Small-colony Variants Strains Research Articles

Chronic Coinfection with Pseudomonas aeruginosa and Normal Colony Staphylococcus aureus Causes Lung Structural Damage in the Cystic Fibrosis Rat

Cystic fibrosis (CF) respiratory outcomes are heavily influenced by complications of infection. Pseudomonas aeruginosa and Staphylococcus aureus are the most common colonizers of the cystic fibrosis lung, and frequently overlap to cause chronic and persistent co-infections associated with severe disease. However, the dynamics of P. aeruginosa and S. aureus co-infection and its impacts on the development of CF lung structural damage are poorly understood. Additionally, small colony variants (SCVs) of S. aureus have been associated with P. aeruginosa infections in people with CF, but their role in disease progression is largely unknown. In this work, the CF rat was used to model chronic lung co-infection with P. aeruginosa and S. aureus, using clinical and laboratory derived normal colony and SCV strains of S. aureus to evaluate the impact of phenotype on clinical outcomes. Rats co-infected with clinically derived S. aureus of both phenotypes experienced increased inflammation in the lung, but only the combination of P. aeruginosa and clinical normal colony S. aureus led to lung structural decline, including mucus obstruction and bronchiectasis. In regression analyses, damage was associated with a higher burden of P. aeruginosa, indicating that chronic co-infection with normal colony S. aureus and P. aeruginosa may support the progression CF lung decline driven by P. aeruginosa, which might be avoided when co-infecting S. aureus exhibits the SCV phenotype.

Biological characterization of lipoic acid- and heme-dependent Escherichia coli small colony variants isolated from sheep in Xinjiang, China.

Escherichia coli (E. coli) small colony variants (SCVs) have garnered attention due to their heightened antibiotic resistance and enhanced cell retention, posing significant risks to public health and food safety. However, understanding of SCVs derived from sheep remains limited. This study aimed to detect the biological characterization of sheep-derived E. coli SCVs and investigate the factors contributing to SCV development with preliminary genomic data. In this study, a lipoic acid-dependent SCV (LA-SCV) and a wild-type (WT) strain were isolated from sheep bile. Then, a heme-dependent SCV (HD-SCV) was induced from WT using amikacin. Initially, we examined factors contributing to SCV formation via comparative genomics. Subsequent comparisons between WT and two SCV strains encompassed antibiotic resistance, hemolytic activity, biofilm formation, motility, and metabolism. Genomic analyses identified a frameshift deletion mutation in the lipA gene in LA-SCV and a stopgain mutation in the hemG gene in HD-SCV, hypothesized as potential triggers for lipoic acid- and heme-dependent SCV development, respectively. Physiological, biochemical, and cultural traits exhibited notable differences between WT and SCVs, including increased antibiotic resistance, hemolytic activity, and biofilm formation, but alongside non-fermentative acetate utilization, slow growth, reduced intracellular ATP, and decreased motility (P < 0.01). The energy and amino acid metabolism were suppressed during the logarithmic phase in LA-SCV, while both logarithmic and stable phases in HD-SCV. These alterations in biological characteristics present significant challenges in managing E. coli pathogenicity and antibiotic resistance.

Iron restriction induces the small-colony variant phenotype in Staphylococcus aureus.

Pathogens such as Staphylococcus aureus must overcome host-induced selective pressures, including limited iron availability. To cope with the harsh conditions of the host environment, S. aureus can adapt its physiology in multiple ways. One of these adaptations is the fermenting small-colony variant (SCV) phenotype, which is known to be inherently tolerant to certain classes of antibiotics and heme toxicity. We hypothesized that SCVs might also behave uniquely in response to iron starvation since one of the major cellular uses of iron is the respiration machinery. In this study, a respiring strain of S. aureus and fermenting SCV strains were treated with different concentrations of the iron chelator, 2,2' dipyridyl (DIP). Our data demonstrate that a major impact of iron starvation in S. aureus is the repression of respiration and the induction of the SCV phenotype. We demonstrate that the SCV phenotype transiently induced by iron starvation mimics the aminoglycoside recalcitrance exhibited by genetic SCVs. Furthermore, prolonged growth in iron starvation promotes increased emergence of stable aminoglycoside-resistant SCVs relative to the naturally occurring subpopulation of SCVs within an S. aureus community. These findings may have relevance to physiological and evolutionary processes occurring within bacterial populations infecting iron-limited host environments.

Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

Bactericidal Activity of the Bacterial ATP Synthase Inhibitor Tomatidine and the Combination of Tomatidine and Aminoglycoside Against Persistent and Virulent Forms of Staphylococcus aureus.

Tomatidine (TO), a steroid alkaloid, exerts a strong bactericidal activity on the infection-persistent phenotype of Staphylococcus aureus, the small-colony variant (SCV), with a minimal inhibitory concentration (MIC) of 0.06 μg/ml. Also, the combination of TO to an aminoglycoside (AMG) shows a strong synergistic effect against prototypical (WT) S. aureus (MIC 0.06 μg/ml), which is otherwise unaffected by TO alone (MIC > 128 μg/ml). We have recently established that the ATP synthase (subunit AtpE) was the molecular target of TO and that TO reduces the production of ATP in S. aureus. The purpose of this study was to understand how TO and the TO-AMG combination exert bactericidal activities against S. aureus SCV and WT strains, respectively. The impact of TO and of the TO-gentamicin (GEN) combination on the membrane potential and generation of reactive oxygen species (ROS) were determined using florescent probes. GEN uptake in WT was assessed in the presence of TO. Virulence of SCV and WT strains as well as of in vitro-selected mutants showing resistance to TO or the TO-GEN combination was evaluated in a murine thigh infection model. TO causes a reduction in membrane potential in both WT and SCV, but significant amounts of ROS are only produced in SCVs. Besides, the presence of TO improves the uptake of GEN by the WT strain and the combination TO-GEN generated 2.5-folds more ROS in WT, compared to that induced by GEN alone. Under anaerobic conditions, WT adopts a fermentative slow-growth phenotype and becomes susceptible to TO even if used alone. In vivo, TO- or TO-GEN-resistant strains were significantly altered in their ability to colonize tissues. These results shed light on the mechanism of action of TO and its synergy with AMGs against S. aureus WT. TO bactericidal activity against SCVs is attributable to both a critical drop in the membrane potential accompanied by a substantial ROS production. In the WT, TO helps GEN uptake and ROS is also important for the synergy. Acquiring resistance to TO significantly impairs virulence. The residual ATP synthase activity of SCVs might represent the Achilles’ heel of persistent S. aureus.

Genotypic variations between wild-type and small colony variant of Staphylococcus aureus in prosthetic valve infectious endocarditis: a comparative genomic and transcriptomic analysis

Staphylococcus aureus small colony variants (SCVs) can cause persistent infections. However, the genomes and transcriptomes of S. aureus SCVs remain poorly understood. A pair of isogenic wild-type and SCV methicillin-resistant S. aureus (MRSA) strains (IE1 and IE2, respectively) were isolated from a patient with prosthetic valve infectious endocarditis. The SCV strain IE2 grew more slowly than the wild-type strain, and serum killing and mouse lethality assays revealed that the virulence of SCV strain IE2 was decreased. Whole-genome sequencing of the SCV and wild-type strains revealed 15 mutations in nine genes associated with metabolism, virulence and DNA repair, including serine/threonine-protein kinase PrkC (prkC), glycerol-3-phosphate acyltransferase (plsY), 2-deoxyribose-5-phosphate aldolase (deoC), extracellular adherence protein (eap), iron compound ABC uptake transporter substrate-binding protein (sstD), RecU Holliday junction resolvase (recU), excinuclease ABC subunit B (uvrB), type I restriction–modification system, M subunit (hsdM) and smooth muscle caldesmon. Sequencing of RNA transcripts revealed that expression levels of 321 genes were upregulated and 582 genes were downregulated in SCV strain IE2 compared with IE1. Most of the differentially expressed genes were involved in metabolism. Expression levels of several genes involved in the pathways to which plsY, deoC, eap and sstD belonged were changed, associated with the metabolism and virulence of S. aureus. In conclusion, the reduced growth rate and decreased virulence of MRSA SCV strains may be related to mutations in and downregulation of genes associated with metabolism and virulence, especially plsY, deoC, eap and sstD.

The role of programmed death ligand 1 pathway in persistent biomaterial-associated infections.

