Genomic investigation and clinical correlates of the in vitro β-lactam: NaHCO3 responsiveness phenotype among methicillin-resistant Staphylococcus aureus isolates from a randomized clinical trial

Abstract

NaHCO₃ responsiveness is a novel phenotype where some methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibit significantly lower minimal inhibitory concentrations (MIC) to oxacillin and/or cefazolin in the presence of NaHCO₃. NaHCO₃ responsiveness correlated with treatment response to β-lactams in an endocarditis animal model. We investigated whether treatment of NaHCO₃-responsive strains with β-lactams was associated with faster clearance of bacteremia. The CAMERA2 trial (Combination Antibiotics for Methicillin-Resistant Staphylococcus aureus) randomly assigned participants with MRSA bloodstream infections to standard therapy, or to standard therapy plus an anti-staphylococcal β-lactam (combination therapy). For 117 CAMERA2 MRSA isolates, we determined by broth microdilution the MIC of cefazolin and oxacillin, with and without 44 mM of NaHCO₃. Isolates exhibiting ≥4-fold decrease in the MIC to cefazolin or oxacillin in the presence of NaHCO₃ were considered "NaHCO₃-responsive" to that agent. We compared the rate of persistent bacteremia among participants who had infections caused by NaHCO₃-responsive and non-responsive strains, and that were assigned to combination treatment with a β-lactam. Thirty-one percent (36/117) and 25% (21/85) of MRSA isolates were NaHCO₃-responsive to cefazolin and oxacillin, respectively. The NaHCO₃-responsive phenotype was significantly associated with sequence type 93, SCCmec type IVa, and mecA alleles with substitutions in positions -7 and -38 in the regulatory region. Among participants treated with a β-lactam, there was no association between the NaHCO₃-responsive phenotype and persistent bacteremia (cefazolin, P = 0.82; oxacillin, P = 0.81). In patients from a randomized clinical trial with MRSA bloodstream infection, isolates with an in vitro β-lactam-NaHCO₃-responsive phenotype were associated with distinctive genetic signatures, but not with a shorter duration of bacteremia among those treated with a β-lactam.

Keywords: MRSA; bacterial genomics; bloodstream-infections; methicillin-resistant Staphylococcus aureus; sodium bicarbonate (NaHCO3); β-lactams.

Fig 1

Isolate selection process. CAMERA2, Combination Antibiotics for Methicillin Resistant Staphylococcus aureus; ST, sequence type.

Fig 2

(a) mecA haplotypes. Scheme of the mecA gene and its regulatory region. SNPs in the regulatory region and amino acid substitutions in the PBP2a protein were used to annotate mecA haplotypes. (b and c) Balloon plot of the intersection between ST, mecA haplotype, and (b) cefazolin-NaHCO₃ responsiveness and (c) oxacillin-NaHCO₃ responsiveness. Cefazolin- and oxacillin-NaHCO₃ responsiveness are defined as a fourfold or greater decrease in the MIC to that agent in the presence of 44 mM of NaHCO₃. RBS, ribosome binding site; MLST, multilocus sequence type; MIC, minimal inhibitory concentration. Created with BioRender.com.

Fig 3

Phylogenetic tree. Maximum likelihood phylogenetic tree of 117 MRSA isolates built from a core genome SNP alignment. STs are shown on the branches. mecA haplotypes (R1, R2, R3, S2, S3, S5) were annotated based on combinations of SNPs in the mecA regulatory region and SNPs in the coding region resulting in amino acid substitution. The MICs of cefazolin and oxacillin alone, and in the presence of 44 mM of NaHCO₃, are presented along with the cefazolin-NaHCO₃- and oxacillin-NaHCO₃-responsive phenotype, which is defined as a greater than fourfold decrease in the MIC measured in the presence compared to the absence of NaHCO₃. Isolates collected from participants with persistent bacteremia are marked with an asterisk (*). MRSA, methicillin-resistant Staphylococcus aureus; SNP, single nucleotide polymorphism; ST, sequence type; MIC, minimal inhibitory concentration; CFZ, cefazolin; OXA, oxacillin.

Fig 4

Cefazolin-NaHCO₃ and oxacillin-NaHCO₃ responsiveness by MLST. (a and c) fold-change in the MIC of cefazolin (a) and oxacillin (c) in the presence of NaHCO₃ compared to MHB alone colored by MLST. The NaHCO₃-responsive phenotype is defined by a greater than fourfold reduction in the MIC in the presence of NaHCO₃ (e.g., from 16 mg/L on MHB to 4 mg/L on MHB with the addition of 44 mM NaHCO₃). (b and d) scatter plot of MIC (mg/L) values of (b) cefazolin and (d) oxacillin with and without NaHCO₃ by BMD. Panels b and d illustrate the relationship between MIC with and MIC without NaHCO₃, with isolates in the lower right corner (below the second dashed line) representing NaHCO₃-responsive groups. MIC, minimal inhibitory concentration; MLST, multilocus sequence type; ST, sequence type; MHB, Mueller Hinton Broth; BMD, broth microdilution.

Fig 5

Clinical correlates of the NaHCO₃ phenotype. (Top) Rate of persistent bacteremia on day 5 among participants who were allocated to standard or combination therapy and had infections caused by cefazolin-NaHCO₃-responsive compared to non-responsive strains. (Bottom) Rate of persistent bacteremia on day 5 among participants who were allocated to standard or combination therapy and had infections caused by oxacillin-NaHCO₃-responsive compared to non-responsive strains.