Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Closed bacterial genomes from whole genome sequencing play an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using 24 hour enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore’s EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken2), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105−108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107−108 CFU/ml and a complete, fragmented MAG was obtained at 105−106 CFU/ml. In silico virulence detection for E. coli MAGs for 105−108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow provides proof of concept for faster detection and complete genomic characterization of STECs from a complex microbial sample compared to current reporting protocols and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.

Pleuropneumonia is one of the most relevant respiratory infections in the swine industry. Despite Actinobacillus pleuropneumoniae (APP) being one of the most important pathogens in the pig production, this is the first comparative study including all available whole genome sequencing data from NCBI.

This cross-sectional study determined the serovars, antimicrobial resistance genes, and virulence factors of Salmonella isolated from hatcheries, broiler farms, processing plants, and retail outlets in Trinidad and Tobago. Salmonella in silico serotyping detected 23 different serovars where Kentucky 20.5% (30/146), Javiana 19.2% (28/146), Infantis 13.7% (20/146), and Albany 8.9% (13/146) were the predominant serovars. There was a 76.0% (111/146) agreement between serotyping results using traditional conventional methods and whole-genome sequencing (WGS) in in silico analysis. In silico identification of antimicrobial resistance genes conferring resistance to aminoglycosides, cephalosporins, peptides, sulfonamides, and antiseptics were detected. Multidrug resistance (MDR) was detected in 6.8% (10/146) of the isolates of which 100% originated from broiler farms. Overall, virulence factors associated with secretion systems and fimbrial adherence determinants accounted for 69.3% (3091/4463), and 29.2% (1302/4463) counts, respectively. Ten of 20 isolates of serovar Infantis (50.0%) showed MDR and contained the blaCTX-M-65 gene. This is the first molecular characterization of Salmonella isolates detected along the entire broiler production continuum in the Caribbean region using WGS. The availability of these genomes will help future source tracking during epidemiological investigations associated with Salmonella foodborne outbreaks in the region and worldwide.

Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Whole genome sequencing generation of closed bacterial genomes plays an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore's EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105-108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107-108 CFU/ml and a complete, fragmented MAG was obtained at 105-106 CFU/ml. In silico virulence detection for E. coli MAGs for 105-108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow could be used for detection and complete genomic characterization of STEC from a complex microbial sample and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.

Salmonella enterica is a highly important foodborne pathogen worldwide. We report the complete genome sequence of a sequence type 14 Salmonella enterica serotype Senftenberg strain carrying the mcr-9 gene in a plasmid isolated from broken chicken eggshells in Trinidad and Tobago, obtained by using a combination of long- and short-read sequencing.

Salmonella enterica is an important foodborne pathogen worldwide. We used long and short-read sequencing to close genomes of eight multidrug-resistant (MDR) S. enterica strains, belonging to serovars Infantis (2), Albany, Oranienburg, I 4,[5],12:i:-, Javiana, Schwarzengrund, and Kentucky from broiler chicken farms and processing plants in Trinidad and Tobago. They also belonged to seven different sequence types (STs- 32, 292, 1510, 19, 24, 152, and 96). Among the strains, seven had demonstrated multi-drug resistance with the presence of at least three AMR genes, whereas three isolates contained the quinolone resistance gene qnrB19 in plasmids (CFSAN103840, CFSAN103854, and CFSAN103872). The extended-spectrum β-lactamase genes blaCTX−M−65 (CFSAN103796) and blaTEM−1 (CFSAN103852) were detected in this study. The genomes closed in this study will be useful for future source tracking and outbreak investigations in Trinidad and Tobago and worldwide.

Leafy greens are responsible for nearly half of the produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States and recent investigations have implicated agricultural water as a potential source. Current FDA detection protocols require extensive analysis time. We aimed to use Oxford Nanopore rapid sequencing kits for an in-field determination of agricultural water microbiome and possible detection and characterization of STECs strain(s) in these samples. We tested the performance of the nanopore rapid sequencing kit (RAD004) for fast microbiome determination using the well characterized ZymoBIOMICS mock microbial community and the number of reads for each identified species was present in the expected proportion. Rapid sequencing kit (LRK001 and RAD004) library preparation of DNA extracted from agricultural water resulted in poor nanopore sequencing reactions, with low output (0.3–1.7 M reads), a high proportion of failed reads (50–60%), and highly sheared DNA before and after a magnetic bead clean up. To improve performance, we prepared a DNA library with the ligation kit (LSK109), which includes multiple cleaning steps, reducing inherent inhibitors and producing a better outcome (2.2 M reads, 15% failed reads). No definitive presence of STEC could be confirmed in any of the sites. Approximately 100 reads from each site (0.02% of total reads) were identified as Escherichia coli, but the specific strain or their virulence genes could not be detected. Sites 9, 10, and 12 were found to be positive for STEC presence by microbiological techniques after enrichment. The rapid sequencing kits can be appropriate for genus or species level microbial identification, but we recommend the use of the ligation kit for increased sequencing depth and removal of contaminants in agricultural water. However, we were not able to identify any STEC strains in these nanopore microbiome samples, due to low initial concentrations. The results from this pilot study provide preliminary evidence that MinION sequencing of agricultural water using the ligation kit has the potential to be used for rapid microbiome determination in the field with optimal results for water quality surveillance.

No abstract