rapidly identify, prevent, and treat food and waterborne diseases that threaten public health. The network includes Immunology (IRU), Microbiology (MRU), Zoonoses (ZRU) and Clinical (CRU) Research Units. The Network is supported by a Coordinating and Biostatistics Center. One of the MRUs will emphasize research aimed at developing and evaluating therapies for botulism.
• Results: Innovative projects that address this action item include: Retrospective study of the emergence of AR Salmonella enteritidis, and Two additional studies focused on the emergence and transmission of AR zoonotic bacteria.

Project Title: NIAID intramural Laboratory of Clinical Infectious Diseases, Tuberculosis Research Section

• Agency: NIH
• Description: The Tuberculosis Research Section is an integrated group of chemists, clinicians, and microbiologists dedicated to improving the chemotherapy of tuberculosis. Projects in the section include evaluation and validation of drug targets, understanding the mechanisms of resistance to specific drugs, and understanding the basic mechanisms of pathogenesis at a molecular level. Research is also focused on understanding how current TB drugs work using the most modern technologies and using this information to develop new and improved therapies and therapeutic approaches.
• Results: In 2007, section scientists and collaborators reported the first description of a series of phenomena unique to M. tuberculosis Beijing W strains that may help to explain their epidemic spread and increased likelihood of developing drug resistance. J Bacteriol. 2007 Apr;189(7):2583-9.

Project Title: The Clinical Mycology Section conducts research to determine molecular mechanisms of azole resistance in clinical isolates of the pathogenic yeast, Candida glabrata.

• Agency: NIH
• Description: NIAID intramural Laboratory of Clinical Infectious Diseases, Clinical Mycology Section
• Results: In 2006, section scientists described a mechanism by which fluconazole resistance in Candida glabrata arises during therapy. In ten patients a single nucleotide mutation in the gene coding for the transcriptional regulator, CgPDR1, increased the transcription of two drug transporters and increased drug efflux so significantly that fluconazole susceptibility decreased at least four fold. Further study of this mechanism continues in 2008.

Project Title: NIAID intramural Laboratory of Human Bacterial Pathogenesis, Pathogen Molecular Genetics Section

• Agency: NIH
• Description: The Pathogen Molecular Genetics Section studies the pathogen-polymorphonuclear neutrophil interface at both the cell and molecular levels to provide information critical to our understanding, treatment, and control of human diseases caused by bacteria. The section's overarching goal is to develop and/or promote development of enhanced diagnostics and better prophylaxis and therapeutics for pathogens such as community-associated methicillin-resistant S. aureus (CA-MRSA).
• Results: In 2006-2007, section scientists reported that a bacterial toxin called PVL, epidemiologically linked to CA-MRSA outbreaks and the presumptive reason for its virulence, is not responsible for the increased incidence and severity of CA-MRSA disease. In 2008, section scientists reported that the USA300 group of CA-MRSA strains comprises nearly identical clones that have emerged from a single bacterial strain with extraordinary transmissibility They also reported new details about the complex mechanisms MRSA uses to avoid destruction by neutrophils.

Project Title: NIAID intramural Laboratory of Human Bacterial Pathogenesis, Pathogen-Host Cell Biology Section

• Agency: NIH
• Description: The Pathogen-Host Cell Biology Section studies the mechanisms of the formation of biofilms in chronic infections with staphylococci with a long-term objective to provide the scientific basis for the development of drugs interfering with these mechanisms. Such drugs would be useful in anti-staphylococcal therapy to both enable the immune system fight the infection and increase the efficiency of common antibiotics.
• Results: In 2007, the section reported the identification of novel cytolytic peptides as key virulence determinants for CA-MRSA. Section research indicates that phenol soluble modulin (PSM) peptides are major determinants of S. aureus virulence, and their increased production in CA-MRSA likely contributes to the enhanced virulence of CA- compared to HA-MRSA. These newly identified peptides encoded by the PSM gene cluster have a significant effect on the ability of CA-MRSA strains to cause disease in animal infection models.
  

