Project : USDA ARS

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

Location: Meat Safety and Quality

2020 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
Under Objective 1, substantial progress has been made comparing genomes of Shiga toxin-containing Escherichia coli (STEC) O157:H7 strains and defining various phenotypes associated with specific genotypes. Previous experiments to compare STEC O157:H7 strains mainly used short-read sequencing technologies and would leave the genome fragmented into an average of 250 pieces. Fifty-three fully closed STEC O157:H7 genomes were generated to use for comparison to each other. The core genome of the STEC O157:H7 strain in this study was comprised of just over 4,000 genes while there were twice as many non-core genes, indicating the genomic diversity between strains. Many of the non-core genes were found in phage regions with the STEC O157:H7 genomes made up of an average of 20 phages which corresponded to 14.6% of the genome. All STEC O157:H7 genomes contain a plasmid of between 90 kb and 100 kb in size. This plasmid was found to evolve at the same rate as the chromosome indicating its close association with the chromosome. Several strains had integration of DNA typically associated with other bacteria and evidence of previous infection with bacteriophage. These results demonstrate the plasticity of the genome and the ability to gain new traits. When comparing strains that differ in their ability to cause disease, 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. These results provide a large-scale view into the complete genomes of STEC O157:H7 and provides insight into potential weaknesses of the bacteria that can be exploited by interventions to produce safer food. Additional research included a comparative genomic analysis of a variety of Salmonella strains with the goal of identifying targets for the rapid identification of Salmonella strains that are more pathogenic to humans (i.e. Highly Pathogenic Salmonella or HPS), and thus able to infect at a lower infective dose. This analysis revealed molecular targets for identifying four of the leading disease-causing Salmonella serotypes in the U.S. namely, Enteritidis, Typhimurium, (1,4,[5],12:i:-), and Newport, as well as a noted invasive serotype, S. Dublin. These data were used to design a molecular assay targeting these markers. To date, the HPS assay has been tested with over 1700 Salmonella isolates encompassing 78 serotypes. A grant was awarded by the Foundation for Meat and Poultry Research and Education (FMPRE) in fiscal year 2020 for continued validation of the HPS assay for identifying Salmonella of greater concern for human health from enrichment samples of a variety of ground meat or trim samples. The result of this research is an assay that will identify HPS in food so contaminated food can be diverted from the food chain and prevent Salmonella illnesses in humans. Under Subobjective 1A, total DNA was isolated from 720 fecal swab samples that had been collected from 180 calves that had (n=90 calves) or had not (n=90 calves) been mass-medicated with a macrolide antibiotic (gamithromycin) for the prevention of bovine respiratory disease complex. Fecal swabs were collected pre-mass-medication (day 0), and post-mass-medication on days 1, 9 and 28 of the study. In all, 72 pooled DNA samples were sequenced using Oxford Nanopore MinION sequencing technology. Libraries yielded on average 330K reads with an average N50 of 8.2kb, and a total of 128.7 Gbases of sequence data collected. Changes in microbial community structure were examined using the Oxford Nanopore analysis portal EPI2ME and the What's In My Pot workflow. These analyses 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 the macrolide antibiotic 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 majority of pools was Methanobrevibacter. These sequence data will be further analyzed using the EPI2ME Antimicrobial Resistance Analysis workflow, to examine changes in distribution of resistance genes in the feces of medicated and non-medicated calves over the 28-day study. The long-read metagenomic sequence data collected represents a rich dataset capturing the microbial diversity present in bovine fecal microbiomes, and the impact of antibiotic exposure on this diversity. Under Objective 2, ARS researchers at Clay Center, Nebraska, have been working with the meat industry to investigate the impact of bacterial biofilms on meat safety at commercial plants. In addition to biofilm cell inactivation, biofilm matrix removal and post-sanitization pathogen prevalence control are all essential for reducing biofilm-related cross contamination and enhancing meat safety. To that end, the multi-faceted approach using multiple sanitizers or combining sanitizer usage with other cleaning methods has been proposed. The effectiveness of a multicomponent sanitizer was evaluated based on the synergistic combination of hydrogen peroxide and quaternary ammonia compounds, deployed as a foam or liquid solution at recommended or diluted concentrations against biofilms formed by Salmonella enterica