Research Project: Monitoring and Molecular Characterization of Antimicrobial Resistance in Foodborne Bacteria

Location:

2020 Annual Report

Objectives
1. Provide data and characterize pathogen prevalence, unique characteristics and trends on antibiotic resistance, subtyping and molecular characterization of foodborne pathogens in food animals. 2. Identify and characterize potential genetic markers within and across serotypes for Salmonella isolated from poultry for rapid identification and diagnosis. 3. Evaluate the role of innovative chemical and/or biological treatments including arsenicals, prebiotics, or ammonium compounds and how they impact the prevalence and type of antimicrobial resistant pathogens or resistance genes. 4. Develop, evaluate and optimize processing treatments to reduce, control and potentially eliminate foodborne pathogens in poultry processing. 5. Evaluate and define the potential role of protozoa in shaping the ecology of bacterial pathogens in controlling foodborne pathogens in poultry processing environments. 6. Develop algorithms for interpreting and handling sequencing data to aid in epidemiological tracking, defining differences in isolates of foodborne pathogens, including antibiotic resistance patterns, and predicting and determining the source of the isolate.

Approach
The goals of this project fit into four major approaches: 1) analysis of antimicrobial resistance mechanisms and genetic elements in foodborne bacteria from poultry, 2) analysis of innovative chemical and/or biological treatments used for poultry processing on resistance in foodborne bacteria, 3) development of alternative methods for processing poultry products, and 4) development of methods that accurately monitor the microbial quality of poultry products processed by alternative methods. Studies will focus on the molecular aspects of antimicrobial resistance to identify and characterize new and emerging resistance phenotypes and genotypes of high priority type bacteria from poultry [categorized as urgent and serious threat level antimicrobial-resistant pathogens by the Centers for Disease Control and Prevention (CDC)]. Those high priority bacteria will be evaluated for resistance to biocides. This project will target foodborne pathogens including Salmonella, Campylobacter, and Listeria and commensals including Escherichia coli and Enterococcus, for their role as reservoirs of resistance. The alternative processing methods in this project include testing several novel chemical and physical decontamination procedures. The approach for most of this work is to apply the intervention strategy and compare the microbial quality of the treated poultry product with control product treated by standard methods. Intervention strategies will include studies on the microbial ecology in and around poultry processing and further processing plants, such as floor drains, to determine a particular ecological niche or reservoir for a specific pathogen in the processing environment. These studies will improve understanding of sources and harborage points for human pathogens and how best to combat colonization of a processing plant with those pathogens. A long term objective is to develop systems using protozoa as natural controllers of foodborne pathogens. This will involve studying the ecology of protozoa that feed on the pathogens and determining methods to enrich the processing environment with effective protozoa. Approaches for monitoring microbial quality will include enhancing the sensitivity and specificity of microbial detection. The project will also use genetic typing methods including whole genome sequencing and metagenomics sequencing to characterize antimicrobial and biocide resistance and track specific clones of pathogens in and around poultry processing environments. Data from this research will be used to assist other Federal agencies in assessing antimicrobial resistance in food animal populations as well as to address a direct need outlined by the National Action Plan for Combating Antibiotic-Resistant Bacteria (CARB) in evaluating potential alternatives to antimicrobials. Data generated on biocide resistance and resistance genes active against chemicals specific to poultry production and processing is a specific concern to USDA-FSIS. Development of technologies for detection of microbial contaminants is a critical need for Federal regulatory agencies.

