Staphylococcal cassette chromosome mec amplification as a mechanism for ceftobiprole resistance in clinical methicillin-resistant Staphylococcus aureus isolates

In this study, we evaluated the ceftobiprole (BPR) susceptibilities of 472 methicillin-resistant Staphylococcus aureus (MRSA) isolates, and investigated the mechanisms underlying BPR resistance.For all MRSA isolates, BPR MIC was determined by agar dilution. We sequenced the BPR-resistant isolates through Illumina short- and MinION long-read sequencing. We also selected MRSA isolates of ST5, ST59, and ST239, and exposed them to increasing BRP concentrations. The isolated mutants developing BPR resistance were sequenced.A total of 471 MRSA isolates were susceptible to BPR, with MICs ranging from 0.25 to 2 mg/L. Compared with HA-MRSA isolates (MIC50 = 2 mg/L; MIC90 = 2 mg/L), CA-MRSA isolates (MIC50 = 0.5; MIC90 = 2 mg/L) were more susceptible to BPR (p < 0.001). Compared with isolates with staphylococcal cassette chromosome mec (SCCmec) type II or III (MIC50 = 2 mg/L; MIC90 = 2 mg/L), isolates with SCCmec type IV (MIC50 = 1 mg/L; MIC90 = 1 mg/L) or V (MIC50 = 0.5 mg/L; MIC90 = 1 mg/L) were more susceptible to BPR (p < 0.001). Nanopore sequencing revealed two copies of SCCmec repeats in the BPR-resistant MRSA isolate. In addition, SCCmec amplification could be induced by BPR exposure in ST239 MRSA isolates; however, no amplification was observed in the other lineages. The induced BPR-resistant MRSA isolates also acquired mutations in mecA and other genes, such as guaA, guaB, relA, rpoA, and oatA, which were speculated as factors contributing to BPR-resistance development.BPR showed significant antibacterial activity against MRSA isolates in China; however, the emergence of a BPR-resistant isolate before its launch was a cause for concern. Multiple genes and pathways are potentially involved in the development of BPR resistance in MRSA, and our data demonstrated the role of nanopore-sequencing in revealing the tandem repeat-mediated resistance mechanism in MRSA.