Staphylococcus epidermidis is commonly involved in biomaterial-associated infections. Bacterial small colony variants (SCV) seem to be well adapted to persist intracellularly in professional phagocytes evading the host immune response. We studied the expression of PD-L1/L2 on macrophages infected with clinical isolates of S. epidermidis SCV and their parent wild type (WT) strains. The cytokine pattern which is triggered by the examined strains was also analysed. In the study, we infected macrophages with S. epidermidis WT and SCV strains. Persistence and release from macrophages were monitored via lysostaphin protection assays. Moreover, the effect of IFN-γ pre-treatment on bacterial internalisation was investigated. Expression of PD-L1/L2 molecules was analysed with the use of FACS. Inflammatory reaction was measured by IL-10, TNF-α ELISAs, and transcriptional induction of TNF-α. Our study revealed that clinical SCV isolates were able to persist and survive in macrophages for at least 3 days with a low cytotoxic effect and a reduced proinflammatory response as compared to WT strains. Bacteria upregulated PD-L1/L2 expression on macrophages as compared to non-stimulated cells. The results demonstrated that the ability of S. epidermidis SCVs to induce elevated levels of anti-inflammatory cytokine, IL-10, and reduced transcriptional induction of TNF-α, together with expression of PD-L1 on macrophages and the ability to persist intracellularly without damaging the host cell could be the key factor contributing to chronicity of SCV infections.

148 Direct detection, clonal analysis, and characterization of wild type and small colony variant strains of Staphylococcus aureus from the sputa of CF patients of the Prague CF centre

148 Direct detection, clonal analysis, and characterization of wild type and small colony variant strains of Staphylococcus aureus from the sputa of CF patients of the Prague CF centre

SigB Is a Dominant Regulator of Virulence in Staphylococcus aureus Small-Colony Variants

Staphylococcus aureus small-colony variants (SCVs) are persistent pathogenic bacteria characterized by slow growth and, for many of these strains, an increased ability to form biofilms and to persist within host cells. The virulence-associated gene expression profile of SCVs clearly differs from that of prototypical strains and is often influenced by SigB rather than by the agr system. One objective of this work was to confirm the role of SigB in the control of the expression of virulence factors involved in biofilm formation and intracellular persistence of SCVs. This study shows that extracellular proteins are involved in the formation of biofilm by three SCV strains, which, additionally, have a low biofilm-dispersing activity. It was determined that SigB activity modulates biofilm formation by strain SCV CF07-S and is dominant over that of the agr system without being solely responsible for the repression of proteolytic activity. On the other hand, the expression of fnbA and the control of nuclease activity contributed to the SigB-dependent formation of biofilm of this SCV strain. SigB was also required for the replication of CF07-S within epithelial cells and may be involved in the colonization of lungs by SCVs in a mouse infection model. This study methodically investigated SigB activity and associated mechanisms in the various aspects of SCV pathogenesis. Results confirm that SigB activity importantly influences the production of virulence factors, biofilm formation and intracellular persistence for some clinical SCV strains.

Prevalence and genetic diversity of Staphylococcus aureus small-colony variants in cystic fibrosis patients

Staphylococcus aureus small-colony variants (SCVs) are being isolated more frequently in cystic fibrosis (CF) patients. We aimed to determine the prevalence of S. aureus SCVs and their phenotypic and genotypic properties in CF patients admitted to a university hospital. Specimens of 248 patients were examined during a period of 11 months. Colonies supposed to be SCVs were evaluated on Columbia blood agar, mannitol salt agar, and brain–heart infusion agar with 5% NaCl (BHIA 5% NaCl). Strains were confirmed by S. aureus nucA PCR. Antibiotic susceptibilities of SCVs and simultaneously isolated S. aureus strains were determined for oxacillin, gentamicin, trimethoprim–sulphamethoxazole, vancomycin, ciprofloxacin, linezolid, and tigecycline. Genetic relatedness between SCVs and normal S. aureus strains was determined with a pulsed-field gel electrophoresis (PFGE) method. S. aureus SCVs were detected in 20 of 248 patients (8.1%). The highest SCV isolation rate was obtained with MSA, followed by BHIA 5% NaCl. Auxotrophism for thymidine was demonstrated in six SCVs. The tigecycline susceptibilities of 48 SCV strains isolated in this study showed higher MIC values than those of 33 simultaneously isolated normal S. aureus strains. Whereas SCVs and normal S. aureus strains showed identical genotypes in 14 of the patients, five patients showed different genotypes. This first study from Turkey evaluating S. aureus SCVs in CF patients has indicated the importance of considering and reporting SCVs in chronic infections such as CF. The presence of SCVs will probably indicate persistent infection, and this might impact on antibiotic treatment decisions, as they are more resistant to antibiotics.

