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


2018 Annual Report

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.

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
Using whole-genome sequencing, the common genomic regions of 194 multidrug resistant Salmonella from food animals and food production facilities were analyzed. A total of 922 antimicrobial resistance genes were identified from the isolates and were located on different replicon types. Specific regional clusters of the genomes harbored multiple resistance genes and indicated that antimicrobial resistance of multiple classes of antibiotics were linked and could be selective for each other. Knowledge of which resistance genes are linked in these regions of the bacterial genomes could predict useful interventions to slow the spread of resistance. Further evaluation of resistance, resistance genes, and mobile genetic elements in foodborne pathogens and commensals continued. One hundred isolates each of drug-resistant Salmonella, Escherichia coli, and Enterococcus were characterized phenotypically and genotypically and selected for whole-genome sequencing. The genomes of streptogramin resistant Enterococcus spp. from dairy cattle and poultry were sequenced and analyzed. Multiple resistance genes were located on mobile genetic elements that were able to transfer to other bacteria. As streptogramin antibiotics are used in clinical medicine, this study provided valuable data on dissemination of resistance genes among enterococci from food animals. In an international collaborative study, ciprofloxacin-resistant Salmonella serotype Kentucky was identified from poultry from Egypt. Resistance to other antibiotics in the isolates was identified using susceptibility testing and whole-genome sequence analysis. Detection of this clone in retail poultry in Egypt indicated that the clone continues to persist as a global threat to public health by potential dissemination to the human population through poultry products. The fourth year of seasonal monitoring of surface water as a reservoir and potential vehicle for the 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, near Athens, Georgia. Salmonella, E. coli, and Enterococcus isolated from the samples were subtyped to species and serotype, characterized for resistance phenotype, DNA fingerprinted, and tested for the presence of virulence genes. Using multi-locus sequence typing, a sequence type associated with multidrug resistance and virulence in E. coli was found. This global clone is well-known for its ability to exchange genetic material which further complicates treatment in humans. Enterococci resistant to newer drugs, daptomycin and tigecycline, both used to treat human illness were also found in the surface water. Because these drugs have only been used in clinical medicine and no analogs exist for use in food animal production, the study indicated a human origin for the resistant isolates. Progress on an international collaboration was made. E. coli from humans and companion animals from Egypt were characterized for antimicrobial resistance and presence of plasmids. Results indicated that the E. coli from humans and companion animals might be shared between the two sources based upon co-existence of resistance phenotypes, plasmid replicons, and genetic patterns. Resistant isolates in companion animals may impact human health by serving as a reservoir of resistant bacteria. Research on microbial contamination of eggs from Pakistan continued. Multidrug resistant Staphylococcus xylosus was the predominant staphylococcal species from eggs of domesticated chickens. The bacteria were multidrug resistant, harbored resistance genes, and produced bacteriocins that killed Salmonella, E. coli, and drug-resistant S. aureus. The bacteriocins are being further investigated for potential use in food production. The genomes of S. Abortusequi, a host-adapted pathogen responsible for abortions in mares, were compared. Core genome phylogeny analysis revealed that these isolates clustered together with other host specific isolates in which genome reduction was reported. Gene gain or loss analysis revealed that these microbes evolved for better adaptation than invasion. The genomes from 14 Salmonella serotypes associated with swine were compared to understand the common genetic features and variations among those serotypes. Pan-genome analysis revealed that more than 75% of genes were conserved among the isolates and 85% of the conserved genes were commonly distributed to different functional orthologous groups. Genome sequences were further examined for resistance genes and most of the resistance genes were associated with class 1 integrons and transposable elements that confirmed their mobile nature. The genes were detected on both plasmids and genomic islands. High concordance was noticed when antibiotic resistance phenotypes were compared with detected resistance genes. Virulence associated genes were commonly detected in all 14 serotypes with few exceptions. An array of highly diverse phages among the serotypes indicated the high adaptability of the serovars. CRISPR loci detected in most of the isolates contained several spacers that matched with phage target genes and shared common ancestral end genes that indicated the common infection history and origin. This data provided evidence of changes in resistance and virulence in Salmonella over time. Progress was made on comparative antimicrobial resistance and genomics of non-pathogenic and pathogenic Enterococcus cecorum associated with outbreaks of enterococcal spondylitis in poultry. Non-pathogenic isolates contained more resistance genes than pathogenic isolates. No mutations or known resistance genes were found for isolates resistant to either linezolid or chloramphenicol suggesting possible new mechanisms of resistance to these drugs. Genome reduction was present in genomes from pathogenic isolates suggesting better host adaptability. Study of the unique core genes showed that the pathogenic genomes were more conserved and non-pathogenic genomes were comparatively diverse. Development of a high-throughput assay to determine susceptibility of Salmonella to 17 biocides was completed. Selection of biocides and appropriate concentrations for use against E. coli was done and testing of the isolates initiated. Production of a susceptibility plate for testing bacterial resistance to heavy metals (zinc, silver, chromate, nickel, lead, cobalt, mercury, copper, cadmium, and iron) was started. Genomic analysis of the 194 Salmonella genomes showed genetic linkage between antimicrobial resistance genes and metal and biocide resistance genes. Major metal and biocide resistance genes were detected in all 194 Salmonella isolates. Most of those genes were categorized as transporters, efflux pumps, or two component system genes and were located on the chromosome. Findings from the study suggested that the selection or maintenance of resistance in Salmonella isolated from food animal sources could be due to exposure to metals in addition to antibiotics.

