Haem toxicity provides a competitive advantage to the clinically relevant Staphylococcus aureus small colony variant phenotype

Microorganisms encounter toxicities inside the host. Many pathogens exist as subpopulations to maximize survivability. Subpopulations of Staphylococcus aureus include antibiotic-tolerant small colony variants (SCVs). These mutants often emerge following antibiotic treatment but can be present in infections prior to antibiotic exposure. We hypothesize that haem toxicity in the host selects for respiration-deficient S. aureus SCVs in the absence of antibiotics. We demonstrate that some but not all respiration-deficient SCV phenotypes are more protective than the haem detoxification system against transient haem exposure, indicating that haem toxicity in the host may contribute to the dominance of menaquinone-deficient and haem-deficient SCVs prior to antibiotic treatment.

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The MpsB protein contributes to both the toxicity and immune evasion capacity of Staphylococcus aureus

Microbiology ◽

10.1099/mic.0.001096 ◽

2021 ◽

Vol 167 (10) ◽

Author(s):

Edward J. A. Douglas ◽

Seána Duggan ◽

Tarcisio Brignoli ◽

Ruth C. Massey

Keyword(s):

Staphylococcus Aureus ◽

Immune Evasion ◽

Type Species ◽

Severe Disease ◽

Toxin Production ◽

Content Type ◽

Small Colony Variant ◽

Link Type ◽

Small Colony

Understanding the role specific bacterial factors play in the development of severe disease in humans is critical if new approaches to tackle such infections are to be developed. In this study we focus on genes we have found to be associated with patient outcome following bacteraemia caused by the major human pathogen Staphylococcus aureus . By examining the contribution these genes make to the ability of the bacteria to survive exposure to the antibacterial factors found in serum, we identify three novel serum resistance-associated genes, mdeA, mpsB and yycH. Detailed analysis of an MpsB mutant supports its previous association with the slow growing small colony variant (SCV) phenotype of S. aureus , and we demonstrate that the effect this mutation has on membrane potential prevents the activation of the Agr quorum sensing system, and as a consequence the mutant bacteria do not produce cytolytic toxins. Given the importance of both toxin production and immune evasion for the ability of S. aureus to cause disease, we believe that these findings explain the role of the mpsB gene as a mortality-associated locus during human disease.

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A novel hemA mutation is responsible for a small-colony-variant phenotype in Escherichia coli

Microbiology ◽

10.1099/mic.0.000962 ◽

2020 ◽

Cited By ~ 1

Author(s):

Alasdair T. M. Hubbard ◽

Issra Bulgasim ◽

Adam P. Roberts

Keyword(s):

Escherichia Coli ◽

Mammalian Cells ◽

Type Species ◽

Fitness Cost ◽

Growth Media ◽

Content Type ◽

Small Colony Variant ◽

Link Type ◽

Small Colony ◽

Tract Infections

We identified a small colony variant (SCV) of an amoxicillin/clavulanic acid-resistant derivative of a clinical isolate of Escherichia coli from Malawi, which was selected for in vitro in a subinhibitory concentration of gentamicin. The SCV was auxotrophic for hemin and had impaired biofilm formation compared to the ancestral isolates. A single novel nucleotide polymorphism (SNP) in hemA, which encodes a glutamyl-tRNA reductase that catalyses the initial step of porphyrin biosynthesis leading to the production of haem, was responsible for the SCV phenotype. We showed the SNP in hemA resulted in a significant fitness cost to the isolate, which persisted even in the presence of hemin. However, the phenotype quickly reverted during sequential sub-culturing in liquid growth media. As hemA is not found in mammalian cells, and disruption of the gene results in a significant fitness cost, it represents a potential target for novel drug development specifically for the treatment of catheter-associated urinary tract infections caused by E. coli .