Staphylococcus aureus Persisters Tolerant to Bactericidal Antibiotics

Bacterial persister cells are non- or slow-growing reversible phenotypic variants of the wild type, tolerant to bactericidal antibiotics. We analyzed here Staphylococcus aureus persister levels by monitoring colony-forming unit counts of planktonically grown cells treated with six different antimicrobials over time. The model laboratory strains HG001–HG003, SA113 and the small colony variant (SCV) strains hemB and menD were challenged by the compounds at different logs of minimal inhibitory concentration (MIC) in exponential or stationary growth phase. Antibiotic tolerance was usually elevated in SCV strains compared to normally growing cells and in stationary versus exponential phase cultures. Biphasic killing kinetics, typical for persister cell enrichment, were observed in both growth phases under different selective conditions. Treatment of exponential phase cultures of HG001–HG003 with 10-fold MIC of tobramycin resulted in the isolation of persisters which upon cultivation on plates formed either normal or phenotypically stable small colonies. Trajectories of different killing curves indicated physiological heterogeneity within persister subpopulations. Daptomycin added at 100-fold MIC to stationary phase SA113 cells rapidly isolated very robust persisters. Fractions of antibiotic-tolerant cells were observed with all S. aureus strains and mutants tested. Our results refute the hypothesis that S. aureus stationary phase cells are equivalent to persisters, as not all of these cells showed antibiotic tolerance. Isolation of S. aureus persisters of different robustness seems to depend on the kind and concentration of the antibiotic, as well as on the strain used.

Characterization of an Enterococcus faecium small-colony variant isolated from blood culture

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not. In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment.

Infection of Polarized Airway Epithelial Cells by Normal and Small-Colony Variant Strains of Staphylococcus aureus Is Increased in Cells with Abnormal Cystic Fibrosis Transmembrane Conductance Regulator Function and Is Influenced by NF-κB

The infection of nonphagocytic host cells by Staphylococcus aureus and more particularly by small-colony variants (SCVs) may contribute to the persistence of this pathogen in the lungs of cystic fibrosis (CF) patients. The development of chronic infections is also thought to be facilitated by the proinflammatory status of CF airways induced by an activation of NF-κB. The aim of this study was to compare the infection of non-CF and CF-like airway epithelial cells by S. aureus strains (normal and SCVs) and to determine the impact of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and NF-κB on the infection level of these cells by S. aureus. We developed an S. aureus infection model using polarized airway epithelial cells grown at the air-liquid interface and expressing short hairpin RNAs directed against CFTR to mimic the CF condition. A pair of genetically related CF coisolates with the normal and SCV phenotypes was characterized and used. Infection of both cell lines (non-CF and CF-like) was more productive with the SCV strain than with its normal counterpart. However, both normal and SCV strains infected more CF-like than non-CF cells. Accordingly, inhibition of CFTR function by CFTRinh-172 increased the S. aureus infection level. Experimental activation of NF-κB also increased the level of infection of polarized pulmonary epithelial cells by S. aureus, an event that could be associated with that observed when CFTR function is inhibited or impaired. This study supports the hypothesis that the proinflammatory status of CF tissues facilitates the infection of pulmonary epithelial cells by S. aureus.

Interleukin-6 and Interleukin-8 Secretions by Polarized Airway Epithelial Cells Infected by Normal and Small-Colony Variant Staphylococcus aureus Strains are Similar Despite Differences in Infection Levels

Staphylococcus aureus small-colony variants (SCVs) can efficiently infect non-professional phagocytes and are often referred to as facultative intracellular pathogens. The ability to hide and persist within host cells is likely to contribute to the development of chronic S. aureus infections such as those observed in the lungs of cystic fibrosis patients. Polarized human pulmonary Calu-3 cells were used to confirm that S. aureus small-colony variants (SCVs) persist within epithelial cells without exacerbating the innate immune response. Whereas all studied S. aureus strains significantly induced the secretion of Interleukin-6 (IL-6) and Interleukin-8 (IL-8) by Calu-3 cells 48 hours after cellular invasion, dead bacteria did not. Surprisingly, no difference in the secretion of these interleukins was detected between cells infected with normal and SCV strains despite the marked difference in infection levels. This study supports the hypothesis that despite their increased ability to persist inside epithelial cells, SCVs do not over activate the host immune response in comparison to normal strains. SCVs may thus help to perpetuate infection without exacerbation of the host immune response.

