93 Detection of AmpC beta-lactamases in E. coli using different screening agars pAmpC-encoding clinical E. coli isolates from Amphia hospital In total 9 pAmpC producing E. coli isolates were selected retrospectively from our laboratory database based upon the presence of pAmpC encoding genes. In the laboratory protocol for AmpC screening on clinical samples, E. coli isolates with cefotaxime and/ or ceftazidime MIC > 1mg/l combined with cefoxitin MIC ≥8mg/l are screened for AmpC production using D68C AmpC & ESBL Detection Set (Mastdiscs, Mastgroup Ltd, Bootle, United Kingdom). The presence of pampC genes and/or alterations in the promoter/attenuator region were evaluated similar as in the Amphia prevalence screening described by Den Drijver et al. (E. Den Drijver et al. 2018). AmpC E. coli isolates from Wageningen University prevalence study In total 18 cAmpC hyperproducing E. coli isolates were selected retrospectively from a former prevalence study among livestock (e.g. cattle, pigs and broilers) by the Wageningen University. Screening swabs were cultured on MacConkey agar (product no. 212123, Becton Dickinson)+1 mg/L cefotaxime (Sigma-Aldrich, Germany)] and inoculated in a selective pre-enrichment broth (Luria–Bertani broth containing 1 mg/L cefotaxime). Phenotypic and genotypic confirmation with micro-array and sanger sequencing were performed similar as described by Dierikx et al. and Hordijk et al. (C. M. Dierikx et al. 2012; C. Dierikx et al. 2013; Hordijk, Wagenaar, Kant, et al. 2013). ESBL-encoding clinical E. coli isolates from Amphia hospital In total 40 ESBL producing E. coli isolates were selected retrospectively from our laboratory database of blood culture E. coli isolates containing of CTX-M encoding genes. In the in-laboratory protocol for ESBL screening on clinical samples, E. coli isolates with cefotaxime and/or ceftazidime MIC > 1mg/l are screened for ESBL production using combination disk diffusion method for cefotaxime, ceftazidime, and cefepime with and without clavulanic acid (Rosco, Taastrup, Denmark) and interpreted according to manufacturer’s instructions. WGS was performed in UMCG using MiSeq (Illumina, San Diego, United States) and assembled with CLC Genomics Workbench 9.0, 9.0.1 or 9.5.2 (Qiagen, Hilden, Germany) as was previously described in more detail by Kluytmans-van den Bergh et al. (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016). Assembly, identification of resistance genes and MLST type and analysis of promoter/ attenuator region were performed as described for the SOM study isolates. 5
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