Progress Report
Under Objective 1, work investigating Salmonella serotype Infantis plasmid pESI (plasmid for epidemic Salmonella Infantis) continued. The pan and core genome of this plasmid in U.S. meat samples, U.S. clinical isolates, and internationally collected isolates was calculated and a dendrogram was created based on the presence and absence of these genes. The dendrogram predicted two lineages of the plasmid: one circulating only in South America and one circulating in both North and South America. It also showed that isolates from U.S poultry tended to cluster together as did U.S. clinical isolates, while other isolates from both sources and international isolates did not cluster meaningfully. A collaboration with the Centers for Disease Control and Prevention yielded data on the prevalence of the plasmid in U.S. clinical Salmonella isolates. It was also determined that while the prevalence of the plasmid is increasing in U.S. poultry isolates, the ESBL gene blaCTX-M-65 is only present on 49% of plasmids. This research has been presented to poultry stakeholders. Through a collaboration with ARS researchers in Wyndmoor, Pennsylvania, the genomes of eight isolates of Salmonella and Escherichia coli containing large plasmids capable of transferring small kanamycin resistant plasmids were sequenced, assembled, and annotated. These plasmids were identified and compared to determine characteristics that would allow transfer of the plasmid at different rates. Analysis of whole-genome sequences of drug-resistant Salmonella, E. coli, and Enterococcus (100 isolates each) continued in order to populate a database of resistance phenotype, resistance genes, and mobile genetic elements in these foodborne pathogens and commensals. This database will allow rapid searches for identification of unique antimicrobial resistance genes including co-linkage of genes and associations with mobile and extrachromosomal elements. A study on antimicrobial resistant populations on “no antibiotic ever” poultry continued to determine if products contain antimicrobial resistant foodborne pathogens and commensals that may pose a risk to the consumer. “No antibiotic ever” chicken and turkey samples, including whole and ground parts, were purchased from local grocery stores in the Athens, Georgia, area. Salmonella, E. coli, Enterococcus, and Staphylococcus were isolated from the samples and tested for resistance to antibiotics. Preliminary data showed that Enterococcus faecalis was resistant to 10 of the 16 drugs tested including tigecycline and linezolid, two of the newer antibiotics used in human medicine. Five different incubation methods using different media were tested for isolation of Salmonella. Up to six Salmonella serotypes were isolated including multiple serotypes from the same product. Salmonella Infantis was isolated regardless of the method used. The sixth year of seasonal monitoring of surface water as a reservoir and potential vehicle for development of resistant bacteria was conducted by collecting water samples through a partnership with the Upper Oconee Watershed Network at 100 different locations along the Oconee River. Fall 2019 and Winter 2020 sampling were done and Salmonella, E. coli, and Enterococcus were isolated and confirmed. E. coli isolates were specifically examined for extended-spectrum beta-lactamase and carbapenem resistance. Research on a collaborative extramural-funded project with Mississippi State University was initiated. Fifty-eight samples from three sample collections were verified as Mannheimia haemolytica, the bovine respiratory pathogen; isolates were tested for antimicrobial susceptibility. Results of the research will bring clarity to the ecology of respiratory antimicrobial resistance and help minimize risk of establishing antimicrobial resistance in other bacterial species and systems integral to disease control in humans and animals. Methicillin-resistant Staphylococcus aureus from chicken meat was characterized in a project with Tuskegee University. Resistant isolates were tested for the presence of resistance genes and genetic elements associated with transfer of resistance among staphylococci. Resistant isolates also contained a host of enterotoxin genes responsible for staphylococcal food poisoning in humans. Progress continued on international collaborations with Pakistan and Egypt. With Pakistan, whole-genome analysis of Salmonella Paratyphi transconjugants was performed. Resulting transconjugants contained resistance genes to aminoglycosides, beta-lactams, phenicols, sulfonamides, and trimethoprim showing that the extended-drug resistance phenotype was able to transfer to other Salmonella serotypes. Carbapenem resistance among E. coli from humans and dogs from Egypt was examined. Whole-genome sequencing was used to compare plasmids containing the resistance genes from the two sources. Two new international collaborations were established with Burkina Faso and Japan. Salmonella originating from humans, animals, and food from Burkina Faso were serotyped, tested for antimicrobial resistance, and sequenced using whole-genome sequencing. In preliminary results, common antimicrobial resistance was observed among the 33 identified serotypes. With Japan, carbapenem-resistant Enterobacteriaceae were sequenced and analyzed for the presence of carbapenem resistance genes and mobile genetic elements. Transfer of colistin resistance on plasmid replicons was demonstrated. Under Objective 3, production of a susceptibility plate for testing bacterial resistance to heavy metals continued. Lead was added to the plate which also contains arsenate, arsenite, cobalt, chromate, nickel, tellurite, and zinc. Concentrations of metals were modified after preliminary testing of 35 E. coli. Under Objective 4, the use of several common antimicrobial broiler processing aids for lessening numbers of naturally occurring Campylobacter on raw chicken livers was tested. Peracetic acid, sodium hypochlorite and cetylpyridinium chloride were moderately effective on liver but less so than normally seen when the same chemicals are applied to muscle. More research is planned to further examine chicken liver decontamination. A field-ready drain heater for decontamination of Listeria-positive floor drains in commercial food processing plants was developed and tested. Previously published study showed that heat is effective to eliminate Listeria in lab scale model floor drains. Preliminary laboratory data with the field heater suggests that this treatment will scale well to floor drains in food processing plants. A collaborator has been secured for field testing. A seven-year, on-going long-term study to measure Campylobacter prevalence in the intestine of broiler chickens at a commercial processing facility was continued. Gut contents were secured from each carcass and saved to allow study of the overall gut microbiome relative to Campylobacter prevalence. Genomic analysis is planned with >400 samples representing Campylobacter positive and negative flocks in all seasons across the seven years. Data will assist in discovery of members of the gut microflora that may be related to the presence or absence of Campylobacter colonization. Under Objective 5, protozoa as an alternative to antimicrobials to control Listeria monocytogenes was studied. L. monocytogenes is an important foodborne pathogen that commonly contaminates processed foods from the factory environment and is a difficult organism to remove by sanitation. In order to identify traits that may influence resistance to sanitation, ARS researchers at Athens, Georgia, collaborated with the University of Georgia to compare the whole-genome sequences of isolates that were persistent in two poultry further-processing plants against isolates that were transient. There were 111 genes that were identified as possibly linked to whether a strain was persistent or transient in processing plants. These include genes related to metabolism, membrane transport, oxidative stress, and chemotaxic functions. Chemototaxis is important for niche localization that may help the bacteria set up persistent colonization. Under Objective 6, the microbiome of retail chicken was investigated. The microbiome is the sum of all the microorganisms that live in a given habitat. The cross section of the identity of these species can influence the presence of other species. Knowing this can be useful as predictive surrogates and may be manipulated to control presence of certain pathogens. For this study, chicken breasts were sampled from four processing establishments under five brands over seven months and the microbiomes were characterized by metagenomic DNA sequencing. The character of the microbiomes correlated with the processing establishment and even more with whether the product was vacuum packed or not. Birds from three establishments were raised "No Antibiotic Ever"; one of those establishments had a substantially elevated presence of antibiotic resistance genes compared to all the other establishments. The chicken microbiome is a robust and multifaceted food microbiology attribute that could provide a variety of safety and quality information with a fraction of sequencing capacity on a common DNA sequencing platform.

