Emergence Of Small-colony Variants Research Articles

Genomic Modification of TonB and Emergence of Small-Colony Phenotype in VIM- and NDM-Producing Escherichia coli following Cefiderocol Exposure In Vitro.

Knowledge on resistance mechanisms toward cefiderocol, a novel siderophore-conjugated cephalosporin antibiotic, is still limited. Although the presence of New-Delhi metallo-β-lactamase has been demonstrated to facilitate the resistance development toward cefiderocol via siderophore receptor mutations in Enterobacter cloacae and Klebsiella pneumoniae, the impact of metallo-β-lactamases on facilitating such mutations in Escherichia coli is not yet elucidated. Our study aimed to study the effect of the presence of various β-lactamases, such as NDM-5, VIM-1, KPC-2, and OXA-48, on the development of cefiderocol resistance in E. coli. To this end, we performed liquid mating to transfer these β-lactamases onto a defined K-12 E. coli background (J53) and exposed these transconjugants to increasing cefiderocol concentrations in a serial passage experiment. Cefiderocol-resistant isolates were genotyped by whole-genome sequencing to investigate the underlying resistance mechanism. Cefiderocol-resistant isolates emerged only in isolates producing VIM-1 and NDM-5 metallo-β-lactamase, but not in those producing the serine β-lactamases KPC-2 and OXA-48. We observed two distinct morphological changes of the J53 E. coli strain exhibiting reduced colony size after insertions of transposable elements in the tonB gene leading to alterations in the TonB binding site and morphological changes consistent with the small-colony variant (SCV) phenotype due to mutations in the hemB and hemH genes. Passaging experiments suggested that these phenotypes were highly plastic. The SCV phenotype is attributed to immune evasion and decreased susceptibility toward antibiotics. The emergence of SCV following cefiderocol exposure may have clinical implications for bacterial clearance and warrants further investigation.

Emergence of Small Colony Variants Is an Adaptive Strategy Used by Pseudomonas aeruginosa to Mitigate the Effects of Redox Imbalance.

The ability to generate a subpopulation of small colony variants (SCVs) is a conserved feature of Pseudomonas aeruginosa and could represent a key adaptive strategy to colonize and persist in multiple niches. However, very little is known about the role of the SCV phenotype, the conditions that promote its emergence, and its possible involvement in an adaptive strategy. In the present work, we investigated the in vitro selective conditions promoting the emergence of SCVs from the prototypical strain PA14, which readily forms SCVs in nonagitated standing cultures. We found that O2 limitation, which causes a redox imbalance, is the main factor selecting for the SCV phenotype, which promotes survival of the population via formation of a biofilm at the air-liquid interface to access the electron acceptor. When this selective pressure is relieved by aeration or supplementation of an alternative electron acceptor, SCVs are barely detectable. We also observed that SCV emergence contributes to redox rebalancing, suggesting that it is involved in an adaptive strategy. We conclude that selection for the SCV phenotype is an adaptive solution adopted by P. aeruginosa to access poorly available O2. IMPORTANCE The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because it is a causative agent of nosocomial infections and the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is often related to the emergence of an alternative phenotype known as small colony variant (SCV). Identification of conditions selecting for the SCV phenotype contributes to knowledge regarding adaptive mechanisms exploited by P. aeruginosa to survive in multiple niches and persist during infections. Hindering this adaptation strategy could help control persistent P. aeruginosa infections.

Pseudomonas aeruginosa Strains from Both Clinical and Environmental Origins Readily Adopt a Stable Small-Colony-Variant Phenotype Resulting from Single Mutations in c-di-GMP Pathways.