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Impact of pH on growth of Staphylococcus epidermidis and Staphylococcus aureus in vitro

Journal of Medical Microbiology ◽

10.1099/jmm.0.001421 ◽

2021 ◽

Vol 70 (9) ◽

Author(s):

Vidula Iyer ◽

Janhavi Raut ◽

Anindya Dasgupta

Keyword(s):

Staphylococcus Aureus ◽

Growth Kinetics ◽

Staphylococcus Epidermidis ◽

Type Species ◽

Ph Dependence ◽

Skin Microbiome ◽

Content Type ◽

Link Type ◽

Kinetics Of

The pH of skin is critical for skin health and resilience and plays a key role in controlling the skin microbiome. It has been well reported that under dysbiotic conditions such as atopic dermatitis (AD), eczema, etc. there are significant aberrations of skin pH, along with a higher level of Staphylococcus aureus compared to the commensal Staphylococcus epidermidis on skin. To understand the effect of pH on the relative growth of S. epidermidis and S. aureus , we carried out simple in vitro growth kinetic studies of the individual microbes under varying pH conditions. We demonstrated that the growth kinetics of S. epidermidis is relatively insensitive to pH within the range of 5–7, while S. aureus shows a stronger pH dependence in that range. Gompertz’s model was used to fit the pH dependence of the growth kinetics of the two bacteria and showed that the equilibrium bacterial count of S. aureus was the more sensitive parameter. The switch in growth rate happens at a pH of 6.5–7. Our studies are in line with the general hypothesis that keeping the skin pH within an acidic range is advantageous in terms of keeping the skin microbiome in balance and maintaining healthy skin.

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Fatal exudative dermatitis in island populations of red squirrels (Sciurus vulgaris): spillover of a virulent Staphylococcus aureus clone (ST49) from reservoir hosts

Microbial Genomics ◽

10.1099/mgen.0.000565 ◽

2021 ◽

Vol 7 (5) ◽

Author(s):

Kay Fountain ◽

Tiffany Blackett ◽

Helen Butler ◽

Catherine Carchedi ◽

Anna-Katarina Schilling ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Type Species ◽

Small Ruminants ◽

Sciurus Vulgaris ◽

Red Squirrels ◽

Content Type ◽

Island Populations ◽

Link Type ◽

Bacterial Cell Membrane ◽

Reservoir Hosts

Fatal exudative dermatitis (FED) is a significant cause of death of red squirrels (Sciurus vulgaris) on the island of Jersey in the Channel Islands where it is associated with a virulent clone of Staphylococcus aureus, ST49. S. aureus ST49 has been found in other hosts such as small mammals, pigs and humans, but the dynamics of carriage and disease of this clone, or any other lineage in red squirrels, is currently unknown. We used whole-genome sequencing to characterize 228 isolates from healthy red squirrels on Jersey, the Isle of Arran (Scotland) and Brownsea Island (England), from red squirrels showing signs of FED on Jersey and the Isle of Wight (England) and a small number of isolates from other hosts. S. aureus was frequently carried by red squirrels on the Isle of Arran with strains typically associated with small ruminants predominating. For the Brownsea carriage, S. aureus was less frequent and involved strains associated with birds, small ruminants and humans, while for the Jersey carriage S. aureus was rare but ST49 predominated in diseased squirrels. By combining our data with publicly available sequences, we show that the S. aureus carriage in red squirrels largely reflects frequent but facile acquisitions of strains carried by other hosts sharing their habitat (‘spillover’), possibly including, in the case of ST188, humans. Genome-wide association analysis of the ruminant lineage ST133 revealed variants in a small number of mostly bacterial-cell-membrane-associated genes that were statistically associated with squirrel isolates from the Isle of Arran, raising the possibility of specific adaptation to red squirrels in this lineage. In contrast there is little evidence that ST49 is a common carriage isolate of red squirrels and infection from reservoir hosts such as bank voles or rats, is likely to be driving the emergence of FED in red squirrels.