Accomplishments
1. Flowing steam decontaminates soiled broiler transport cage flooring. Live haul cages are used to transport broilers from the farm to a processing facility. These cages are large and expensive; therefore, companies have a limited supply and continually reuse the same cages. Campylobacter, a leading human foodborne pathogen, can be readily detected in the feces of broilers from a Campylobacter positive flock. Feces left in a cage by Campylobacter positive broilers can result in the next broilers placed in the same cage to also become contaminated. Water spray and sanitizing of broiler transport cages is logistically complicated, physically difficult, water intense and largely ineffective to eliminate Campylobacter. ARS researchers in Athens, Georgia, tested steam as a means to decontaminate transport cage flooring. Purposely soiled transport cage flooring was treated by 15 seconds of flowing steam with and without a preceding water spray. The steam treatment resulted in an approximately 99% reduction in the number of Campylobacter. When the steam treatment was preceded by a 15 second water spray, the Campylobacter reduction was improved to 99.99% compared to untreated controls. While Campylobacter was not eliminated, steam shows potential as an effective method to sanitize broiler transport cages and to control transfer to previously negative broilers. Lowering the number of Campylobacter on live broilers entering the processing plant would be expected to lessen contamination of fully processed poultry meat products and reduce consumer exposure to Campylobacter.