A subpopulation of small-colony variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolates obtained from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread the ability of P. aeruginosa strains to develop this phenotype is and what the genetic mechanism(s) behind the emergence of SCVs are according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to spontaneously adopt an SCV-like smaller alternative morphotype distinguishable from that of the ancestral parent strain under laboratory culture conditions. We found that all the P. aeruginosa strains tested could adopt an SCV phenotype, regardless of their origin. Whole-genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, the Wsp and YfiBNR pathways. We conclude that the ability to switch to an SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain. IMPORTANCE P. aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because this bacterium is the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is considered related to the emergence of an alternative small-colony-variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various nonclinical environments and the involvement of c-di-GMP in SCV emergence from both clinical and environmental strains, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to adapt readily in all environments. Hindering this adaptation strategy could help control persistent infection by P. aeruginosa.

Assessing the interspecies relationship betweenPseudomonas aeruginosa and Staphylococcus aureus in mixed biofilms grown in alginate beads and collagen scaffolds

Polymicrobial biofilms in chronic infected wounds harbour different bacterial species that interact with each other, competing or co-operating to survive. The two most common pathogens co-isolated from chronic wound biofilms are Pseudomonas aeruginosa and Staphylococcus aureus. Evidence from in vitrobiofilms models have shown these two bacteria interactand data suggests that P. aeruginosa inhibits the growth of S. aureusin mixed biofilms. This study aimed to assess the growth of these two species in a complex polymicrobial biofilm in a 3D matrix comprised of either alginate (1.5% w/v) or a collagen scaffold. Using a five-species biofilm (S. aureus, P. aeruginosa, Citrobacter freundii, Enterococcus faecalisand Escherichia coli), with all bacteria inoculated at time zero, it was consistently observed that P. aeruginosa was not recoverable over a 72h period, with sampling every 24h. However, P. aeruginosagrew well if it was added to a pre-formed four-species biofilm. Further, P. aeruginosa was seen to inhibit the growth of S. aureus after 24h subsequent co-culture in the pre-formed biofilm, which resulted in the emergence of small colony variants of S. aureus. Interestingly when P. aeruginosawas co-inoculated in a four species biofilm that did not contain S. aureus,its growth was not inhibited, suggesting a competitive interaction between these two bacteria during establishment of the early biofilm. These data were consistent in alginate beads and collagen scaffolds. In a chronic wound P. aeruginosais regarded as a late coloniser and the phenomena observed in this study might be reflective of this.

Persistence of Staphylococcus aureus: Multiple Metabolic Pathways Impact the Expression of Virulence Factors in Small-Colony Variants (SCVs).

Staphylococcus aureus is able to survive within host cells by switching its phenotype to the small-colony variant (SCV) phenotype. The emergence of SCVs is associated with the development of persistent infections, which may be both chronic and recurrent. This slow-growing subpopulation of S. aureus forms small colonies on solid-medium agar, is induced within host cells, presents a non-homogenous genetic background, has reduced expression of virulence factors and presents a variable phenotype (stable or unstable). While virtually all SCVs isolated from clinical specimens can revert to the parental state with rapid growth, the stable SCVs recovered in clinical specimens have been found to contain specific mutations in metabolic pathways. In contrast, other non-stable SCVs are originated from regulatory mechanisms involving global regulators (e.g., sigB, sarA, and agr) or other non-defined mutations. One major characteristic of SCVs was the observation that SCVs were recovered from five patients with infections that could persist for decades. In these five cases, the SCVs had defects in electron transport. This linked persistent infections with SCVs. The term “persistent infection” is a clinical term wherein bacteria remain in the host for prolonged periods of time, sometimes with recurrent infection, despite apparently active antibiotics. These terms were described in vitro where bacteria remain viable in liquid culture medium in the presence of antibiotics. These bacteria are called “persisters”. While SCVs can be persisters in liquid culture, not all persisters are SCVs. One mechanism associated with the metabolically variant SCVs is the reduced production of virulence factors. SCVs have consistently shown reduced levels of RNAIII, a product of the accessory gene regulatory (agrBDCA) locus that controls a quorum-sensing system and regulates the expression of a large number of virulence genes. Reduced Agr acitivity is associated with enhanced survival of SCVs within host cells. In this review, we examine the impact of the SCVs with altered metabolic pathways on agr, and we draw distinctions with other types of SCVs that emerge within mammalian cells with prolonged infection.

