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 Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant

2010 ◽  
Vol 59 (5) ◽  
pp. 521-527 ◽  
Author(s):  
Rachna Singh ◽  
Pallab Ray ◽  
Anindita Das ◽  
Meera Sharma
Keyword(s):  
Staphylococcus Aureus ◽  
Tricarboxylic Acid Cycle ◽  
Surface Hydrophobicity ◽  
Tricarboxylic Acid ◽  
Polysaccharide Intercellular Adhesin ◽  
Wild Type ◽  
Small Colony Variant ◽  
Enhanced Production ◽  
Small Colony

The role of Staphylococcus aureus small-colony variants (SCVs) in the pathogenesis of biofilm-associated infections remains unclear. This study investigated the mechanism behind increased biofilm-forming potential of a menadione-auxotrophic Staphylococcus aureus SCV compared with the wild-type parental strain, as recently reported by our laboratory. SCVs displayed an autoaggregative phenotype, with a greater amount of polysaccharide intercellular adhesin (PIA), significantly reduced tricarboxylic acid cycle activity and a decreased susceptibility to aminoglycosides and cell-wall inhibitors compared with wild-type. The biofilms formed by the SCV were highly structured, consisting of large microcolonies separated by channels, and contained more biomass as well as significantly more PIA than wild-type biofilms. The surface hydrophobicity of the two phenotypes was similar. Thus, the autoaggregation and increased biofilm-forming capacity of menadione-auxotrophic Staphylococcus aureus SCVs in this study was related to the enhanced production of PIA in these variants.

scholarly journals Attenuated Virulence and Biofilm Formation in Staphylococcus aureus following Sublethal Exposure to Triclosan

2012 ◽  
Vol 56 (6) ◽  
pp. 3092-3100 ◽  
Author(s):  
Joe Latimer ◽  
Sarah Forbes ◽  
Andrew J. McBain
Keyword(s):  
Staphylococcus Aureus ◽  
Growth Rate ◽  
Growth Rates ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Concentration Gradients ◽  
Content Type ◽  
Small Colony Variant ◽  
Small Colony ◽  
Repeated Passage

ABSTRACTSubeffective exposure ofStaphylococcus aureusto the biocide triclosan can reportedly induce a small-colony variant (SCV) phenotype.S. aureusSCVs are characterized by low growth rates, reduced pigmentation, and lowered antimicrobial susceptibility. While they may exhibit enhanced intracellular survival, there are conflicting reports regarding their pathogenicity. The current study reports the characteristics of an SCV-like strain ofS. aureuscreated by repeated passage on sublethal triclosan concentrations.S. aureusATCC 6538 (the passage 0 [P0] strain) was serially exposed 10 times to concentration gradients of triclosan to generate strain P10. This strain was then further passaged 10 times on triclosan-free medium (designated strain ×10). The MICs and minimum bactericidal concentrations of triclosan for P0, P10, and ×10 were determined, and growth rates in biofilm and planktonic cultures were measured. Hemolysin, DNase, and coagulase activities were measured, and virulence was determined using aGalleria mellonellapathogenicity model. Strain P10 exhibited decreased susceptibility to triclosan and characteristics of an SCV phenotype, including a considerably reduced growth rate and the formation of pinpoint colonies. However, this strain also had delayed coagulase production, had impaired hemolysis (P< 0.01), was defective in biofilm formation and DNase activity, and displayed significantly attenuated virulence. Colony size, hemolysis, coagulase activity, and virulence were only partially restored in strain ×10, whereas the planktonic growth rate was fully restored. However, ×10 was at least as defective in biofilm formation and DNase production as P10. These data suggest that although repeated exposure to triclosan may result in an SCV-like phenotype, this is not necessarily associated with increased virulence and adapted bacteria may exhibit other functional deficiencies.

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 Rapidly Correcting Frameshift Mutations in the Mycobacterium tuberculosis orn Gene Produce Reversible Ethambutol Resistance and Small-Colony-Variant Morphology

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Hassan Safi ◽  
Subramanya Lingaraju ◽  
Shuyi Ma ◽  
Seema Husain ◽  
Mainul Hoque ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Frameshift Mutation ◽  
Relative Increase ◽  
Wild Type ◽  
Content Type ◽  
Reading Frame ◽  
Small Colony Variant ◽  
Large Colony ◽  
Small Colony ◽  
High Level

ABSTRACT We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis. However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 μg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

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 Adaptation Genomics of a Small-Colony Variant in a Pseudomonas chlororaphis 30-84 Biofilm

2014 ◽  
Vol 81 (3) ◽  
pp. 890-899 ◽  
Author(s):  
Dongping Wang ◽  
Robert J. Dorosky ◽  
Cliff S. Han ◽  
Chien-chi Lo ◽  
Armand E. K. Dichosa ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Genetic Alterations ◽  
Whole Genome Sequence ◽  
Phenotypic Switching ◽  
Whole Genome ◽  
Pseudomonas Chlororaphis ◽  
Wild Type ◽  
Content Type ◽  
Small Colony Variant ◽  
Small Colony

ABSTRACTThe rhizosphere-colonizing bacteriumPseudomonas chlororaphis30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from aP. chlororaphis30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations inyfiR(cyclic-di-GMP production),fusA(elongation factor), andcyoE(heme synthesis) and a 70-kb deletion. Genetic analysis revealed that theyfiRlocus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in thefusAgene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching inP. chlororaphisis not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles inP. chlororaphisnatural persistence.

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 Phenotypic Stability of Staphylococcus aureus Small Colony Variants (SCV) Isolates from Cystic Fibrosis (CF) Patients

2019 ◽  
Vol 16 (11) ◽  
pp. 1940 ◽  
Author(s):  
Clemens Kittinger ◽  
Daniela Toplitsch ◽  
Bettina Folli ◽  
Lilian Masoud Landgraf ◽  
Gernot Zarfel
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Point Mutations ◽  
High Variability ◽  
Wild Type ◽  
Phenotypic Stability ◽  
Small Colony Variant ◽  
Gene Coding ◽  
Wild Type Phenotype ◽  
Small Colony

One of the most interesting features of Staphylococcus aureus is its ability to switch to a small colony variant (SCV). This switch allows the pathogen to survive periods of antibiotic treatment or pressure from the immune system of the host and further enables it to start the infection once again after the environmental stress declines. However, so far only little is known about this reversion back to the more virulent wild type phenotype. Therefore, this study aimed to analyze the frequency of reversion to the wild type phenotype of thymidine auxotroph S. aureus SCV isolates (TD-SCVs) obtained from patients with cystic fibrosis (CF). With the use of single cell starting cultures, the occurrence of the thymidine prototroph revertants was monitored. The underlying mutational cause of the SCVs and subsequent revertants were analyzed by sequencing the gene coding for thymidylate synthase (ThyA), whose mutations are known to produce thymidine auxotroph S. aureus SCV. In our study, the underlying mutational cause for the switch to the TD-SCV phenotype was primarily point mutations. Out of twelve isolates, seven isolates showed an occurrence of revertants with a frequency ranging from 90.06% to 0.16%. This high variability in the frequency of reversion to the wild type was not expected. However, this variability in the frequency of reversion may also be the key to successful re-infection of the host. Sometimes quick reversion to the wild type proves necessary for survival, whereas other times, staying hidden for a bit longer leads to success in re-colonization of the host.

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