84 Chapter 5 screening nor in positive family contacts of index patients. All patients (both adults and children) and healthcare workers of whom WGS of an MRSA isolate was performed were retrospectively identified and were screened to meet the selection criteria. Healthcare workers will be also addressed as ‘patients’ from now on in this manuscript, since they were treated as patients for this matter. Inclusion criteria were≥1 visit to the outpatient infectious diseases clinic because of MRSA carriage or infection,≥1 positive MRSA culture from any site, and available WGS data of the MRSA isolate. Exclusion criterion was the absence of follow-up cultures. Only the first available MRSA isolate per patient was included in the analysis. The patients had been assessed by the outpatient clinicians using protocols based on the national MRSA eradication guideline [16]. This includes in case of an MRSA infection, adequately treating the infection first, and subsequently screen for persistent colonization. Data collection Clinical data were extracted from the electronic patient files. This included demographics, complicated versus uncomplicated carriage, treatment regimen, duration of therapy, and follow-up cultures. MRSA culture results were extracted from the laboratory information system. This included initial and follow-up MRSA cultures, including minimal inhibitory concentrations (MICs) of antibiotics, phenotypic susceptibility results, and WGS results. Microbiological methods Culturing using BHI broth with 2.5% saline and MRSAid chromagar (bioMérieux, Lyon, France), susceptibility determination by automated susceptibility testing by VITEK2 (bioMérieux, Lyon, France), and cefoxitin disk diffusion were performed according to the Dutch Society of Medical Microbiology guideline for laboratory detection of highly resistant microorganisms as part of routine diagnostic procedures [17]. MIC breakpoints and zone diameter breakpoints for resistance and intermediate sensitivity were based on EUCAST criteria [18]. The isolates were identified as S. aureus by matrix-assisted laser desorption/ionization–time of flight mass spectrometry (Bruker Daltonics, Billerica, US). First MRSA isolates per patient were genotypically confirmed by Xpert MRSA NxG based on the detection of the mecA or mecC targets (Cepheid, Sunnyvale, US). A total DNA extraction for whole-genome sequencing was performed directly from colonies of the respective isolates using the Ultraclean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, US) according to the manufacturer’s protocol. DNA concentrations were determined using a Qubit® 2.0 fluorometer and the dsDNA HS and/or BR assay kit (Life Technologies, Carlsbad, CA, US). Subsequently, DNA libraries
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