Molecular ecology and risk factors for third-generation cephalosporin-resistant Escherichia coli carriage by dogs living in urban and nearby rural settings.

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.

Evidence that faecal carriage of resistant Escherichia coli by 16-week-old dogs in the United Kingdom is associated with raw feeding.

We report a survey (August 2017 to March 2018) and risk factor analysis of faecal carriage of antibacterial-resistant (ABR) Escherichia coli in 223 16-week-old dogs in the United Kingdom. Raw feeding was associated with the presence of fluoroquinolone-resistant (FQ-R) E. coli and those resistant to tetracycline, amoxicillin, and streptomycin, but not to cefalexin. Whole genome sequencing of 36 FQ-R E. coli isolates showed a wide range of sequence types (STs), with almost exclusively mutational FQ-R dominated by ST744 and ST162. Comparisons between E. coli isolates from puppies known to be located within a 50 × 50 km region with those isolated from human urinary tract infections (isolated in parallel in the same region) identified an ST744 FQ-R lineage that was carried by one puppy and caused one urinary tract infection. Accordingly, we conclude that raw feeding is associated with carriage of ABR E. coli in dogs even at 16 weeks of age and that bacteria carried by puppies are shared with humans. We therefore suggest that those who feed their dogs raw meat seriously consider the potential ABR-transmission threat their pet may become as a result and deploy appropriate hygiene practices in mitigation.

Factors influencing the detection of antibacterial-resistant Escherichia coli in faecal samples from individual cattle.

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.

Limited phylogenetic overlap between fluoroquinolone-resistant Escherichia coli isolated on dairy farms and those causing bacteriuria in humans living in the same geographical region.

BACKGROUND: Our primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms are closely phylogenetically related to those causing bacteriuria in humans living in the same 50 × 50 km geographical region suggestive of farm-human sharing. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms. METHODS: FQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularization approach. RESULTS: Of 489 FQ-R human isolates, 255 were also third-generation-cephalosporin-resistant, with strong genetic linkage between aac(6')Ib-cr and blaCTX-M-15. We identified possible farm-human sharing for pairs of ST744 and ST162 isolates, but minimal core genome SNP distances were larger between farm-human pairs of ST744 and ST162 isolates (71 and 63 SNPs, respectively) than between pairs of isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli, while total dry cow therapy use showed a negative association. CONCLUSIONS: This work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm.

Reduced Antibacterial Drug Resistance and blaCTX-M ß-Lactamase Gene Carriage in Cattle-Associated Escherichia coli at Low Temperatures, at Sites Dominated by Older Animals, and on Pastureland: Implications for Surveillance.

Little is known about the drivers of critically important antibacterial resistance in species with zoonotic potential present on farms (e.g., CTX-M ß-lactamase-positive Escherichia coli). We collected samples monthly between January 2017 and December 2018 on 53 dairy farms in South West England, along with data for 610 variables concerning antibacterial usage, management practices, and meteorological factors. We detected E. coli resistant to amoxicillin, ciprofloxacin, streptomycin, and tetracycline in 2,754/4,145 (66%), 263/4,145 (6%), 1,475/4,145 (36%), and 2,874/4,145 (69%), respectively, of samples from fecally contaminated on-farm and near-farm sites. E. coli positive for blaCTX-M were detected in 224/4,145 (5.4%) of samples. Multilevel, multivariable logistic regression showed antibacterial dry cow therapeutic choice (including use of cefquinome or framycetin) to be associated with higher odds of blaCTX-M positivity. Low average monthly ambient temperature was associated with lower odds of blaCTX-ME. coli positivity in samples and with lower odds of finding E. coli resistant to each of the four test antibacterials. This was in addition to the effect of temperature on total E. coli density. Furthermore, samples collected close to calves had higher odds of having E. coli resistant to each antibacterial, as well as E. coli positive for blaCTX-M Samples collected on pastureland had lower odds of having E. coli resistant to amoxicillin or tetracycline, as well as lower odds of being positive for blaCTX-MIMPORTANCE Antibacterial resistance poses a significant threat to human and animal health and global food security. Surveillance for resistance on farms is important for many reasons, including tracking impacts of interventions aimed at reducing the prevalence of resistance. In this longitudinal survey of dairy farm antibacterial resistance, we showed that local temperature-as it changes over the course of a year-was associated with the prevalence of antibacterial-resistant E. coli We also showed that prevalence of resistant E. coli was lower on pastureland and higher in environments inhabited by young animals. These findings have profound implications for routine surveillance and for surveys carried out for research. They provide important evidence that sampling at a single time point and/or single location on a farm is unlikely to be adequate to accurately determine the status of the farm regarding the presence of samples containing resistant E. coli.

