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Background Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. Results VRE isolates, including E. faecalis ( n  = 24), E. faecium ( n  = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium ( p  < 0.001) and E. faecalis ( p  < 0.001) and with the number of AMR genes in E. faecium ( p  = 0.005). Genes conferring vancomycin resistance, including van A and van M ( E. faecium ), van G ( E. faecalis ), and van C ( E. casseliflavus / E. gallinarum ), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium , E. faecalis , E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium , than E. casseliflavus and E. gallinarum . A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium . Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. Conclusions There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp .

Background Enterococcus is ubiquitous in nature and is a commensal of both the bovine and human gastrointestinal (GI) tract. It is also associated with clinical infections in humans. Subtherapeutic administration of antibiotics to cattle selects for antibiotic resistant enterococci in the bovine GI tract. Antibiotic resistance genes (ARGs) may be present in enterococci following antibiotic use in cattle. If located on mobile genetic elements (MGEs) their dissemination between Enterococcus species and to pathogenic bacteria may be promoted, reducing the efficacy of antibiotics. Results We present a comparative genomic analysis of twenty-one Enterococcus spp. isolated from bovine feces including Enterococcus hirae ( n  = 10), Enterococcus faecium ( n  = 3), Enterococcus villorum ( n  = 2), Enterococcus casseliflavus ( n  = 2), Enterococcus faecalis ( n  = 1), Enterococcus durans ( n  = 1), Enterococcus gallinarum ( n  = 1) and Enterococcus thailandicus ( n  = 1). The analysis revealed E. faecium and E. faecalis from bovine feces share features with human clinical isolates, including virulence factors. The Tn 917 transposon conferring macrolide-lincosamide-streptogramin B resistance was identified in both E. faecium and E. hirae , suggesting dissemination of ARGs on MGEs may occur in the bovine GI tract. An E. faecium isolate was also identified with two integrative conjugative elements (ICEs) belonging to the Tn 916 family of ICE, Tn 916 and Tn 5801 , both conferring tetracycline resistance. Conclusions This study confirms the presence of enterococci in the bovine GI tract possessing ARGs on MGEs, but the predominant species in cattle, E. hirae is not commonly associated with infections in humans. Analysis using additional complete genomes of E. faecium from the NCBI database demonstrated differential clustering of commensal and clinical isolates, suggesting that these strains may be specifically adapted to their respective environments.

To reduce the transmission risk of bovine spongiform encephalopathy prions (PrP BSE ), specified risk materials (SRM) that can harbour PrP BSE are prevented from entering the feed and food chains. As composting is one approach to disposing of SRM, we investigated the inactivation of PrP BSE in lab-scale composters over 28 days and in bin composters over 106–120 days. Lab-scale composting was conducted using 45 kg of feedlot manure with and without chicken feathers. Based on protein misfolding cyclic amplification (PMCA), after 28 days of composting, PrP BSE seeding activity was reduced by 3–4 log 10 with feathers and 3 log 10 without. Bin composters were constructed using ~ 2200 kg feedlot manure and repeated in 2017 and 2018. PMCA results showed that seeding activity of PrP BSE was reduced by 1–2 log 10 in the centre, but only by 1 log 10 in the bottom of bin composters. Subsequent assessment by transgenic (Tgbov XV) mouse bioassay confirmed a similar reduction in PrP BSE infectivity. Enrichment for proteolytic microorganisms through the addition of feathers to compost could enhance PrP BSE degradation. In addition to temperature, other factors including varying concentrations of PrP BSE and the nature of proteolytic microbial populations may be responsible for differential degradation of PrP BSE during composting.

Rapid and accurate diagnosis of bovine respiratory disease (BRD) presents a substantial challenge to the North American cattle industry. Here we utilize recombinase polymerase amplification (RPA), a fast and sensitive isothermal DNA-based technology for the detection of four BRD pathogens ( ), genes coding antimicrobial resistance (AMR) and integrative conjugative elements (ICE) which can harbor AMR genes. Eleven RPA assays were designed and validated including: a) one conventional species-specific multiplex assay targeting the 4 BRD pathogens, b) two species-specific real-time multiplex RPA assays targeting / and / , respectively with a novel competitive internal amplification control, c) seven conventional assays targeting AMR genes ( ), and d) one real-time assay targeting ICE. Each real-time RPA assay was tested on 100 deep nasopharyngeal swabs (DNPS) collected from feedlot cattle previously assessed for targets using either culture methods and/or polymerase chain reaction (PCR) verification (TC-PCR). The developed RPA assays enabled sensitive and accurate identification of BRD agents and AMR/ICE genes directly from DNPS, in a shorter period than TC-PCR, showing considerable promise as a tool for point-of-care identification of BRD pathogens and antimicrobial resistance genes.

NRC publication: Yes

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Bacillus anthracis Sterne strain spores were analyzed for their resistance against five disinfectants: commercial sodium hypochlorite, Spor-Klenz Ready-to-Use Cold Sterilant, Accelerated Hydrogen Peroxide (AHP), Virkon, and Surface Decontamination Foam (SDF). The aim of this study was to find an effective disinfectant that would reduce the viability of B. anthracis Sterne spores at ≥6 log in the presence of variables such as animal grease and fat, stainless steel, and temperature (room temperature and 4°C). SDF and 10% sodium hypochlorite consistently reduced the growth of viable B. anthracis Sterne spores after 5 min in the presence of stainless steel at room temperature. It took at least 10 min of contact time for AHP to consistently reduce spore growth by ≥6 log, while it took at least 20 min for 5% bleach and Spor-Klenz to consistently inactivate ≥6 log spores in the presence of stainless steel at room temperature. AHP was the only disinfectant that reduced the viability of B. anthracis Sterne spores at ≥6 log in the presence of stainless steel and animal grease, both at room temperature and 4°C after 24 h of contact time.

Real-time PCR has been used previously to detect Yersinia pestis; this study applies this rapid, specific, and sensitive nucleic acid-based method to the detection and quantitation of Y. pestis specifically in food. Five sets of primers and corresponding TaqMan dual-labelled fluorogenic hybridization probes for Y. pestis were designed and optimized for specificity testing using genomic DNA from 71 bacterial strains. Four Y. pestis-specific primer and probe sets were developed, based on the virulence plasmid targets, and used to distinguish this bacterium from the various Yersinia and other bacterial species tested. An additional primer and probe set, based on a chromosomal gene target, distinguished Y. pestis and Yersinia pseudotuberculosis from the various Yersinia and other bacterial species tested. With optimized conditions, the quantitative detection limit of the probes for Y. pestis pure cultures ranged from 13 to 220 CFU. Standard curves were generated for the probes and used to determine the amplification efficiencies. The primers and probes demonstrated high amplification efficiencies, and their performance was evaluated using spiked milk and ground beef samples. The quantitative detection limit was 10(1) to 10(3) CFU/ml in milk and 10(2) to 10(5) CFU/g in ground beef without any preenrichment step. Testing the hybridization probes on food samples demonstrated the detection of Y. pestis in a foodborne application; this is the first such report, to our knowledge.

ABSTRACTUsing a combination of Illumina paired-end sequencing, Pacific Biosciences RS II sequencing, and OpGen Argus whole-genome optical mapping, we report here the first complete genome sequence of Yersinia massiliensis. The completed genome consists of a 4.99-Mb chromosome, a 121-kb megaplasmid, and a 57-kb plasmid.

Here, we report the first draft genome sequence of Enterococcus thailandicus isolated from the feces of feedlot cattle in Southern Alberta.