Location: Meat Safety and Quality
2021 Annual Report
Objectives
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.
Approach
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.
Progress Report
This is the final report for the project 3040-42000-017-00D terminated in February 2021, which has been replaced by new Project 3040-42000-020-00D. For additional information, see the new project report.
Objective 1: Research continued on whole genome sequencing to characterize Shiga Toxin-Producing Escherichia coli (STEC) and Salmonella strains to better understand how genetic variation associates with their ability to cause disease in humans. Comparative genomic analysis of a variety of Salmonella strains identified targets that can identify Salmonella strains that are more pathogenic to humans. A Cooperative Research and Development agreement has been established with a commercial diagnostic company to develop a rapid real-time assay using these Salmonella markers. For STEC O157:H7, genome sequence was used with computer modeling to successfully predict the human infection potential of cattle STEC O157:H7 isolates. Only a small subset of strains (<10%) from the bovine reservoir are likely to cause human disease, even within the previously defined groups. Most of the targets used for the prediction were in mobile elements of the genome. This finding has potentially important implications for public health as it means that herds infected only with these virulent strains could be targeted for control.
Comparisons of Salmonella Montevideo core genome sequences showed strains of this serotype fall into four distinct clades. While strains from all four clades have been isolated from humans, cattle isolates were all found to group in clade I, while the majority of strains associated with human illness were members of clades II, III and IV, with the majority residing in clade IV. Results further showed distinct differences in gene content among members of the four clades, especially with regard to prophage distribution, secreted effector and virulence factors, metabolic island and fimbrial operon content. Taken together, these differences suggest that the success of clade IV strains as human pathogens may be attributed to a combination of fitness advantages for colonizing and persisting within food types that are not normally cooked, and virulence traits that aid in modulating the host immune system and avoiding clearance.
When comparing the complete closed genome of STEC O157:H7 strains that differ in their ability to cause disease in humans using the core genome, there were very few differences in gene content between the strains indicating that most of the difference between these two pathotypes is related to nucleotide base changes and not addition or loss of genes. To increase our understanding of this observation, STEC O157:H7 of different pathogenicity levels were phenotyped using phenotype microarray technology. These data showed a strong correlation between metabolic rates in STEC O157 that associated with strains being from an environmental or clinical source. To better understand the described phenotypes, comparison of mRNA transcription was completed to determine the correlation between metabolic phenotype and mRNA abundance for the environmental and clinical strains. The majority of the transcriptional differences were from the clinical strain. These involved carbon and amino acid substrates that correlated with the metabolic phenotyping. The differentially expressed genes in the clinical strain are involved in utilization of different carbohydrates associated with plants and degradation of amino acids, specifically lysine, threonine and glutamic acid. Also, the virulence genes, intimin, espD, espA and Shiga toxin had greater expression in the clinical strain versus the environmental strain. The environmental strains showed an increased ability to utilize n-acetyl-D-Galactosamine compared to the clinical strain but there was no difference by mRNA transcription between the two strains.
Objective 2: The first study determined the prevalence and concentrations of antimicrobial resistant (AMR) E. coli, Salmonella spp., Enterococcus spp. and Staphylococcus aureus in ground beef and pork chops from animals produced with no restrictions on antimicrobial use ("conventional") and animals with no antimicrobials used ("raised without antimicrobials"). Generally, levels of antimicrobial resistance were similar between meats from animals raised with and without antimicrobials. Other studies conducted showed similar AMR levels between products with and without “raised without antibiotics” claims. This study also demonstrated that antibiotic uses during beef cattle production minimally impact AMR occurrence and that processing interventions effectively remove antimicrobial resistant bacteria (AMB). ARS researchers at Clay Center, Nebraska, also found that tylosin inclusion in feed to prevent liver abscesses had no impact on detection of AMB. These results suggest that this application of tylosin has no or minimal impact on antimicrobial resistance in cattle production and processing environments.
