Campylobacter is one of the leading bacterial causes of gastroenteritis worldwide. It frequently contaminates poultry and other raw meat products, which are the primary sources of Campylobacter infections in humans. Plasmids, known as important mobile genetic elements, often carry genes for antibiotic resistance, virulence, and self-mobilization. They serve as the main vectors for transferring genetic material and spreading resistance and virulence among bacteria. In this study, we identified 34 new plasmids from 43 C. jejuni and C. coli strains isolated from retail meat using long-read and short-read genome sequencing. Pangenomic analysis of the plasmid assemblies and reference plasmids from GenBank revealed five distinct groups, namely, pTet, pVir, mega plasmids (>80 kb), mid plasmids (~30 kb), and small plasmids (<6 kb). Pangenomic analysis identified the core and accessory genes in each group, indicating a high degree of genetic similarity within groups and substantial diversity between the groups. The pTet plasmids were linked to tetracycline resistance phenotypes in host strains. The mega plasmids carry multiple genes (e.g., aph(3’)-III, type IV and VI secretion systems, and type II toxin–antitoxin systems) important for plasmid mobilization, virulence, antibiotic resistance, and the persistence of Campylobacter. Together, the identification and comprehensive genetic characterization of new plasmids from Campylobacter food isolates contributes to understanding the mechanisms of gene transfer, particularly the spread of genetic determinants of virulence and antibiotic resistance in this important pathogen.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies’ MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Campylobacter is a leading bacterial cause of gastrointestinal infections in humans and has imposed substantial medical and public health burdens worldwide. Among a total of 39 species in the Campylobacter genus, C. jejuni is the most important species responsible for approx. 90% of human Campylobacter illness. Most cases of the infection were acquired by ingesting undercooked poultry meat due to the high prevalence of Campylobacter in the products. Here, we reported the dataset of raw sequences, de novo assembled and annotated genomes of C. jejuni strains S27, S33, and S36 recently isolated from retail chicken by using PacBio highly accurate long-read sequencing technology combined with bioinformatics tools. Our data revealed several virulence and antibiotic resistance genes in each of the chromosomes, a type IV secretion system in the plasmid (pCjS33) of C. jejuni S33, and a type VI secretion system and a phage in the plasmid (pCjS36) of C. jejuni S36. This study not only provides new sequence data but also extends the knowledge pertaining to the genomic and functional aspects of this important foodborne pathogen, including the genetic determinants of virulence and antibiotic resistance.

Introduction: Backyard poultry (BYP) are increasingly linked to cases of campylobacteriosis and salmonellosis.Methods: Between 2022 and 2024, soiled bedding samples from 70 BYP farms were tested for Campylobacter spp. and/or Salmonella enterica.Results: Nine farms (12.86%) had at least one sample positive for S. enterica, while 19.05% (12/63) tested positive for Campylobacter spp. We sequenced 54 S. enterica isolates from eight farms in this sample and four farms from previous sampling in 2021 (n = 12 total farms) to determine the genetic characteristics of S. enterica from backyard poultry. Salmonella Schwarzengrund was the most common serovar (33%; 18/54) found, followed by Kentucky (16.7%; 9/54) and serovars Hadar (14.8%; 8/54) and Enteritidis (14.8%; 8/54). Though over half of isolates (51.9%; 28/54) exhibited no predicted genotypic or phenotypic resistance to antimicrobials, some serovars such as Salmonella Hadar were resistant to multiple antimicrobials. Four isolates had intermediate phenotypic resistance to ciprofloxacin and two were resistant to ampicillin.Conclusions: In summary, the frequency of Campylobacter and Salmonella in BYP populations of Vermont may pose a significant public health risk. Although the rate of antimicrobial resistance was low among S. enterica isolates, resistance to medically important antibiotics was observed, and isolate serovars aligned with serovars implicated in human illness in Vermont.

Escherichia coli strain FEX669 was isolated from retail ground chicken and shown to contain the extraintestinal pathogenic E. coli (ExPEC) virulence genes sfaD/focC and iutA . Because this presumptive ExPEC strain was isolated from a retail food item and it was a weak biofilm former, it was characterized using whole-genome sequencing using the PacBio RS II platform.

Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes are routinely responsible for severe foodborne illnesses in the United States. Current identification methods utilized by the U.S. Food Safety Inspection Service require at least four days to identify STEC and six days for L. monocytogenes. Adoption of long-read, whole genome sequencing for food safety testing could significantly reduce the time needed for identification, but method development costs are high. Therefore, the goal of this project was to use NanoSim-H software to simulate Oxford Nanopore sequencing reads to assess the feasibility of sequencing-based foodborne pathogen detection and guide experimental design. Sequencing reads were simulated for STEC, L. monocytogenes, and a 1:1 combination of STEC and Bos taurus genomes using NanoSim-H. At least 2500 simulated reads were needed to identify the seven genes of interest targeted in STEC, and at least 500 reads were needed to detect the gene targeted in L. monocytogenes. Genome coverage of 30x was estimated at 21,521, and 11,802 reads for STEC and L. monocytogenes, respectively. Approximately 5–6% of reads simulated from both bacteria did not align with their respective reference genomes due to the introduction of errors. For the STEC and B. taurus 1:1 genome mixture, all genes of interest were detected with 1,000,000 reads, but less than 1x coverage was obtained. The results suggested sample enrichment would be necessary to detect foodborne pathogens with long-read sequencing, but this would still decrease the time needed from current methods. Additionally, simulation data will be useful for reducing the time and expense associated with laboratory experimentation.