scholarly journals Staphylococcus aureus Adapts to Oxidative Stress by Producing H2O2-Resistant Small-Colony Variants via the SOS Response

2015 ◽  
Vol 83 (5) ◽  
pp. 1830-1844 ◽  
Author(s):  
Kimberley L. Painter ◽  
Elizabeth Strange ◽  
Julian Parkhill ◽  
Kathleen B. Bamford ◽  
Darius Armstrong-James ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Time Course ◽  
Strand Break ◽  
Host Cells ◽  
Wild Type ◽  
Small Colony Variants ◽  
Content Type ◽  
Dna Double Strand Break ◽  
Small Colony

The development of chronic and recurrentStaphylococcus aureusinfections is associated with the emergence of slow-growing mutants known as small-colony variants (SCVs), which are highly tolerant of antibiotics and can survive inside host cells. However, the host and bacterial factors which underpin SCV emergence during infection are poorly understood. Here, we demonstrate that exposure ofS. aureusto sublethal concentrations of H2O2leads to a specific, dose-dependent increase in the population frequency of gentamicin-resistant SCVs. Time course analyses revealed that H2O2exposure caused bacteriostasis in wild-type cells during which time SCVs appeared spontaneously within theS. aureuspopulation. This occurred via a mutagenic DNA repair pathway that included DNA double-strand break repair proteins RexAB, recombinase A, and polymerase V. In addition to triggering SCV emergence by increasing the mutation rate, H2O2also selected for the SCV phenotype, leading to increased phenotypic stability and further enhancing the size of the SCV subpopulation by reducing the rate of SCV reversion to the wild type. Subsequent analyses revealed that SCVs were significantly more resistant to the toxic effects of H2O2than wild-type bacteria. With the exception of heme auxotrophs, gentamicin-resistant SCVs displayed greater catalase activity than wild-type bacteria, which contributed to their resistance to H2O2. Taken together, these data reveal a mechanism by whichS. aureusadapts to oxidative stress via the production of a subpopulation of H2O2-resistant SCVs with enhanced catalase production.

scholarly journals Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

2014 ◽  
Vol 82 (4) ◽  
pp. 1600-1605 ◽  
Author(s):  
Melissa A. Dean ◽  
Randall J. Olsen ◽  
S. Wesley Long ◽  
Adriana E. Rosato ◽  
James M. Musser
Keyword(s):  
Staphylococcus Aureus ◽  
Pathway Analysis ◽  
Molecular Mechanisms ◽  
Point Mutations ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Small Colony Variants ◽  
Content Type ◽  
Genes Encoding ◽  
Small Colony

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of fiveS. aureusSCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

scholarly journals Inactivation of thyA in Staphylococcus aureus Attenuates Virulence and Has a Strong Impact on Metabolism and Virulence Gene Expression

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Andre Kriegeskorte ◽  
Desiree Block ◽  
Mike Drescher ◽  
Nadine Windmüller ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Thymidylate Synthase ◽  
Molecular Basis ◽  
De Novo ◽  
Strong Impact ◽  
Small Colony Variants ◽  
Content Type ◽  
Long Term Treatment ◽  
Small Colony ◽  
The Impact

ABSTRACTStaphylococcus aureusthymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronicS. aureusinfections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS;thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations inthyAwere leading to inactivity of TS proteins, and TS inactivity led to tremendous impact onS. aureusphysiology and virulence. Whole DNA microarray analysis of the constructed ΔthyAmutant identified severe alterations compared to the wild type. Important virulence regulators (agr,arlRS,sarA) and major virulence determinants (hla,hlb,sspAB, andgeh) were downregulated, while genes important for colonization (fnbA,fnbB,spa,clfB,sdrC, andsdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyAmutant was strongly attenuated in virulence models, including aCaenorhabditis eleganskilling model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed thatthyAactivity has a major role forS. aureusvirulence and physiology.IMPORTANCEThymidine-dependent small-colony variants (TD-SCVs) ofStaphylococcus aureuscarry mutations in the thymidylate synthase (TS) gene (thyA) responsible forde novosynthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistantS. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact onS. aureusvirulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

scholarly journals Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin

2016 ◽  
Vol 23 (6) ◽  
pp. 442-450 ◽  
Author(s):  
Luigi Fiaschi ◽  
Benedetta Di Palo ◽  
Maria Scarselli ◽  
Clarissa Pozzi ◽  
Kelly Tomaszewski ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcal Infection ◽  
Host Cells ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Content Type ◽  
Single Particle Reconstruction ◽  
Alpha Hemolysin ◽  
Human Sera ◽  
Membrane Spanning