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Detection and partial characterization of extracellular inducers of persistence in Staphylococcus epidermidis and Staphylococcus aureus

Journal of Medical Microbiology ◽

10.1099/jmm.0.001392 ◽

2021 ◽

Vol 70 (6) ◽

Author(s):

Elyse C. Curry ◽

Ryan G. Hart ◽

Danni Y. Habtu ◽

Neal R. Chamberlain

Keyword(s):

Molecular Weight ◽

Staphylococcus Aureus ◽

Staphylococcus Epidermidis ◽

Type Species ◽

Proteinase K ◽

Partial Characterization ◽

Content Type ◽

Link Type ◽

Inducing Factors

Introduction. This study describes the identification and partial characterization of persistence-inducing factors (PIFs) from staphylococci. Hypothesis/Gap Statement. Increases in persisters during mid-log phase growth indicate that quorum-sensing factors might be produced by staphylococci. Aim. To identify and partially characterize PIFs from Staphylococcus epidermidis RP62A and Staphylococcus aureus SH1000. Methodology. Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were obtained at various OD600s and following incubation at 16 h. The CCFs were used to develop a PIF assay. The PIF assay was used to partially characterize PIF from S. epidermidis and S. aureus for sizing of PIF activity, temperature and protease sensitivity and inter-species communications. Results. The optimal OD600s for S. epidermidis and S. aureus PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis ’ PIF activity was decreased by storage at 4 oC but not at 20 oC (16 h), 37 oC (1 h) or 100 oC (15 min). S. aureus ’ PIF activity was decreased following storage at 4 oC (2 weeks) and after boiling at 100 oC for 5 min but not after incubation at 37 oC (1 h). PIF activity from both species went through a 3000 molecular weight cutoff ultrafilter. Proteinase K treatment of S. aureus PIF decreased activity but did not decrease the PIF activity of S. epidermidis . PIF from S. epidermidis did not increase persisters when used to treat S. aureus cells and nor did PIF from S. aureus increase persisters when used to treat S. epidermidis cells. Conclusions. Attempts to discover PIFs for staphylococci were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species produce extracellular, low-molecular-weight inducers of persistence when assayed using an OD600 -based PIF assay.

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Staphylococcus singaporensis sp. nov., a new member of the Staphylococcus aureus complex, isolated from human clinical specimens

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.005067 ◽

2021 ◽

Vol 71 (10) ◽

Author(s):

Ka Lip Chew ◽

Sophie Octavia ◽

Deborah Lai ◽

Raymond T. P. Lin ◽

Jeanette W. P. Teo

Keyword(s):

Staphylococcus Aureus ◽

Type Species ◽

Clinical Laboratory ◽

Core Gene ◽

Separate Species ◽

New Members ◽

Content Type ◽

Link Type ◽

Flight Mass Spectrometry ◽

Genotypic Analysis

Staphylococcus argenteus and Staphylococcus schweitzeri are the newest members of the Staphylococcus aureus complex. The number of clinical reports attributed to these new S. aureus complex members is limited. In a retrospective clinical laboratory study conducted over a 4-month period investigating the prevalence of S. argenteus and S. schweitzeri , a total of 43 isolates were selected. Phylogeny based on core-gene multilocus sequence typing (MLST) analysis confirmed that 37 were S. argenteus but a genetically distinct clade of six isolates was identified. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses further supported the classification of these six isolates as a separate species. When compared to S. aureus complex reference genomes, the ANI values were ≤94 % and the dDDH values were <53 %. Based on the seven-gene S. aureus MLST scheme, the six isolates belong to five novel allelic profiles (ST6105, ST6106, ST6107, ST6108 and ST109). Their clinical infection features were similar to S. aureus . Skin and soft tissue infections presented in four out of the six cases. Routine clinical diagnostic identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and biochemical profiling does not differentiate these new members from the rest of the complex. Genotypic analysis suggests that the six isolates belong to a novel species, Staphylococcus singaporensis sp. nov. with isolate SS21T (=DSM 111408T=NCTC14419T) designated as the type strain.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05904-11 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3092-3100 ◽

Cited By ~ 47

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.