Denture stomatitis associated with small-colony variants of Staphylococcus aureus: a case report

BackgroundThe small-colony variants (SCVs) of Staphylococcus aureus were isolated from persistent and recurrent infections, especially after placement of medical devices having direct contact with human tissues. The emergence of SCVs is a survival strategy of S. aureus which enables them to hide inside host’s cells and induces a less severe immune response than to wild-type S. aureus. However, contrary to other medical devices, dental prosthesis as a surface potentially colonized by SCVs of S. aureus has not been examined thus far. We reported the first case of SCVs - S. aureus infection in denture wearer.Case presentationA 62-year-old woman with a complete removable acrylic denture presented extensive elevated erythematous lesions on the palate, compatible with denture stomatitis. The patient had a history of arterial hypertension, cigarette smoking and wearing denture at night. The fungal colonies, identified as Candida albicans, were cultured on Sabouraud agar. From three swabs (from hard palate mucosa, denture surface and angular cheilitis lesions) were cultured of pinpoint, clear, non-pigmented, and non-haemolytic colonies on Columbia agar. The small colonies turned out to be Gram-positive cocci, catalase-, Pastorex Staph Plus -, and clumping factor-positive, and oxidase-negative. Suspected phenotypically SCVs forms were definitively identified as S. aureus based on PCR amplification of species specific nuc and coa genes. Methicillin-resistance was verified by mecA gene detection. The isolates turned out to be susceptible to methicillin (MSSA) and resistant to gentamicin. The isolate was identified as menadione-auxotrophic variant.ConclusionsThis case demonstrated that oral cavity in denture wearers may be a reservoir of small-colony variants of S. aureus, besides C. albicans. The prevalence of these bacteria and their role in the pathogenesis of oral diseases are not understood. Due to problems with their detection and identification, the true prevalence of oral SCVs may be underestimated.

Intraosteoblastic activity of levofloxacin and rifampin alone and in combination against clinical isolates of meticillin-susceptible Staphylococcus aureus causing prosthetic joint infection

Staphylococcus aureus may invade and persist intracellularly in prosthetic joint infections (PJIs). Despite optimized treatments with levofloxacin plus rifampin, the intracellular reservoir may lead to infection relapse. This study assessed the intracellular activity of levofloxacin and rifampin in an in-vitro model of human osteoblastic infection. Ten meticillin-susceptible S. aureus strains were used to infect osteoblastic MG63 cells. Osteoblasts were challenged with rifampin and levofloxacin at cortical and cancellous bone concentrations. Efficacy was measured as the intracellular counts of colony-forming units (log10CFU) compared with untreated controls. The emergence of small colony variants (SCVs) was determined, and the results were stratified according to the patient's prognosis (six cured and four with persistence/relapse). All regimes led to a significant decrease in CFU count compared with controls (1-2 log10CFU). Levofloxacin was the most effective treatment at both cortical and cancellous bone concentrations (-2.4 to -1.9 log10CFU, respectively). The addition of rifampin to levofloxacin did not improve performance (-1.9 log10CFU for cortical concentration and -1.8 log10 CFU for cancellous concentration). An increase in SCVs was observed in the presence of rifampin. The efficacy of antimicrobials was higher and the formation of SCVs was lower against strains belonging to PJIs with a favourable outcome. Levofloxacin plus rifampin had good intracellular activity against S. aureus. However, from the intracellular perspective, the addition of rifampin to levofloxacin showed no benefit but could account for an increased number of SCVs.

Antimicrobial activity against intraosteoblastic Staphylococcus aureus.

