Genomic Analysis of Stealthy MRSA Isolates

Total genomic DNA of these two “stealth” MRSA isolates and their revertants were extracted using the TaKaRa MiniBEST Bacteria GenomicDNA Extraction Kit (TaKaRa Bio Inc., Beijing, China). The whole genomes of wild-type “stealth” MRSA isolates and one revertant of the nasal swab strain were sequenced at the Beijing Genomics Institute using the long-reads PacBio third-generation sequencing method (Pacific Biosciences of California, Inc., Menlo Park, CA, USA) and the short-reads Illumina NovaSeq PE150 platform (Illumina, San Diego, CA, USA). After filtering low-quality reads, the clean data were obtained, which were preliminarily assembled using Link v5.0.1 and subsequently corrected with the Illumina data. Then, Rapid Annotation using the Subsystem Technology v2.0 server was used for gene annotation. Using BLAST, comparative genome analysis was performed among the parental and variant strains. The mutated genes were then confirmed using Sanger sequencing, and the mecA gene of revertants from the clinical strain was analyzed using conventional sequencing, as previously described by us (1 (link)). The sequences were aligned and further analyzed using the Qiagen CLC Genomics Workbench (33 ).