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The Persister Character of Clinical Isolates of Staphylococcus aureus Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin.

ABSTRACT: Staphylococcus aureus may cause relapsing infections. We previously showed that S. aureus SH1000 surviving intracellularly to bactericidal antibiotics are persisters. Here, we used 54 non-duplicate clinical isolates to assess links between persistence, resistance evolution, and intracellular survival, using moxifloxacin throughout as test bactericidal antibiotic. The relative persister fraction (RPF: percentage of inoculum surviving to 100× MIC moxifloxacin in stationary phase culture for each isolate relative to ATCC 25923) was determined to categorize isolates with low (?10) or high (>10) RPF. Evolution to resistance (moxifloxacin MIC ? 0.5 mg/L) was triggered by serial passages at 0.5× MIC (with daily concentration readjustments). Intracellular moxifloxacin maximal efficacy (E_max) was determined by 24 h concentration-response experiments [pharmacodynamic model (Hill-Langmuir)] with infected THP-1 monocytes exposed to moxifloxacin (0.01 to 100× MIC) after phagocytosis. Division of intracellular survivors was followed by green fluorescence protein dilution (FACS). Most (30/36) moxifloxacin-susceptible isolates showed low RPF but all moxifloxacin-resistant (n = 18) isolates harbored high RPF. Evolution to resistance of susceptible isolates was faster for those with high vs. low RPF (with SOS response and topoisomerase-encoding genes overexpression). Intracellularly, moxifloxacin E_max was decreased (less negative) for isolates with high vs. low RPF, independently from resistance. Moxifloxacin intracellular survivors were non-dividing. The data demonstrate and quantitate persisters in clinical isolates of S. aureus, and show that this phenotype accelerates resistance evolution and is associated with intracellular survival in spite of high antibiotic concentrations. Isolates with high RPF may represent a possible cause of treatment failure not directly related to resistance in patients receiving active antibiotics.

SUBMITTER: Nguyen TK

PROVIDER: S-EPMC7719683 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The Persister Character of Clinical Isolates of Staphylococcus aureus Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin.

Nguyen Tiep K TK Peyrusson Frédéric F Dodémont Magali M Pham Nhung H NH Nguyen Hoang A HA Tulkens Paul M PM Van Bambeke Françoise F

Frontiers in microbiology 20201123

Staphylococcus aureus may cause relapsing infections. We previously showed that S. aureus SH1000 surviving intracellularly to bactericidal antibiotics are persisters. Here, we used 54 non-duplicate clinical isolates to assess links between persistence, resistance evolution, and intracellular survival, using moxifloxacin throughout as test bactericidal antibiotic. The relative persister fraction (RPF: percentage of inoculum surviving to 100× MIC moxifloxacin in stationary phase cult ...[more]

PMID: 33329458

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Project description:BackgroundPersister cells comprise a phenotypic variant that shows extreme antibiotic tolerance resulting in treatment failures of bacterial infections. While this phenomenon has posed a great threat in public health, mechanisms underlying their formation in Staphylococcus aureus remain largely unknown. Increasing evidences of the presence of persister cells in recalcitrant infections underscores the great urgency to unravel the mechanism by which these cells develop. Previously, we characterized msaABCR operon that plays roles in regulation of virulence, biofilm development and antibiotic resistance. We also characterized the function of MsaB protein and showed that MsaB is a putative transcription factor that binds target DNA in response to nutrients availability.ResultsIn this study, we compared the number of persister cell in wild type, msaABCR deletion mutant and the complemented strain in two backgrounds USA300 LAC and Mu50. Herein, we report that msaABCR deletion mutant forms significantly less number of persister cells relative to wild type after challenge with various antibiotics in planktonic and biofilm growth conditions. Complementation of the msaABCR operon restored wild type phenotype. Combined antibiotic therapy along with msaABCR deletion significantly improves the killing kinetics of stationary phase and biofilm S. aureus cells. Transcriptomics analysis showed that msaABCR regulates several metabolic genes, transcription factors, transporters and enzymes that may play role in persister cells formation, which we seek to define in the future.ConclusionsThis study presented a new regulator, msaABCR operon, that is involved in the persister cells formation, which is a poorly understood in S. aureus. Indeed, we showed that msaABCR deletion significantly reduces the persister cells formation in all growth phases tested. Although, we have not yet defined the mechanism, we have shown that msaABCR regulates several metabolic, transporters, and extracellular proteases genes that have been previously linked with persister cells formation in other bacterial systems. Taken together, this study showed that inactivation of the msaABCR operon enhances the effectiveness of antibiotics for the treatment of S. aureus infections, especially in context of persister cells.

Project description:PhoU homologs are one of the determinant factors in the regulation of persister formation and phosphate metabolism in many bacterial species; however, the functions of PhoU homologs exhibit species-specific characteristics. The pathogenesis of Staphylococcus aureus is closely correlated with persister formation and virulence factors. The functions of two PhoU homologs, PhoU1 and PhoU2, in S. aureus are unclear yet. In this study, single- and double-deletion mutants of phoU1 and phoU2 were generated in strain USA500 2395. The ΔphoU1 or ΔphoU2 mutants displayed a change in persister formation and virulence compared to the parent strain; the persisters to vancomycin and levofloxacin were decreased at least 1,000-fold, and the number of intracellular bacteria surviving in the A549 cells for 24 h decreased to 82 or 85%. The α-hemolysin expression and activity were increased in the ΔphoU2 mutants. Transcriptome analysis revealed that 573 or 285 genes were differentially expressed by at least 2.0-fold in the ΔphoU1 or ΔphoU2 mutant vs. the wild type. Genes involved in carbon and pyruvate metabolism were up-regulated, and virulence genes and virulence regulatory genes were down-regulated, including type VII secretion system, serine protease, leukocidin, global regulator (sarA, rot), and the two-component signal transduction system (saeS). Correspondingly, the deletion of the phoU1 or phoU2 resulted in increased levels of intracellular pyruvate and ATP. Deletion of the phoU2, but not the phoU1, resulted in the up-regulation of inorganic phosphate transport genes and increased levels of intracellular inorganic polyphosphate. In conclusion, both PhoU1 and PhoU2 in S. aureus regulate virulence by the down-regulation of multiple virulence factors (type VII secretion system, serine protease, and leucocidin) and the persister generation by hyperactive carbon metabolism accompanied by increasing intracellular ATP. The results in S. aureus are different from what we have previously found in Staphylococcus epidermis, where only PhoU2 regulates biofilm and persister formation. The different functions of PhoU homologs between the two species of Staphylococcus warrant further investigation.

Dataset Information

The Persister Character of Clinical Isolates of Staphylococcus aureus Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin.

Publications

The Persister Character of Clinical Isolates of <i>Staphylococcus aureus</i> Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin.

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