Although Staphylococcus aureus persistence in osteoblasts, partly as small-colony variants (SCVs), can contribute to bone and joint infection (BJI) relapses, the intracellular activity of antimicrobials is not currently considered in the choice of treatment strategies for BJI. Here, antistaphylococcal antimicrobials were evaluated for their intraosteoblastic activity and their impact on the intracellular emergence of SCVs in an ex vivo osteoblast infection model. Osteoblastic MG63 cells were infected for 2 h with HG001 S. aureus. After killing the remaining extracellular bacteria with lysostaphin, infected cells were incubated for 24 h with antimicrobials at the intraosseous concentrations reached with standard therapeutic doses. Intracellular bacteria and SCVs were then quantified by plating cell lysates. A bactericidal effect was observed with fosfomycin, linezolid, tigecycline, oxacillin, rifampin, ofloxacin, and clindamycin, with reductions in the intracellular inocula of -2.5, -3.1, -3.9, -4.2, -4.9, -4.9, and -5.2 log10 CFU/100,000 cells, respectively (P < 10(-4)). Conversely, a bacteriostatic effect was observed with ceftaroline and teicoplanin, whereas vancomycin and daptomycin had no significant impact on intracellular bacterial growth. Ofloxacin, daptomycin, and vancomycin significantly limited intracellular SCV emergence. Overall, ofloxacin was the only molecule to combine an excellent intracellular activity while limiting the emergence of SCVs. These data provide a basis for refining the choice of antibiotics to prioritise in the management of BJI, justifying the combination of a fluoroquinolone for its intracellular activity with an anti-biofilm molecule, such as rifampin.

Nitrogen gas flushing can be bactericidal: the temperature-dependent destiny of Bacillus weihenstephanensis KBAB4 under a pure N2 atmosphere.

Gram-negative Pseudomonas and Gram-positive Bacillus are the most common spoilage bacteria in raw and pasteurized milk, respectively. In previous studies, nitrogen (N2) gas flushing treatments of raw and pasteurized milk at cold chain-temperatures inhibited bacterial spoilage and highlighted different susceptibilities to the N2 treatment with the exclusion of certain bacterial types. Here, we investigated the effects of pure N2 gas flushing on representative strains of these genera grown in mono- or co-cultures at 15 and 25°C. Bacillus weihenstephanensis, a frequent inhabitant of fluid dairy products, is represented by the genome-sequenced KBAB4 strain. Among Pseudomonas, P. tolaasii LMG 2342T and strain C1, a raw milk psychrotroph, were selected. The N2 gas flushing treatment revealed: (1) temperature-dependent responses; (2) inhibition of the growth of both pseudomonads; (3) emergence of small colony variants (SCVs) for B. weihenstephanensis strain KBAB4 at 15°C induced by the N2 treatment or when grown in co-culture with Pseudomonas strains; (4) N2 gas flushing modulates (suppressed or stimulated) bacterial antagonistic reactions in co-cultures; (5) most importantly, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed that at 25°C the majority of the KBAB4 cells were killed by pure N2 gas flushing. This observation constitutes the first evidence that N2 gas flushing has bactericidal effects.

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain.

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain.

Small colony variants (SCVs) of Staphylococcus aureus – A bacterial survival strategy

Small colony variants (SCVs) of Staphylococcus aureus have been implicated in chronic recurrent infections and have therefore gained renewed interest during the last decade. Moreover, SCVs have been shown to be part of the regular growth cycle, are highly dynamic or stable and can be selected during various harsh conditions. As such, the emergence of SCVs has been described not only in human, but also in veterinary medicine as well as in food microbiology. SCVs are characterized by impaired growth, down-regulation of genes for metabolism and virulence, while sigB and genes important for persistence and biofilm formation are up-regulated. Furthermore, SCVs are resistant to various antibiotics such as aminoglycosides, trimethoprim-sulfamethoxazole, fluorquinolones, fusidic acid or even to antiseptics such as triclosan. An underlying mechanism has been determined for hemin-, menadione- and thymidine-dependent SCVs as well as for SCVs which are impaired in their stress response. SCVs are optimized for persistence in the host. They are able to reverse and thereby constitute a highly dynamic subpopulation of S. aureus. Such phenotype switching constitutes an integral part of the infection process enabling the bacteria to hide inside the host cell without eliciting a strong host response.

