Biology subjects

Chambers, H. F.

Publications and source records attributed to Chambers, H. F..

4 recordsLinked to original sources

bioRxiv2023

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum β-lactam resistance in Staphylococcus aureus

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to Next Generation {beta}-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum {beta}- lactam resistance is classically mediated by PBP2a, a Penicillin-Binding Protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as Methicillin-Resistant Lacking mec (MRLM) are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum {beta}-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of {beta}-lactam resistant S. aureus infections.

microbiology↗

bioRxiv2021

Loss of GdpP function in Staphylococcus aureus leads to β-lactam tolerance and enhanced evolution of β-lactam resistance

SynopsisO_ST_ABSBackgroundC_ST_ABSWe previously reported the presence of mutations in gdpP among Staphylococcus aureus strains that were obtained by serial passaging in {beta}-lactam drugs. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. ObjectivesWe sought to identify the role of gdpP in {beta}-lactam resistance of S. aureus. MethodsCDA concentrations in bacterial cytosol were measured through mass-spectrometric analysis. gdpP deletion mutagenesis and their complemented strains were created in clinically relevant S. aureus strains to characterize its function. ResultsgdpP associated mutations among passaged strains were identified to cause loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a {beta}-lactam challenge (two to three log increase in bacterial colony forming units) by promoting tolerance without enhancing MICs of {beta}-lactam antibiotics. Our results demonstrate that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level {beta}-lactam resistance and could lead to {beta}-lactam treatment failure of S. aureus infections. ConclusionsLoss of GdpP function increases tolerance to {beta}-lactams that can lead to its therapy failure and can permit {beta}-lactam resistance to occur more readily.

microbiology↗

bioRxiv2019

Ability of Bicarbonate Supplementation to Sensitize Selected Methicillin-Resistant Staphylococcus aureus (MRSA) Strains to β-Lactam Antibiotics in an Ex Vivo Simulated Endocardial Vegetation Model

Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) significantly increases {beta}-lactam susceptibility of selected MRSA strains (\"NaHCO3-responsive\"). This \"sensitization\" phenomenon translated to enhanced {beta}-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated {beta}-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro \"NaHCO3-responsive\" or \"NaHCO3-nonresponsive\" phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB{+/-}NaHCO3. Intra-SEV MRSA killing was determined over 72 hr exposure. In both NaHCO3-responsive strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced {beta}-lactam MICs to below the OXA susceptibility breakpoint ([≤] 4 mg/L) resulting in bactericidal activity ([≥] 3 log kill) in the model for both OXA and CFZ. In contrast, neither in vitro-defined NaHCO3-nonresponsive MRSA strains showed significant sensitization in the SEV model to either {beta}-lactam. At both NaHCO3 concentrations, the fractional time-above-MIC was >50% for both CFZ and OXA in the NaHCO3-responsive MRSA. Also, in RPMI+10% LB media (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against each NaHCO3-responsive strain in the SEV model. Neither CFZ nor OXA exposures selected for high-level {beta}-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.

microbiology↗

bioRxiv2019

Bicarbonate Resensitization of Methicillin-Resistant Staphylococcus aureus to β-Lactam Antibiotics

Endovascular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major healthcare concern, especially infective endocarditis (IE). Standard antimicrobial susceptibility testing (AST) defines most MRSA strains as resistant to {beta}-lactams, often leading to use of costly and/or toxic treatment regimens. In this investigation, five prototype MRSA strains, representing the range of genotypes in current clinical circulation, were studied. We identified two distinct MRSA phenotypes upon AST using standard media, with or without sodium bicarbonate (NaHCO3) supplementation: one highly susceptible to the anti-staphylococcal {beta}-lactams, oxacillin and cefazolin ( NaHCO3-responsive) and one resistant to such agents ( NaHCO3-nonresponsive). These phenotypes accurately predicted clearance profiles of MRSA from target tissues in experimental MRSA IE treated with each {beta}-lactam. Mechanistically, NaHCO3 reduced expression of two key genes involved in the MRSA phenotype, mecA and sarA, leading to decreased production of penicillin-binding protein (PBP) 2a (that mediates methicillin resistance), in NaHCO3-responsive (but not in NaHCO3-nonresponsive) strains. Moreover, both cefazolin and oxacillin synergistically killed NaHCO3-responsive strains in the presence of the host defense antimicrobial peptide (LL-37) in NaHCO3-supplemented media. These findings suggest that AST of MRSA strains in NaHCO3-containing media may potentially identify infections caused by NaHCO3-responsive strains that are appropriate for {beta}-lactam therapy.

microbiology↗