2. Heat and cold treatment decreases Campylobacter on chicken liver. Foodborne outbreaks of campylobacteriosis traced to pâté or mousse prepared from undercooked chicken liver have become more prevalent in the last several years prompting the USDA Food Safety and Inspection Service to list Campylobacter contamination of chicken liver as a critical research need. Campylobacter is readily detected on fresh raw chicken livers in the processing plant and at retail. ARS researchers in Athens, Georgia, tested a mild heat and/or freeze treatment to lessen Campylobacter contamination of fresh chicken livers. Heat and cold treatments at various time periods were tested. Immersion in 60oC water for five minutes was effective to significantly lower Campylobacter numbers on chicken liver. Forty-eight hours at -25oC in a household freezer was moderately effective. When heat and freezing were combined in series, a nearly 99% decrease in the number of naturally occurring Campylobacter on both the surface and within inner tissue of chicken liver was achieved. A mild heat process followed by freezing livers for presentation at retail may be useful to lessen consumer exposure to Campylobacter.

3. Campylobacter contamination in a commercial duck processing plant. Duck meat, while not as commonly eaten in the U.S as chicken, is enjoyed by a substantial population. There is little information available regarding the microbiological safety of U.S. processed duck meat. ARS researchers in Athens, Georgia, examined duck samples collected once a month for one year in a large U.S. commercial duck slaughter and processing facility. Internal organ samples, ceca and crop, collected at evisceration represent what is carried into the slaughter plant with the live ducks. Campylobacter was detected in 80% of ceca samples. Results showed that Campylobacter, and to a lesser degree Salmonella, can be expected to accompany live animals into the plant. Campylobacter was detected on 67% of whole duck carcasses examined prior to carcass chilling. Pathogen prevalence was reduced significantly due to antimicrobial processes applied. Overall, 2% and 3% of final oven ready cut-up duck leg quarters yielded detectable numbers of Salmonella and Campylobacter, respectively. Numbers of indicator bacteria on duck carcasses were similarly lessened due to the antimicrobial nature of commercial duck processing including immersion chilling and chlorinated spray treatment. Overall, current commercial duck processing techniques are effective to lessen bacterial prevalence and numbers on duck meat products.

4. Controlling Listeria with protozoa. Listeria monocytogenes is a bacterium that lives in food processing habitats. The organism can contaminate the food product and become a health hazard. Protozoa that eat and destroy bacteria may provide a safe method of controlling Listeria, but protozoa tested to date have not been effective in killing the bacterium. ARS researchers in Athens, Georgia, tested a bacteria-eating protozoan known as Tetrahymena pyriformis for its ability to kill Listeria. The two organisms were cultured together and once the protozoa had ingested a share of the bacteria, the residual bacteria that were outside of the protozoa were killed with antibiotic. The amount of time for Tetrahymena to kill the ingested Listeria was then measured. Almost all the ingested Listeria were killed within 24 hours. Nineteen different strains of Listeria were tested, and all were equally killed. The Tetrahymena showed ability to kill adherent Listeria as well as those in culture suspension. Thus, Tetrahymena may be a useful candidate for treating food processing plants to control Listeria.

5. Genomic comparison of diverse Salmonella serovars isolated from swine Salmonella. Salmonella has the ability to survive at different stages of food production, processing, and storage and also grow in improperly cooked food. Some Salmonella serotypes are host-specific while others have broad host range due to genetic factors associated with colonization and infection. To understand the genetic variations among different Salmonella serotypes, ARS researchers in Athens, Georgia, analyzed the whole-genome sequence of fourteen Salmonella serotypes. The data was assessed for resistance and virulence genes, and their association with mobile genetic elements was predicted. Resistance determinants were mainly located on mobile genetic elements or integrated into the chromosome. Most known and putative virulence genes were part of the core genome, but a small fraction was detected on the mobile elements. Integrated viruses (phage) were highly diverse and many harbored virulence, antibiotic or metal resistance genes. Analysis determined that although all isolates were swine-associated, much of the genetic diversity detected in the different Salmonella serotypes was due to acquired genes that enable the bacterium to thrive and survive during infection. The findings of this study will help researchers understand genetic diversity in Salmonella serotypes, and how specific genes help adapt some serotypes to certain host animals. The processes these genes perform can be further studied to determine how they could be blocked to prevent colonization and infection of food animals.