Molecular Epidemiology of Escherichia coli Producing CTX-M and pAmpC ß-Lactamases from Dairy Farms Identifies a Dominant Plasmid Encoding CTX-M-32 but No Evidence for Transmission to Humans in the Same Geographical Region.

Third-generation cephalosporin resistance (3GC-R) in Escherichia coli is a rising problem in human and farmed-animal populations. We conducted whole-genome sequencing analysis of 138 representative 3GC-R isolates previously collected from dairy farms in southwest England and confirmed by PCR to carry acquired 3GC-R genes. This analysis identified blaCTX-M (131 isolates encoding CTX-M-1, -14, -15, -and 32 and the novel variant CTX-M-214), blaCMY-2 (6 isolates), and blaDHA-1 (1 isolate). A highly conserved plasmid was identified in 73 isolates, representing 27 E. coli sequence types. This novel ∼220-kb IncHI2 plasmid carrying blaCTX-M-32 was sequenced to closure and designated pMOO-32. It was found experimentally to be stable in cattle and human transconjugant E. coli even in the absence of selective pressure and was found by multiplex PCR to be present on 26 study farms representing a remarkable range of transmission over 1,500 square kilometers. However, the plasmid was not found among human urinary E. coli isolates we recently characterized from people living in the same geographical location, collected in parallel with farm sampling. There were close relatives of two blaCTX-M plasmids circulating among eight human and two cattle isolates, and a closely related blaCMY-2 plasmid was found in one cattle and one human isolate. However, phylogenetic evidence of recent sharing of 3GC-R strains between farms and humans in the same region was not found.IMPORTANCE Third-generation cephalosporins (3GCs) are critically important antibacterials, and 3GC resistance (3GC-R) threatens human health, particularly in the context of opportunistic pathogens such as Escherichia coli There is some evidence for zoonotic transmission of 3GC-R E. coli through food, but little work has been done examining possible transmission via interaction of people with the local near-farm environment. We characterized acquired 3GC-R E. coli found on dairy farms in a geographically restricted region of the United Kingdom and compared these with E. coli from people living in the same region, collected in parallel. While there is strong evidence for recent farm-to-farm transmission of 3GC-R strains and plasmids-including one epidemic plasmid that has a remarkable capacity to be transmitted-there was no evidence that 3GC-R E. coli found on study farms had a significant impact on circulating 3GC-R E. coli strains or plasmids in the local human population.

Characterization of AmpC-hyperproducing Escherichia coli from humans and dairy farms collected in parallel in the same geographical region.

OBJECTIVES: To characterize putative AmpC-hyperproducing third-generation cephalosporin-resistant E. coli from dairy farms and their phylogenetic relationships; to identify risk factors for their presence; and to assess evidence for their zoonotic transmission into the local human population. METHODS: Proteomics was used to explain differences in antimicrobial susceptibility. WGS allowed phylogenetic analysis. Multilevel, multivariable logistic regression modelling was used to identify risk factors. RESULTS: Increased use of amoxicillin/clavulanate was associated with an increased risk of finding AmpC hyperproducers on farms. Expansion of cephalosporin resistance in AmpC hyperproducers was seen in farm isolates with marR mutations (conferring cefoperazone resistance) or when AmpC was mutated (conferring fourth-generation cephalosporin and cefoperazone resistance). Phylogenetic analysis confirmed the dominance of ST88 amongst farm AmpC hyperproducers but there was no evidence for acquisition of farm isolates by members of the local human population. CONCLUSIONS: Clear evidence was found for recent farm-to-farm transmission of AmpC-hyperproducing E. coli and of adaptive mutations to expand resistance. Whilst there was no evidence of isolates entering the local human population, efforts to reduce third-generation cephalosporin resistance on dairy farms must address the high prevalence of AmpC hyperproducers. The finding that amoxicillin/clavulanate use was associated with an increased risk of finding AmpC hyperproducers is important because this is not currently categorized as a highest-priority critically important antimicrobial and so is not currently targeted for specific usage restrictions in the UK.