1. Antimicrobial effect of plant-derived compounds on Salmonella. Salmonella is often found in poultry and can be the cause of foodborne human infections. Many strategies have been developed to reduce the level of Salmonella in poultry to prevent those infections. One strategy is to inhibit the growth of Salmonella by adding natural plant extracts to chicken feed or water. Previous work found that two plant extracts, trans-cinnamaldehyde (TC) and eugenol (EG), reduced colonization of chickens with Salmonella. To understand how TC and EG reduced colonization of chickens, ARS researchers at Athens, Georgia, grew Salmonella in the laboratory with a sub-inhibitory concentration of TC or EG added to the culture. TC and EG reduced the expression of genes required for motility, pathogenicity, invasion of intestinal cells, transport systems, and outer membrane proteins in Salmonella. The plant-derived compounds, TC and EG, exerted antimicrobial effects on Salmonella by multiple mechanisms required for growth in the host environment. Alternatives to antimicrobials combat both antimicrobial resistance and Salmonella which is important to food safety and human health.

2. Pathogenic and antimicrobial resistant Escherichia coli in surface water. Surface waters are important sources of water for drinking, industrial, agricultural, and recreational uses; hence, contamination of water by fecal bacteria, such as Escherichia coli, is a major environmental and public health concern. Very little data is available on pathogenic or antimicrobial resistant E. coli in surface water. To address this, ARS researchers at Athens, Georgia, collected water samples quarterly from 2015 to 2017 from the Upper Oconee River Watershed, Athens, Georgia. E. coli counts to assess the water quality were occasionally above the United States Environmental Protection Agency (U.S. EPA) threshold for recreational water. Pathogenic E. coli were detected in the water including those which cause diarrhea in humans. Antimicrobial resistant and multidrug resistance in E. coli to antibiotics used to treat human infections was also detected. Results from this study demonstrated that E. coli is prevalent in high levels in the Upper Oconee Watershed, indicating possible widespread fecal contamination within the water. The study emphasizes the role of environmental water as a reservoir of resistant and pathogenic E. coli that may be transferred to humans through drinking and recreational activities.

3. Multidrug resistant Salmonella in sub-Saharan Africa. Salmonella is an important foodborne infection that can cause gastroenteritis or invasive systemic infections. In Africa, it is a major cause of child mortality. Multidrug resistant (MDR) strains of Salmonella are more difficult to treat during an invasive infection. In this study, ARS researchers at Athens, Georgia, isolated the genomes of MDR S. Typhimurium strains from feces of humans and poultry in Burkina Faso were sequenced to characterize resistance in the isolates. The genetic similarity of these isolates suggested transmission from poultry to humans of a specific Salmonella Typhimurium strain. This study provided new data to better understand the geographical spread of S. Typhimurium genotypes and their potential host reservoirs in sub-Saharan Africa. The epidemic caused by S. Typhimurium in sub-Saharan Africa shows that next-generation sequencing facilities should be available also to the scientists in the resource-limited African countries to detect epidemics in their early phase. The present study highlights the need for surveillance systems of foodborne pathogens to prevent enteric diseases in sub-Saharan Africa that may also impact food safety and antimicrobial resistance in countries worldwide.