6. Antimicrobial resistance and genomic analysis of Enterococcus cecorum from poultry. Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but has been detected in both diseased (clinical) and healthy (non-clinical) poultry. Differences between the two groups of isolates have not been defined. ARS researchers in Athens, Georgia, studied antimicrobial resistance genes and the genome of clinical and non-clinical E. cecorum from chickens were analyzed using whole-genome sequencing to better define their differences. The comparison confirmed that non-clinical isolates contained more resistance genes than clinical isolates. Resistance genes were both shared and exclusive to each group indicating varying genetic characteristics among E. cecorum isolates. The genome analysis revealed that the non-clinical E. cecorum genomes were comparatively diverse due to acquisition of additional genes while genome reduction in the conserved clinical genomes suggested better host adaptability. This data is important for human health due to clinical disease potential in humans and antimicrobial resistance in the bacterium that may be passed to humans through food. Identification of unknown virulence determinants is important for animal health for determination of the pathogenesis of E. cecorum infections in poultry and for production of a vaccine candidate for protection of poultry flocks. This research is useful for food safety scientists as well as veterinarians who treat poultry for infections caused by E. cecorum.

7. Escherichia coli isolated from a mixed-use watershed in Northeast Georgia, United States. Surface water may play a role in the development and spread of antibiotic resistant bacteria. To address this, ARS researchers in Athens, Georgia, characterized antibiotic resistance mechanisms in Escherichia coli isolated in a study of quarterly water samples from the upper Oconee watershed in Northeast Georgia. Genes conferring resistance to azithromycin, ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole were identified. Most of the isolates also carried plasmids and 11 different plasmids were detected among the isolates. Twenty-nine sequence types were identified, including the epidemic urinary tract infection associated sequence type 131. One of the sequence type 131 E. coli isolates produced an extended-spectrum beta-lactamase rendering the isolate resistant to antibiotics like ceftriaxone used to treat dangerous infections. This is the first report of pathogenic extended-spectrum beta-lactamase-producing sequence type 131 isolated from environmental water in the United States. Resistance mechanisms to commonly used antimicrobials were found in E. coli isolated from surface water, and they also often carried mobile genetic elements like plasmids, which may play a role in the transmission of resistance genes among bacteria in the water environment. This study confirmed the presence of antibiotic resistance and pathogenic E. coli in recreational waters.

8. Pathogenic strain of Escherichia coli in surface water. Surface waters such as streams, lakes, and rivers can be contaminated with bacteria, including bacteria that are antibiotic resistant. Contaminated water may be responsible for spreading resistant bacteria, including potential pathogens, to humans through drinking untreated water, recreational contact, or irrigation of fresh fruits or vegetables. ARS researchers in Athens, Georgia, sampled from streams in the Upper Oconee watershed near Athens, Georgia, from 2015-2017 for the presence of antibiotic resistant bacteria. Escherichia coli, a bacterium that can be a harmless resident of the human gut or in some cases a human pathogen, were isolated from the water samples. Of the E. coli isolated, 6.9% were antibiotic resistant; six isolates that were resistant to multiple antibiotics were investigated by whole-genome sequencing. One of the E. coli isolates was a human pathogenic strain and carried a gene conferring resistance to extended-spectrum beta-lactam antibiotics which are used to treat invasive E. coli infections. The gene was located on a very rare phage-like plasmid suggesting that it may be a new emerging mechanism of antibiotic resistance gene transmission.