Small colony variants of Staphylococcus aureus — review

Bacterial variants of Staphylococcus aureus called small colony variants (SCVs) originate by mutations in metabolic genes, resulting in emergence of auxotrophic bacterial subpopulations. These variants are not particularly virulent but are able to persist viable inside host cells. SCVs show their characteristic auxotrophic growth deficiency and depressed α-cytotoxin activity. Environmental pressure such as antibiotics, select for isogenic SCV cells that are frequently found coexisting with their parent wild-type strains in a mixed bacterial culture. SCV strains often grow on blood agar as non-pigmented or pinpoint pigmented colonies and their key biochemical tests are often non-reactive. Their altered metabolism or auxotrophism can result in long generation time and thus SCV phenotype, more often than not SCV can be overgrown by their wild-type counterparts and other competitive respiratory flora. This could affect laboratory detection. Thus, molecular methods, such as 16S rRNA partial sequencing or amplification of species-specific DNA targets (e.g. coagulase, nuclease) directly from clinical material or isolated bacterial colonies, become the method of choice. Patients at risk of infection by S. aureus SCVs include cystic fibrosis patients (CF), patients with skin and foreign-body related infections and osteomyelitis, as they suffer from chronic staphylococcal infections and are subject to long-term antibiotic therapy. Molecular evidence of SCV development has not been found except for some random mutations of the thymidylate synthase gene (thyA) described in SCV S. aureus strains of CF patients. These variants are able to bypass the antibiotic effect of folic acid antagonists such as sulfonamides and trimethoprim. Resistance to gentamicin and aminoglycosides in the hemin or menadione auxotrophic SCVs was hypothesized as being due to decreased influx of the drugs into cells as a result of decreased ATP production and decreased electrochemical gradient on cell membranes.

Intracellular Activity of Antibiotics in a Model of Human THP-1 Macrophages Infected by a Staphylococcus aureus Small-Colony Variant Strain Isolated from a Cystic Fibrosis Patient: Study of Antibiotic Combinations

In a companion paper (H. A. Nguyen et al., Antimicrob. Agents Chemother. 53:1434-1442, 2009), we showed that vancomycin, oxacillin, fusidic acid, clindamycin, linezolid, and daptomycin are poorly active against the intracellular form of a thymidine-dependent small-colony variant (SCV) strain isolated from a cystic fibrosis patient and that the activity of quinupristin-dalfopristin, moxifloxacin, rifampin, and oritavancin remains limited (2- to 3-log CFU reduction) compared to their extracellular activity. Antibiotic combination is a well-known strategy to improve antibacterial activity, which was examined here against an intracellular SCV strain using combinations with either rifampin or oritavancin. Time-kill curve analysis using either concentrations that caused a static effect for each antibiotic individually or concentrations corresponding to the maximum concentration in human serum showed largely divergent effects that were favorable when antibiotics were combined with rifampin at low concentrations only and with oritavancin at both low and high concentrations. The nature of the interaction between rifampin, oritavancin, and moxifloxacin was further examined using the fractional maximal effect method, which allows categorization of the effects of combinations when dose-effect relationships are not linear. Rifampin and oritavancin were synergistic at all concentration ratios investigated. Oritavancin and moxifloxacin were also synergistic but at high oritavancin concentrations only. Rifampin and moxifloxacin were additive. This approach may help in better assessing and improving the activity of antibiotics against intracellular SCV strains.

Somatic cell scores and clinical signs following experimental intramammary infection of dairy cows with a Staphylococcus aureus small colony variant ( S. aureus SCV) in comparison to other bovine strains

A recently isolated bovine Staphylococcus aureus small colony variant (SCV) was shown to persist within cultured endothelial cells. However, the clinical importance of S. aureus SCV in persistent infection of the mammary gland is unknown. The hypothesis tested here was a naturally occurring bovine S. aureus SCV, Heba3231, and a Newbould hemB mutant that was an artificially created SCV would establish mild intramammary infection and induce a different host response compared to their isogenic parental strains, 3231 and Newbould 305. Four groups of clinically healthy cows, 5 cows/treatment group, were infected by the intramammary route with SCV Heba3231, strain 3231, the hemB mutant or strain Newbould 305. Three quarters of the mammary gland of each cow were challenged with ∼5000 colony-forming units of bacteria and the fourth quarter was infused with PBS. Cows were monitored and assigned clinical scores for the first 5 days post-challenge based on rectal temperature, appetite, milk yield, udder uniformity, milk appearance, bacterial culture of foremilk samples and somatic cell score (SCS). All cows were examined for chronic infection up to day 36 post-challenge. Cows challenged with SCV strains developed mastitis that was mild compared with that induced by the parent strains. Cows challenged with strain 3231 developed the most severe clinical mastitis. SCS from all treatment groups were significantly ( P < 0.05) higher at days 2–5 and 14–36 post-infection compared to day 0 before challenge. This milder host response to SCVs may represent an adaptation of S. aureus to the mammary gland environment.

