92 Chapter 5 Supplementary data Supplement 1. Detailed supplementary “Materials and Methods” AmpC E. coli isolates from SOM study In total 27 E. coli isolates were selected from the SOM-study based upon the presence of pAmpC encoding genes in the whole genome sequence (WGS) data (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016; Kluytmans-van den Bergh et al. 2019). In this study perianal swabs taken from hospital patients, were pre-enriched using selective tryptic soy broth (TSB) and subsequently cultured on EbSA screening agar (Cepheid Benelux, Apeldoorn, the Netherlands). Sequencing was performed as described by Kluytmans-van den Bergh et al. (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016). The assembled genomes were uploaded to the online bioinformatics tools ResFinder v2.1 and MLST v1.8 (Center for Genomic Epidemiology, Technical University of Denmark, Lingby, Denmark (version 2.1) (Zankari et al. 2012; Alessandra Carattoli et al. 2014) . ESBL- and pAmpC-encoding genes were reported when at least 60% of the length of the best matching gene in the ResFinder database was covered with a sequence identity of at least 90%. Conventional MLST sequence types were based on the Achtman MLST scheme (Wirth et al. 2006). In addition, the assembled sequences of each isolate were aligned against the promoter/attenuator region of the cAmpC gene of the E. coli K-12 strain MG1655 (GenBank database accession number U00096 (https://www.ncbi. nlm.nih.gov/nuccore/U00096)) using CLC Genomic Workbench version 8.5 (CLC Bio, Qiagen, Hilden, Germany). The level of hyperproduction of AmpC was based on a former study by Tracz et al. (Tracz et al. 2007). AmpC E. coli isolates from Amphia prevalence screening In total 22 cAmpC hyperproducing and 4 pAmpC containing E. coli isolates were selected from 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 pre-enriched using selective TSB and subsequently cultured on MacConkey agar plate containing cefotaxime (1 mg/L) or MacConkey double agar plate containing cefotaxime (1 mg/L) with cefoxitin (8 mg/L) one side and ceftazidime (1 mg/L) with cefoxitin (8mg/L) other side (Mediaproducts, Groningen, The Netherlands). Presence of pAmpC was analyzed with micro-array MDR CT103 (Check-Points, Wageningen, the Netherlands), alterations in the promoter and attenuator region were analysed with Sanger sequencing as previously described (Stéphane Corvec et al. 2003).
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