A Combined Phenotypic-Genotypic Predictive Algorithm for In Vitro Detection of Bicarbonate: β-Lactam Sensitization among Methicillin-Resistant Staphylococcus aureus (MRSA)

Abstract

Antimicrobial susceptibility testing (AST) is routinely used to establish predictive antibiotic resistance metrics to guide the treatment of bacterial pathogens. Recently, a novel phenotype termed “bicarbonate (NaHCO3)-responsiveness” was identified in a relatively high frequency of clinical MRSA strains, wherein isolates demonstrate in vitro “susceptibility” to standard β-lactams (oxacillin [OXA]; cefazolin [CFZ]) in the presence of NaHCO3, and in vivo susceptibility to these β-lactams in experimental endocarditis models. We investigated whether a targeted phenotypic-genotypic screening of MRSA could rule in or rule out NaHCO3 susceptibility upfront. We studied 30 well-characterized clinical MRSA bloodstream isolates, including 15 MIC-susceptible to CFZ and OXA in NaHCO3-supplemented Mueller–Hinton Broth (MHB); and 15 MIC-resistant to both β-lactams in this media. Using a two-tiered strategy, isolates were first screened by standard disk diffusion for susceptibility to a combination of amoxicillin-clavulanate [AMC]. Isolates then underwent genomic sequence typing: MLST (clonal complex [CC]); agr; SCCmec; and mecA promoter and coding region. The combination of AMC disk susceptibility testing plus mecA and spa genotyping was able to predict MRSA strains that were more or less likely to be NaHCO3-responsive in vitro, with a high degree of sensitivity and specificity. Validation of this screening algorithm was performed in six strains from the overall cohort using an ex vivo model of endocarditis. This ex vivo model recapitulated the in vitro predictions of NaHCO3-responsiveness vs. nonresponsiveness above in five of the six strains.

The NaHCO 3 -Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus (MRSA) Is Influenced by mecA Genotype

“…The parental responsive MW2 and nonresponsive C36 stains ( 10 , 15 , 30 ) and their swap constructs were then assessed for susceptibility to the anti-staphylococcal β-lactam, oxacillin (OXA), in the presence or absence of NaHCO 3 . Upon minimum inhibitory concentration (MIC) assessment of the MW2 parent and its mecA swap mutants, alteration of the RBS -7 site from T-to-G eliminated the NaHCO 3 -responsiveness phenotype to OXA in this background, such that mutants harboring the RBS -7G allele (ALC9200 and ALC9196) were highly resistant to OXA in the presence of NaHCO 3 ( Table 1 ).…”

“…The recent discoveries of the impact of specific mecA alleles on β-lactam susceptibility phenotypes appear to reveal novel paradigms for further understanding of β-lactam resistance in MRSA. Thus, mutations within the mecA promoter at the -7 site (a part of the RBS) have now been linked to both altered susceptibility to a combination of β-lactam/β-lactamase inhibitors, as well as to the NaHCO 3 -β-lactam-responsive phenotype ( 27 , 30 ).…”

“…One possible explanation for the differential ability of NaHCO 3 to repress mecA transcription in the MW2 versus C36 background strains may be intrinsic differences in the native SCC mec cassettes in each strain background. NaHCO 3 -responsive strain, MW2, possesses SCC mec type IV, whereas the nonresponsive strain C36 possesses SCC mec type II ( 30 ). Although linkage between specific SCC mec types and the NaHCO 3 -responsive versus-nonresponsive phenotypes has not been established ( 24 ), the underlying genetic differences between these two cassettes may contribute to the ability of specific mecA genotypes to confer NaHCO 3 -responsiveness.…”

“…The primary parental strains utilized in this study were the prototypical and well-characterized NaHCO 3 -responsive strain, MW2 ( 10 , 20 , 30 , 38 ) and the nonresponsive strain, C36 ( 15 , 30 ) ( Table 2 ). Strains MW2 and C36 have previously been identified as having either the “susceptible 2” or “resistant 2” mecA genotypes, respectively ( 30 ), as defined by Harrison et al ( 27 ). Strains were stored at −80°C and isolated on tryptic soy agar (TSA) at 37°C in ambient air when ready for use.…”

Recent Developments in Phenotypic and Molecular Diagnostic Methods for Antimicrobial Resistance Detection in Staphylococcus aureus: A Narrative Review

“…The routine implementation of WGS is carried out in a few settings and mostly for outbreak investigation or molecular surveillance [ 106 ]. WGS is the method with the highest resolution, since it can detect single nucleotide polymorphisms (SNP)s and it can detect all known resistance-associated genes and their variants.…”