Listeria monocytogenes remains a threat to the food system and has led to numerous foodborne outbreaks worldwide. L. monocytogenes can establish itself in food production facilities by adhering to surfaces, resulting in increased resistance to environmental stressors. The aim of this study was to evaluate the adhesion ability of L. monocytogenes at 8 °C and to analyse associations between the observed phenotypes and genetic factors such as internalin A (inlA) genotypes, stress survival islet 1 (SSI-1) genotype, and clonal complex (CC). L. monocytogenes isolates (n = 184) were grown at 8 °C and 100% relative humidity for 15 days. The growth was measured by optical density at 600 nm every 24 h. Adherent cells were stained using crystal violet and quantified spectrophotometrically. Genotyping of inlA and SSI-1, multi-locus sequence typing, and a genome-wide association study (GWAS) were performed to elucidate the phenotype-genotype relationships in L. monocytogenes cold adhesion. Among all inlA genotypes, truncated inlA isolates had the highest mean adhered cells, ABS595nm = 0.30 ± 0.15 (Tukey HSD; P < 0.05), while three-codon deletion inlA isolates had the least mean adhered cells (Tukey HSD; P < 0.05). When SSI-1 was present, more cells adhered; less cells adhered when SSI-1 was absent (Welch's t-test; P < 0.05). Adhesion was associated with clonal complexes which have low clinical frequency, while reduced adhesion was associated with clonal complexes which have high frequency. The results of this study support that premature stop codons in the virulence gene inlA are associated with increased cold adhesion and that an invasion enhancing deletion in inlA is associated with decreased cold adhesion. This study also provides evidence to suggest that there is an evolutionary trade off between virulence and adhesion in L. monocytogenes. These results provide a greater understanding of L. monocytogenes adhesion which will aid in the development of strategies to reduce L. monocytogenes in the food system.

Escherichia coli is a Gram-negative bacterium that is ubiquitous in animals and humans, with some strains capable of causing disease. The aim of this study was to perform a comparative genomic analysis of 2732 generic E. coli isolates that were recovered from poultry samples collected from six regions in Canada as part of the National Microbiological Baseline study in Broiler Chicken. Isolates were subjected to whole genome sequencing and a subset (1122/2732) were tested for phenotypic resistance to 15 antimicrobials. These E. coli isolates were highly diverse, representing 376 serotypes, 236 sequence types and 21 pathotypes, of which 19 were hybrid pathotypes. A high concordance (>85%) between resistance phenotype and the presence of antimicrobial resistance genes and point mutations (resistance determinants) was observed for 13/15 antimicrobials. Over 95% of the β-lactam, fluoroquinolone, and phenicol resistance genes were predicted to be plasmid-borne. The number of resistance determinants per genome was highest in Quebec, while resistance genes associated with β-lactam resistance were more frequently detected in isolates from British Columbia. Generic E. coli in Canadian poultry are highly diverse, can carry pathotype-associated virulence factors and resistance determinants of clinical significance with a risk of emerging into pathogenic strains.

Plant cell walls are comprised of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade these components to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine ( Erethizon dorsatum ) is replete with novel lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four genes from the porcupine microbiome encoding putative novel lignocellulose-degrading enzymes, including a β-xylanase, endoxylanase, β-glucosidase, and an ⍺-L-arabinofuranosidase. These genes were identified via conserved catalytic domains associated with cellulose and hemicellulose degradation. We cloned the putative β-xylanase into the pET26b(+) plasmid, enabling inducible gene expression in Escherichia coli ( E. coli ) and periplasmic localization. We demonstrated IPTG-inducible accumulation of β-xylanase protein but failed to detect xylobiose degrading activity in a reporter assay. Alternative assays may be required to measure activity of this putative β-xylanase. In this report, we describe how a synthetic metagenomic pipeline can be used to identify novel microbial lignocellulose-degrading enzymes and take initial steps to introduce a hemicellulose-degradation pathway into E. coli to enable biofuel production from wood pulp feedstock.