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01031-12 ◽

2012 ◽

Vol 56 (12) ◽

pp. 6166-6174 ◽

Cited By ~ 11

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.

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Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms

Microbiology ◽

10.1099/mic.0.000933 ◽

2020 ◽

Vol 166 (8) ◽

pp. 695-706 ◽

Cited By ~ 1

Author(s):

Kevin H. Martin ◽

Grace I. Borlee ◽

William H. Wheat ◽

Mary Jackson ◽

Bradley R. Borlee

Keyword(s):

Staphylococcus Aureus ◽

Mycobacterium Bovis ◽

Type Species ◽

Acanthamoeba Castellanii ◽

Methicillin Resistant ◽

Content Type ◽

Host Immune Responses ◽

Link Type ◽

Abiotic Surfaces ◽

Low Concentrations

Biofilm-associated infections are difficult to eradicate because of their ability to tolerate antibiotics and evade host immune responses. Amoebae and/or their secreted products may provide alternative strategies to inhibit and disperse biofilms on biotic and abiotic surfaces. We evaluated the potential of five predatory amoebae – Acanthamoeba castellanii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, Vermamoeba vermiformis and Dictyostelium discoideum – and their cell-free secretions to disrupt biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis . The biofilm biomass produced by MRSA and M. bovis was significantly reduced when co-incubated with A. castellanii, A. lenticulata and A. polyphaga, and their corresponding cell-free supernatants (CFS). Acanthamoeba spp. generally produced CFS that mediated biofilm dispersal rather than directly killing the bacteria; however, A. polyphaga CFS demonstrated active killing of MRSA planktonic cells when the bacteria were present at low concentrations. The active component(s) of the A. polyphaga CFS is resistant to freezing, but can be inactivated to differing degrees by mechanical disruption and exposure to heat. D. discoideum and its CFS also reduced preformed M. bovis biofilms, whereas V. vermiformis only decreased M. bovis biofilm biomass when amoebae were added. These results highlight the potential of using select amoebae species or their CFS to disrupt preformed bacterial biofilms.

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The general stress response of Staphylococcus aureus promotes tolerance of antibiotics and survival in whole human blood

Microbiology ◽

10.1099/mic.0.000983 ◽

2020 ◽

Vol 166 (11) ◽

pp. 1088-1094 ◽

Cited By ~ 1

Author(s):

Nisha Ranganathan ◽

Rebecca Johnson ◽

Andrew M. Edwards

Keyword(s):

Staphylococcus Aureus ◽

Stress Response ◽

Human Blood ◽

Chronic Infection ◽

Type Species ◽

Content Type ◽

General Stress Response ◽

Link Type ◽

General Stress ◽

Immune Defences

Staphylococcus aureus is a frequent cause of invasive human infections such as bacteraemia and infective endocarditis. These infections frequently relapse or become chronic, suggesting that the pathogen has mechanisms to tolerate the twin threats of therapeutic antibiotics and host immunity. The general stress response of S. aureus is regulated by the alternative sigma factor B (σB) and provides protection from multiple stresses including oxidative, acidic and heat. σB also contributes to virulence, intracellular persistence and chronic infection. However, the protective effect of σB on bacterial survival during exposure to antibiotics or host immune defences is poorly characterized. We found that σB promotes the survival of S. aureus exposed to the antibiotics gentamicin, ciprofloxacin, vancomycin and daptomycin, but not oxacillin or clindamycin. We also found that σB promoted staphylococcal survival in whole human blood, most likely via its contribution to oxidative stress resistance. Therefore, we conclude that the general stress response of S. aureus may contribute to the development of chronic infection by conferring tolerance to both antibiotics and host immune defences.

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