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

Location:

2019 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
Recently, Salmonella enterica serotype Infantis has emerged as a dominant strain in poultry in the United States. This strain of Infantis has caused numerous human infections and carries an extended spectrum beta-lactamase gene, a concern to the poultry industry and consumers as some beta-lactam antibiotics would no longer be useful for treating diseases. Using whole-genome sequencing data, prevalence of the plasmid (pES1) harboring the gene in U.S. Hazard Analysis and Critical Control Point isolates was determined. The pan and core genome of this plasmid in U.S. meat samples was also evaluated to compare with human and international isolates. Sequencing of historic Infantis isolates from 1997-2013 collected by the National Antimicrobial Resistance Monitoring System was also initiated. This project involves collaboration between multiple federal agencies including the Food Safety and Inspection Service (FSIS), the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA) and the Animal and Plant Health Inspection Service (APHIS). This research will provide poultry stakeholders with prevalence and history of the pES1 plasmid in the U.S. and understanding of why S. Infantis remains persistent in flocks. A survey of Escherichia coli whole genome sequences from food animal carcasses at FSIS inspected plants was performed to determine the characteristics of plasmids that were carried. As expected, IncF and IncCol plasmids were the most common. Unexpectedly, IncB/O/K/Z plasmids were prevalent, especially in shiga-toxin positive strains of E. coli. The IncB/O/K/Z plasmids were found to occur in three evolutionarily cohesive groups that did not correlate with the replication initiation gene, which is the basis for Inc-group typing. The groups were labeled Coh-1, Coh-2 and Coh-3. There was only one gene out of 332 genes that was common to all three groups. Antimicrobial resistance genes were uncommon, but extremely rare in Coh-3. Coh-1 and Coh-2 had genes for transfer of the plasmids between bacterial strains, but these were mostly missing in Coh-3. Coh-3 plasmids carried many more toxin genes (average of about 7 versus 0.57) and many more transposases (average of 27 versus 3.2) than Coh-1 and Coh-2 plasmids. The nomenclature of IncB/O/K/Z plasmids needs to be clarified, but results show that the plasmid group is more important for virulence than for antimicrobial resistance. Having discovered evolutionarily cohesive groups among IncB/O/K/Z plasmids, it was hypothesized that classification of all plasmids could be clarified by analyzing cohesive groups. To date, it has been found that some groups, such as the IncA/C type, are highly cohesive, but the majority of plasmids from Enterobacteriaceae have not yet been found to be in cohesive groups. Approximately 42% of 15,702 genes could be found on both chromosomes and plasmids, 58% only on plasmids, but the genes that were found on both chromosomes and plasmids were found almost 1.65 times more often. Thus, the amount of genetic exchange among plasmids and chromosomes overrides cohesive grouping, but additional investigation is being performed to determine if the plasmids have backbone structures that correlate with cohesive groups. One hundred isolates each of drug-resistant Salmonella, E. coli, and Enterococcus were sequenced and the genome sequences analyzed 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. The fifth 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. Salmonella, E. coli, and Enterococcus isolated from the samples were collected and stored for future research to characterize resistance phenotype, genotypes, and virulence. Progress on two international collaborations was made. E. coli from humans, retail food, and poultry from Egypt were characterized for antimicrobial resistance phenotype, genotype and genetic relatedness using pulsed-field gel electrophoresis and multi-locus sequence typing. Results from the three sources were compared and showed some shared characters which may indicate circulation of E. coli clones between the populations. Research on prevalence and antimicrobial resistance of staphylococci from household and farmed poultry in Pakistan was initiated. Staphylococci isolated from the samples were identified to species and tested for resistance to antibiotics of human importance; resistant isolates were tested for the presence of resistance genes. Thirteen staphylococcal species from both household and farmed poultry were identified indicating a diverse staphylococcal population. Resistance ranging up to ten antimicrobials and eight antimicrobial classes was detected indicating a high level of multidrug resistance. Resistance to the macrolides, beta-lactams, tetracycline, and the human use drug, tigecycline, ranged from approximately 30-70% in the isolates. Statistical analysis linking resistance phenotype to genotype was also performed. A study on antimicrobial resistant populations of “no antibiotic ever” poultry was initiated 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 are being isolated from the samples and tested for resistance to antibiotics. Of note, Salmonella serotype Reading has been isolated from ground turkey samples and will be further tested using whole-genome sequencing for possible linkage to the on-going Salmonella Reading foodborne outbreak in raw turkey products. Both methicillin-resistant Staphylococcus aureus and erythromycin-resistant Enterococcus have been isolated from the products suggesting that “no antibiotic ever” poultry retail meat may still contain antimicrobial resistant bacteria. Genomic comparisons of diverse Salmonella serovars isolated from swine was completed to understand the genetic variations among different Salmonella serovars. High concordance (97%) was detected between phenotypically confirmed antibiotic resistances; antibiotic resistance genes were identified using whole-genome sequencing. 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 were detected on the mobile elements. Predicted integrated phage were highly diverse and many harbored virulence, metal resistance, or antibiotic resistance genes. Overall genomic analysis revealed a great deal of diversity among Salmonella serovars due to acquired genes that enable them to thrive and survive during infection. This data will provide evidence of changes in resistance and virulence in Salmonella over time. Eighty E. coli from food animals were tested on the biocide susceptibility panel initially developed for Salmonella. One percent of isolates was resistant to acidified sodium chlorite, sodium metasilicate, and arsenite, 2% to lactic acid, and 19% to sodium hypochlorite. Production of a susceptibility plate for testing bacterial resistance to heavy metals (arsenite, arsenate, zinc, chromate, nickel, cobalt, tellurite) continued. Twenty-two E. coli that were tested on the biocide susceptibility plate were also tested on the metal plate. Re-testing of the isolates is on-going to confirm the metal resistance phenotypes. It was determined that the rate of destruction of Listeria by the protozoa, Tetrahymena pyriformis, was approximately three-quarters of a million Listeria cells per cell of Tetrahymena in 24 hours. The destruction of Listeria was the same for all nineteen diverse strains of Listeria that were used. Current experiments are being conducted to determine if destruction of Listeria improves after several generations of Tetrahymena growth when fed Listeria. If Tetrahymena can be selected for improved destruction of Listeria, it may be effective in reducing the organism in food processing plant floor drains.

