A Nonclassical Mechanism of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus and Its Effect on Virulence

Satishkumar, Nidhi; Lai, Li-Yin; Mukkayyan, Nagaraja; Vogel, Bruce E.; Chatterjee, Som S.

doi:10.1128/spectrum.02284-22

Cited by 3 publications

(10 citation statements)

References 47 publications

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“…Infection of C. elegans in the absence of drug depicted a significant decrease in virulence of the triple mutant. This phenotype was consistent with previous studies demonstrating decreased C. elegans infection by strains possessing pbp4 - associated mutations (28). Further, accumulation of intracellular CDA has also been previously associated with decreased virulence (39, 40).…”

Section: Discussionsupporting

confidence: 93%

“…We thus sought to assess the effects of alterations in both, PBP4 and GdpP by studying the effect of mutations detected in CRB (16, 17). The pbp4 regulatory site (P pbp4 *) and missense ( pbp4 **) mutations were introduced in SF8300ex (Wtex); a mecA and blaZ excised wild-type (Wt) SF8300, a USA300, a prominent community-associated strain (Table S1, Fig 2A) (2, 19, 28). Additionally, the loss-of-function effect of gdpP mutations was introduced by deletion of gdpP , thus resulting in the strain Wtex P pbp4 * pbp4 ** Δ gdpP , or, the triple mutant.…”

Section: Resultsmentioning

confidence: 99%

“…Growth assays were performed as previously described using Bioscreen C, an automated growth curve analysis system (Growth Curves USA) (28). Briefly, overnight cultures of bacteria were diluted to an OD 600nm of 0.1 in TSB with or without antibiotics and 200 µL of the bacterial dilution was pipetted into each well of a Bioscreen C plate in triplicates.…”

Section: Methodsmentioning

confidence: 99%

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

Lai,

Satishkumar,

Cardozo

et al. 2023

Preprint

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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 β-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum β-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 β-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of β-lactam resistant S. aureus infections.

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Section: Discussionsupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Lai,

Satishkumar,

Cardozo

et al. 2023

Preprint

Self Cite

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show abstract

“…Growth assays were performed as previously described using Bioscreen C, an automated growth curve analysis system (Growth Curves USA) ( 29 ). Briefly, overnight cultures of bacteria were diluted to an OD 600 nm of 0.1 in TSB with or without antibiotics, and 200 µL of the bacterial dilution was pipetted into each well of a Bioscreen C plate in triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…Infection of C. elegans DH26 was performed as described previously ( 29 ). Briefly, after age synchronization, 15 L4 young adult worms were incubated with 1.5 × 10 6 S. aureus in each well of a 96-well flat plate.…”

Section: Methodsmentioning

confidence: 99%

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

Lai,

Satishkumar,

Cardozo

et al. 2024

mBio

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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 β-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum β-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 spp. 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 toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum β-lactam resistance in S. aureus . It thus suggests reconsideration of the current approach toward diagnosis and treatment of β-lactam-resistant S. aureus infections. IMPORTANCE In Staphylococcus aureus , high-level, broad-spectrum resistance to β-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA . This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to β-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA , as seen by increased bacterial survival in the presence of β-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and G GDEF d omain p rotein containing p hosphodiesterase ( gdpP ) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of β-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

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Staphylococcus aureus AbcA transporter enhances persister formation under β-lactam exposure

Truong-Bolduc,

Wang,

Ferrer-Espada

et al. 2024

Antimicrob Agents Chemother

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We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6–14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

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A Nonclassical Mechanism of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus and Its Effect on Virulence

Cited by 3 publications

References 47 publications

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

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

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

Staphylococcus aureus AbcA transporter enhances persister formation under β-lactam exposure

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