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

Abstract

Objectives: In this study, we evaluated the ceftobiprole (BPR) susceptibilities of 472 methicillin-resistant Staphylococcus aureus (MRSA) isolates, and investigated the mechanisms underlying BPR resistance.

Methods: 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.

Results: 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.

Discussion: 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.

Keywords: Amplification; Ceftobiprole; MRSA; Resistance; SCCmec.