Whole genome sequencing WGS was performed for all identified highly resistant P. aeruginosa, -A. baumannii, CPE, ESBL-E, MRSA, and VRE isolates from universal screening samples. Total genomic DNA was extracted using the MagNA Pure 96 platform (Roche Applied Science, Mannheim, Germany). Genomic DNA was sent to Novogene (HongKong, China) where it 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 creating >100x coverage using Illumina technology. Fastq data were provided and de novo genomic assemblies were generated using CLC Genomics Workbench (Qiagen, Hilden, Germany) with default parameters (2, 3). Presence of antimicrobial resistance (AMR) genes was determined using the web-based comprehensive antimicrobial resistance database (CARD) (including perfect and strict hits)(https://card.mcmaster.ca/analyze/rgi) (4). Conventional multi locus sequence types (MLST) and core genome multi locus sequence type (cgMLST) were determined based on each species’ corresponding (cg)MLST scheme (https://cgmlst.org/ncs) available in SeqSphere+ software (Ridom, Munster, Germany). Isolates were identified to the species level by analysing their de-novo assemblies using the Tyge Strain Genome Server (TYGS - https://tygs.dsmz.de/) (5). References 1. van der Zwaluw K, de Haan A, Pluister GN, Bootsma HJ, de Neeling AJ, Schouls LM. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods. PLoS One. 2015;10(3):e0123690. 2. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. 3. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-9. 4. Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 2017;45(D1):D566-D73. 94 Chapter 2.3
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