Project Title: NIAID intramural Laboratory of Malaria and Vector Research, Malaria Genetics Section

• Agency: NIH
• Description: Section research addresses malaria drug resistance, antigenic variation, and disease virulence. Discoveries in these areas will support the development and evaluation of new diagnostic tools, antimalarial strategies, and candidate molecules for vaccines. Strains of malaria that are resistant to chloroquine have become a major problem and section scientists are seeking the exact resistance mechanism to support searches for new antimalarial compounds that can reverse or circumvent it.
• Results: Research is ongoing to characterize molecules that determine chloroquine and quinine responses in Plasmodium parasites; dissect structure-function relationships of the P. falciparum chloroquine resistance transporter (CRT); and evaluate candidate genes in a chromosome locus recently shown to affect the quinine response of P. falciparum. In 2007, section scientists and collaborators reported that mutations in transmembrane domains 1, 4 and 9 of the P. falciparum CRT alter susceptibility to chloroquine, quinine and quinidine (Mol Microbiol 2007. 63:270-82).

Project Title: Impact of Diet and Gut Microbial Ecology on Foodborne bacterial pathogens and autimicrobial resistance in farm animals.

• Agency: USDA
• Description: The project goal is to identify factors affecting persistence of antibiotic resistance genes and other genetic determinants among normal and pathogenic enteric bacteria.
• Results: We have found that low (sub-Mic) levels of the antimicrobial carbadox stimulate 100-fold increases in the in vitro transfer of natural resistance to the antibiotic tylosin.

Project Title: Surveillance of antibiotic resistance in normal enteric bacteria

• Agency: USDA
• Description: The project goal is to determine tetracycline resistant genotypes, species identities, and resistance "baseline' levels of commensal bacteria in the swine intestinal tract. Current research aims to evaluate the transmissibility of resistance between Megaspaera elsdenii strains and other intestinal bacteria.
• Results: We found that Megasphaera elsdenii strains are multiply drug resistant. Further, strains contain hybrid (recombinant) tetracycline resistant genes. Thus M. elsdenii is a potential site for evolution of antibiotic resistance as well as for the persistence of resistance in the swine intestinal tract.

Project Title: To assess the gene variability associated with resistant versus susceptible strains of Salmonella, Campylobacter, Entercocci and E. coli

• Agency: USDA
• Description: A microarray chip has been developed that can screen for almost 800 resistance and virulence genes among the four bacterial species. Additional genes are being added for other bacteria. The microarray chip was also successfully tested on Listeria, another important food-borne pathogen.
• Results: Ongoing. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA. A DNA microarray to detect 100 resistance genes was successfully tested and has now been expanded to detect 775 resistance and virulence genes simultaneously.

Project Title: Prevalence of food borne and commensal pathogens in wild birds

• Agency: USDA
• Description: Fecal samples from approximately 175 wild birds were tested for the presence of Campylobacter, E. coli, Enterococcus, and Salmonella. Samples were negative for Salmonella but a few isolates of Arcobacter sp. were detected.
• Results: Completed-2007. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA. From the study, 103 samples were positive for Enterococcus, 53 were positive for Campylobacter, 35 were positive for E. coli, and 19 were positive for Arcobacter. None of the samples contained Salmonella.

Project Title: Assess the ability temperature has on survival of resistant versus sensitive bacteria.

• Agency: USDA
• Description: A pan-susceptible and multiple-resistant strains were compared for their ability to survive following challenge of poultry exposed to various room temperatures.
• Results: Ongoing. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA.

Project Title: Determine the effect of antimicrobial selective pressure on the rate of spread of Salmonella typhimurium in poultry

• Agency: USDA
• Description: Salmonella strains have arisen that are resistant to multiple antimicrobials including 3rd generation cephalosporins. The ability of those strains to be transmitted between hosts and under antimicrobial selective pressure is presently unknown.
• Results: Two Salmonella strains (one pan-susceptible and one resistant to 12 antimicrobials used in the NARMS program) were compared by a natural transmission study in chickens in the presence of MIC levels of chlortetracycline (tet). The percentage of positive cloacal swabs from birds exposed to the resistant strain indicated that more birds were positive when tet treatment was administered. Cloacal swabs from the susceptible strain exposed birds indicated that more birds were positive in the absence of tet treatment. The same results were observed for tissues at necropsy on D10. Results indicated that resistant strain did not transmit faster in the presence of tet, and suggested that use of tet had a protective effect on tissue colonization.