The impact of soil amendment with cattle manure on levels of AMB E. coli, Enterococcus, and Salmonella was determined. AMR levels in the genera tested were not impacted by the application of manure to the soil. Although, the level of two antimicrobial resistance genes were slightly higher immediately following application of cattle manure in the fall but returned to normal levels prior to planting in the spring. We looked at AMR presence in the livestock-wildlife interface to determine the risk of other animals to move AMB between cattle feedlots. E. coli isolates with high-priority AMR (resistance to cephalosporins and ciprofloxacin) were isolated from the feces of cattle, raccoons,and mice. This study showed that cattle and wildlife harbored highly similar AMR E. coli, and that nearly identical isolates were present at multiple farms within livestock and wildlife hosts. The genetic conservation observed between AMR E. coli isolates from cattle and wildlife suggest a complex AMR livestock ecology that has inputs from multiple sources. Lastly, whole genome sequencing was used to compare AMB over two years at a commercial cattle feeding operation. Commensal third-generation cephalosporin resistant (3GCr) E. coli are theorized to contribute to the occurrence of 3GCr Salmonella through horizontal gene transfer of plasmids harboring genes. 3GCs are critically important for treating serious human Salmonella infections. Results suggest that the predominant means of 3GCr Salmonella occurrence in this feeding operation is the persistence of adapted Salmonella strains. Horizontal gene transfer from 3GCr E. coli isolates appears to play a minor role.
Metagenomic sequence analysis of fecal swabs collected from calves that either had or had not been mass-medicated with a macrolide antibiotic (gamithromycin) for the prevention of Bovine Respiratory Disease Complex, was conducted. Oxford Nanopore MinION sequencing technology showed the dominant genera for sample days 0 and 1 were consistently Bacteroides, Clostridium, Lactobacillus and Faecalibacterium, but that post mass-medication on Day 9, calves that had received gamithromycin showed an average 10-fold increase in the number of reads corresponding to the genus Escherichia. By Day 28 however Escherichia read counts decreased to pre-treatment levels and the dominant genus in the majority of pools was Methanobrevibacter. The long-read metagenomic sequence data collected in this study represents a rich dataset capturing the microbial diversity present in bovine fecal microbiomes over a four-week time period, and the impact of antibiotic exposure on this diversity.
Objective 3: STEC and Salmonella were studied to understand the mechanisms and genetic basis for biofilm formation and sanitizer resistance. The specific genetic markers responsible for biofilm forming ability and high sanitizer resistance were clustered according to previously determined STEC and Salmonella genotypes, indicating that a one size fits all approach to remove biofilm or combat high sanitizer resistance might not be effective against all strains. Rather, a multicomponent approach combining sanitizing reagents with different functional mechanisms might be better. The novel multicomponent sanitizers significantly reduced the amount of viable biofilm cells even when they were applied at diluted concentrations with short exposure periods. Furthermore, multiple consecutive treatments at the product’s recommended concentration combined with sufficient exposure time effectively controlled pathogen post-sanitization regrowth. 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. The higher biofilm-forming ability and stress tolerance did not affect the strains’ antibiotic resistance profile which indicates that the two resistance mechanisms are different. When comparing the genome of strains before and after exposure to low concentration sanitizer, the only observed differences were in two regions of the chromosome that can be inverted. In other bacteria, inverting regions of DNA is used to control gene expression or create a different version of the affected protein. Additional experiments will be needed to determine the role of these inversions with increased ability to form biofilm or enhance sanitizer resistance. These data provide phenotypic and genotypic information that improved biofilm removal and increased our understanding of the genetic basis for biofilm formation and sanitizer resistance.
Overall, the accomplishments of this project provide evidence-based, scientifically justified information and technology that has been, and will continue to be, useful for understanding and designing pre-harvest control of STEC, Salmonella, and AMR bacterial organisms in livestock and beef production systems.
Accomplishments
Review Publications
Harhay, D.M., Weinroth, M.D., Bono, J.L., Harhay, G.P., Bosilevac, J.M. 2021. Rapid estimation of Salmonella enterica contamination level in ground beef – application of the time-to-positivity method using a combination of molecular detection and direct plating. Food Microbiology. 93. Article 103615. https://doi.org/10.1016/j.fm.2020.103615.
Wang, R., Zhou, Y., Kalchayanand, N., Harhay, D.M., Wheeler, T.L. 2021. Consecutive treatments with a multicomponent sanitizer inactivate biofilms formed by Escherichia coli O157:H7 and Salmonella enterica and remove biofilm matrix. Journal of Food Protection. 84(3):408-417. https://doi.org/10.4315/JFP-20-321.