9. Antimicrobial resistance of Enterococcus from the Upper Oconee Watershed, Georgia. It is well-known that enterococci are abundant in the environment; however, the role of surface water as a reservoir of antimicrobial resistant enterococci remains largely undefined. ARS researchers in Athens, Georgia, collected surface water samples during each of four seasons from 2015 to 2016 from the Upper Oconee Watershed, located in Athens, Georgia. Enterococci were isolated, identified to species, and tested for susceptibility against a panel of antimicrobials. At least ten enterococcal species were detected during the sampling period. Regardless of species, the highest levels of resistance were to lincomycin and tetracycline; isolates also exhibited resistance to two newer antimicrobials, daptomycin and tigecycline. Multidrug resistance was observed to as many as five classes of antimicrobials. Resistant enterococci appeared to be randomly dispersed over the seasons rather than clustered by species or antimicrobial resistance. These results may indicate the potential of human intestinal illness and/or colonization of the human gut with resistant enterococci as enterococci are considered an indicator of water health and correlate with increased disease risk to humans during recreational exposure to water. This information is useful to environmental scientists and state and federal environmental regulatory agencies as they develop policy regarding conservation of water sources.

10. Escherichia coli from humans, retail chicken, and ground beef in Egypt. Contamination of retail foods with foodborne pathogens, particularly those that are resistant to antimicrobials, poses a persistent threat to human health. Little is known about Escherichia coli clones circulating among retail food and humans in Egypt which may be similar to those in the United States. ARS researchers in Athens, Georgia, and Mansoura University, Egypt determined antimicrobial resistance and diversity of E. coli from humans, retail chicken, and ground beef from Mansoura, Egypt. Higher antimicrobial resistance levels were found among chicken isolates compared to beef and human isolates. Regardless of isolate source, the predominant antimicrobial resistances were to ampicillin, tetracycline, sulfisoxazole, and streptomycin and the prevalent genes detected corresponded with resistance phenotypes. The majority of E. coli from all three sources were commensal instead of pathogenic and genetic analysis showed that isolates were distributed among both distinct and shared groups. The existence of common genetic determinants among isolates from retail foods and humans in Egypt as well as the circulation of shared genetic types indicates a possible epidemiological link with potential zoonotic hazards. This data is useful for scientists from various disciplines in order to determine environmental niches of bacterial clones for development of mitigation strategies for foodborne illness.

11. Genomic sequence of Escherichia coli Sequence Type 131. Pathogenic bacteria known as extraintestinal pathogenic Escherichia coli are important causes of infections. Spread of multidrug resistant E. coli globally can also be attributed to clones, such as E. coli sequence type 131. Three sequence type 131 isolates were sequenced by ARS researchers in Athens, Georgia, and Wyndmoor, Pennsylvania. Isolates contained resistance genes to different antibiotics including aminoglycosides, beta-lactams, chloramphenicol, fluoroquinolones, sulfonamides, tetracycline, and trimethoprim as well as virulence genes. The presence of antibiotic and virulence genes in these clones provides greater propensity to transfer genetic material compared to non-sequence type 131 E. coli. The genomic information from these strains will be useful for understanding the dissemination and pathogenicity of E. coli sequence type 131, as well as for facilitating the development of novel antimicrobial therapies.

12. Carbapenem resistant Escherichia coli among humans and dogs in Egypt. The emergence of carbapenem-resistant Escherichia coli has considerably threatened human and animal health worldwide. ARS researchers in Athens, Georgia, investigated the carbapenem-resistance gene, New Delhi Metalloprotease, and genetic relatedness of E. coli from humans and dogs in Egypt. Using whole-genome sequencing, two different New Delhi Metalloprotease variants were identified; one in E. coli from urine of a healthy person and an environmental sample of dogs, and a different variant in E. coli from urine from a human patient. The New Delhi Metalloprotease gene was associated with the same plasmid type in E. coli from both humans and dogs but could not transfer to other E. coli by conjugation. This study showed, for the first time in Egypt, the emergence of a New Delhi Metalloprotease variant in E. coli in dogs that share similar genetic features with human E. coli in the same area. This study will be useful for regulatory agencies as it shows that periodic surveillance and monitoring of emerging antimicrobial resistance using whole-genome sequencing are warranted for setting control interventions.

