Escherichia coli 045 isolates associated with swine postweaning diarrhea in Quebec were characterized with respect to virulence determinants genetically and investigated for their attaching and effacing (A/E) activities by experimental inoculation of gnotobiotic piglets and by the HEp-2 cell adherence assay. All of 32 isolates tested were negative for enterotoxigenic and verotoxigenic E. coli virulence determinants, heat-labile enterotoxin (LT), heat-stable enterotoxins (STap, STb), verotoxins (VT1, VT2), and F4 (K88), F5 (K99), F6 (987P), and F41, except one STh-positive and two F4-positive isolates. A total of 25 isolates hybridized with an EaeA probe, and 11 hybridized with an enteropathogenic E. coli adherence factor (EAF) probe. None of 32 isolates hybridized with a bundle-forming pilus (BFP) probe. The EAF, EaeA, and BFP factors have been associatedwith human enteropathogenic E. coli strains. A total of 10 of 12 eaeA-positive porcine 045 isolates induced A/E lesions characterized by intimate adherence of bacteria to the intestinal epithelial cell membrane with effacement of the microvilli, similar to those of human attaching-effacing E. coli. However, A/E lesions were not observed in the piglets inoculated with any one of three eaeA-negative 045 isolates. All E. coli 045 isolates were non-adherent to HEp-2 cells. Thus, we have demonstrated the production of typical A/E lesions by nonenterotoxigenic E. coli 045 isolates from swine postweaning diarrhea. The results indicate the significance of the eaeA gene in A/E activities of these isolates and suggest that EAF and BFP are not involved in 045 E. coli infection of weaning piglets.

The chicken gastrointestinal tract harbors microorganisms that play a role in the health and disease status of the host. The cecum is the part of the gut that carries the highest microbial densities, has the longest residence time of digesta, and is a vital site for urea recycling and water regulation. Therefore, the cecum provides a rich environment for bacteria to horizontally transfer genes between one another via mobile genetic elements such as plasmids and bacteriophages. In this study, we used broiler chicken cecum as a model to investigate antibiotic resistance genes that can be transferred in vitro from cecal flora to Salmonella enterica serovar Heidelberg. We used whole-genome sequencing and resistome enrichment to decipher the interactions between S. Heidelberg, the gut microbiome, and acquired antibiotic resistance. After 48 h of incubation of ceca under microaerophilic conditions, we recovered one S. Heidelberg isolate with an acquired IncK2 plasmid (88 kb) carrying an extended-spectrum-β-lactamase gene (blaCMY-2). In vitro, this plasmid was transferable between Escherichia coli and S. Heidelberg strains but transfer was unsuccessful between S. Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with P1-like phages. This study contributes to our understanding of horizontal gene transfer between an important foodborne pathogen and the chicken gut microbiome. IMPORTANCE S. Heidelberg is a clinically important serovar, linked to foodborne illness and among the top 5 serovars isolated from poultry in the United States and Canada. Acquisition of new genetic material from the microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding of the interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. In this report, we show that the native flora in broiler ceca were capable of transferring mobile genetic elements carrying the AmpC β-lactamase (blaCMY-2) gene to an important foodborne pathogen, S. Heidelberg. The potential role for bacteriophage transduction is also discussed.

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E. coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid. These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro. Nine of these 13 isolates were found to be eaeA positive by PCR; four of these nine were eaeB positive. The 5 end, but not the 3 end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used. Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively. The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present. Only one canine E. coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test. The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement. However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract. Our results indicate that canine E. coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group.

Medical demands during the COVID-19 pandemic have triggered a grave shortage of medical-grade personal protective equipment (PPE), especially, N95 respirators. N95 respirators are critical for the personal protection of medical providers and others when being exposed to individuals with infections caused by the SARS-CoV-2 coronavirus. To address the shortage of N95 respirators, innovative methods are needed to decontaminate coronaviruses from N95 respirators, allowing them to be safely reused by healthcare workers. For this research, we use a commercial ozone disinfecting cabinet to examine the efficacy of ozone-based disinfection of a conservative surrogate virus for SARS-CoV-2, the MS2 bacteriophage. Treatment of mask materials with enhanced ozone treatment resulted in 2.38-log 10 (>99%) reduction of phage from household dust masks and a range of 1.43-log 10 (96.2%) to 4-log 10 (99.99%) reductions of phage from common N95 mask materials.