Project Title: To phenotypically and genotypically characterize Salmonella serotype Newport identified from NARMS 2000 and 2001 collection of isolates

• Agency: USDA
• Description: To phenotypically and genotypically characterize Salmonella serotype Newport identified from NARMS 2000 and 2001 collection of isolates
• Results: Between 2000 and 2001, the animal arm of NARMS recovered a total of 241 Salmonella newport non-diagnostic (slaughter and on-farm) isolates. MDR S. newport isolates were recovered more frequently than pan-susceptible isolates and most of the MDR isolates were resistant to > nine antimicrobials. None of the Newport isolates contained Class 2, Class 3, or Class 4 integrons (intI2, intI3, or int4, respectively). However, Class I (intI1) integrons were identified in most of the animal species regardless of whether they were MDR or pan-susceptible. Large and small plasmids were identified mainly in the MDR Newport isolates. By PFGE analysis, Newport appears to be heterogeneous among multiple animal species, but homogeneous in a particular species. These data can be used for comparison with isolates obtained from human outbreaks to determine if a particular animal species served as the source of infection.

Project Title: Evaluate the prevalence and antimicrobial susceptibility of E. coli isolated from fruits and vegetables

• Agency: USDA
• Description: Although a number of studies have determined levels of resistant bacteria on meat items from grocery stores, few studies have been conducted on the prevalence of bacteria from fruits and vegetables. In collaboration with scientists from USDA-AMS, we evaluated the prevalence and antimicrobial susceptibility of generic E. coli isolated from fruits and vegetables collected from different regions of the US and determined that resistance to 17 different antimicrobials among these E. coli is low.
• Results: Completed-2004. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA.

Project Title: Assess the prevalence of E. coli 0157:H7 in downer cows

• Agency: USDA
• Description: This study assessed the prevalence of E. coli 0157:H7 in downer cows.
• Results: Data indicated that 4.9% of downer cows versus 1.5% of health cows harbor E. coli 0157:H7 in their colons. Not all isolates were clonal, resistance to antimicrobials was low and very little multiple resistance was observed. These data implicate downer cows as having a higher prevalence of E. coli 0157:H7 than healthy cows and may affect the use of downer cows as sources of meat.

Project Title: Determine the effect of three feed-based antimicrobials (apramycin, carbadox, and tetracycline) on the development of antimicrobial resistance in generic E. coli

• Agency: USDA
• Description: Study to determine the effect of three feed-based antimicrobials (apramycin, carbadox, and tetracycline) on the development of antimicrobial resistance in generic E. coli.
• Results: Resistance to tetracycline in E. coli varied widely by sample, group, and trial. However, a significant increase in the percentage of resistant isolates was observed in piglets fed antimicrobials when compared to controls. Resistance to apramycin also increased in piglets when compared to controls. However, upon removal of apramycin, resistance in E. coli declined. Resistance to carbadox remained unchanged after feeding carbadox when compared to controls. Piglets fed low doses of antimicrobials demonstrated improved growth when compared to controls. These data are useful for veterinarians, pharmaceutical manufacturers, and scientists as they devise ways to limit the development of resistance to antimicrobials while maintaining animal health.

Project Title: Characterize antimicrobial resistance, species, and genetic diversity of Campylobacter isolated from feedlot cattle

• Agency: USDA
• Description: In collaboration with scientists from USDA-APHIS-VS-CEAH, antimicrobial resistance was examined in Campylobacter isolates from feedlot cattle as part of a NAHMS study. Results indicate that a majority of the isolates were susceptible to the antimicrobials that were tested and that there is significant genetic diversity among isolates. These data provided a significant overview of antibiotic resistance among Campylobacter from healthy beef cattle across the US. This work will be useful to beef producers, regulatory agencies and researchers in antimicrobial resistance.
• Results: Completed. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA.

Project Title: Study the role of tetracycline resistance in Campylobacter species.

