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 South West England and confirmed by PCR to carry acquired 3GC-R genes. This analysis identified blaCTX-M (131 isolates: encoding CTX-M-1, -14, -15, -32 and the novel variant, CTX-M-214), blaCMY-2 (6 isolates) and blaDHA-1 (one 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 1500 square kilometres. However, the plasmid was not found amongst human urinary E. coli we have recently characterised from people living in the same geographical location, collected in parallel with farm sampling. There were close relatives of two blaCTX-M plasmids circulating amongst eight human and two cattle isolates, and a closely related blaCMY-2 plasmid 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 (e.g. via interaction of people with the local near-farm environment). We characterised 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. Whilst 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 transmit – there was no evidence that 3GC-R found on study farms had a significant impact on circulating 3GC-R E. coli strains or plasmids in the local human population.

Little is known about the drivers of critically important antibacterial resistance in species with zoonotic potential present on farms (e.g. CTX-M β-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, respectively, in 2754/4145 (66%), 263/4145 (6%), 1475/4145 (36%) and 2874/4145 (69%) of all samples from faecally contaminated on-farm and near-farm sites. E. coli positive for blaCTX-M were detected in 224/4145 (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-M E. coli positivity in samples and with lower odds of finding E. coli resistant to each of the four test antibacterials. This was additional 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 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-M. Importance 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 to track the 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.

16Little is known about the drivers of critically important antibacterial resistance in 17 species with zoonotic potential present on farms (e.g. CTX-M ꞵ-lactamase-positive 18 Escherichia coli). There is also debate about the influence of farms on the circulation 19 of resistance in local human populations. This was a two-year surveillance study on 20 53 dairy farms. E. coli positive for blaCTX-M were detected in 224/4145 (5.4%) of all 21 samples from faecally-contaminated sites. E. coli positive for blaCTX-M were more 22 prevalent (98/631; 15.5%) in calf samples and less prevalent (12/630; 1.9%) in 23 samples collected from pastureland, including publicly accessible sites. Multilevel, 24 multivariable logistic regression showed antibiotic dry cow therapeutic choice to be 25 associated with risk of blaCTX-M positivity, including use of cefquinome or framycetin; 26 74% of blaCTX-M-positive E. coli were framycetin-resistant. Low temperature was 27 associated with low risk of blaCTX-M positivity. This was additional to the effect of 28 temperature on total E. coli density, a finding with profound implications for 29 surveillance. There was no evidence that study farms had a significant impact on 30 circulating blaCTX-M plasmids in the local human population: across 296 fully 31 sequenced E. coli isolates, two cattle isolates shared blaCTX-M plasmids with eight 32 urinary isolates collected in parallel.33 34 Antimicrobial resistance (AMR) -and particularly antibacterial resistance (ABR) -is a 35 significant global challenge. Many countries are implementing plans to reduce the 36 use of antibacterial drugs (ABs) in food-producing animals. For example, the most 37 recent UK five-year National Action Plan includes a target to reduce AB use (ABU) in 38the treatment of food-producing animals by 25% (1). In Europe, AB sales for food-39 producing animals fell by 20% from 2011 to 2016 (2). In the UK dairy industry, 40 overall ABU dropped from 24 mg/kg in 2015 to 17 mg/kg in 2017 (3, 4). In 2018, 41 additional industry-led policies were enforced in the UK that aim to almost eliminate 42 the use of highest priority critically important antimicrobials (HP-CIAs) such as third-43 and fourth-generation cephalosporins (3GCs and 4GCs) and fluoroquinolones on 44 dairy farms. One reason for reducing ABU in farming is the belief that such 45 measures will reduce the prevalence of ABR bacteria carried by farm animals. 46 However, there is a need for better data on drivers of ABR in farming. More 47 granularity of understanding is required concerning the risks of using individual ABs 48 and other management practices. This is especially important in terms of drivers of 49 HP-CIA resistance. One key focus is on 3GC resistance in Escherichia coli, a 50 species commonly found in animal faeces and considered one of the most significant 51 potential zoonotic threats to humans (5). 523GC-resistance is increasingly common in E. coli causing infections in humans (6) 53 and is also found in farmed and domestic animals around the world (7). The...

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.

J. WHYTE, A.D.G. ROBERTS, K.A. MORLEY, R.J. SHARP and P.D. MARSH.2000.A human‐derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50–60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence‐activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h.