4. Human-associated antimicrobial resistant Staphylococcus aureus in table eggs. Staphylococcus aureus is a bacterium that can be commonly found on the skin or in the nasal passages of most humans and animals. It can cause a number of diseases in humans including staphylococcal food poisoning characterized by vomiting and diarrhea. Eggs are usually considered safe and are naturally protected by the egg shell and a semi-permeable membrane; however, bacteria such as S. aureus may enter and contaminate the eggs by crossing both the egg shell and the membrane. In addition, there is increasing interest in the presence of antimicrobial resistance in S. aureus, specifically methicillin-resistant S. aureus (MRSA). In this study, ARS researchers at Athens, Georgia, isolated staphylococci from table eggs in Pakistan and MRSA from the products were characterized. MRSA were recovered from a portion of the eggs and all were resistant to multiple antimicrobials. Using molecular analysis, the MRSA were characteristic of MRSA known to cause human infections globally. Results from this study showed that MRSA are present in table eggs which may be transmitted to humans. The genetic similarities of MRSA present in the eggs to that of humans may suggest human to poultry transmission of MRSA via contamination. This information is of importance to consumers and personnel who handle eggs as safe handling and cooking methods are critical to avoid colonization and infection with MRSA.

5. Streptogramin-resistant enterococci from food animals and the environment. The streptogramin antimicrobials, Quinupristin/Dalfopristin and virginiamycin, have been used in both human and veterinary medicine. Quinupristin/Dalfopristin is used in human medicine to treat vancomycin-resistant Enterococcus faecium bacteremia while virginiamycin was used in food animal production for decades. Streptogramin resistance in enterococci from both humans and animals has been documented and many mechanisms of resistance have been described. To identify genes for streptogramin resistance, ARS researchers in Athens, Georgia, sequenced the genomes of resistant enterococci from food animals and the environment. Antimicrobial resistance genes were identified conferring resistance not only to the streptogramin antibiotics, but also to several other classes of antibiotics used in treatment of human infections. The long use of virginiamycin in food animals may have contributed to streptogramin resistance in enterococci from those sources. The routine collection and analysis of animal and environmental associated bacteria will help identify novel mechanism of resistance. Monitoring and surveillance of antimicrobial resistance to drugs used in food production will identify the effect of antimicrobials used in animals on resistance in humans and the environment.

6. Genomic sequencing of antimicrobial resistant Escherichia coli from Nigeria. Widespread use of antimicrobials in economically challenged countries is a driver of worldwide antimicrobial resistance. In order to effectively combat antimicrobial resistance, monitoring of resistance in these regions is necessary. Escherichia coli is a common commensal of the intestinal tract of humans and animals, but can also be an opportunist pathogen associated with illnesses caused by food-producing animals. In order to effectively treat these infections, it is important to understand the mechanisms of resistance in E. coli. To determine resistance, ARS researchers at Athens, Georgia, isolated the genomes of multidrug resistant E. coli from humans and chicken carcasses were sequenced. Multiple genes conferring resistance to the critical human use B-lactam drug class were identified in those isolates. The isolates also harbored genes conferring resistance to other drugs used to treat human infections. This information is essential to food safety and human health for development of control strategies for combating antimicrobial resistance to ensure continued effectiveness of antimicrobials.