• Agency: USDA
• Description: Tetracycline resistance appear to be common among bacteria particularly when multiple resistance is detected. Our goal is to study the presence of, and characterize, tetracycline resistant genes among Campylobacter species.
• Results: Ongoing. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA. Plasmids carrying genes that confer resistance to tetracycline and kanamycin in Campylobacter were identified and characterized. Two additional, novel plasmids were also identified and sequenced. Jesse, T.W., L.G. PIttenger-Alley, and M.D. Englen. 2006. Sequence analysis of two cryptic plasmids from an agricultural isolate of Campylobacter coli. Plasmid. 55: 64-69.

Project Title: Characterize antimicrobial resistance and species of Campylobacter isolated from dairy cattle

• Agency: USDA
• Description: In collaboration with scientists from USDA-APHIS-VS-CEAH, antimicrobial resistance was examined in Campylobacter isolates from US dairy cattle as part of a NAHMS study.
• Results: Completed. Results indicate that a majority of the isolates were susceptible to the antimicrobials tested. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA.

Project Title: To increase recovery of Campylobacter from various sources

• Agency: USDA
• Description: Because of the fastidious nature of Campylobacter, recovery from meat or other sources is difficult. We developed an enhanced methods for recovering Campylobacter from chicken carcass rinsates by employing a centrifugation step of the rinsate prior to enrichment in culture media. This resulted in a >50% increase in the recovery of Campylobacter. This is significant in that previous methods were leading to the isolation and under reporting of Campylobacter in samples. This work will be useful to scientists involved in Campylobacter research.
• Results: Completed. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA.

Project Title: Determine the prevalence and level of Campylobacter in parents (breeders) and offspring (broilers) of commercially reared pigs

• Agency: USDA
• Description: Studies were conducted to determine the prevalence and level of Campylobacter in parents (breeders) and offspring (broilers) of commercially reared pigs Prevalence of Campylobacter ranged from 42 to 100% positive in three broiler offspring flocks (90% of breeders were shedding).
• Results: Completed. Bacterial Epidemiology and Antimicrobial Resistance Research Unit, ARS, Athens, GA.

Project Title: To evaluate the prevalence and antimicrobial resistance of enterococci isolated from retail food items

• Agency: USDA
• Description: In a study of retail food (meat, vegetables, and fruit) collected from grocery stores in NE Georgia, enterococci were isolated, identified to species, and tested for antimicrobial susceptibility. Results indicated that although enterococci were prevalent among food items, resistance to antimicrobials used in human medicine was very low (linezolid, gentamicin, ciprofloxacin) or nonexistent (vancomycin). This was the first study analyzing enterococci isolated not only from meats, but fruits and vegetables as well. This work will be useful to scientists involved in Enterococcus research as well as regulatory agencies and the industry as they develop and implement mitigation strategies.
• Results: Results indicated that although enterococci were prevalent among food items, resistance to antimicrobials used in human medicine was very low (linezolid, gentamicin, ciprofloxacin) or nonexistent (vancomycin). This was the first study analyzing enterococci isolated not only from meats, but fruits and vegetables as well. This work will be useful to scientists involved in Enterococcus research as well as regulatory agencies and the industry as they develop and implement mitigation strategies.

Project Title: To evaluate antimicrobial resistance and virulence of enterococci isolated from retail food items

• Agency: USDA
• Description: In a study of retail food (meat, vegetables, and fruit) collected from grocery stores in NE Georgia, enterococci were isolated, identified to species, and tested for antimicrobial susceptibility and presence of virulence determinants.
• Results: Results indicated positive statistical associations (significance level = 0.05) between several virulence genes and bacitracin resistance, erythromycin resistance, lincomycin resistance and tetracycline resistance. Negative correlations were observed among many of the virulence attributes and ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, and tylosin resistance.

Project Title: Determine molecular genetics of streptogramin resistance in enterococci from animals.

• Agency: USDA
• Description: In this study, mechanisms of streptogramin resistance in enterococci from animals and the environment was investigated. From 2000-2004, enterococci were isolated from poultry carcass rinsates, fruits, vegetables, retail meats, and environmental rinsates or from swine and cattle fecal samples collected on-farm.
• Results: One Q/D resistance gene, vatD, which had not been found previously in enterococci from animals, was detected in three enterococcal isolates from the study. Two other Q/D resistance genes, vatB and vgaB, had never been previously reported in enterococci. To date, this is the first report of vatD from enterococci from animals in the U.S. and the first report of vatB and vgaB in enterococci.

