Prevalence of Cefotaxime-Resistant Escherichia coli Isolates from Healthy Cattle and Sheep in Northern Spain: Phenotypic and Genome-Based Characterization of Antimicrobial Susceptibility

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Abstract
In order to estimate herd-level prevalence of ESBL-/AmpC- and carbapenemase-producing commensal Escherichia coli in ruminants in the Basque Country (Northern Spain), a cross-sectional survey was conducted in 2014–2016 in 300 herds using selective isolation. ESBL-/AmpC-producing E. coli were isolated in 32.9% of dairy cattle herds, 9.6% of beef cattle herds and 7.0% of sheep flocks. No carbapenemase-producing E. coli were isolated. Phenotypic antimicrobial susceptibility determined by broth microdilution using EUCAST epidemiological cut-off values identified widespread co-resistance to extended spectrum cephalosporins and other antimicrobials (110/135 isolates), particularly tetracycline, sulfamethoxazole, trimethoprim, and ciprofloxacin. All isolates were susceptible to tigecycline, imipenem, meropenem and colistin. The genome of 66 isolates was sequenced using Illumina NovaSeq6000 and screened for antimicrobial resistance determinants against ResFinder and PointFinder. Plasmid/chromosomal location of resistance genes was predicted with PlasFlow and plasmid replicons were identified using PlasmidFinder. Fifty-two acquired resistance genes and point mutations in another four genes that coded for resistance to eleven antimicrobial classes were identified. Fifty-five genomes carried ESBL-encoding genes, blaCTX-M-14 being the most common, and 11 carried determinants of the AmpC phenotype, mostly blaCMY-2 gene. Additionally, genes coding for β-lactamases of the CTX-M group 9 were detected as well as the sporadic presence of blaSHV-12, blaCMY-4, and a point mutation in the ampC promoter. Only a bovine isolate co-harbored more than one ESBL/AmpC genetic determinant (blaCTX-M-14 and a mutation in the ampC promoter), confirming its ESBL+AmpC phenotype. Most ESBL/AmpC genes were located in IncI1 plasmids, which also carried a great variety of other AMR genes. Importance: Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC) producing E. coli have emerged in recent years as one of the fastest spreading antimicrobial resistance determinants in humans and food-producing animals, becoming a concern for animal and public health. This study provided an insight into the prevalence of cefotaxime-resistant E. coli in cattle and sheep in the Basque Country and the associated genetic determinants of antimicrobial resistance. These constituted an important contribution to the limited repository of such data for cattle in the region and for sheep worldwide. Antimicrobial susceptibility testing by phenotypic and molecular methods is key in surveillance programs to enhance early detection of resistance development, monitor resistance trends and provide guidance to clinicians in selecting the adequate therapy.
Funding Information
  • Ekonomiaren Garapen eta Lehiakortasun Saila, Eusko Jaurlaritza (Predoctoral Fellowship)

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