Phenotypic and genotypic correlates of the sodium bicarbonate-responsive phenotype among methicillin-resistant Staphylococcus aureus isolates from skin and soft-tissue infections

Objectives: The objective of this study is to assess the frequency of the novel sodium bicarbonate (NaHCO₃)-responsive phenotype, wherein clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates are rendered susceptible to standard-of-care β-lactams in the presence of NaHCO₃, in a collection of 103 clinical U.S. MRSA skin and soft-tissue infection (SSTI) isolates and 22 clinical European SSTI isolates. This study determined the correlation between specific phenotypic and genotypic metrics and the NaHCO₃-responsive phenotype among U.S. SSTI isolates. Methods: Antimicrobial susceptibility testing was performed to determine susceptibility phenotypes. Targeted and whole-genome sequencing with a genome-wide sequence analysis were conducted to identify specific and novel genotypes of interest that may be associated with the NaHCO₃-responsive phenotype. Gene expression analysis and targeted gene deletion were performed to assess the role of a specific novel genetic locus in the NaHCO₃-responsive phenotype. Results: The NaHCO₃-responsive phenotype was identified in 78/103 U.S. isolates and 4/22 UK isolates to cefazolin (CFZ), and in 17/103 U.S. isolates and 1/22 UK isolates to oxacillin. In U.S. isolates, a significant association was identified between NaHCO₃-responsiveness to CFZ and: (a) susceptibility to amoxicillin–clavulanate; (b) a specific mecA genotype; (c) clonal complex type 8; and (d) spa type t008. Genome-wide sequence analysis identified single nucleotide polymorphisms (SNPs) in an AraC family regulator (SAUSA300_RS00540) to be exclusively found in NaHCO₃-non-responsive SSTI strains. In vitro HCO₃ exposures of NaHCO₃-responsive strains, but not -non-responsive strains, caused >2-fold upregulated expression of this gene. Deletion of this gene rendered NaHCO₃-responsive strain MRSA 11/11 no longer NaHCO₃-responsive to CFZ; we have termed this gene the staphylococcal AraC bicarbonate-response regulator. Discussion: NaHCO₃-responsiveness is highly associated with clonal complex type 8/spa type t008, a commonly circulating genetic background in North America. The AraC bicarbonate-response regulator, staphylococcal AraC bicarbonate-response regulator, appears to be associated with the mechanism of NaHCO₃-responsiveness, but more work is needed to verify.