Characterizing Antimicrobial Resistant Escherichia coli and Associated Risk Factors in a Cross-Sectional Study of Pig Farms in Great Britain

Abstract

Combatting antimicrobial resistant (AMR) using a One-Health approach is essential as various bacteria, including Escherichia coli, a common bacteria, are becoming increasingly resistant and livestock may be a reservoir. The AMR gene content of 492 E. coli, isolated from 56 pig farms across Great Britain in 2014-2015, and purified on antibiotic selective and non-selective plates, was determined using whole genome sequencing (WGS). The E. coli were phylogenetically diverse harboring a variety of AMR profiles with widespread resistance to "old" antibiotics; isolates harbored up to seven plasmid Inc-types. None showed concurrent resistance to third-generation cephalosporins, fluoroquinolones and clinically relevant aminoglycosides, although ∼3% harbored AMR genes to both the former two. Transferable resistance to carbapenem and colistin were absent, and six of 117 E. coli STs belonged to major types associated with human disease. Prevalence of genotypically MDR E. coli, gathered from non-selective media was 46.9% and that of extended-spectrum-beta-lactamase E. coli was low (∼4% from non-selective). Approximately 72.6% of E. coli from ciprofloxacin plates and only 8.5% from the other plates harbored fluoroquinolone resistance due to topoisomerase mutations; the majority were MDR. In fact, multivariable analysis confirmed E. coli purified from CIP enrichment plates were more likely to be MDR, and suggested MDR isolates were also more probable from farms with high antibiotic usage, specialist finisher farms, and farms emptying their manure pits only after each batch. Additionally, farms from the South East were more likely to have MDR E. coli, whereas farms in Yorkshire and the Humber were less likely. Future investigations will determine whether suggested improvements such as better biosecurity or lower antimicrobial use decreases MDR E. coli on pig farms. Although this study focuses on pig farms, we believe the methodology and findings can be applied more widely to help livestock farmers in the United Kingdom and elsewhere to tackle AMR.

Keywords: Escherichia coli; Great Britain; antimicrobial resistance; epidemiology; multidrug resistance; pig farms; plasmids; risk factor analysis.

FIGURE 1

Correlation of AMR genes harbored by isolates with the selection plate. The number of AMR genes detected by WGS in 492 isolates from 56 pigs farms. The colored bars indicate the number of isolates selected on agar supplemented with: antibiotic-free plate (green); 1 mg/L ciprofloxacin (blue); 1 mg/L cefotaxime (orange); or from Brilliance carbapenem -resistant Enterobacteriaceae Agar plates (CRE; pink).

FIGURE 2

Relationship between numbers of AMR genes, farm region, and production type. Box plot representing the interquartile range or midspread of AMR genes found in isolates from a farm (numbered as MSG01–MSG35 and MSG37–MSG57). Farms were ordered according to their region and production type (farrow-to-finisher = blue and finisher-only = red). Number to left of bars indicate the number of isolates from farm. Center lines show the median; box limits indicate the 25th and 75th percentiles as determined by R software. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, with outliers represented by dots.

FIGURE 3

Phylogenetic tree of Escherichia coli isolates. A SNP-based maximum-likelihood phylogenetic tree was constructed in RaxML-NG using 272,385 single nucleotide polymorphisms (SNP) present in the core genome. Clades and subclades are labeled on the relevant branches. Isolate names are colored according to farm production type (blue, farrow-to-finish; pink, finisher-only) and nodes at the end of the leaves are colored according to the six regions in England from where the E. coli isolates originate. The circles at the edge indicate the following information (working from inner circle to outer circle): (1) the top 8 MLST clonal complexes identified; (2) MLST ST744; (3) QRDR mutations in gyrA; (4) QRDR mutations in parC; (5) presence of plasmid quinolone resistance (qnr genes); (6) ciprofloxacin susceptibility using EUCAST ECOFF; (7) ciprofloxacin susceptibility using EUCAST clinical breakpoints; (8) presence of ESBL gene variants; and (9) the numbers EFSA AMR classes harboring different resistance genes in each isolate (genes conferring resistant to the same antimicrobial were grouped within the same class).