Phenotype Switching Is a Natural Consequence of Staphylococcus aureus Replication

The pathogen Staphylococcus aureus undergoes phenotype switching in vivo from its normal colony phenotype (NCP) to a slow-growing, antibiotic-resistant small-colony-variant (SCV) phenotype that is associated with persistence in host cells and tissues. However, it is not clear whether phenotype switching is the result of a constitutive process that is selected for under certain conditions or is triggered by particular environmental stimuli. Examination of cultures of diverse S. aureus strains in the absence of selective pressure consistently revealed a small gentamicin-resistant SCV subpopulation that emerged during exponential-phase NCP growth and increased in number until NCP stationary phase. Treatment of replicating bacteria with the antibiotic gentamicin, which inhibited NCP but not SCV replication, resulted in an initial decrease in SCV numbers, demonstrating that SCVs arise as a consequence of NCP replication. However, SCV population expansion in the presence of gentamicin was reestablished by selection of phenotype-stable SCVs and subsequent SCV replication. In the absence of selective pressure, however, phenotype switching was bidirectional and occurred at a high frequency during NCP replication, resulting in SCV turnover. In summary, these data demonstrate that S. aureus phenotype switching occurs via a constitutive mechanism that generates a dynamic, antibiotic-resistant subpopulation of bacteria that can revert to the parental phenotype. The emergence of SCVs can therefore be considered a normal part of the S. aureus life cycle and provides an insurance policy against exposure to antibiotics that would otherwise eliminate the entire population.

Staphylococcus aureus sigma B-dependent emergence of small-colony variants and biofilm production following exposure to Pseudomonas aeruginosa 4-hydroxy-2-heptylquinoline-N- oxide

BackgroundStaphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.ResultsWe demonstrated that HQNO stimulates S. aureus to form a biofilm in association with the formation of SCVs. The emergence of SCVs and biofilm production under HQNO exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor B (SigB). Analysis of gene expression revealed that exposure of a prototypical S. aureus strain to HQNO activates SigB, which was leading to an increase in the expression of the fibronectin-binding protein A and the biofilm-associated sarA genes. Conversely, the quorum sensing accessory gene regulator (agr) system and the α-hemolysin gene were repressed by HQNO. Experiments using culture supernatants from P. aeruginosa PAO1 and a double chamber co-culture model confirmed that P. aeruginosa stimulates biofilm formation and activates SigB in a S. aureus strain isolated from a CF patient. Furthermore, the supernatant from P. aeruginosa mutants unable to produce HQNO induced the production of biofilms by S. aureus to a lesser extent than the wild-type strain only in a S. aureus SigB-functional background.ConclusionsThese results suggest that S. aureus responds to HQNO from P. aeruginosa by forming SCVs and biofilms through SigB activation, a phenomenon that may contribute to the establishment of chronic infections in CF patients.

A role for sigma factor B in the emergence of Staphylococcus aureus small-colony variants and elevated biofilm production resulting from an exposure to aminoglycosides

Staphylococcus aureus small-colony variants (SCVs) and biofilms are linked to chronic infections. It is known that the presence of aminoglycoside antibiotics may contribute to the emergence of SCVs and it is thought that molecular mechanisms are involved in the ability of S. aureus to adopt this phenotype. No study has addressed the possible role of the stress- and colonization-related alternative sigma factor B (SigB) in the emergence of SCVs, although a sustained SigB activity was reported in these variants. Here, we demonstrate that SigB is involved in the emergence of SCVs resulting from an exposure to a sub-inhibitory concentration of aminoglycosides. Monitoring of gene expression in an aminoglycoside-treated prototypical strain or in clinical SCVs showed the activation of SigB, whereas the accessory gene regulator ( agr) system was not. Furthermore, gentamicin-treated prototypical bacteria and SCVs had an increased ability to form biofilm only in a SigB functional background. The administration of a sub-inhibitory concentration of gentamicin significantly increased the formation of SCVs for a prototypical strain but not for the sigB mutant in a mouse model of S. aureus-induced mastitis. Collectively, our results show that SigB may positively influence the appearance of S. aureus SCVs and the production of biofilm upon aminoglycoside exposure.