ABSTRACTStaphylococcus aureusalpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineeredS. aureusalpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent fromS. aureusinfection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate againstS. aureus.

scholarly journals Pharmacodynamic Evaluation of the Activity of Antibiotics against Hemin- and Menadione-Dependent Small-Colony Variants of Staphylococcus aureus in Models of Extracellular (Broth) and Intracellular (THP-1 Monocytes) Infections

2012 ◽  
Vol 56 (7) ◽  
pp. 3700-3711 ◽  
Author(s):  
L. G. Garcia ◽  
S. Lemaire ◽  
B. C. Kahl ◽  
K. Becker ◽  
R. A. Proctor ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Concentration Effect ◽  
Intracellular Bacteria ◽  
Maximal Effect ◽  
Small Colony Variants ◽  
Content Type ◽  
Pharmacodynamic Profile ◽  
Lower Amplitude ◽  
Small Colony ◽  
Antibiotic Failure

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate ofmenDandhemBmutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistantS. aureus(MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for themenDSCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for themenDmutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

scholarly journals Influence of the Protein Kinase C Activator Phorbol Myristate Acetate on the Intracellular Activity of Antibiotics against Hemin- and Menadione-Auxotrophic Small-Colony Variant Mutants of Staphylococcus aureus and Their Wild-Type Parental Strain in Human THP-1 Cells

2012 ◽  
Vol 56 (12) ◽  
pp. 6166-6174 ◽  
Author(s):  
Laetitia G. Garcia ◽  
Sandrine Lemaire ◽  
Barbara C. Kahl ◽  
Karsten Becker ◽  
Richard A. Proctor ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Parental Strain ◽  
Wild Type ◽  
Content Type ◽  
Small Colony Variant ◽  
Intracellular Activity ◽  
Kinase C ◽  
Pharmacodynamic Profile ◽  
Small Colony ◽  
Intracellular Fate

ABSTRACTIn a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700–3711, 2012), we evaluated the intracellular fate ofmenDandhemBmutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistantStaphylococcus aureusstrain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, itshemBmutant, and the genetically complemented strain in PMA-activated cells and against themenDstrain in both activated and nonactivated cells. This effect was inhibited when cells were incubated withN-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H2O2. In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition ofN-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H2O2. Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

scholarly journals Tomatidine Inhibits Replication of Staphylococcus aureus Small-Colony Variants in Cystic Fibrosis Airway Epithelial Cells

2011 ◽  
Vol 55 (5) ◽  
pp. 1937-1945 ◽  
Author(s):  
Gabriel Mitchell ◽  
Mariza Gattuso ◽  
Gilles Grondin ◽  
Éric Marsault ◽  
Kamal Bouarab ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Electron Transport ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Plant Secondary Metabolite ◽  
Airway Epithelial ◽  
Small Colony Variants ◽  
Content Type ◽  
Small Colony

ABSTRACTSmall-colony variants (SCVs) often are associated with chronicStaphylococcus aureusinfections, such as those encountered by cystic fibrosis (CF) patients. We report here that tomatidine, the aglycon form of the plant secondary metabolite tomatine, has a potent growth inhibitory activity against SCVs (MIC of 0.12 μg/ml), whereas the growth of normalS. aureusstrains was not significantly altered by tomatidine (MIC, >16 μg/ml). The specific action of tomatidine was bacteriostatic for SCVs and was clearly associated with their dysfunctional electron transport system, as the presence of the electron transport inhibitor 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) caused normalS. aureusstrains to become susceptible to tomatidine. Inversely, the complementation of SCVs' respiratory deficiency conferred resistance to tomatidine. Tomatidine provoked a general reduction of macromolecular biosynthesis but more specifically affected the incorporation of radiolabeled leucine in proteins of HQNO-treatedS. aureusat a concentration corresponding to the MIC against SCVs. Furthermore, tomatidine inhibited the intracellular replication of a clinical SCV in polarized CF-like epithelial cells. Our results suggest that tomatidine eventually will find some use in combination therapy with other traditional antibiotics to eliminate persistent forms ofS. aureus.