Characterization of cefotaxime-resistant urinary Escherichia coli from primary care in South-West England 2017-18.

OBJECTIVES: Third-generation cephalosporin-resistant Escherichia coli from community-acquired urinary tract infections are increasingly reported worldwide. We sought to determine and characterize the mechanisms of cefotaxime resistance employed by urinary E. coli obtained from primary care, over 12 months, in Bristol and surrounding counties in South-West England. METHODS: Cefalexin-resistant E. coli isolates were identified from GP-referred urine samples using disc susceptibility testing. Cefotaxime resistance was determined by subsequent plating onto MIC breakpoint plates. ß-Lactamase genes were detected by PCR. WGS was performed on 225 isolates and analyses were performed using the Center for Genomic Epidemiology platform. Patient information provided by the referring general practices was reviewed. RESULTS: Cefalexin-resistant E. coli (n=900) isolates were obtained from urines from 146 general practices. Following deduplication by patient approximately 69% (576/836) of isolates were cefotaxime resistant. WGS of 225 isolates identified that the most common cefotaxime-resistance mechanism was blaCTX-M carriage (185/225), followed by plasmid-mediated AmpCs (pAmpCs) (17/225), AmpC hyperproduction (13/225), ESBL blaSHV variants (6/225) or a combination of both blaCTX-M and pAmpC (4/225). Forty-four STs were identified, with ST131 representing 101/225 isolates, within which clade C2 was dominant (54/101). Ciprofloxacin resistance was observed in 128/225 (56.9%) of sequenced isolates, predominantly associated with fluoroquinolone-resistant clones ST131 and ST1193. CONCLUSIONS: Most cefalexin-resistant E. coli isolates were cefotaxime resistant, predominantly caused by blaCTX-M carriage. The correlation between cefotaxime resistance and ciprofloxacin resistance was largely attributable to the high-risk pandemic clones ST131 and ST1193. Localized epidemiological data provide greater resolution than regional data and can be valuable for informing treatment choices in the primary care setting.

Coordinate hyperproduction of SmeZ and SmeJK efflux pumps extends drug resistance in Stenotrophomonas maltophilia.

A Stenotrophomonas maltophilia mutant that coordinately hyper-expresses three resistance nodulation division-type efflux pump genes, smeZ, smeJ, and smeK, has been identified. SmeZ is responsible for elevating aminoglycoside MICs; SmeJ and SmeK are jointly responsible for elevating tetracycline, minocycline, and ciprofloxacin MICs and conferring levofloxacin resistance. One clinical isolate with this same phenotype was identified from a sample of six, and the isolate also coordinately hyper-expresses smeZ and smeJK, confirming the clinical relevance of our findings.

Long-term patient-reported outcomes after total hip replacement: comparison to the general population.

Hip replacement is one of the most common elective procedures performed in the NHS, with patients generally reporting good mid-term outcomes. However, patient-reported long-term outcomes have been less well studied. The aim of this study was to explore the extent of variation in long-term patient reported outcomes after total hip replacement (THR), and to compare outcomes to a control population without THR. All patients who had undergone primary THR at one centre 12-16 years ago and who had previously completed an Oxford hip score (OHS) 5-8 years post-operatively were invited to complete a postal OHS. Participants in the control group who had not undergone hip or knee replacement also completed an OHS. The Oxford hip score (OHS) was completed by 407 THR patients and 927 controls. The median score of 18 for the THR patients was significantly worse than the median score of 12 for the control group (p <0.001). Similar results were found when comparisons were stratified by age. There was considerable variation in change in OHS from 5-8 years to 12-16 years post-operatively, although a significant worsening in outcome was found only in patients over 80 years old. Patients continue to report good functional outcomes at 12-16 years after THR, although function is signficantly worse than the general population who have not undergone THR.

