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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety & Quality Research » Research » Research Project #430418

Research Project: Genomic and Metagenomic Differences in Foodborne Pathogens and Determination of Ecological Niches and Reservoirs

Location: Meat Safety & Quality Research

2017 Annual Report

1a. Objectives (from AD-416):
Objective 1. Molecular characterization including whole genome sequencing and transcriptomic characterization of foodborne bacteria, including pathogens and commensals, exposed to various physiologically relevant conditions reflective of the production continuum. Sub-objective 1.A: De novo, whole genome sequencing and metagenomic profiling of the microbial community present in bovine rectoanal mucosa (RAM) swab samples. Sub-objective 1.B: Characterize the genomic, phenotypic and transcriptional differences present in clinically important STEC and Salmonella serotypes exposed to different physiological relevant conditions in order to identify virulence and regulatory control mechanisms. Objective 2. Characterize the ecological niches and reservoirs to identify mechanisms of foodborne pathogen, especially biofilms, for their ability to colonize and persist leading to the development of intervention strategies. Sub-objective 2.A: Molecular mechanisms of biofilm formation. Sub-objective 2.B: Association between biofilm formation, antibiotic resistance, and sanitizer tolerance. Objective 3. Development and validation of various antimicrobial resistance detection methodologies including culture and genomic techniques, such as whole genome sequencing. Sub-objective 3.A: Evaluation of culture based methods for the detection of bacteria resistant to antimicrobials important to human medicine. Sub-objective 3.B: Development of genomic methods for the detection of antimicrobial resistance elements.

1b. Approach (from AD-416):
The cost of food borne illness and the loss of productivity in the United States is reported to be greater than $14 billion a year. While research efforts have resulted in great strides in tracking contamination entry points and identifying mitigation strategies, outlier events continue to occur and complete prevention of foodborne pathogens entering the food chain remains an elusive goal. Attaining this goal is challenging in part because many of the target pathogens live in dynamic and complicated communities, likely not even causing disease in their host reservoir. In addition, a better understanding of the use of antimicrobial agents in animal production and the possible impact on foodborne pathogens acquiring resistance has become a top priority for many government agencies and health care advocates. The project described here will provide new information about these issues by helping to better understand the different colonization sites and how various pathogens survive and interact with their respective bacterial communities. Further, we will characterize population differences within these foodborne bacteria, focusing on those that enhance an organism’s ability to cause human illness. Ultimately, the overall aim of this project is to provide new information about pathogen (predominantly Shiga toxin-containing Escherichia coli (STEC) and Salmonella enterica) persistence and survival in a variety of environments that position them for entry into the food supply.

3. Progress Report:
Under Objective 1, we have made substantial progress in whole genome sequencing of bacterial members of the microbial community present in swab samples collected from the bovine rectoanal mucosa (RAM). RAM samples were validated for their diagnostic accuracy in the detection and enumeration of the pathogen Salmonella enterica, and the microbial communities present in these samples were investigated. Over 5000 bacterial isolates have been collected from bovine RAM samples and just over 2000 have been identified. In all, genomic sequence data has been collected from 60 bacterial strains representing 15 different genera present in bovine RAM samples. Mock RAM communities will be constructed from the sequenced bacterial strains, and total genomic DNA will be isolated and sequenced from these communities. Also, we completed sequencing 24 strains from STEC O157, STEC O26 and Salmonella spp. Genome comparison show that there is no correlation with nucleotide polymorphisms, prophage content or chromosomal rearrangements. STEC O157 and STEC O26 strains had more chromosomal rearrangements than what was observed in Salmonella. For STEC O157, these chromosomal rearrangements were almost always at the terminus of replication. Bacteriophages are important mobile elements that can integrate into a bacteria’s chromosome and often carry virulence factors. STEC O157 and STEC O26 both had more prophage/cryptic prophage integrated in their chromosomes than Salmonella. The phages in STEC O157 typically integrate at the same location on the chromosome, but there were also phage that integrated in previously undefined locations. To better understand what effect genome variability has on STEC O157, Biolog phenotyping microarrays were used to determine growth characteristic with different carbon, sulfur, phosphorous and nitrogen sources along with different pH and osmolality. There was a strong correlation between metabolic rates in STEC O157 and STEC O26 that associated with a strain being from an environmental or clinical source. Under Objective 2, we continue to make progress towards understanding the molecular mechanisms of biofilm formation and sanitizer resistance by common foodborne pathogens. We have tested STEC O157:H7 strains isolated from “High Event period” contamination for their biofilm forming ability on food contact surfaces of materials commonly used in the meat industry, as well as their survival and recovery ability after sanitization. Furthermore, selected STEC and Salmonella strains isolated from beef trim contamination were tested for cell surface structure expression, sanitizer susceptibility, and antibiotic resistance profiles. More importantly, the effect of residual amount of common sanitizers on bacterial biofilm formation and sanitizer susceptibility was evaluated. Our results showed that repeated exposure to low concentrations of common sanitizers could enhance biofilm formation and reduce sanitizer susceptibility by certain STEC O157:H7 and Salmonella strains. However in most cases, the developed resistance phenotype was not stable. Furthermore, we have initiated testing of novel sanitizer products for their effectiveness to penetrate biofilm structure, dissolve bacterial cell surface matrix, and thus remove biofilms from colonized food contact surfaces.