Background: The rising prevalence of Extended-spectrum β-lactamase-producing (ESBL) Escherichia coli poses a significant threat to human and animal health. Methods: To address this, we conducted a longitudinal two-year One Health study to assess ESBL E. coli occurrence and distribution across dairy farms, surrounding environments, and urban wastewater in a peri-urban region of Western Canada. Results: A total of 546 presumptive ESBL E. coli were recovered, with the highest occurrence in wastewater influent (75.9%) and calf feces (73.6%), and lowest in soil (6.3%) and surface water (18.8%). Seasonal analysis showed a significantly higher occurrence in summer compared to spring. The bla_CTX-M-15 gene predominated (79%), followed by bla_TEM (28%) and bla_SHV (9%), with most isolates harboring multiple ESBL genes. Whole-genome sequencing of 387 isolates identified 75 resistance determinants spanning nine antimicrobial classes, including 24 β-lactamase genes and 10 CTX-M variants. Ninety-four sequence types (STs), including nine novel STs, were detected. The most common STs were ST648, ST69, and ST10, with distinct distributions across sources. Plasmid analysis revealed extensive diversity, with approximately half of the plasmid types shared across multiple sample types, indicating potential horizontal gene transfer. Over 200 virulence factors were identified, including toxin genes and Shiga toxin-associated genes, primarily in calf and surface water isolates. Phylogroups A and B1 dominated samples from dairy farms, and phylogroup B2 was restricted to wastewater and surface water. Conclusions: These findings identify the environment as a reservoir for ESBL E. coli and reveal the unexpected predominance of the emerging MDR ST648 lineage, rather than ST131, and reinforce the need for comprehensive integrated One Health surveillance.

Staphylococcus shinii is a recently proposed staphylococcal species identified in animal-associated microbiomes and fermented foods, with promising probiotic and biotechnological applications. However, its genomic diversity, functional traits and safety profile need to be well established. Here, we report the first recovery and genome sequencing of 41 S . shinii isolates from blueberry fruit washes and surrounding air. Phylogenomic analysis, including reference genomes, revealed at least 15 well-supported intra-species lineages (genomovars) defined by phylogenetic relationship and average nucleotide identity (>99.5% within genomovars; <99.5% between them). Pangenome analysis showed a highly conserved core genome comprising 83.3% of genes, alongside diverse accessory genes across genomovars. Core functions in S. shinii included carbohydrate metabolism, vitamin biosynthesis and short-chain fatty acid production, traits potentially linked to probiotic properties. Biosynthetic gene clusters for bioactive compounds were broadly conserved, although genes encoding ribosomally synthesized and post-translationally modified peptides varied among genomovars. About 10–18 antimicrobial resistance genes (ARGs) were identified per S. shinii genome, several of which were located on the chromosome. Phenotypic testing confirmed susceptibility to ciprofloxacin, gentamicin and ampicillin, while resistance to other antimicrobials varied and was often plasmid-associated. Resistance patterns differed across genomovars, emphasizing the need for intra-species resolution in risk assessment. Although major virulence factors listed in the Virulence Factor Database were absent, putative pathogenicity-related genes were found in the core genome using the Pathogen–Host Interaction Database. Their presence, along with ARGs, underscores the need for caution in clinical contexts. Nonetheless, the absence of major virulence determinants and the presence of beneficial traits in some S. shinii isolates support their potential use in the agri-food system or environmental biotechnology, pending strain-specific safety evaluation.

(Narrative): Hybrid whole genome sequencing was used to investigate if nosocomial Verona integron-encoded metallo-β-lactamase (VIM) carbapenemase transmission occurred between two patients without epidemiological links or common pathogens. Challenges in genomic methodology and appropriate analytical depth for mobile carbapenemase outbreaks are described including how inappropriate choices can mislead results and impact infection control practices.