Accomplishments
1. Neutralization of residual antimicrobial processing chemicals in broiler carcass rinse. The USDA-Food Safety and Inspection Service (FSIS) has established pathogen reduction performance standards for Campylobacter on broiler carcasses. Processors may apply antimicrobial processing aids as a spray or immersion to lower contamination on carcasses. In the United States, broiler carcasses are generally sampled by whole carcass rinse and the potential exists for residual levels of antimicrobial processing aids to be carried over into the rinsate. It has been shown that, if unmitigated, such carryover can interfere with the detection of Salmonella. ARS researchers in Athens, Georgia, demonstrated that unmitigated carryover of antimicrobial treatment can also interfere with the detection and recovery of Campylobacter in broiler carcass rinse samples. Traditional buffered peptone water was tested and found that it did not offer enough neutralizing capability to counteract residual antimicrobial activity of some post-chill processing aids (peroxyacetic acid, cetylpyridinium chloride, acidified sodium chloride, or a blend of acids) to allow full recovery of Campylobacter. A recently reported formulation for a neutralizing buffered peptone water (developed at the U.S. National Poultry Research Center and currently being used by FSIS) outperformed the traditional carcass rinse medium and allowed significantly improved recovery of Campylobacter even in the presence of 3 of the 4 tested antimicrobial processing aids. Performance of the new carcass rinse medium with the fourth antimicrobial processing aid (acidified sodium chloride) was not different from the traditional formulation. Neutralizing buffered peptone water represents a significant improvement in the broiler carcass rinse method for detection of Campylobacter.

2. A susceptibility assay for salmonella to commercial and household biocides. Antimicrobial interventions, known as biocides, can be used to reduce bacterial contamination during processing of retail meat; however, little is known about how bacteria may develop resistance to these compounds and there is no systematic way to detect changes in susceptibility to these compounds that may indicate the development of resistance. ARS researchers in Athens, Georgia designed a high throughput panel of 17 common household and commercially-used biocides to determine the minimum inhibitory concentration of Salmonella for these compounds and to determine if resistance to biocides correlates with resistance to antibiotics. Multidrug resistant Salmonella isolates were tested on the panel and the minimum inhibitory concentration of Salmonella to each of the chemicals was calculated. Isolates were resistant to cetylpyridinium chloride, hexadecyltrimethylammonium bromide, citric acid, acidified sodium chlorite, chlorhexidine, arsenite, and arsenate. No correlation was detected between susceptibility of Salmonella to the biocides and their antibiotic resistance. As the overall aim in the food industry is to reduce antimicrobial use, this assay and the minimum inhibitory concentration data will be important to help monitor the effect of biocides on both biocide and antimicrobial resistance.

