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Molecular mechanisms of thioridazine resistance in Staphylococcus aureus.

ABSTRACT: Staphylococcus aureus has developed resistance towards the most commonly used anti-staphylococcal antibiotics. Therefore, there is an urgent need to find new treatment opportunities. A new approach relies on the use of helper compounds, which are able to potentiate the effect of antibiotics. A well-studied helper compound is thioridazine, which potentiates the effect of the ?-lactam antibiotic dicloxacillin against Methicillin-resistant Staphylococcus aureus (MRSA). In order to identify thioridazine's mechanism of action and how it potentiates the effect of dicloxacillin, we generated thioridazine resistant strains of MRSA USA300 by serial passage experiments. Selected strains were whole-genome sequenced to find mutations causing thioridazine resistance. Genes observed to be mutated were attempted deleted in MRSA USA300. The cls gene encoding a cardiolipin synthase important for synthesis of the membrane lipid cardiolipin was found to be mutated in thioridazine resistant strains. Deletion of this gene resulted in a two-fold increased Minimum inhibitory concentrations (MIC) value for thioridazine compared to the wild type and decreased susceptibility similar to the thioridazine resistant strains. Since cardiolipin likely plays a role in resistance towards thioridazine, it might also be important for the mechanism of action behind the potentiating effect of thioridazine. TDZ is known to intercalate into the membrane and we show here that TDZ can depolarize the plasma membrane. However, our results indicate that the membrane potential reducing effect of TDZ is independent of the resistance mechanism.

SUBMITTER: Wassmann CS

PROVIDER: S-EPMC6082566 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Molecular mechanisms of thioridazine resistance in Staphylococcus aureus.

Wassmann Claes Søndergaard CS Lund Lars Christian LC Thorsing Mette M Lauritzen Sabrina Prehn SP Kolmos Hans Jørn HJ Kallipolitis Birgitte Haahr BH Klitgaard Janne Kudsk JK

PloS one 20180808 8

Staphylococcus aureus has developed resistance towards the most commonly used anti-staphylococcal antibiotics. Therefore, there is an urgent need to find new treatment opportunities. A new approach relies on the use of helper compounds, which are able to potentiate the effect of antibiotics. A well-studied helper compound is thioridazine, which potentiates the effect of the β-lactam antibiotic dicloxacillin against Methicillin-resistant Staphylococcus aureus (MRSA). In order to identify thiorida ...[more]

PMID: 30089175

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Project description:Pseudomonas aeruginosa and Staphylococcus aureus are commonly isolated from polymicrobial infections, such as wound infections and chronic respiratory infections of persons with cystic fibrosis. Despite their coisolation, P. aeruginosa produces substances toxic to S. aureus, including pyocyanin, a blue-pigmented molecule that functions in P. aeruginosa virulence. Pyocyanin inhibits S. aureus respiration, forcing it to derive energy from fermentation and adopt a small-colony variant (SCV) phenotype. The mechanisms by which S. aureus sustains infection in the presence of pyocyanin are not clear. We sought to clarify the mechanisms of pyocyanin toxicity in S. aureus as well as identify the staphylococcal factors involved in its resistance to pyocyanin toxicity. Nonrespiring S. aureus SCVs are inhibited by pyocyanin through pyocyanin-dependent reactive oxygen species (ROS) production, indicating that pyocyanin toxicity is mediated through respiratory inhibition and ROS generation. Selection on pyocyanin yielded a menadione auxotrophic SCV capable of growth on high concentrations of pyocyanin. Genome sequencing of this isolate identified mutations in four genes, including saeS, menD, NWMN_0006, and qsrR QsrR is a quinone-sensing repressor of quinone detoxification genes. Inactivation of qsrR resulted in significant pyocyanin resistance, and additional pyocyanin resistance was achieved through combined inactivation of qsrR and menadione biosynthesis. Pyocyanin-resistant S. aureus has an enhanced capability to inactivate pyocyanin, suggesting QsrR-regulated gene products may degrade pyocyanin to alleviate toxicity. These findings demonstrate pyocyanin-mediated ROS generation as an additional mechanism of pyocyanin toxicity and define QsrR as a key mediator of pyocyanin resistance in S. aureus IMPORTANCE Many bacterial infections occur in the presence of other microbes, where interactions between different microbes and the host impact disease. In patients with cystic fibrosis, chronic lung infection with multiple microbes results in the most severe disease manifestations. Staphylococcus aureus and Pseudomonas aeruginosa are prevalent cystic fibrosis pathogens, and infection with both is associated with worse outcomes. These organisms have evolved mechanisms of competing with one another. For example, P. aeruginosa produces pyocyanin, which inhibits S. aureus growth. Our research has identified how pyocyanin inhibits S. aureus growth and how S. aureus can adapt to survive in the presence of pyocyanin. Understanding how S. aureus sustains infection in the presence of P. aeruginosa may identify means of disrupting these microbial communities.

Project description:BACKGROUND:Antimicrobial resistance is an increasingly serious problem in public health globally. Monitoring resistance levels within healthcare and community settings is critical to combat its ongoing increase. This study aimed to describe the rates and molecular mechanisms of mupirocin resistance in clinical Staphylococcus aureus isolates from Tygerberg Hospital, and to describe its association with strain types. METHODS:We retrospectively selected 212?S. aureus isolates which were identified from blood samples and pus swabs during the years 2009-2011 and 2015-2017. The isolates were identified using conventional microbiological methods and genotyping was done using spa typing. Cefoxitin (30??g) disc diffusion and the two disc strategy (5??g and 200??g) were used to determine susceptibility to methicillin and mupirocin, respectively. Isolates with high-level resistance were screened for the plasmid mediated genes mupA and mupB by PCR, and sequencing of the ileS gene was done for all isolates exhibiting low-level resistance to describe the mutations associated with this phenotype. Chi-square test was used to assess the associations between mupirocin resistance and S. aureus genotypes. RESULTS:Of 212?S. aureus isolates, 12% (n?=?25) were resistant to mupirocin, and 44% (n?=?93) were methicillin resistant. Strain typing identified 73 spa types with spa t045 being the most predominant constituting 11% of the isolates. High-level mupirocin resistance was observed in 2% (n?=?5), and low-level resistance in 9% (n?=?20) of the isolates. The prevalence of high-level mupirocin resistance amongst MRSA and MSSA was 4 and 1% respectively, while the prevalence of low-level mupirocin resistance was significantly higher in MRSA (18%) compared to MSSA (3%), (p?=?0.032). mupA was the only resistance determinant for high-level resistance, and the IleS mutation V588F was identified in 95% of the isolates which showed low-level resistance. A significant association was observed between spa type t032 and high-level mupirocin resistance, and types t037 and t012 and low-level resistance (p?<? 0.0001). CONCLUSION:The study reported higher rates of low-level mupirocin resistance compared to high-level resistance, and in our setting, mupirocin resistance was driven by certain genotypes. Our study advocates for the continuous screening for mupirocin resistance in S. aureus in clinical settings to better guide treatment and prescribing practices.

Project description:Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by ?-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and ?-lactams is not fully understood. In the present study, we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan (PGN) synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs PGN biosynthesis at a stage that precedes transpeptidation by penicillin-binding proteins (PBPs). In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced ?-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal PGN precursors with pentaglycine branches, the correct substrate of S. aureus PBPs. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards ?-lactam antibiotics.

Dataset Information

Molecular mechanisms of thioridazine resistance in Staphylococcus aureus.

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Molecular mechanisms of thioridazine resistance in Staphylococcus aureus.

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