53 Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC algorithm was selected as the final algorithm and validated in two validation sets. Validation set 1 was used to validate the algorithm and represents the epidemiology in a Dutch hospital setting. Due to a low number of pampC-positive strains and restricted geographical background we broadened the representation of suspect AmpC-producing isolates in a second validation set (validation set 2). An extensive description of the selection of samples in the training set, validation set 1 and validation set 2 can be found in the Supplementary Materials and methods (available at JAC Online). Etests and AmpC disc-diffusion confirmation tests Deep-frozen samples of the selected strains were recultured on Columbia III agar (BD Diagnostic Systems, Sparks, MD, USA) or blood agar (Media production, Elizabeth-Tweesteden Hospital, Tilburg, the Netherlands) prior to testing. Strains were tested using Etest (bioMérieux, Marcy-l’Étoile, France) to determine the MICs of cefotaxime, ceftazidime and cefoxitin. Etests were placed on Mueller–Hinton (Oxoid Ltd, Altrincham, Cheshire, England) culture plates, which were placed in the oven within 15 min and incubated for 16–20 h under an O2 atmosphere at 36°C. Exact MIC values were noted. The presence of AmpC was phenotypically confirmed using the AmpC Confirm Kit (Rosco Diagnostica A/S, Taastrup, Denmark) according to the manufacturer’s guidelines. A second phenotypical confirmation with the D68C AMPC + ESBL detection set (MAST Group Ltd, Bootle, UK) was performed according to the manufacturer’s guidelines. From both confirmation tests the zone inhibition differences, measured in millimetres, were recorded for further use. DNA isolation, library preparation and DNA sequencing For logistical reasons DNA isolation, library preparation and DNA sequencing were performed at two different centres. For training and validation set 2, bacterial DNA was extracted by a CTAB-based method and a paired-end 2 × 150 bp library was sequenced using an Illumina NextSeq500 sequencer (Illumina, San Diego, CA, USA) (see the Supplementary Materials and methods). For validation set 1, bacterial DNA was extracted using the MagNA Pure LC Total Nucleic Acid Kit - High Performance on a MagNA Pure LC instrument (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) according to the manufacturer’s protocol. A 2 × 300 bp paired-end library was sequenced on an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA) (Supplementary Materials and methods). 4
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