3. Mobile genetic element of antimicrobial resistant Salmonella from food animals. Treatment of human Salmonella infections is becoming more difficult due to increasing antimicrobial resistance in the bacteria. Mobile genetic DNA, such as plasmids, carry the resistance genes which can be transferred among Salmonella. To fill the limited data gap on plasmids, ARS researchers in Athens, Georgia isolated approximately 200 antibiotic resistant Salmonella from food-animals, which were analyzed for plasmids and antibiotic resistance genes by whole genome sequencing. Results showed that a variety of different types of plasmids were present in these Salmonella and almost all of the antibiotic resistance genes were found on plasmids. Common arrangements of these antibiotic resistance genes were found on many different types of plasmids. These gene arrangements were called cassettes because identical genes were found in many different plasmids as if a cassette had been inserted into them. Six cassettes were identified in this study and when compared to the database of all Salmonella whole genome sequences, many of them were found to be widespread in isolates from other studies. This improves understanding of antibiotic resistance in Salmonella associated with food animals and can provide answers on how the bacteria become resistant posing a threat to human health.

4. Control of Campylobacter contamination during broiler defeathering using shredded sponge or paper. Numbers of Campylobacter on broiler carcass skin surface increase due to leakage of gut contents during automated defeathering. To control contamination of poultry carcasses during processing, multiple novel methods for plugging the cloaca of broiler carcasses prior to scalding were designed and tested. A tampon sewed into the cloaca of a carcass between killing and picking is an effective means of plugging cloacae for experimental purposes, but this is not logistically practical in a commercial setting. ARS researchers in Athens, Georgia, developed a shredded absorbent sponge material as a means to plug the cloaca. Results showed that the high surface area and high friction shredded sponge material can be placed in the cloaca and much of it stayed in place while plugged carcasses were in the feather-picking machinery. A plug made of 50 cc shredded sponge material was effective to occlude broiler carcass vents during automated defeathering. Significantly fewer Campylobacter cells were detected on the breast skin of post-pick carcasses plugged with 50 mL of shredded sponge than on post-pick, unplugged control carcasses. This system may be a practical means to prevent Campylobacter escape from the cloaca during defeathering.

5. Improved detection and culture of Campylobacter. ARS researchers in Athens, Georgia undertook studies to improve detection and culture of Campylobacter. Results from the study showed that a 0.45 or 0.65 micron pore size nitrocellulose filter, placed on top of solid plating medium, is useful as a means to eliminate non-Campylobacter contamination from samples with high numbers of both Campylobacter and background bacteria such as broiler feces or gut contents. The rate of six subtypes of Campylobacter passage through filters and onto the growth medium below was determined and the proportion of an inoculum that makes passage through the filter was measured. The mean difference between direct plating and filter plating was 2.4 log colony forming units. These data demonstrate the use of the filter method improves the detection of Campylobacter and can quantitatively estimate the cellular density in a complex, highly contaminated sample.

6. Detection of Campylobacter on the surface and from internal tissue of retail chicken livers. Foodborne campylobacteriosis has been traced to undercooked chicken liver. ARS researchers in Athens, Georgia, determined the prevalence of Campylobacter associated with chicken livers at retail and which subtypes were detected on the surface versus internal tissues of livers. Fresh livers were purchased at retail and the surface, internal tissue, and whole blended livers were cultured for Campylobacter. Campylobacter was found from the surface of 64% of livers; significantly less on internal tissue (31%), and on 67% of blended whole liver. Multiple subtypes were detected from some livers. In several livers, a different subtype was dominant on the surface than was dominant internally. In one liver, three subtypes were detected. Various subtypes of Campylobacter can be readily isolated from fresh retail chicken livers confirming the food safety risk to consumers associated with undercooked chicken livers.

7. Circulating ciprofloxacin-resistant Salmonella Kentucky ST198 from retail chicken in Egypt. Ciprofloxacin-resistant Salmonella serotype Kentucky was identified from human patients in Egypt at the beginning of the last decade and rapidly disseminated to other African and Middle East countries. Available information about the ciprofloxacin-resistant S. Kentucky clone from poultry and poultry products in Egypt is still limited. ARS researchers in Athens, Georgia, in collaboration with a visiting scientist, identified S. Kentucky isolates that were multidrug resistant to ciprofloxacin, ampicillin, chloramphenicol, levofloxacin, nalidixic acid, sulfisoxazole, and tetracycline; one isolate also exhibited reduced susceptibility to trimethoprim/sulfamethoxazole. Whole-genome sequence analysis identified resistance genes to those drugs. Detection of this clone on retail poultry in Egypt indicates that the clone continues to persist as a threat to public health. This information is useful for epidemiologists and regulatory agencies and warrants the implementation of national and international control strategies to limit the global spread of this strain.

