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For a One-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Whole-genome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. Phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. For E. faecium from cattle, resistance to β-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. Clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all β-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.

Several areas of the world suffer a notably high incidence of Shiga toxin-producing Escherichia coli . To assess the impact of persistent cross-species transmission systems on the epidemiology of E. coli O157:H7 in Alberta, Canada, we sequenced and assembled E. coli O157:H7 isolates originating from collocated cattle and human populations, 2007–2015. We constructed a timed phylogeny using BEAST2 using a structured coalescent model. We then extended the tree with human isolates through 2019 to assess the long-term disease impact of locally persistent lineages. During 2007–2015, we estimated that 88.5% of human lineages arose from cattle lineages. We identified 11 persistent lineages local to Alberta, which were associated with 38.0% (95% CI 29.3%, 47.3%) of human isolates. During the later period, six locally persistent lineages continued to be associated with human illness, including 74.7% (95% CI 68.3%, 80.3%) of reported cases in 2018 and 2019. Our study identified multiple locally evolving lineages transmitted between cattle and humans persistently associated with E. coli O157:H7 illnesses for up to 13 y. Locally persistent lineages may be a principal cause of the high incidence of E. coli O157:H7 in locations such as Alberta and provide opportunities for focused control efforts.

Background Plasmids play a major role in the transfer of antimicrobial resistance (AMR) genes among bacteria via horizontal gene transfer. The identification of plasmids in short-read assemblies is a challenging problem and a very active research area. Plasmid binning aims at detecting, in a draft genome assembly, groups (bins) of contigs likely to originate from the same plasmid. Several methods for plasmid binning have been developed recently, such as PlasBin-flow, HyAsP, gplas, MOB-suite, and plasmidSPAdes. This motivates the problem of evaluating the performances of plasmid binning methods, either against a given ground truth or between them. Results We describe PlasEval, a novel method aimed at comparing the results of plasmid binning tools. PlasEval computes a dissimilarity measure between two sets of plasmid bins, that can originate either from two plasmid binning tools, or from a plasmid binning tool and a ground truth set of plasmid bins. The PlasEval dissimilarity accounts for the contig content of plasmid bins, the length of contigs and is repeat-aware. Moreover, the dissimilarity score computed by PlasEval is broken down into several parts, that allows to understand qualitative differences between the compared sets of plasmid bins. We illustrate the use of PlasEval by benchmarking four recently developed plasmid binning tools—PlasBin-flow, HyAsP, gplas, and MOB-recon—on a data set of 53 E. coli bacterial genomes. Conclusion Analysis of the results of plasmid binning methods using PlasEval shows that their behaviour varies significantly. PlasEval can be used to decide which specific plasmid binning method should be used for a specific dataset. The disagreement between different methods also suggests that the problem of plasmid binning on short-read contigs requires further research. We believe that PlasEval can prove to be an effective tool in this regard. PlasEval is publicly available at https://github.com/acme92/PlasEval

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.

The biocide triclosan is in many consumer products and is a frequent contaminant of wastewater (WW) such that there is concern that triclosan promotes resistance to important antibiotics. This study identified functional mechanisms of triclosan resistance (TCSR) in WW metagenomes, and assessed the frequency of TCSR in WW-derived and clinical isolates of Escherichia coli and Enterococcus spp. Metagenomic DNA extracted from WW was used to profile the microbiome and construct large-insert cosmid libraries, which were screened for TCSR. Resistant cosmids were sequenced and the TCSR determinant identified by transposon mutagenesis. Wastewater Enterococcus spp. (N = 94) and E. coli (N = 99) and clinical Enterococcus spp. (N = 146) and vancomycin-resistant E. faecium (VRE; N = 149) were collected and tested for resistance to triclosan and a comprehensive drug panel. Functional metagenomic screening revealed diverse FabV homologs as major WW TCSR determinants. Resistant clones harboured sequences likely originating from Aeromonas spp., a common WW microbe. The triclosan MIC90s for E. coli, E. faecalis, and E. faecium isolates were 0.125, 32, and 32 mg/L, respectively. For E. coli, there was no correlation between the triclosan MIC and any drug tested. Negative correlations were detected between the triclosan MIC and levofloxacin resistance for E. faecalis, and between triclosan and vancomycin, teicoplanin, and ampicillin resistance for E. faecium. Thus, FabV homologs were the major contributor to the WW triclosan resistome and high-level TCSR was not observed in WW or clinical isolates. Elevated triclosan MICs were not positively correlated with antimicrobial resistance to any drug tested.

