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37 Decline in AmpC beta-lactamase-producing E. coli in a Dutch teaching hospital region of the campC gene of the E. coli K-12 strain MG1655 (GenBank database accession number U00096) using Vector NTI Advance 11 software (ThermoFisher Scientific, Waltham, USA) or CLC Genomic Workbench version 8.5 (CLC Bio, Qiagen, Hilden, Germany). Hyperproduction of chromosome-encoded AmpC was assumed when similar alterations in the promoter/attenuator region were found as described by Tracz et al. (Tracz et al. 2007). Amplified Fragment Length Polymorphism Isolates from patients who were admitted to the same ward and revealed the same AmpC mechanism were selected for Amplified Fragment Length Polymorphism (AFLP) to determine clonal relatedness. If a patient harboured more than one AmpC-producing isolate from different genus or species, or with different resistance mechanisms or genes, all isolates were typed. AFLP typing was performed and interpreted as described by Savelkoul et al (Savelkoul et al. 1999). Statistical methods All data were pseudonymized and subsequently analysed with Statistical Package for Social Science software (SPSS; IBM Corp., Armonk, New York, US; version 19). The 95% confidence intervals of proportions were calculated using the modified Wald method. A trend analysis for the prevalence of AmpC genes was performed using the Mantel-Haenzsel test for linear association. The trend result was adjusted for gender using logistic regression. Statistical significance was accepted at p <0.05. Ethical considerations The yearly PPS for AmpC-producing E. coli and Klebsiella spp. rectal carriage is part of the routine hospital infection control policy and is approved by the management of the hospital. This is in accordance with the current regulations in the Netherlands and requires verbal consent from participating patients. According to the Dutch regulation for research with human subjects, neither medical nor ethical approval, was required to conduct the surveillance since it was part of the local hospital policy, and all data were processed anonymously. Patients who indicated that they did not want to participate in the survey were excluded (opt-out) (Willemsen et al. 2015). According to the patient’s preferences, samples were taken by nursing staff or by patients themselves. The Infection Control Practitioner collected information on the patient’s characteristics from the medical record. 3

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