Activation of the SOS response increases the frequency of small colony variants

BACKGROUND: In Staphylococcus aureus sub-populations of slow-growing cells forming small colony variants (SCVs) are associated with persistent and recurrent infections that are difficult to eradicate with antibiotic therapies. In SCVs that are resistant towards aminoglycosides, mutations have been identified in genes encoding components of the respiratory chain. Given the high frequencies of SCVs isolated clinically it is vital to understand the conditions that promote or select for SCVs. RESULTS: In this study we have examined how exposure to sub-inhibitory concentrations of antibiotics with different mechanism of action influence the formation of SCVs that are resistant to otherwise lethal concentrations of the aminoglycoside, gentamicin. We found that exposure of S. aureus to fluoroquinolones and mitomycin C increased the frequency of gentamicin resistant SCVs, while other antibiotic classes failed to do so. The higher proportion of SCVs in cultures exposed to fluoroquinolones and mitomycin C compared to un-exposed cultures correlate with an increased mutation rate monitored by rifampicin resistance and followed induction of the SOS DNA damage response. CONCLUSION: Our observations suggest that environmental stimuli, including antimicrobials that reduce replication fidelity, increase the formation of SCVs through activation of the SOS response and thereby potentially promote persistent infections that are difficult to treat

PasT of <i>Escherichia coli</i> sustains antibiotic tolerance and aerobic respiration as a bacterial homolog of mitochondrial Coq10

International audienc

Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus

The emergence of antimicrobial resistance severely threatens our ability to treat bacterial infections. While acquired resistance has received considerable attention, relatively little is known of intrinsic resistance that allows bacteria to naturally withstand antimicrobials. Gene products that confer intrinsic resistance to antimicrobial agents may be explored for alternative antimicrobial therapies, by potentiating the efficacy of existing antimicrobials. In this study, we identified the intrinsic resistome to a broad spectrum of antimicrobials in the human pathogen, Staphylococcus aureus. We screened the Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2, for increased susceptibility to the anti-staphylococcal antimicrobials (ciprofloxacin, oxacillin, linezolid, fosfomycin, daptomycin, mupirocin, vancomycin and gentamicin). 68 mutants were confirmed by E-test to display at least two-fold increased susceptibility to one or more antimicrobial agents. The majority of the identified genes have not previously been associated with antimicrobial susceptibility in S. aureus. For example, inactivation of genes encoding for subunits of the ATP synthase, atpA, atpB, atpG and atpH, reduced the minimum inhibitory concentration (MIC) of gentamicin 16-fold. To elucidate the potential of the screen, we examined treatment efficacy in the Galleria mellonella infection model. Gentamicin efficacy was significantly improved, when treating larvae infected with the atpA mutant compared to wild type cells with gentamicin at a clinically relevant concentration. Our results demonstrate that many gene products contribute to the intrinsic antimicrobial resistance of S. aureus. Knowledge of these intrinsic resistance determinants provides alternative targets for compounds that may potentiate the efficacy of existing antimicrobial agents against this important pathogen

Inhibition of the ATP synthase sensitizes <i>Staphylococcus aureus</i> towards human antimicrobial peptides

Abstract Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human β-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system

Human antimicrobial peptide, LL-37, induces non-inheritable reduced susceptibility to vancomycin in <i>Staphylococcus aureus</i>

Abstract Antimicrobial peptides (AMPs) are central components of the innate immune system providing protection against pathogens. Yet, serum and tissue concentrations vary between individuals and with disease conditions. We demonstrate that the human AMP LL-37 lowers the susceptibility to vancomycin in the community-associated methicillin-resistant S. aureus (CA-MRSA) strain FPR3757 (USA300). Vancomycin is used to treat serious MRSA infections, but treatment failures occur despite MRSA strains being tested susceptible according to standard susceptibility methods. Exposure to physiologically relevant concentrations of LL-37 increased the minimum inhibitory concentration (MIC) of S. aureus towards vancomycin by 75%, and resulted in shortened lag-phase and increased colony formation at sub-inhibitory concentrations of vancomycin. Computer simulations using a mathematical antibiotic treatment model indicated that a small increase in MIC might decrease the efficacy of vancomycin in clearing a S. aureus infection. This prediction was supported in a Galleria mellonella infection model, where exposure of S. aureus to LL-37 abolished the antimicrobial effect of vancomycin. Thus, physiological relevant concentrations of LL-37 reduce susceptibility to vancomycin, indicating that tissue and host specific variations in LL-37 concentrations may influence vancomycin susceptibility in vivo

Novel pathways for ameliorating the fitness cost of gentamicin resistant small colony variants

Small colony variants (SCVs) of the human pathogen Staphylococcus aureus are associated with persistent infections. Phenotypically, SCVs are characterized by slow growth and they can arise upon interruption of the electron transport chain that consequently reduce membrane potential and thereby limit uptake of aminoglycosides (e.g. gentamicin). In this study, we have examined the pathways by which the fitness cost of SCVs can be ameliorated. Five gentamicin resistant SCVs derived from S. aureus JE2 were independently selected on agar plates supplemented with gentamicin. The SCVs carried mutations in the menaquinone and hemin biosynthesis pathways, which caused a significant reduction in exponential growth rates relative to wild type (0.59-0.72) and reduced membrane potentials. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged for up to 500 generations with or without sub-lethal concentrations of gentamicin. Amelioration of the fitness cost followed three evolutionary trajectories and was dependent on the initial mutation type (point mutation vs. deletion) and the passage condition (absence or presence gentamicin). For SCVs evolved in the absence of gentamicin, 12 out of 15 lineages derived from SCVs with point mutations acquired intra-codonic suppressor mutations restoring membrane potential, growth rate, gentamicin susceptibility and colony size to wild type levels. For the SCVs carrying deletions, all lineages enhanced fitness independent of membrane potential restoration without alterations in gentamicin resistance levels. By whole genome sequencing, we identified compensatory mutations in genes related to the σB stress response (7 out of 10 lineages). Inactivation of rpoF that encode for the alternative sigma factor SigB (σB) partially restored fitness of SCVs. For all lineages passaged in the presence of gentamicin, fitness compensation via membrane potential restoration was suppressed, however selected for secondary mutations in fusA and SAUSA300_0749. This study is the first to describe fitness compensatory events in SCVs with deletion mutations and adaptation of SCVs to continued exposure to gentamici