Macrolides are crucial for the management and treatment of bovine respiratory disease (BRD). However, antimicrobial resistance (AMR) threatens the efficacy of these and other antimicrobials. We developed real-time recombinase polymerase amplification (RPA) assays targeting three clinically relevant macrolide antimicrobial resistance genes (ARGs)—msrE-mphE and erm42—in ≤30 min using extracted DNA. A set of 199 deep nasopharyngeal swabs (DNPS) collected from feedlot calves near the time of arrival were selected based on bacterial culture (BC) results for Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni and antimicrobial susceptibility testing (AST) for tulathromycin, tilmicosin, tildipirosin, or gamithromycin. Samples were also tested for the same targets using RPA and polymerase chain reaction (PCR). In samples that were culture-positive for one or more macrolide-resistant BRD-associated bacteria (n = 101), msrE-mphE and/or erm42 were detected in 95% of cases using RPA. The remaining 98 samples were either culture-negative, or the recovered bacteria were macrolide-susceptible: 43% of these were RPA-positive for at least one macrolide ARG. Together with BC-AST and PCR, Bayesian latent class modelling estimated the clinical sensitivity of RPA for macrolide ARGs to be 95% and specificity to be 58%, with moderate agreement between RPA and BC-AST (κ = 0.52) or PCR (κ = 0.55). The estimated sensitivity of the RPA multiplex assay for the targeted macrolide ARGs was very good, although estimated specificity was limited. However, Sanger sequencing confirmed RPA detection of msrE-mphE in BC-AST/PCR-negative samples (n = 23), reflecting the presence of this locus in non-target bacteria, as well as potential ARG variants among BRD bacteria. These findings support the potential of RPA for rapid ARG detection from extracted DNA. Continued assay optimization and evaluation for detection of respiratory bacteria and ARGs will further enhance its diagnostic utility.

Foodborne illness is exacerbated by novel and emerging pathotypes, persistent contamination, antimicrobial resistance, an ever-changing environment, and the complexity of food production systems. Sporadic and outbreak events of common foodborne pathogens like Shiga toxigenic E. coli (STEC), Salmonella, Campylobacter, and Listeria monocytogenes are increasingly identified. Methods of controlling human infections linked with food products are essential to improve food safety and public health and to avoid economic losses associated with contaminated food product recalls and litigations. Bacteriophages (phages) are an attractive additional weapon in the ongoing search for preventative measures to improve food safety and public health. However, like all other antimicrobial interventions that are being employed in food production systems, phages are not a panacea to all food safety challenges. Therefore, while phage-based biocontrol can be promising in combating foodborne pathogens, their antibacterial spectrum is generally narrower than most antibiotics. The emergence of phage-insensitive single-cell variants and the formulation of effective cocktails are some of the challenges faced by phage-based biocontrol methods. This review examines phage-based applications at critical control points in food production systems with an emphasis on when and where they can be successfully applied at production and processing levels. Shortcomings associated with phage-based control measures are outlined together with strategies that can be applied to improve phage utility for current and future applications in food safety.

Enterococcus species are used as One Health indicators of antimicrobial resistance (AMR) in humans, animals, and the environment. A surveillance study in beef cows and calves isolated Enterococcus casseliflavus along with E. faecium, E. faecalis, and E. hirae. Given the high prevalence of E. casseliflavus, we elected to characterize this species to better understand its role in the antimicrobial resistance of enterococci in cows and calves. Almost 12% of E. casseliflavus isolates exhibited multidrug resistance with the majority being resistant to lincomycin (99%), followed by quinupristin–dalfopristin (34%), ciprofloxacin (9.6%), tylosin (4.5%), erythromycin (2.7%), tetracycline (1.8%), tigecycline (1.5%), daptomycin (0.6%), streptomycin (0.3%), and kanamycin (0.3%). All E. casseliflavus were susceptible to chloramphenicol, penicillin, streptomycin, nitrofurantoin, gentamicin, and linezolid. Whole genome antimicrobial resistance gene profiling identified vanC-type intrinsic vancomycin resistance genes in all E. casseliflavus, with the vanC4XYT gene cluster being dominant (67%) followed by vanC2XYT (31%) and vanC3XYT (1.5%). Resistance genes for erythromycin (ermB) and tetracycline (tetM) were rarely identified (2.1% and 1.2%, respectively) within E. casseliflavus genomes. No resistance genes were identified to explain either the quinupristin–dalfopristin or ciprofloxacin resistance in these isolates. A core genome phylogenetic tree revealed two clades that exhibited no distinct association with the age of the host, time of sample collection, or the farm sampled. The open nature of the E. casseliflavus pan-genome highlighted its intraspecies diversity. These findings suggest that E. casseliflavus is likely a low-risk species in terms of contributing to antimicrobial resistance in the cow–calf sector.

Numerous antimicrobial resistance (AMR) surveillance studies have been conducted in North American feedlot cattle to investigate the major bacterial pathogens of the bovine respiratory disease (BRD) complex, specifically: Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. While most bacterial isolates recovered from healthy cattle are susceptible to a repertoire of antimicrobials, multidrug resistance is common in isolates recovered from cattle suffering from BRD. Integrative and conjugative elements (ICE) have gained increasing notoriety in BRD-Pasteurellaceae as they appear to play a key role in the concentration and dissemination of antimicrobial resistant genes. Likewise, low macrolide susceptibility has been described in feedlot isolates of M. bovis. Horizontal gene transfer has also been implicated in the spread of AMR within mycoplasmas, and in-vitro experiments have shown that exposure to antimicrobials can generate high levels of resistance in mycoplasmas via a single conjugative event. Consequently, antimicrobial use (AMU) could be accelerating AMR horizontal transfer within all members of the bacterial BRD complex. While metagenomics has been applied to the study of AMR in the microbiota of the respiratory tract, the potential role of the respiratory tract microbiome as an AMR reservoir remains uncertain. Current and prospective molecular tools to survey and characterize AMR need to be adapted as point-of-care technologies to enhance prudent AMU in the beef industry.