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AbstractResistance to third generation cephalosporins and carbapenems in Gram-negative bacteria is chiefly mediated by beta-lactamases including ESBL, AmpC and carbapenemase enzymes. Routine phenotypic detection methods do not provide timely results, and there is a lack of comprehensive molecular panels covering all important markers.An ESBL/carbapenemase HRM assay (SHV, TEM, CTX-M ESBLs, and NDM, IMP, KPC, VIM and OXA-48-like carbapenemases) and an AmpC HRM assay (16S rDNA control, FOX, MOX, ACC, EBC, CIT and DHA) were designed, and evaluated on 111 Gram-negative isolates with mixed resistance patterns.The sensitivity for carbapenemase, ESBL and AmpC genes was 96.7% (95%CI:82.8-99.9%), 93.6% (95%CI:85.7-97.9%) and 93.8% (95%CI:82.8-98.7%), respectively with a specificity of 100% (95%CI:95.6-100%), 93.9% (95%CI:79.8-99.3%) and 93.7% (95%CI:84.5-98.2%).The HRM assays enable the simultaneous detection of the fourteen most important ESBL, carbapenemase and AmpC genes and could be used as a molecular surveillance tool or to hasten detection of AMR for treatment management.

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Objectives To compare faecal third-generation cephalosporin-resistant (3GC-R) Escherichia coli isolates from dogs living in a city and in a rural area ∼30 km away; to compare isolates from dogs, cattle and humans in these regions; and to determine risk factors associated with 3GC-R E. coli carriage in these two cohorts of dogs. Methods Six hundred dogs were included, with faecal samples processed to recover 3GC-R E. coli using 2 mg/L cefotaxime. WGS was by Illumina and risk factor analyses were by multivariable linear regression using the results of an owner-completed survey. Results 3GC-R E. coli were excreted by 20/303 rural and 31/297 urban dogs. The dominant canine 3GC-R ST was ST963 (blaCMY-2), which also accounted for 25% of CMY-2-producing E. coli in humans. Phylogenetic overlap between cattle and rural dog CTX-M-14-producing E. coli ST117 was observed as well as acquisition of pMOO-32-positive E. coli ST10 by a rural dog, a plasmid common on cattle farms in the area. Feeding raw meat was associated with carrying 3GC-R E. coli in rural dogs, but not in urban dogs, where swimming in rivers was a weak risk factor. Conclusions Given clear zoonotic potential for resistant canine E. coli, our work suggests interventions that may reduce this threat. In rural dogs, carriage of 3GC-R E. coli, particularly CTX-M producers, was phylogenetically associated with interaction with local cattle and epidemiologically associated with feeding raw meat. In urban dogs, sources of 3GC-R E. coli appear to be more varied and include environments such as rivers.

Background: Bacteria adapted to live within animals can protect their hosts against harmful infections. Beyond antagonism with pathogens, a 'defensive' bacterial symbiont could engage in additional interactions with other colonizing microorganisms. A single bacterium might thus have cascading ecological impacts on the whole microbiome that are rarely investigated. Here, we assess the role of a defensive symbiont as a driver of hostassociated microbiota composition by using a bacterial species (Enterococcus faecalis) that was previously experimentally adapted to a nematode host model (Caenorhabditis elegans). Results: An analysis of 16S rRNA data from C. elegans exposed to E. faecalis and subsequently reared in soil, reveal that symbiont adaptation to host environment or its protective potential had minimal impact on microbiota diversity. Whilst the abundance of Pseudomonas was higher in the microbiota of hosts with protective E.faecalis (and another protective species tested), a few other generaincluding Serratia and Salinisporawere less abundant in hosts colonized by all E. faecalis strains. In addition, the protective effect of E. faecalis against virulent Staphylococcus aureus pathogens was maintained despite multi-species interactions within the microbiota. Conclusions: Our results reveal the degree to which a new, evolving symbiont can colonise and maintain pathogen-resistance with minimal disruption to host microbiota diversity.