and Escherichia coli O157:H7 strains under meat processing conditions. The impact of multiple consecutive treatments with the sanitizer product, as recommended by the manufacture, on post-sanitization prevention of pathogen prevalence and biofilm reoccurrence was investigated as well. Results showed that the multicomponent sanitizer significantly reduced the amount of viable biofilm cells even when it was 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 prevalence. Examination with a scanning electron microscope showed that sanitizer treatment significantly dissolved bacterial connecting substances and removed the majority of the biofilm matrix. No intact biofilm structure was detected, but instead the treated bacteria exhibited indented and distorted shape with shortened cell length and increased surface roughness, indicating severe cell injury and death. Results indicated that consecutive treatments with this multicomponent sanitizer is effective in inactivating/removing Salmonella enterica and E. coli O157:H7 biofilms. Additionally under Objective 2, the genetic basis of Salmonella enterica associated with enhanced biofilm formation and sanitizer tolerance after repeated exposure to residual amounts of common sanitizers was investigated. A Salmonella Montevideo daughter strain with an enhanced biofilm-forming ability and sanitizer tolerance was obtained after repeated exposure to low concentrations of the sanitizer, likely due to genomic alternation after such exposure. The higher biofilm-forming ability and stress tolerance did not affect the strains' antibiotic resistance profile. Whole genome sequencing analysis and comparison between the parental and daughter strains identified differences in two inverted regions, both associated with a DNA invertase. Further studies are ongoing to confirm the observation in additional Salmonella serotypes/strains, and to investigate the association between the altered DNA regions and the phenotype changes. Under Objective 3, whole genome sequences have been assembled and annotated for 447 isolates obtained over two years at a commercial cattle feeding operation. Third-generation cephalosporins (3GCs) are critically important for treating serious human Salmonella infections. Although rare, Salmonella isolated from human patients are increasingly resistant to 3GCs. Commensal 3GC-resistant (3GCr) E. coli are theorized to contribute to the occurrence of 3GCr Salmonella through horizontal gene transfer of plasmids harboring genes (blaCMY-2 and blaCTX-M) conferring 3GC resistance. In all, 3GCr Salmonella isolates, 3GC-susceptible (3GCs) Salmonella isolates, and 3GCr E. coli isolates were sequenced. The 3GCs Salmonella were predominantly serotypes Muenchen or Montevideo. All 3GCr Salmonella harbored the blaCMY-2 gene with 97.5% containing an IncC plasmid origin of replication (ori). The IncC 3GCr Salmonella were predominantly Montevieo. The IncC ori was not present in any of the 3GCs Montevideo isolates. Of the 3GCr E. coli only 36.2% contained the blaCMY-2 gene with 16 different plasmid ori detected. The most frequently detected ori was IncFIB, followed by IncFIC, IncC was the sixth most frequently detected. Eleven sequence types (akin to serotypes) were identified for 20 blaCMY-2 IncC ori 3GC-resistant E. coli, with no sequence type frequency > 3. These results suggest that the predominant means of 3GCr Salmonella occurrence in this cattle feeding operation is the persistence of adapted Salmonella strains. Horizontal gene transfer from 3GCr E. coli isolates appears to play a minor role.

Accomplishments
1. A novel strategy for estimating Salmonella contamination levels in raw ground beef. Salmonella is a leading cause of foodborne illness worldwide with an estimated 93.8 million cases caused by over 1,700 type. In spite of the use of numerous process controls in food production industries, there has been little progress in decreasing the occurrence of Salmonella food poisoning over the past decade. This is in part because current testing methods only show the presence or absence of Salmonella, but do not measure how much contamination is in the tested product. Rapid methods for identifying more highly contaminated products are needed in order to allow food producers to investigate the root causes of practices that result in higher levels of contamination. To address this need, ARS scientists at Clay Center, Nebraska, developed a novel strategy for estimating Salmonella contamination levels in raw ground beef based on the time to detect Salmonella in enrichments. This approach was successfully used to estimate Salmonella contamination levels in ground beef samples using two different commercial molecular detection methods. The ability to detect high levels of Salmonella contamination will enable meat companies to improve their process control as well as remove more highly contaminated products from the food chain. This will improve the safety of beef and decrease the incidence of human exposure to levels of Salmonella that cause disease.

U.S. DEPARTMENT OF AGRICULTURE

Meat Safety and Quality: Clay Center, NE