148 Chapter 8 2016). The underlying assumption is that differences in coverage between chromosomal and plasmid-related DNA sequence data may be used as a derivative of the plasmid copy number. A similar method was used by Pena-Gonzalez et al. to estimate the copy number of pXO1/pXO2-like plasmids in Bacillus anthracis isolates, revealing consistent results compared to quantitative PCR analysis (Pena-Gonzalez et al. 2018). The present study uses the sequencing depth of the blaCMY-2 containing scaffolds to estimate the copy number by comparing this data with the sequencing depth of the chromosome-encoded household genes of the E. coli isolates. The present explorative study aims to evaluate whether the susceptibility for cefotaxime, ceftazidime and piperacillin-tazobactam in CMY-2-producing E. coli is associated with the copy number of the blaCMY-2-containing plasmid. Materials and methods Selection of isolates A national collection of 2,005 phenotypic ESBL-E isolates was prospectively gathered in the SoM study, a multicenter cluster-randomized study isolation strategies for ESBL-E positive patients in 14 Dutch hospitals between 2011 and 2014 (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016; Kluytmans-van den Bergh et al. 2019). Isolates were obtained from routine clinical cultures and perianal screening cultures that were taken during ward-based prevalence surveys as part of the project. The methods used to detect ESBL-E have been described previously (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016). In 160 (8%) of the 2,005 phenotypic ESBL-E isolates, no ESBLencoding gene could be detected. Of those, twenty (13%) were E. coli isolates that contained blaCMY-2 and were included in the present study. Antimicrobial susceptibility testing The susceptibility for cefotaxime (CTX), ceftazidime (CAZ) and piperacillin-tazobactam (TZP) was measured with the antibiotic gradient on a strip method (CTX and CAZ: E-test (bioMérieux, Marcy l’Etoile, France); TZP: MIC test strip (Liofilchem, Roseto degli Abruzzi, Italy)). Short-read sequence data and de novo assembly Raw short-read sequence data were previously generated on a MiSeq or a HiSeq 2500 sequencer (Illumina, San Diego, CA, USA) as described previously (Marjolein F Q
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