Review Publications
Adenipekun, E., Jackson, C.R., Ramadan, H., Iwalokun, B., Oyedeji, K., Frye, J.G., Barrett, J.B., Hiott, L.M., Woodley, T.A., Oluwadun, A. 2016. Prevalence and multidrug resistance of Escherichia coli from community acquired infections in Lagos, Nigeria. Journal of Infection. 10(9):920-931.
Bono, A., Hartman, C., Solaimanpour, S., Tong, H., Porwollik, S., McClelland, M., Frye, J.G., Mrazek, J., Karls, A. 2017. Novel DNA binding and regulatory activities for s54 (RpoN) in Salmonella Typhimurium 14028s. Journal of Bacteriology. doi:10.1128/JB.00816-16.
Gupta, S., McMillan, E., Jackson, C.R., Desai, P., Porwollik, S., McClelland, M., Hiott, L.M., Humayoun, S.B., Frye, J.G. 2016. Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Putten Strain CRJJGF_00159 (Phylum Gammaproteobacteria). Genome Announcements. 4(5):e00895-16.
Gupta, S., Williams, L., McMillan, E., Jackson, C.R., Desai, P., Porwollik, S., McClelland, M., Hiott, L.M., Humayoun, S.B., Frye, J.G. 2016. Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Blockley Strain CRJJGF_00147 (Phylum Gammaproteobacteria). Genome Announcements. 4(5): e00954-16.
Chen, C., Strobaugh Jr, T.P., Nguyen, L.T., Abley, M.J., Lindsey, R.L., Jackson, C.R. 2018. Isolation and characterization of two novel groups of Kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals. PLoS One.
Gupta, S., McMillan, E., Jackson, C.R., Desai, P., Porwollik, S., McClelland, M., Hiott, L.M., Humayoun, S.B., Barrett, J.B., Frye, J.G. 2016. Draft Genome Sequence of Salmonella enterica subspecies diarizonae Serovar IIIb 61:k:1,5,(7) Strain CRJJGF_00165 (Phylum Gammaproteobacteria). Genome Announcements. 4(6):e01322-16.
Cox Jr, N.A., Cosby, D.E., Thippareddi, H., Ritz, C.W., Berrang, M.E., Jackson, J.S., Mize, S.C., Kumar, S., Howard, A.K., Rincon, A.M., Ukidwe, M., Landrum, M., Frye, J.G., Plumblee Lawrence, J.R., Hiott, L.M., Jackson, C.R., Hinton Jr, A., Cook, K.L. 2018. Incidence, species and antimicrobial resistance of naturally occurring Campylobacter isolates from quail carcasses sampled in a commercial processing facility. Journal of Food Safety. 2018:38:e12438.
Sharma, P., Gupta, S., Barrett, J.B., Hiott, L.M., House, S.L., Woodley, T.A., Frye, J.G., Jackson, C.R. 2017. Draft genome sequence analysis of eight streptogramin-resistant Enterococcus species isolated from animal and environmental sources in the US. Genome Announcements. 16;5(46). pii: e01287-17. doi: 10.1128/genomeA.01287-17.
Gupta, S., Mcmillan, E., Jackson, C.R., Desai, P., Porwollik, S., Mcclelland, M., Hiott, L.M., Humayoun, S.B., Frye, J.G. 2016. Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Orion Strain CRJJGF_00093 (Phylum Gammaproteobacteria). Genome Announcements. 4(5):e01063-16.
Kollanoor Johny, A., Frye, J.G., Donoghue, A.M., Donoghue, D., Venkitanarayanan, K. 2017. Gene expression response of Salmonella enterica serotype Enteritidis phage type 8 to the subinhibitory concentrations of the plant-derived compounds,trans-cinnamaldehyde,and eugenol. Frontiers in Microbiology. 8(1828):1-10. doi: 10.3389/fmicb.2017.01828.
Syed, M., Shah, S., Sherafzal, Y., Shafi-Ur-Rehman, S., Khan, M., Barrett, J.B., Woodley, T.A., Jamil, B., Abbasi, S., Jackson, C.R. 2018. Detection and molecular characterization of methicillin resistant Staphylococcus aureus from table eggs. Foodborne Pathogens and Disease. 15(2):86-93.
Kagambega, A., Lienemann, T., Frye, J.G., Barro, N., Haukka, K. 2018. Whole genome sequencing of multidrug-resistant Salmonella enterica serovar Typhimurium isolated from humans and poultry in Burkina Faso. Tropical Medicine and Health. 46:4. doi: 10.1186/s41182-018-0086-9.
Sharma, P., Gupta, S., Adenipekun, E., Barrett, J.B., Hiott, L.M., Woodley, T.A., Iwalokun, B., Oyedeji, K., Oluwadun, A., Ramadan, H., Frye, J.G., Jackson, C.R. 2017. Draft genome sequence analysis of multidrug-resistant Escherichia coli strains isolated in 2013 from humans and chickens in Nigeria. Genome Announcements. 5(40):e01073-17. doi:10.1128/genomeA.01073-17.
Cho, S., Hiott, L.M., Barrett, J.B., Mcmillan, E., House, S.L., Humayoun, S.B., Adams, E.S., Jackson, C.R., Frye, J.G. 2018. Prevalence and Characterization of Escherichia coli Isolated from the Upper Oconee Watershed, in Northeast Georgia. PLoS One. 13(5):e0197005. doi:10.1371/journal.pone.0197005.