8. Genomic analysis of human-associated streptogramin-resistant Staphylococcus aureus. Methicillin-resistant Staphylococcus aureus (MRSA) infections are difficult to treat because they are not only resistant to clinically relevant beta-lactam antibiotics, but may also exhibit multidrug resistance. As MRSA clones emerged with decreased susceptibility to glycopeptides, the streptogramin compound, Quinupristin-Dalfopristin, came into use. However, the streptogramin, virginiamycin, was used in food animal production for decades, and common resistance mechanisms for the two drugs from both human and animal sources is cause for concern. In this study, ARS researchers in Athens, Georgia, analyzed the genome sequence of a human-associated streptogramin-resistant MRSA was to provide valuable insight into its resistome and virulence factors. The isolate was resistant to ampicillin, ceftriaxone, gentamicin, kanamycin, oxacillin, penicillin, Quinupristin-Dalfopristin, and tetracycline and contained resistance genes to the aminoglycosides, beta-lactams, fosfomycin, macrolides, streptogramins, and tetracycline. The isolate also harbored numerous plasmids and virulence genes. This data will provide a basis for comparison to Gram-positive bacteria of food animal origin which will be useful for researchers and policy makers as antimicrobial use in food animal production is evaluated.

9. Antimicrobial resistant staphylococci from household chicken eggs. Eggs are a healthy and nutritious food source, but may be contaminated by bacteria. Previous studies have reported the presence of staphylococci in eggs of farmed chickens, but no study has evaluated the staphylococcal population of eggs from household chickens. In this study, ARS researchers in Athens, Georgia, isolated and characterized staphylococci from eggs of household chickens in Pakistan. Staphylococcus xylosus was the most frequently detected species; isolates were resistant to antimicrobials used in human medicine and harbored antimicrobial resistance genes. The isolates also produced an inhibitory substance capable of preventing the growth of a broad spectrum of bacteria including methicillin-susceptible and methicillin-resistant S. aureus, Salmonella enterica serotype Typhi, and Escherichia coli. Like farmed table eggs, eggs of household chickens also contain staphylococci that may be resistant to antimicrobials used to treat human infections, a food safety concern. Since S. xylosus is a documented opportunistic human pathogen and is also used in food production as starter cultures for meat fermentation, this information is useful for consumers and personnel who handle eggs as safe handling and cooking methods should be followed to avoid colonization and infection with these resistant bacteria. This information is also useful for scientists as they develop prevention and control strategies.

10. Beta-lactam resistant Escherichia coli in humans and chickens from Egypt. Beta-lactam resistant Escherichia coli in both humans and food animals is cause for concern on a worldwide level as resistant bacteria may be passed to humans from food animals limiting treatment options for human infections. In this study, ARS researchers in Athens, Georgia collected clinical samples from patients with extraintestinal infections and healthy broiler chickens from Egypt during the 2nd half of 2015 and examined for the presence of E. coli. The isolates were tested for susceptibility against beta-lactam antimicrobials and screened for the presence of extended-spectrum beta-lactamase and virulence genes; clonal and phylogenetic types were also determined. While both human and chicken samples were positive for E. coli, more isolates from humans were resistant to the beta-lactam antimicrobials than those from chickens and extended-spectrum beta-lactamase genes were prevalent among all E. coli isolates. The majority of resistant E. coli isolates from human and chicken belonged to commensal rather than virulent groups. Using pulsed-field gel electrophoresis, some E. coli clustered based upon source, but the majority of clusters contained E. coli from both humans and chickens. Results of this study suggest that although no single clone appeared to be circulating among the beta-lactam resistant E. coli, some shared characteristics exist among isolates from the two sources. Increased study will aide researchers and regulatory personnel to determine the role of humans and food animals in dissemination of beta-lactam resistant E. coli for effective use of antimicrobials intervention strategies.