Bacteriophage and Phenotypic Variation in Pseudomonas aeruginosa Biofilm Development

A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon.

Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus

AbstractStaphylococcus aureus small colony variants (SCVs) occur frequently after local treatment with aminoglycosides and cause persistent as well as recurrent infections. So far, the molecular mechanism of the emergence of SCVs is not understood and regulatory as well as genetic mechanisms seem conceivable. To screen for possible mutations, the hemin biosynthetic gene cluster of a gentamicin-induced SCV was sequenced and was found to contain a deletion in the gene hemH. To further assess the influence of a high mutation rate on the development of SCVs, we tested the emergence of SCVs in a strain that had been inactivated in the DNA proofreading enzyme MutS. In the mutant, spontaneous SCVs emerged 556-fold more frequently than in the parent strain. By incubation in the presence of subinhibitory concentrations of gentamicin, the SCV frequency in the parent strain could be increased to 9.7×10−6, whereas it remained rather stable in the mutant (1.8×10−5). Eighty percent of the gentamicin-induced SCVs were hemin auxotrophic in contrast to only 20% of the spontaneous SCVs which may explain the large proportion of hemin auxotrophs among clinical SCVs from patients previously treated with aminoglycosides. Additionally, a clinical S. aureus SCV isolate with a mutator phenotype, indicated by the generation of rifampicin-resistant mutants at a 16-fold higher frequency than in the reference strain S. aureus NCTC 8325, was characterized. The results demonstrate that a high mutation rate favours the emergence of SCVs, and suggest that mutations in general play an important role in the development of SCVs.

Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus

Bacterial antibiotic resistance is often associated with a fitness cost in the absence of the antibiotic [1, 2]. We have examined a resistance mechanism in Staphylococcus aureus that negates these costs. Exposure to gentamicin both in vitro and in vivo has been reported to result in the emergence of a gentamicin-resistant small colony variant (SCV) [3–8]. We show that the emergence of SCVs following exposure to gentamicin results from a rapid switch and that bacteria exposed to cycles of gentamicin followed by antibiotic-free medium repeatedly switched between a resistant SCV and a sensitive parental phenotype (revertants). The fitness of revertants relative to S. aureus with stable gentamicin resistance was greater in drug-free media, which suggests that S. aureus has evolved an inducible and reversible resistance mechanism that circumvents a permanent cost to fitness.

Characteristics of quinolone-induced small colony variants in Staphylococcus aureus.

Exposure of Staphylococcus aureus to 1 x MIC of the quinolone antibiotic pazufloxacin for 24 h, followed by plating on drug-free media, led to the emergence of small colony variants (SCVs) in addition to large colony variants (LCVs). However, following incubation with 0.25 or 4 x MIC of pazufloxacin, only LCVs were obtained. The SCVs were half as susceptible to pazufloxacin or ciprofloxacin as wild-type S. aureus, while the susceptibilities of LCVs were essentially unchanged. The reduced susceptibilities of SCVs did not result from mutations in the quinolone-resistance-determining regions of DNA gyrase and topoisomerase IV, since the sequences of these genes were identical to those of the wild-type. However, the SCVs accumulated pazufloxacin and ciprofloxacin to a lesser degree than did wild-type. Furthermore, their susceptibility to quinolones was almost unaffected by reserpine or verapamil, suggesting that the reduced uptake resulted from decreased permeability, rather than from an active efflux pump. The ability of various quinolones to induce emergence of SCVs in S. aureus, correlated with the presence of carbon-bonded substituents at the C-7 position of a quinoline or naphthyridine nucleus, or with the presence of a benzoxazine nucleus. In conclusion, pazufloxacin-induced SCVs represent a mutant that one might expect to be rapidly eliminated in vivo and, hence, not to survive as a quinolone-resistant pathogen. This finding suggests a novel approach for development of future quinolones.