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Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB, making it extremely difficult to predict nitrofurantoin resistance from whole-genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here, we report a WGS survey of 200 oqxAB-negative E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterized individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and NfsA/B enzyme assays in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, and A112T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E, and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D, and A188V in NfsA and G66D, M75I, V93A, and A174E in NfsB are functionally silent in this context. We identified that 9/166 (5.4%) nitrofurantoin-susceptible isolates were "pre-resistant," defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme assays and proteomics, we demonstrated that 9/34 (26.5%) ribE wild-type nitrofurantoin-resistant isolates also carried functionally wild-type nfsB or nfsB/nfsA. In these cases, NfsA/B activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis but is still insufficient to allow its reliable prediction from WGS data.

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Rates of fluoroquinolone resistance in Escherichia coli, a key opportunistic human pathogen, are problematic. Taking a One Health approach, we investigated the excretion of fluoroquinolone-resistant (FQ-R) E. coli by 600 dogs (303 from rural and 297 from urban environments) recruited from a 50 × 50 km region where we have also surveyed FQ-R E. coli from cattle and from human urine. FQ-R E. coli were detected in faeces from 7.3% (rural) and 11.8% (urban) of dogs. FQ-R E. coli from rural dogs tended to be of sequence types (STs) commonly excreted by cattle, whilst those from urban dogs tended to carry plasmid-mediated quinolone resistance genes, common in human E. coli in our study region. Phylogenetic evidence was obtained for sharing FQ-R E. coli - particularly for STs 10, 162 and 744 - between cattle, dogs and humans. Epidemiological analysis showed a strong association between feeding dogs uncooked meat and the excretion of FQ-R E. coli, particularly for STs 10, 162 and 744. This practice, therefore, could serve as a transmission link for FQ-R E. coli from farmed animals entering the home so we suggest that dogs fed uncooked meat should be handled and housed using enhanced hygiene practices.

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OBJECTIVES: Zoos are environments where species of highly valued animals are kept largely separated from others and the wider world. We report the molecular ecology of critically important antibiotic resistant (ABR) Escherichia coli carried by 28 mammalian species housed in a zoo located in an urban residential district. METHODS: Over 3 months we collected 167 faecal samples from captive mammals and processed for E. coli resistant to third-generation cephalosporins (3GC-R) and fluoroquinolones (FQ-R). Isolates were sequenced using Illumina. RESULTS: We identified high rates of faecal sample-level positivity, with 50%, 57% and 36% of mammalian species excreting 3GC-R, FQ-R or dual 3GC-R/FQ-R E. coli, respectively. Isolates represented multiple ST and ABR mechanisms; CTX-M-15 and CMY-2 dominated for 3GC-R, and target-site mutation caused 75% of FQ-R. We identified multiple examples of ABR E. coli transmission between mammalian species in separate enclosures, and a variant of the epidemic plasmid pCT within the zoo. There was no evidence for ABR E. coli leaving the zoo, based on comparative analysis with E. coli from humans, cattle and dogs isolated from the 50 × 50 km region in which the zoo is located. Amoxicillin/clavulanate was the most widely used antibiotic in the zoo, and we identified four widely disseminated amoxicillin/clavulanate resistance mechanisms, including a previously unreported inhibitor-resistant TEM, and the carbapenemase OXA-181. CONCLUSIONS: We conclude that the zoo studied here is a 'melting pot' for the selection and circulation of 3GC-R and FQ-R E. coli, but these circulating E. coli appear captive within the zoo.

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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.

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Coevolution between hosts and parasites is a major driver of rapid evolutionary change1 and diversification.2,3 However, direct antagonistic interactions between hosts and parasites could be disrupted4 when host microbiota form a line of defense, a phenomenon widespread across animal and plant species.5,6 By suppressing parasite infection, protective microbiota could reduce the need for host-based defenses and favor host support for microbiota colonization,6 raising the possibility that the microbiota can alter host-parasite coevolutionary patterns and processes.7 Here, using an experimental evolution approach, we co-passaged populations of nematode host (Caenorhabditis elegans) and parasites (Staphylococcus aureus) when hosts were colonized (or not) by protective bacteria (Enterococcus faecalis). We found that microbial protection during coevolution resulted in the evolution of host mortality tolerance-higher survival following parasite infection-and in parasites adapting to microbial defenses. Compared to unprotected host-parasite coevolution, the protected treatment was associated with reduced dominance of fluctuating selection dynamics in host populations. No differences in host recombination rate or genetic diversity were detected. Genomic divergence was observed between parasite populations coevolved in protected and unprotected hosts. These findings indicate that protective host microbiota can determine the evolution of host defense strategies and shape host-parasite coevolutionary dynamics.

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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.

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