Determinants of fracture risk in a UK-population-based cohort of older women: a cross-sectional analysis of the Cohort for Skeletal Health in Bristol and Avon (COSHIBA).

BACKGROUND: Identification of individuals with high fracture risk from within primary care is complex. It is likely that the true contribution of falls to fracture risk is underestimated. METHODS: Cross-sectional analysis of a population-based cohort of 3,200 post-menopausal women aged 73 ± 4 years. Self-reported data were collected on fracture, osteoporosis clinical risk factors and falls/mobility risk factors. Self-reported falls were compared with recorded falls on GP computerised records. Multivariable logistic regression was used to identify independent risk factors for fracture. RESULTS: A total of 838 (26.2%) reported a fracture after aged 50; 441 reported falling more than once per year, but 69% of these had no mention of falls on their computerised GP records. Only age [odds ratios (OR): 1.37 per 5 year increase, 95% confidence interval (CI): 1.23-1.53], height (1.02 per cm increase, 95% CI: 1.01-1.04), weight (OR: 0.99 per kg increase, 95% CI: 0.98-0.99) and falls (OR: 1.49 for more than once per year compared with less, 95% CI: 1.13-1.94) were independent risk factors for fracture. Falls had the strongest association. CONCLUSION: When identifying individuals with high fracture risk we estimate that more than one fall per year is at least twice as important as height and weight. Furthermore, using self-reported falls data is essential as computerised GP records underestimate falls prevalence.

Patient-reported history of leg ulceration 12-16 years after total primary knee or hip replacement.

BACKGROUND AND PURPOSE: Deep vein thrombosis is common after total joint replacement. It is frequently asymptomatic, and it is unclear whether this leads to longer-term problems such as post-thrombotic syndrome and leg ulceration. We investigated whether the postoperative prevalence of ulceration in patients who had undergone primary total hip replacement (THR) or total knee replacement (TKR) was higher than that found in a control group who had not undergone total joint replacement. METHODS: The study group consisted of patients who had undergone THR or TKR at one orthopedic center 12-16 years previously without routine chemothromboprophylaxis, and who had not undergone revision surgery. The control group was recruited via primary care. All participants were recruited by post and asked to complete a questionnaire. Age- and sex-adjusted prevalence of self-reported leg ulceration was calculated, and logistic regression was used to determine whether there were any associations between THR or TKR and leg ulceration. RESULTS: Completed questionnaires were received from 441 THR patients (54% response rate), 196 TKR patients (48%) and 967 control participants (36%). No statistically significant differences in age- and sex-adjusted prevalence of ulceration were found between the groups, for either lifetime prevalence or prevalence over the previous 15 years. INTERPRETATION: Patients who undergo THR and TKR without chemothromboprophylaxis are unlikely to be at a higher risk of long-term venous ulceration than the normal population.

The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants.

BACKGROUND: Stenotrophomonas maltophilia is a nosocomial opportunistic pathogen of the Xanthomonadaceae. The organism has been isolated from both clinical and soil environments in addition to the sputum of cystic fibrosis patients and the immunocompromised. Whilst relatively distant phylogenetically, the closest sequenced relatives of S. maltophilia are the plant pathogenic xanthomonads. RESULTS: The genome of the bacteremia-associated isolate S. maltophilia K279a is 4,851,126 bp and of high G+C content. The sequence reveals an organism with a remarkable capacity for drug and heavy metal resistance. In addition to a number of genes conferring resistance to antimicrobial drugs of different classes via alternative mechanisms, nine resistance-nodulation-division (RND)-type putative antimicrobial efflux systems are present. Functional genomic analysis confirms a role in drug resistance for several of the novel RND efflux pumps. S. maltophilia possesses potentially mobile regions of DNA and encodes a number of pili and fimbriae likely to be involved in adhesion and biofilm formation that may also contribute to increased antimicrobial drug resistance. CONCLUSION: The panoply of antimicrobial drug resistance genes and mobile genetic elements found suggests that the organism can act as a reservoir of antimicrobial drug resistance determinants in a clinical environment, which is an issue of considerable concern.

nalD encodes a second repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa.