scholarly journals Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study

2009 ◽  
Vol 58 (8) ◽  
pp. 1067-1073 ◽  
Author(s):  
Rachna Singh ◽  
Pallab Ray ◽  
Anindita Das ◽  
Meera Sharma
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Wild Type ◽  
Planktonic Cells ◽  
Intrinsic Resistance ◽  
Persister Cells ◽  
Small Colony Variants ◽  
Small Colony ◽  
Wild Type Parent

The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms. This study investigated whether persisters and/or SCVs contribute to the antibiotic resistance of Staphylococcus aureus biofilms. A detailed dose-dependent killing of biofilms and planktonic cells with five antibiotics (oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin) was analysed by treating them with each antibiotic at a concentration of 0–100 μg ml−1 at 37 °C for 48 h. The killing of biofilm cells by all of the antibiotics showed the presence of persister cells – most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re-exposure to the antibiotics. SCVs were observed in biofilms only after treatment with ciprofloxacin, and these SCVs were of a transient nature. The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. The treatment of planktonic cells with amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. Thus the high resistance of S. aureus biofilms to multiple unrelated antibiotics is largely dependent on the presence of persister cells. Biofilms harbour a large number of persisters in comparison with planktonic cultures, which either do not harbour persisters or harbour only a small number. SCVs, although not specifically associated with S. aureus biofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associated infections. The intrinsic resistance of these variants may in turn contribute to the enhanced antibiotic resistance of the biofilms thus formed.

scholarly journals Evolution of Staphylococcus aureus under Vancomycin Selective Pressure: the Role of the Small-Colony Variant Phenotype

2014 ◽  
Vol 59 (2) ◽  
pp. 1347-1351 ◽  
Author(s):  
Justin R. Lenhard ◽  
Christof von Eiff ◽  
Irene S. Hong ◽  
Patricia N. Holden ◽  
Michael D. Bear ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Therapy ◽  
Selective Pressure ◽  
Type Model ◽  
Methicillin Resistant ◽  
Small Colony Variants ◽  
Content Type ◽  
Small Colony Variant ◽  
Small Colony

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) often persist despite antibiotic therapy. Against a 108-CFU/ml methicillin-resistantS. aureus(MRSA) (strain COL) population of which 0%, 1%, 10%, 50%, or 100% was an isogenichemBknockout (Ia48) subpopulation displaying the SCV phenotype, vancomycin achieved maximal reductions of 4.99, 5.39, 4.50, 3.28, and 1.66 log10CFU/ml over 48 h. Vancomycin at ≥16 mg/liter shifted a population from 50% SCV cells at 0 h to 100% SCV cells at 48 h, which was well characterized by a Hill-type model (R2> 0.90).

scholarly journals Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Franziska Kleinert ◽  
René Kallies ◽  
Michael Hort ◽  
Annegret Zweynert ◽  
Christiane Szekat ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Acquired Resistance ◽  
Laboratory Strain ◽  
Resistance Mechanisms ◽  
Transposition Frequency ◽  
Related Sequence ◽  
Small Colony Variants ◽  
Content Type ◽  
Small Colony ◽  
Insertion Sites

ABSTRACT Staphylococcus aureus has acquired resistance to nearly all antibiotics used in clinical practice. Whereas some resistance mechanisms are conferred by uptake of resistance genes, others evolve by mutation. In this study, IS256 has been shown to play a role, e.g., in S. aureus strains displaying intermediate resistance to vancomycin (VISA). To characterize the IS256 insertion sites in the genomes of two closely related sequence type 247 (ST247) VISA strains, all insertions were mapped in both VISA and a susceptible control strain. The results showed that the three ST247 strains contained the highest number so far of IS256 insertions for all sequenced S. aureus strains. Furthermore, in contrast to the case with the other IS elements in these genomes, the IS256 insertion sites were not identical in the closely related strains, indicating a high transposition frequency of IS256. When IS256 was introduced into a laboratory strain which was then cultured in the presence of antibiotics, it was possible to isolate small-colony variants (SCVs) that possessed IS256 insertions in guaA and hemY that displayed increased resistance to vancomycin and aminoglycosides, respectively. For these clones, a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs was observed. The reversion was caused by excision of IS256 in a small number of fast-growing clones that quickly outcompeted the SCVs in broth cultures. In conclusion, the presence of IS256 confers a strong genomic plasticity that is useful for adaptation to antibiotic stress.

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