4. Accomplishments
1. Levels of antimicrobial resistance in meats from animals produced with and without antibiotics. Meats produced from animals "raised without antibiotics" (RWA) are perceived to harbor lower levels of antimicrobial resistance than meats from animals produced with no restrictions on antimicrobial use ("Conventional"). Additionally, risk-benefit modeling requires concentrations of antimicrobial resistant bacteria and antimicrobial resistance genes. ARS scientists in Clay Center, Nebraska determined prevalence and concentrations of antimicrobial resistant bacteria and the levels of 10 antimicrobial resistance genes in ground beef and pork chops from animals either raised without antibiotics or conventionally. Generally, levels of antimicrobial resistance were similar between meats from animals raised with and without antimicrobials. These results demonstrate that conventional beef and pork products do not pose a greater risk of exposure to antimicrobial resistance than RWA products.

2. Computer modeling to predict the zoonotic potential of E. coli O157 cattle isolates. Conventional genome sequence-based analyses predict that the majority of E. coli O157 strains from cattle can cause infections in humans, especially if they belong to one of two established groups. ARS scientist in Clay Center, Nebraska, with collaborators in Scotland used genome sequence with computer modeling to successfully predict the human infection potential of cattle E. coli O157 isolates. Only a small subset of strains (<10%) from the bovine reservoir are likely to cause human disease. This finding has potentially important implications for public health as herds infected only with these virulent strains could be targeted for control.

Review Publications
Shaaban, S., Cowley, L., McAteer, S., Jenkins, C., Dallman, T., Bono, J.L., Gally, D. 2016. Evolution of a zoonotic pathogen: investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing. Microbial Genomics. doi:10.1099/mgen.0.000096.

Nguyen, S.V., Harhay, D.M., Bono, J.L., Smith, T.P.L., Fields, P.I., Dinsmore, B.A., Santovenia, M., Kelley, C.M., Wang, R., Bosilevac, J.M., Harhay, G.P. 2016. Complete, closed genome sequences of 10 Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from human and bovine sources. Genome Announcements. 4(6):e01212-16. doi:10.1128/genomeA.01212-16.

Cowley, L.A., Dallman, T.J., Fitzgerald, S., Irvine, N., Rooney, P.J., McAteer, S., Day, M., Perry, N.T., Bono, J.L., Jenkins, C., Gally, D. 2016. Short-term evolution of Shiga toxin-producing Escherichia coli O157 between two food-borne outbreaks. Microbial Genomics. doi:10.1099/mgen.0.000084.

Lupolova, N., Dallman, T.J., Matthews, L., Bono, J.L., Gally, D.L. 2016. Support vector machine applied to predict the zoonotic potential of E. coli O157 cattle isolates. Proceedings of the National Academy of Sciences. 113(40):11312-11317. doi:10.1073/pnas.1606567113.

Miller, W.G., Yee, E., Lopes, B.S., Chapman, M.H., Huynh, S., Bono, J.L., Parker, C., Strachan, N., Forbes, K.J. 2017. Comparative genomic analysis identifies a Campylobacter clade deficient in selenium metabolism. Genome Biology and Evolution. doi: 10.1093/gbe/evx093.

Miller, W.G., Yee, E., Bono, J.L. 2017. Complete genome sequence of the Campylobacter helveticus type strain ATCC 51209T. Genome Announcements. 5(21): e00398-17.

Nguyen, S.V., Harhay, G.P., Bono, J.L., Smith, T.P., Harhay, D.M. 2017. Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of Loci encoding increased protein quality control mechanisms. mSystems 2:e00190-16. doi:10.1128/mSystems.00190-16.

Miller, W.G., Yee, E., Revez, J., Bono, J.L., Rossi, M. 2017. Complete genome sequence of the Campylobacter cuniculorum type strain LMG 24588. Genome Announcements. 5:e00543-17.