procedures. For isolates growing on the OXA side, an OXA-48-like PCR was performed. When the PCR was negative, a CIM test was performed for P. aeruginosa and Enterobacterales, and an antimicrobial susceptibility test with VITEK®2 (bioMérieux) for A. baumannii. Colonies growing on the ESBL plate were identified to species level using MALDITOF. Antimicrobial susceptibility was determined with VITEK®2, and a combination diskdiffusion method (ESBL + AmpC Screen Kit; Rosco Diagnostica, Taastrup, Denmark) was performed to phenotypically confirm the presence of an ESBL. A CIM test was performed when the presence of a carbapenemase was suspected as well. Antibiotic susceptibility results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (10). All identified MRSA, VRE, ESBL-E, CPE, CP-PA and CR-AB isolates were stored at -80°C. Whole genome sequencing WGS was performed for all identified isolates. DNA was extracted using the MagNA pure 96 platform (Roche Applied Science, Mannheim, Germany) and shipped to Novogene (Hong Kong, China) for sequencing. Genomic DNA was fragmented by shearing to a size of ~ 350 bp. Libraries were prepared using the NEBNext® DNA Library Prep kit (New England Biolabs, Ipswich, MA, USA) and subjected to 150 bp paired-end sequencing generating >100 × coverage using Illumina. Incidental, samples were sequenced using the in-house platform. Library preparation was conducted with the Illumina DNA Prep (Illumina, San Diego, CA, United States). Sequencing was conducted using the iSeq 100 System (Illumina) generating 150 bp paired-end reads. De novo genomic assemblies were generated using CLC Genomics Workbench v21 (Qiagen, Hilden, Germany). Presence of antibiotic resistance genes was analyzed using the web-based interface of the Comprehensive Antimicrobial Resistance Database (CARD - https://card.mcmaster.ca/ accessed on July 4. 2022). The analysis was restricted to include perfect and strict hits (11, 12). Plasmid replicon types were detected using the online Plasmidfinder software v2.1 (https://cge.food.dtu.dk/services/PlasmidFinder accessed on November 16, 2022/) with default settings (13) Identification confirmation was performed using the Type strain genome server (TYGS) (https://tygs.dsmz.de)/ (14). For Enterobacter spp. and P. aeruginosa, conventional Multi Locus Sequence Types (MLST) and core-genome MLST (cgMLST) or whole-genome MLST analysis (wgMLST) was performed using the available schemes available in BioNumerics (Applied Maths, St-Martens-Latem, Belgium) and for K. pneumoniae and E. faecium using the schemes available in SeqSphere (Ridom, Munster, Germany). For Citrobacter freundii an ad hoc wgMLST scheme was created in SeqSphere using the cgMLST Target Definer v1.5 with the genomic sequence of the Type strain (ATCC 8090, accession nr. CP049015.1) as seed genome and 24 NCBI Refseq genomes as penetration query genomes. Genomes improperly assigned to C. freundii and plasmid based genes were excluded. The resulting scheme consisted of 3162 core genes and 1142 accessory genes. The sequence data for this study has been deposited in BioProject ID: PRJNA904531. 3 153 Environmental contamination with MDRO in single-occupancy rooms
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