Aims: To investigate whether on-farm antibacterial usage (ABU), environmental antibacterial-resistant (ABR) Escherichia coli prevalence, sampling and sample handling methodologies are associated with ABR E. coli positivity in individual faecal samples from dairy heifers. Methods and Results: Three hundred and sixty-four heifers from 37 farms were sampled via rectal or faecal pat sampling. Samples were stored at −80°C for variable periods before microbiological analysis. Data analysis was done through a multilevel, multivariable logistic regression approach. Individual rectal samples had increased odds of positivity for amoxicillin-, cefalexin-and tetracycline-resistant E. coli. Sample storage for 6-12 months was associated with decreased odds of finding amoxicillin-and tetracycline-resistant E. coli. On-farm ABU had little influence, and environmental ABR E. coli prevalence had no significant influence on the odds of sample-level positivity for ABR E. coli.Conclusions: Sampling methodology and sample handling have a greater association than on-farm factors with the detection of ABR E. coli in individual faecal samples from dairy heifers.Significance and Impact of the Study: Sampling and storage methodologies should be considered carefully at the point of designing ABR surveillance studies in livestock and their environments and, where possible, these methodologies should be standardized between and within future studies.

SynopsisObjectivesOur aims were to compare faecal third-generation cephalosporin-resistant (3GC-R) Escherichia coli isolates from dogs living in a city and in a rural area ~30 km away; to compare isolates from dogs, cattle, and humans in these regions; to determine risk factors associated with 3GC-R E. coli carriage in these two cohorts of dogs.Methods600 dogs were included, with faecal samples processed to recover 3GC-R E. coli using 2 mg/L cefotaxime. WGS was by Illumina; risk factor analyses were multivariable linear regression using the results of an owner-completed survey.Results3GC-R E. coli were excreted by 20/303 rural and 31/297 urban dogs. Dog/human sharing was evident for the dominant canine 3GC-R sequence type, ST963(blaCMY-2). Cattle/dog sharing was evident for CTX-M-14 and CTX-M-32-producing E. coli from rural dogs, including sharing of plasmid pMOO-32, which is common on cattle farms in the area. Feeding raw meat was associated with carrying 3GC-R E. coli in rural dogs, but not in urban dogs, where swimming in rivers was a weak risk factor.ConclusionsGiven clear zoonotic potential for resistant canine E. coli, our work suggests interventions that may reduce this threat. In rural dogs, carriage of 3GC-R E. coli, particularly CTX-M producers, was phylogenetically associated with interaction with local cattle and epidemiologically associated with feeding raw meat. In urban dogs, sources of 3GC-R E. coli appear to be more varied and include environments such as rivers.

Background: Bacteria adapted to live within animals can protect their hosts against harmful infections. Beyond antagonism with pathogens, a ‘defensive’ bacterial symbiont could engage in additional interactions with other colonizing micro-organisms. A single bacterium might thus have cascading ecological impacts on the whole microbiome that are rarely investigated. Here, we assess the role of a defensive symbiont as a driver of host-associated microbiota composition by using a bacterial species (Enterococcus faecalis) that was previously experimentally adapted (Enterococcus faecalis) to a nematode host model (Caenorhabditis elegans). Results: An analysis of 16S rRNA data from C. elegans exposed to E. faecalis and subsequently reared in soil, reveal that symbiont adaptation to host environment or its protective potential had minimal impact on microbiota diversity. Whilst the abundance of Pseudomonas was higher in the microbiota of hosts with protective E.faecalis (and another protective species tested), three other genera – Serratia, Klebsiella and Salinispora – were less abundant in hosts colonized by all E. faecalis strains. In addition, the protective effect of E. faecalis against opportunistic Staphylococcus aureus pathogens was maintained despite multi-species interactions within the microbiota. Conclusions: Our results reveal the degree to which a new, evolving symbiont can colonise and maintain its conferred phenotype (i.e., pathogen-resistance) with minimal disruption to the host microbiota diversity.