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.

SmeDEF-mediated antimicrobial drug resistance in Stenotrophomonas maltophilia clinical isolates having defined phylogenetic relationships.

OBJECTIVES: To test whether smeDEF overexpression leads to a predictable multi-drug resistance phenotype in Stenotrophomonas maltophilia and to measure the frequency with which smeDEF overexpression occurs in clinical isolates and in spontaneous drug-resistant mutants. METHODS: Overexpression of smeDEF was induced in clinical isolates by the introduction of chromosomal mutations in smeT using a gene-replacement approach. Spontaneous drug-resistant mutants were selected using greater than MIC concentrations of various antimicrobial agents. Levels of smeE and smeF mRNAs were quantified using RT-PCR; MICs were determined using Etest. RESULTS: Of 20 spontaneous S. maltophilia drug-resistant mutants tested, four overexpressed smeDEF, but only two carried mutations within smeT. Of 30 clinical isolates tested, 6 significantly overexpressed smeDEF. One of these had an IS1246-like element embedded within the putative SmeT binding site in the smeDEF promoter. All smeDEF overexpressing derivatives of an isolate had the same resistance profile; derivatives that did not overexpress smeDEF did not share this resistance profile. However, no consistent phenotype could be associated with smeDEF overexpression in S. maltophilia isolates per se. CONCLUSIONS: SmeT is not the only gene product that affects smeDEF expression. IS element insertion is a viable mechanism by which smeDEF expression can be derepressed. There is evidence for a background-specific, predictable effect on resistance profile when smeDEF is overexpressed, but the variability of backgrounds encountered means no general SmeDEF-mediated phenotype can be defined. There is strong evidence for the existence of as yet unidentified multi-drug efflux pumps in this species.

Beta-lactam resistance and beta-lactamase expression in clinical Stenotrophomonas maltophilia isolates having defined phylogenetic relationships.

AIMS: To test the hypothesis that Stenotrophomonas maltophilia isolates from certain phylogenetic groups have predictable beta-lactamase expression and beta-lactam resistance profiles. METHODS: Isolates were grouped using sequences of the 16S rRNA gene and smeT-smeD intergenic region. beta-Lactamase activities in cell extracts were quantified spectrophotometrically and beta-lactam MICs were determined using agar dilution methodology and Etest as appropriate. RESULTS: A collection of 50 clinical S. maltophilia isolates from Europe and North, South and Central America were phylogenetically grouped. Group 'A' (22 out of 50) includes remarkably genetically homogeneous isolates; group 'B' (17 out of 50) includes isolates that are genetically heterogeneous and quite distinct from those of group A. Members of these two groups are, however, indistinguishable in terms of their beta-lactam resistance and beta-lactamase expression phenotypes. In contrast, isolates from group 'C', which are less common (8 out of 50), are considerably more susceptible to beta-lactams owing to reduced inducibility of beta-lactamase expression following beta-lactam challenge. CONCLUSIONS: The majority of S. maltophilia clinical isolates behave similarly in terms of beta-lactamase expression and beta-lactam resistance properties, despite considerable phylogenetic variability.

Analysis of sequence variation among smeDEF multi drug efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophilia isolates.

OBJECTIVES: To determine the level of variation in the smeDEF efflux pump and smeT transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical Stenotrophomonas maltophilia isolates. METHODS: smeDEF sequencing used a PCR genome walking approach. Determination of the sequence surrounding smeDEF used a flanking primer PCR method and specific primers anchored in smeD or smeF together with random primers. RESULTS: smeDEF is chromosomal and located in the same position in the chromosome in all three subgroups of isolates. Flanking smeD is a gene, smeT, encoding a putative transcriptional repressor for smeDEF. Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic beta-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint. The smeD-smeT intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates. CONCLUSIONS: These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, S. maltophilia, based on genotypic properties. Isolate D457, in which most work concerning smeDEF expression has been performed, does not fall into S. maltophilia subgroup A, which is the most typical.