Transposon Mutagenesis Identifies Novel Genes Associated with Staphylococcus aureus Persister Formation.

Wenjie Wang,Gang Chen,Jing Wu,Peng Cui,Wenhong Zhang,Jing Zhao,Xin Du,Ying Zhang,Nan Wu,Jiazhen Chen,Min Li

doi:10.3389/fmicb.2015.01437

Abstract

Pathogenic bacterial persisters are responsible for the recalcitrance of chronic and persistent infections to antimicrobial therapy. Although the mechanisms of persister formation and survival have been widely studied in Escherichia coli, persistence mechanisms in Staphylococcus aureus remain largely unknown. Here, we screened a transposon mutant library of a clinical methicillin-resistant Staphylococcus aureus(MRSA)strain, USA500 (ST8), under antibiotic pressure and identified 13 genes whose insertion mutations resulted in a defect in persistence. These candidate genes were further confirmed by evaluating the survival of the mutants upon exposure to levofloxacin and several other stress conditions. We found 13 insertion mutants with significantly lower persister numbers under several stress conditions, including sdhA, sdhB, ureG, mnhG1, fbaA, ctaB, clpX, parE, HOU_0223, HOU_0587, HOU_2091, HOU_2315, and HOU_2346, which mapped into pathways of oxidative phosphorylation, TCA cycle, glycolysis, cell cycle, and ABC transporters, suggesting that these genes and pathways may play an important role in persister formation and survival. The newly constructed knockout strains of ureG, sdhA and sdhB and their complemented strains were also tested for defect in persisters following exposure to levofloxacin and several other stress conditions. The results from these experiments were consistent with the screening results, which indicated that deletion of these genes in MRSA USA500 leads to persister defect. These findings provide novel insights into the mechanisms of persister formation and survival in S. aureus and offer new targets for the development of persister-directed antibiotics for the improved treatment of chronic and persistent infections.

Highlights

Bacterial persisters were first described by Hobby in 1942 (Hobby et al, 1942) and have since been identified in various bacterial pathogens, such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Pseudomonas aeruginosa (Moyed and Bertrand, 1983; Mulcahy et al, 2010; Zhang et al, 2012)
The parent S. aureus strain used in this study, USA500, is a clinical isolate that is resistant to penicillin, oxacillin, cefazolin, cefuroxime, and cefoxitin and is susceptible to gentamicin, vancomycin, linezolid, levofloxacin, and rifampicin (Table S1)
Levofloxacin had a high bactericidal activity against stationary-phase bacteria, which were decreased by 5-log and 7log after 3- and 5-day exposure, respectively (Figure 1); levofloxacin was used to screen for mutants with reduced persister numbers

Summary

Introduction

Bacterial persisters were first described by Hobby in 1942 (Hobby et al, 1942) and have since been identified in various bacterial pathogens, such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Pseudomonas aeruginosa (Moyed and Bertrand, 1983; Mulcahy et al, 2010; Zhang et al, 2012). Persisters are a small bacterial population that survive a lethal. Genes in S. aureus Persister Formation concentration of antibiotics (Lewis, 2010). This heterogeneous subpopulation of slow- or non-growing bacterial cells regrow after removal of the antibiotic (Zhang, 2014). S. aureus is an opportunistic pathogen that can cause severe wound and nosocomial infections. Persisters pose significant challenges for the effective treatment of various infections

Methods

Results

Conclusion