13. Multidrug resistant Escherichia coli from humans and food animals in Nigeria. Beta-lactam antibiotics are the most commonly used class of antibiotics. Production of beta-lactamases has been reported as one of the most common mechanisms of resistance to these antibiotics and has played a significant role in the global spread of resistance, particularly in community and hospital settings, as well as in agriculture and the environment. ARS researchers in Athens, Georgia, analyzed multidrug resistant Escherichia coli from food animals and humans from Nigeria for the presence of beta-lactamase encoding genes and plasmids which may harbor those genes. A subset of isolates was also tested to determine if the plasmid-associated genes were able to transfer among E. coli. Results showed that beta-lactamase genes were prevalent in multidrug resistant E. coli from both humans and animals and contained multiple, diverse plasmids. As the plasmid-associated genes were mobile, they are likely to continue disseminating among E. coli and facilitating transfer of associated beta-lactamase genes in this region. This information will assist in understanding and monitoring the spread of multidrug resistant E. coli producing beta-lactamases and plasmid prevalence among food animal and community isolates in Nigeria important to both human and animal health. This information is useful for policy makers and scientists as they develop prevention and control strategies for combating antimicrobial resistance in resource-limited countries.

14. Genome analysis of multidrug resistant Escherichia coli isolated from poultry in Nigeria. Escherichia coli is a common commensal of the intestinal tract of humans and animals but can also be an opportunist pathogen associated with illnesses after consumption of food-animal products. In order to effectively treat these infections, it is important to understand the mechanisms of resistance in E. coli. ARS researchers in Athens, Georgia, sequenced and analyzed three multidrug-resistant E. coli isolated from chicken fecal samples from Lagos, Nigeria for the presence of antimicrobial resistance genes, plasmid replicons, virulence genes, and phage. The isolates harbored genes conferring resistance to aminoglycosides, extended-spectrum beta-lactamases, tetracycline, trimethoprim, sulfonamides, fluoroquinolones, and chloramphenicol. Some genes were located on plasmids capable of transferring among bacteria. Data from this study highlights the role of chickens as a potential reservoir of mobile antimicrobial resistance genes as well as virulence genes and phage. Increased efforts of antibiotic surveillance to control and regulate the use of antimicrobial agents is required to reduce risk factors associated with the acquisition of these multidrug resistant isolates. This information is useful for policy makers and scientists as they develop prevention and control strategies to inhibit spread of resistant bacteria to humans and the environment, especially in underfunded countries.

