Screening for β-lactamases

CRE isolates were tested for β-lactamase-encoding genes using next-generation sequencing (NGS). Total genomic DNA was extracted using the fully automated Thermo Scientific™ KingFisher™ Flex Magnetic Particle Processor (Cleveland, OH, USA). To perform NGS, DNA extracts were quantified using the Qubit™ High Sensitivity DS-DNA assay (Invitrogen, Thermo Fisher Inc.) and normalized to 0.2 ng/μL. A total of 1 ng high-quality genomic DNA was used as input material for library construction using the Nextera XT™ DNA library preparation kit (Illumina, San Diego, CA, USA). Libraries were normalized using the bead-based normalization procedure (Illumina) and sequenced on MiSeq. The generated FASTQ files were assembled using SPAdes Assembler and subjected to a proprietary software (JMI Laboratories) for screening of β-lactamase genes.¹⁰