Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit β-lactam susceptibility in vitro , ex vivo , and in vivo in the presence of NaHCO ₃ (NaHCO ₃ -responsive MRSA). Here, we investigate the impact of NaHCO ₃ on factors required for PBP2a functionality.

Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a difficult clinical treatment issue. Recently, a novel phenotype was discovered amongst selected MRSA which exhibited enhanced β-lactam susceptibility in vitro in the presence of NaHCO₃ (termed ‘NaHCO₃-responsiveness’). This increased β-lactam susceptibility phenotype has been verified in both ex vivo and in vivo models. Mechanistic studies to-date have implicated NaHCO₃-mediated repression of genes involved in the production, as well as maturation, of the alternative penicillin-binding protein (PBP) 2a, a necessary component of MRSA β-lactam resistance. Herein, we utilized RNA-sequencing (RNA-seq) to identify genes that were differentially expressed in NaHCO₃-responsive (MRSA 11/11) vs. non-responsive (COL) strains, in the presence vs. absence of NaHCO₃-β-lactam co-exposures. These investigations revealed that NaHCO₃ selectively repressed the expression of a cadre of genes in strain 11/11 known to be a part of the sigB-sarA-agr regulon, as well as a number of genes involved in the anchoring of cell wall proteins in MRSA. Moreover, several genes related to autolysis, cell division, and cell wall biosynthesis/remodeling, were also selectively impacted by NaHCO₃-OXA exposure in the NaHCO₃-responsive strain MRSA 11/11. These outcomes provide an important framework for further studies to mechanistically verify the functional relevance of these genetic perturbations to the NaHCO₃-responsiveness phenotype in MRSA.

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.

MRSA is generally viewed as resistant to standard β-lactam antibiotics. However, a NaHCO ₃ -responsive phenotype is observed in a substantial proportion of clinical MRSA strains in vitro , i.e., isolates which demonstrate enhanced susceptibility to standard β-lactam antibiotics (e.g., oxacillin) in the presence of NaHCO ₃ .

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to β-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many β-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed “NaHCO₃-responsiveness”. This phenotype features β-lactam susceptibility of certain MRSA strains only in the presence of NaHCO₃. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO₃-responsive and NaHCO₃-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on β-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO₃. These investigations revealed that NaHCO₃ differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO₃-β-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO₃. This NaHCO₃-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO₃-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO₃-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO₃-responsiveness. This investigation also provides strong evidence that the NaHCO₃-responsive phenotype in MRSA may involve NaHCO₃-mediated increases in both initial cell surface β-lactam binding, as well as ultimate PBP2a binding of β-lactams.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most β-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed ‘NaHCO ₃ -responsiveness’, is a property whereby strains are susceptible in vitro to many β-lactams in the presence of NaHCO ₃ .