Detection of mutations in the FemXAB protein family in oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates

Methicillin-resistant Staphylococcus aureus (MRSA) strains that express the mecA gene but are oxacillin susceptible (OS-MRSA; oxacillin MIC < 2 mg/L) are increasingly reported. To gain molecular and functional insights on this observation, we focused on additional factors possibly contributing to phenotypic susceptibility. The nucleotide content of mecA, femA, femB and femX genes, which are considered essential for methicillin resistance, was determined in four OS-MRSA clinical isolates and a genetically similar low-level MRSA control (oxacillin MIC 6 mg/L). Gene expression was quantified compared with the low- and a high-level MRSA (MIC 256 mg/L) control. The tertiary structure of Fem proteins was predicted based on protein structure homology modelling, using web-based automated comparative protein modelling. Growth kinetics were tested for the study and control isolates, to determine whether FemXAB mutations lead to reduced fitness. Genes mecA, femA, femB and femX were expressed similarly in the study and the control isolates. Mutations in the gene mecA were not present in any isolate. However, several mutations leading to amino acid substitutions in positions possibly affecting Fem enzyme activity were detected in all fem genes. Two OS-MRSA that had no oxacillin heteroresistance had more mutations in the Fem proteins compared with the remaining isolates that were heteroresistant. The low-level MRSA control had considerably fewer mutations. No differences between growth rates of the OS-MRSA and the MRSA controls were observed. Accumulation of amino acid changes in Fem proteins might affect intact cell wall synthesis, even though not causing reduced viability, thus contributing to atypical oxacillin responsiveness.