Triclosan resistance in methicillin-resistant Staphylococcus aureus expressed as small colony variants: a novel mode of evasion of susceptibility to antiseptics

Triclosan is in widespread use in domestic, commercial and healthcare settings and is used to reduce methicillin-resistant Staphylococcus aureus (MRSA) load in carriers. Triclosan resistance is uncommon, usually being due to mutation in fabI or overexpression of efflux pumps. This study investigated the ability of triclosan-containing silicone elastomer to kill MRSA adherent to its surface. Silicone discs containing triclosan were prepared by a matrix-expansion method previously described. Discs were exposed to three strains of MRSA for 1 h for adhesion to take place. After incubation, discs were removed at intervals, sonicated and the sonicates analysed by chemiluminescence and viable counting. Survivors were found to consist of small colony variants (SCVs). These were then subjected to tests for known SCV characteristics and for susceptibility to triclosan. Viable counts fell until 51 h, when they began to increase, due to SCV. Of the three SCV strains, two showed impaired coagulase production and all showed reduced deoxyribonuclease production. None was auxotrophic. MICs of triclosan in the SCV rose by between 8- and 67-fold. Prolonged exposure of MRSA to triclosan-impregnated silicone, as in 'antimicrobial' plastics or catheters, resulted in the induction of SCV status and triclosan resistance. This has implications for industrial, medical and domestic use of polymers containing triclosan. SCVs are pathogenic and persistent. The widespread use of triclosan could lead to infection with MRSA SCVs, and new antimicrobials with physiological targets similar to that of triclosan might give rise to SCV resistance in clinical use.

The small colony variant (SCV) concept—the role of staphylococcal SCVs in persistent infections

The recovery of small colony variants (SCVs) from clinical specimens was first described at the beginning of the last century. However, not until the past decade was an association of these variants with chronic, recurrent, and persistent infections such as chronic osteomyelitis and persistent skin and softtissue infection described. Since then, a number of reports and prospective studies have supported a pathogenic role for SCVs in disease. Biochemical and other characteristics of SCVs have suggested a link between electron-transport defective SCV strains and persistent infections, however, the strains examined were genetically undefined SCVs. Therefore, a stable mutant in electron transport was generated by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus aureus. This mutant showed characteristics typical of clinical SCVs such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Furthermore, the mutant was able to persist within cultured endothelial cells due to decreased a-toxin production. It was suggested that the intracellular location of this subpopulation might shield the variants from host defenses and antibiotics, thus providing one explanation for the difficulty in removing SCVs from host tissues. Therefore, a defect in the electron-transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly. Because of their fastidious growth characteristics, they are easily missed or misidentified in the clinical laboratory. Therefore, when an infection persists for weeks or months or fails to respond to antimicrobial therapy, clinicians as well as laboratory personnel should consider further efforts to search for this staphylococcal subpopulation.

Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes

Small colony variants (SCVs) of Staphylococcus aureus have been proposed to persist within vascular endothelium, thereby sustaining chronic infections. To identify the intracellular SCV location we infected primary human endothelial cells with various wild type and SCV strains of S. aureus and visualised maturation of phagosomes using live cell imaging. Staphylococci-containing phagosomes were matured by sequential and dynamic interactions with Rab5- and Rab7-positive vesicles. Within 45-60 min all internalised staphylococci accumulated in LAMP-1- and LysoTracker-enriched lysosomal organelles and remained there for up to 5 days. Recovery of most staphyloccocal strains was below 1% after a 24 h intracellular stay, indicating a high bactericidal activity of the endothelial cell lysosomes. However, the menadione auxotroph SCV strain JB1 displayed a recovery rate of 4% and, furthermore, through multiple intracellular passaging a subtype (JB1-P4) with a recovery rate of 25-30% could be generated. Interestingly, both JB1 and JB1-P4 also resided exclusively in lysosomes. Thus, on one hand we document effective bactericidal activity of human endothelial cell lysosomes towards staphylococci, and on the other hand we provide evidence that certain SCVs are capable to withstand this activity.