Review Publications
Cho, S., Nguyen, A., Mcdonald, J., Woodley, T.A., Hiott, L.M., Barrett, J.B., Jackson, C.R., Frye, J.G. 2019. Genetic characterization of antimicrobial resistant Escherichia coli isolated from a mixed-use watershed in northeast Georgia, USA. International Journal of Environmental Research and Public Health. https://doi.org/10.3390/ijerph16193761.
Cho, S., Gupta, S., Mcmillan, E., Sharma, P., Ramadan, H., Jove, T., Jackson, C.R., Frye, J.G. 2019. Whole genome sequencing of multidrug resistant E. coli from surface water in northeast Georgia, USA, including a ST131 epidemic strain with a blaCTX-M-15 extended spectrum beta-lactamase gene encoded on a phage-like plasmid. Microbial Drug Resistance. https://doi.org/10.1089/mdr.2019.0306.
Gupta, S., Sharma, P., Mcmillan, E., Hiott, L.M., Woodley, T.A., Humayoun, S.B., Barrett, J.B., Mcclelland, M., Jackson, C.R., Frye, J.G. 2019. Genomic comparison of diverse Salmonella serovars isolated from swine. PLoS One. https://doi.org/10.1371/journal.pone.0224518.
Berrang, M.E., Meinersmann, R.J., Cox Jr, N.A., Adams, E.S. 2020. Water rinse and flowing steam to kill Campylobacter on broiler transport coop flooring. Food Control. https://doi.org/10.1016/j.foodcont.2020.107214.
Keshri, J., Ramirez, R., Svendsen, M., Keillor, H., Moss, M., Jordan, H., Smith, K., Ball, P., Seal, B., Berrang, M.E., Oakley, B. 2020. Draft genome sequences of two novel Paenibacillus isolates from goose and chicken gastrointestinal material. Microbiology Resource Announcements. http://doi.org/10.1128/MRA.00013-20.
Berrang, M.E., Cox Jr, N.A., Meinersmann, R.J., Bowker, B.C., Zhuang, H., Huff, H. 2020. Mild heat and freezing to lessen bacterial numbers on chicken liver. Journal of Applied Poultry Research 29(1):251-257. https://doi.org/10.1016/j.japr.2019.10.012.
Cho, S., Hiott, L.M., Mcdonald, J., Barrett, J.B., Mcmillan, E., House, S.L., Adams, E.S., Frye, J.G., Jackson, C.R. 2019. Diversity and antimicrobial resistance of Enterococcus from the Upper Oconee Watershed, Georgia. Journal of Applied Microbiology. 128(4):1221-1233. https://doi.org/10.1111/jam.14550.
Cho, S., Jackson, C.R., Frye, J.G. 2020. The prevalence and antimicrobial resistance of Salmonella, Escherichia coli, and Enterococcus sp. in surface water. Letters in Applied Microbiology. https://doi.org/10.1111/lam.13301.
Keshri, J., Ramirez, R., Berrang, M.E., Oakley, B. 2020. Draft genome sequences of two novel Bacillus isolates from backyard and commercial chicken GIT. Microbiology Resource Announcements. https://doi.org/10.1128/MRA.00492-20.
Meinersmann, R.J., Berrang, M.E., Rigsby, L.L. 2020. Recoverability of Listeria monocytogenes after coculture with Tetrahymena pyriformis. Journal of Food Safety. https://doi.org/10.1111/jfs.12778.
Ramadan, H., Jackson, C.R., Frye, J.G., Hiott, L.M., Samir, M., Awad, A., Woodley, T.A. 2020. Antimicrobial resistance, genetic diversity and multilocus sequence typing of Escherichia coli from humans, retail chicken and ground beef in Egypt. Pathogens. https://doi.org/10.3390/pathogens9050357.
Sharma, P., Gupta, S., Adenipekun, E., Barrett, J.B., Hiott, L.M., Woodley, T.A., Iwalokun, B., Oluwadun, A., Ramadan, H., Frye, J.G., Jackson, C.R. 2019. Genome analysis of multidrug resistant Escherichia coli isolated from poultry in Nigeria. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2019.2659.
Sharma, P., Gupta, S., Barrett, J.B., Woodley, T.A., Hiott, L.M., Kariyawasam, S., Jackson, C.R., Frye, J.G. 2020. Comparison of antimicrobial resistance and pan-genome of clinical and non-clinical Enterococcus cecorum from poultry using whole-genome sequencing. Foods. https://doi.org/10.3390/foods9060686.
Yang, Y., Sommers, C.H., Adenipkun, E., Ceruso, M., Jackson, C.R., Woodley, T.A., Barrett, J.B., Hiott, L.M., Liu, Y., Frye, J.G. 2020. Draft genomic sequence of three Escherichia coli Sequence Type 131 isolates (H45, H43ii, and H43iii) from patients in Lagos, Nigeria. Microbiology Resource Announcements. 9:17. https://doi.org/10.1128//mra.asm
Adenipekun, E., Jackson, C.R., Ramadan, H., Iwalokun, B., Frye, J.G., Barrett, J.B., Hiott, L.M., Woodley, T.A., House, S.L., Mcmillan, E., Sharma, P., Oluwadun, A. 2019. Plasmid replicons and B-lactamase-encoding genes of multidrug- resistant Escherichia coli isolated from humans and food animals in Lagos, Southwest Nigeria. Microbial Drug Resistance. https://doi.org/10.1089/mdr.2018.0305.
Berrang, M.E., Meinersmann, R.J., Knapp, S.W. 2020. Presence of bacterial pathogens and levels of indicator bacteria associated with duck carcasses in a commercial processing facility. Journal of Food Protection. https://doi.org/10.4315/0362-028X.JFP-19-397.
Ramadan, H., Gupta, S., Sharma, P., Ahmed, M., Hiott, L.M., Barrett, J.B., Woodley, T.A., Frye, J.G., Jackson, C.R. 2019. Circulation of emerging NDM-5-producing Escherichia coli among humans and dogs in Egypt. Zoonoses and Public Health. https://doi.org/10.1111/zph.12676.