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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

2016 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:
This report documents progress from Project Number 3040-42000-017-00D, which started on February 1, 2016 and continues research from Project Number 3040-42000-015-00D, entitled “Exploring Genomic Differences and Ecological Reservoirs To Control Foodborne Pathogens.” Progress was made on all three objectives. Under Objective 1, we have made substantial progress towards sequencing STEC O157 and STEC O26 strains with more than half our goal finished. Preliminary genome comparison indicated that strains that are similar by single nucleotide polymorphism (SNP) analysis can have different prophage content and contain genomic rearrangement. Biolog phenotyping microarrays for STEC O157 strains is nearly complete. We see tremendous variation between strains with respect to their metabolic profiles. Analogous to the genomic comparison results, strains that are similar by SNP analysis do not have similar metabolic profiles. Also, progress was made in identifying multidrug resistant (MDR) Enterobacteriaceae isolated from feedlot cattle fecal swabs, to be used in whole genome sequence analysis. Isolates (n=2200) were screened using multiplex PCR reactions for particular cephalosporin and macrolide resistance genotypes, and a diverse set of organisms from 15 bacterial genera were selected based on MDR phenotype, as well as macrolide and cephalosporin resistance and genotypes. From this subset of strains (n=412) complete closed reference genome sequence data was collected for two Escherichia coli isolates with unique MDR phenotypes. An additional 28 strains from this subset will be sequenced to fulfill the 2017 milestone for sub-objective 1.A. Under Objective 2, we have been continuously making progress towards understanding the mechanisms and genetic basis for biofilm formation and sanitizer resistance by common foodborne pathogens. In particular, we have been identifying specific genetic markers responsible for biofilm forming ability and high sanitizer resistance that might be clustered according to previously determined STEC and Salmonella genotypes. Representative STEC O157 and Salmonella enterica strains of various genotypes have been selected and are being tested for their biofilm forming ability, survival and recovery growth ability after sanitization, as well as cell surface expression of specific extracellular polymeric substances (EPS). These tests are being conducted using materials and temperature commonly encountered in the meat industry under normal operating conditions. Furthermore, we are currently working with the meat industry to test the effectiveness of novel sanitizer products in terms of bacterial susceptibility, biofilm tolerance level, as well as the unique ability of these products to penetrate biofilm structure, dissolve EPS matrix, thus, effectively remove biofilms from colonized food contact surfaces. Under objective 3, to address concerns regarding impact of antimicrobial use on the occurrence of antimicrobial resistant bacteria and antimicrobial resistance genes, we have made progress on two projects. We collected over 350 ground beef samples and 350 pork chop samples. These samples equally split between meats from animals produced with no restrictions on antimicrobial use ("Conventional") and animals with no antimicrobials used ("Raised without antimicrobial"). We have determined prevalence and concentration of antimicrobial resistant E. coli, Salmonella spp., Enterococcus spp., and Staphylococcus aureus in these samples. We have also developed a protocol for isolation of bacterial metagenomic DNA from these samples that reduces contamination by host (i.e. cattle or swine) DNA. We have begun quantitative polymerase chain reaction (PCR) to determine the concentrations of 10 antimicrobial resistance genes in these samples. We have also collected over 300 fecal, 300 hide, and 300 pen surface material samples from cattle feedlots beginning a project comparing methods of measuring antimicrobial resistance at cattle feedlots. Also, fecal swab samples (n=720) from feedlot cattle were screened for various bacterial genera demonstrating resistance to cephalosporin or macrolide antibiotics. This screening resulted in the collection of over 2000 organisms. The genus and species of these isolates will be determined and a subset will be selected for the whole genome sequencing component of sub-objective 3.B. All fecal swab samples have been stored as glycerol stocks at -80°C and will be used for total genomic DNA isolation as part of the metagenomic sequencing component of sub-objective 1.A.

4. Accomplishments
1. Developed a method for identifying seven different bacterial macrolide resistance determinants. Cattle are frequently administered macrolide antibiotics for the prevention or treatment of various diseases. In order to examine the effect of macrolide exposure on fecal shedding of resistant bacteria, rapid methods for characterizing resistance gene content were needed. To address this need, researchers in Clay Center, Nebraska developed a method to detect seven major antibiotic resistance mechanisms simultaneously. This method was successfully used to screen over 2000 bacterial isolates and revealed subpopulations of bacteria containing antibiotic resistance genes not previously recognized in these organisms.

Review Publications
Dickey, A.M., Loy, J.D., Bono, J.L., Smith, T.P.L., Apley, M.D., Lubbers, B.V., DeDonder, K.D., Capik, S.F., Larson, R.L., White, B.J., Blom, J., Chitko-McKown, C.G., Clawson, M.L. 2016. Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with conjunctivitis versus the deep nasopharynx of asymptomatic cattle. Veterinary Research. 47:31.

Luedtke, B.E., Bosilevac, J.M., Harhay, D.M., Arthur, T.M. 2016. Effect of direct-fed microbial dosage on the fecal concentrations of enterohemorrhagic Escherichia coli in feedlot cattle. Foodborne Pathogens and Disease. 13(4):190-195. doi:10.1089/fpd.2015.2063

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 and closed genome sequences of 10 Salmonella enterica subsp. enterica serovar Anatum isolated from human and bovine sources. Genome Announcements. Vol. 4(3): e00447-16. doi: 10.1128/genomeA.00447-16.