Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most β-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO₃-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care β-lactams in the presence of physiologically relevant concentrations of NaHCO₃, in vitro and ex vivo; moreover, such "NaHCO₃-responsive" isolates can be effectively cleared by β-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO₃ exposure on NaHCO₃-responsive MRSA is to repress WTA synthesis. This NaHCO₃ effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting β-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO₃ exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO₃ and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO₃-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO₃), pre-exposure to TCP prior to infection sensitized the NaHCO₃-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO₃ is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific β-lactam agents.

Keywords: NaHCO3-responsive; experimental infective endocarditis (IE); methicillin-resistant Staphylococcus aureus (MRSA); penicillin-binding proteins (PBPs); wall teichoic acid (WTA) synthesis; β-lactams.

Fig 1

Time-kill curves for NaHCO₃-responsive strains MRSA 11/11 (A) and MW2 (B), and non-responsive strains COL (C) and BMC1001 (D) exposed to CFX, with or without NaHCO₃ and TCP. Cells were incubated without drug for 3 h to enter log phase. For all assays, drug concentrations are 30 µg/mL CFX and 32 µg/mL TCP.

Fig 2

SEV kill curves for NaHCO₃-responsive strain MW2 (A), and non-responsive strains COL (B) and BMC1001 (C) exposed to CFX, with or without NaHCO₃ and TCP. CFX dosing mimics human PK/PD, while TCP dosing was optimized to reach a steady state of 8 µg/mL throughout the experiment.

Fig 3

Tissue burdens (CFU/g) of NaHCO₃-responsive strain MW2 (A) and non-responsive strain COL (B) following infection in a rabbit model of IE. Rabbits were grouped as follows: control = infected with MW2 or COL and untreated; CFX = infected with MW2 or COL and treated with CFX; TCP = infected with MW2 or COL that were exposed to TCP prior to infection and untreated; TCP + CFX = infected with MW2 or COL that were exposed to TCP prior to infection and treated with CFX. Veg. = cardiac vegetation, Kid. = kidney, Spl. = spleen. Statistics were calculated by an unpaired Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4

Schematic of potential impacts of TCP and NaHCO₃ on WTA biosynthesis. Previous studies have demonstrated that TCP inhibits WTA biosynthesis (similarly to tarocins and tunicamycin), potentially via impacts on TarO functionality (13, 15, 17). NaHCO₃ has been demonstrated to specifically inhibit the expression of cap5/8P in a NaHCO₃-responsive MRSA strain (36). Inhibition of cap5/8P expression, coupled with inhibition of TarO enzymatic activity (via direct or indirect mechanisms), could result in disruption of WTA biosynthesis, as previously observed (12), sensitizing NaHCO₃-responsive MRSA to β-lactams. Created with BioRender.com.