11. Antimicrobial metaphylaxis influences multidrug resistant Mannheimia haemolytica in stocker cattle. Stocker calves are especially prone to bovine respiratory disease and are often treated with multiple antibiotics, a practice called metaphylaxis, which can result in multidrug resistant bacteria. ARS researchers in Athens, Georgia, studied the prevalence of multidrug resistant bacteria in stocker cattle over a 21 day period from arrival on the farm. The cattle received tildipirosin metaphylaxis on day 0 and were also treated with up to three additional antimicrobials if they developed respiratory disease. Nasopharyngeal swabs were collected and cultured for bacteria that cause bovine respiratory disease. Mannheimia haemolytica was isolated from cattle and a majority of these isolates were multidrug resistant on multiple days during the sampling period. M. haemolytica isolates were genotyped and found to be highly diverse and up to 14 antibiotic resistance genes were detected in a subset of isolates using genome sequencing. This study indicated that multidrug resistant M. haemolytica is highly prevalent and genetically diverse in stocker cattle. This information is vital to understanding how metaphylaxis affects resistance in bacteria causing bovine respiratory disease and developing methods to prevent it.

Review Publications
Gupta, S., Sharma, P., Barrett, J.B., Hiott, L.M., Woodley, T.A., Frye, J.G., Jackson, C.R. 2019. Draft genome sequence of human-associated streptogramin resistant Staphylococcus aureus. Journal of Global Antimicrobial Resistance. https://doi.org/10.1016/j.jgar.2018.11.021.
Berrang, M.E., Meinersmann, R.J., Cox Jr, N.A., Thompson, T.M. 2018. Multi-locus sequence subtypes of Campylobacter detected on the surface and from internal tissue of retail chicken livers. Journal of Food Protection. 81(9):1535-1539. https://doi.org/10.4315/0362-028X.JFP-18-131.
Berrang, M.E., Meinersmann, R.J., Adams, E.S. 2018. Shredded sponge or paper as a cloacal plug to limit broiler carcass Campylobacter contamination during automated defeathering. Journal of Applied Poultry Research. https://doi.org/10.3382/japr/pfy051.
McMillan, E., Gupta, S., Williams, L., Jove, T., Hiott, L.M., Woodley, T.A., Barrett, J.B., Jackson, C.R., Wasilenko, J., Simmons, M., Tillman, G., McClelland, M., Frye, J.G. 2019. Antimicrobial resistance genes, cassettes, and plasmids present in Salmonella enterica associated with United States food animals. Frontiers in Microbiology. 10:832. https://doi.org/10.3389/fmicb.2019.00832.
Humayoun, S.B., Hiott, L.M., Gupta, S., Barrett, J.B., Woodley, T.A., Johnston, J., Jackson, C.R., Frye, J.G. 2018. A susceptibility assay for salmonella to commercial and household biocides. PLoS One. https://doi.org/10.1371/journal.pone.0209072.
Woolums, A., Karisch, B., Frye, J.G., Epperson, W., Smith, D., Blanton, J., Austin, F., Kaplan, R., Hiott, L.M., Woodley, T.A., Gupta, S., Jackson, C.R., McClelland, M. 2018. Multidrug resistant Mannheimia haemolytica isolated from high-risk beef stocker cattle after antimicrobial metaphylaxis and treatment for bovine respiratory disease. Veterinary Microbiology. 221:143-152. https://doi.org/10.1016/j.vetmic.2018.06.005.
Syed, M., Jackson, C.R., Ramadan, H., Afridi, R., Bano, S., Bibi, S., Fatima, B., Tabassum, S., Jamil, B., Khan, M., Barrett, J.B., Woodley, T.A. 2019. Detection and molecular characterization of staphylococci from eggs of domesticated chickens. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2018.2585.
Ramadan, H., Gupta, S., Sharma, P., Sallam, K., Hiott, L.M., Elsayed, H., Barrett, J.B., Frye, J.G., Jackson, C.R. 2018. Draft genome sequences of two ciprofloxacin-resistant Salmonella enterica subsp. enterica serovar Kentucky ST198 isolates from retail chicken carcasses in Egypt. Journal of Global Antimicrobial Resistance. 14:101-103. https://doi.org/10.1016/j.jgar.2018.06.012.
Ramadan, H., Jackson, C.R., Taha, S., Moawad, A., Barrett, J.B., Woodley, T.A. 2018. Contribution of healthy chickens to antimicrobial-resistant Escherichia coli associated with human extraintestinal infections in Egypt. Vector-Borne and Zoonotic Diseases. 18(8):408-416. https://doi.org/10.1089/vbz.2017.2237.
Berrang, M.E., Meinersmann, R.J., Cox Jr, N.A. 2019. Campylobacter subtypes detected in broiler ceca and livers collected at slaughter. Poultry Science. https://doi.org/10.3382/ps/pez340.