Project Title: Characterize erythromycin resistance in enterococci isolated from swine farms using different regimens of tylosin

• Agency: USDA
• Description: The effect of tylosin use on erythromycin resistant enterococci isolated from farms was investigated.
• Results: Results from the study suggested that although resistance was higher on a farm where tylosin was used as a growth promotant, a few resistant enterococci also persisted on a farm where no antimicrobials were being used. Isolates from farms were analyzed for antimicrobial resistance gene content as well as genetic determinants for dissemination of resistance. These data provide insight as to the development and persistence of resistance on-farm and will be useful to research and industry scientists as they develop and implement Enterococcus mitigation strategies.

Project Title: Characterize aminoglycoside resistance among enterococci isolated from poultry

• Agency: USDA
• Description: Aminoglycoside antimicrobials are of interest due to their use in both animals and humans.
• Results: In this study, resistance to aminoglycosides in enterococci from poultry samples was examined. High-level gentamicin, kanamycin, and streptomycin resistance was found in 23%, 41%, and 19% of the isolates, respectively. Of the ten aminoglycoside resistance genes examined, five were identified in the isolates using PCR. Seven resistant E. faecalis isolates were negative for all genes tested suggesting that additional resistance genes may exist. Phylogenetic analysis revealed that the isolates were genetically different with little clonality. Data from this study suggest that enterococci from poultry are diverse and contain potentially unidentified aminoglycoside resistance genes. This work will be useful to scientists involved in Enterococcus research as well as the industry as they develop and implement mitigation strategies.

Project Title: To characterize 3rd generation cephalosporin resistant Salmonella from animal sources.

• Agency: USDA
• Description: We characterized the strains and resistance mechanisms of 3rd generation cephalosporin resistant Salmonella in the United states.
• Results: CMY-2 is the most common mechanism of B-lactam resistance in salmonellae in the US. This is in contrast to Europe where it is the Extended Spectrum Beta-Lactamase (ESBL). Isolates carrying the CMY-2 gene are significantly more likely to multiple drug resistant, and that certain Salmonella serotypes were more likely to carry the resistance. Third generation cephalosporins are important antimicrobials used to treat severe infections in both humans and animals. The research resulted in a predictive diagnostic test for multiple drug resistant Salmonella. Turkeys, horses, cats and dogs are significantly more likely to have these isolates than cattle, swine, chicken and exotics. The multiple drug resistance identified was found to be encoded on a large transferable plasmid.

Project Title: To study the ability of resistant strains to have a competitive persistence advantage

• Agency: USDA
• Description: Recently, Salmonella strains have arisen that are resistant to multiple antimicrobials including 3rd generation cephalosporins. The ability of those strains to be transmitted between hosts and under antimicrobial selective pressure is presently unknown.
• Results: Two Salmonella strains (one pan-susceptible and one resistant to 12 antimicrobials used in the NARMS program) were compared by a natural transmission study in chickens in the presence of MIC levels of chlortetracycline (tet). The percentage of positive cloacal swabs from birds exposed to the resistant strain indicated that more birds were positive when tet treatment was administered. Conversely, cloacal swabs from the susceptible strain exposed birds indicated that more birds were positive in the absence of tet treatment. The same results were observed for tissues at necropsy on D10. These results indicated that resistant strain did not have an increased transmissibility in the presence of tet and suggested that use of tet had a protective effect on tissue colonization.

Project Title: Isolation and characterization of methicillin-resistant Staphylococcus aureus (MRSA) from retail pork.

• Agency: USDA
• Description: Retail pork products in the local Athens area are being collected and screened for MRSA. These isolates will be compared with human MRSA isolates donated by a local hospital to determine if the MRSA from swine and retail pork products are genetically related to those from humans. The isolates from the various sources will be subjected to antimicrobial susceptibility testing and the antimicrobial resistance genes identified by PCR. Virulence genes from the isolates will also be identified using PCR, multi-locus sequence typing (MLST), and multiple variable number of tandem repeat (MLVA) analysis. Genetic comparisons will be accomplished using PFGE analysis.

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