99 Limited genetic diversity of blaCMY-2-containing IncI1-pST12 plasmids from Enterobacteriaceae AmpC E. coli Isolates from Amphia Prevalence Screening pampC gene containing E. coli isolates were selected from a prevalence screening, which had been performed in the Amphia hospital described by Den Drijver et al. (E. Den Drijver et al. 2018). Rectal swabs taken from hospital patients were preenriched using selective TSB containing cefotaxime (0.25 mg/L) and vancomycin (8 mg/L) and subsequently cultured on a MacConkey agar plate containing cefotaxime (1 mg/L) or a MacConkey double agar plate containing cefotaxime and cefoxitin (1 and 8 mg/mL, respectively) on half the plate and ceftazidime and cefoxitin (1 and 8 mg/mL, respectively) on the other half of the plate (Mediaproducts, Groningen, The Netherlands) (Drijver et al. 2019). For all oxidase-negative isolates that grew on either side of the selective agar plates, species identification was performed by automated mass spectrometry systems (VitekMS, bioMérieux, Marcy l’Etoile, France). Susceptibility testing was performed using Vitek 2 (bioMérieux, Marcy l’Etoile, France). The presence of AmpC in all oxidase-negative isolates was phenotypically confirmed using the D68C AmpC & ESBL Detection Set (Mastdiscs, Mastgroup Ltd., Bootle, UK) and interpreted according to the manufacturer’s instructions. All phenotypically confirmed isolates were sequenced in the University of Groningen Medical Center (UMCG) using MiSeq (Illumina, San Diego, CA, USA) 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). pAmpC-encoding Clinical Isolates from Elisabeth-Tweesteden Hospital Suspected pampC gene containing E. coli isolates from blood cultures were selected retrospectively from our laboratory database based upon the presence of a phenotype (cefoxitin minimal inhibitory concentration (MIC) > 8 mg/L and/or cefotaxime MIC ≥ 1mg/L and/or ceftazidime MIC ≥ 1mg/L. One Salmonella enterica serotype Kentucky isolate from a faecal sample was selected from our laboratory database based upon the presence of an AmpC suspected phenotype (cefoxitin MIC > 8 mg/L and/or cefotaxime MIC ≥ 1mg/L and/or ceftazidime MIC ≥ 1mg/L). The isolates were recultured from deep frozen samples on blood agar and identified using the MALDI-TOF MS (BD Diagnostic Systems, Sparks, MD, USA). Susceptibility testing was performed using a Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, MD, USA). The isolates were sequenced using an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). DNA isolation and sequencing were performed as described by Coolen et al. (Coolen et al. 2019). De novo assembly and error correction were performed using SPAdes version 3.9.1 (Bankevich et al. 2012). 6
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