Stable Small-colony Variants Research Articles

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages.

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

The Controversial Effect of Antibiotics on Methicillin-Sensitive S. aureus: A Comparative In Vitro Study.

Methicillin-sensitive Staphylococcus (S.) aureus (MSSA) bacteremia remains a global challenge, despite the availability of antibiotics. Primary treatments include β-lactam agents such as cefazolin and flucloxacillin. Ongoing discussions have focused on the potential synergistic effects of combining these agents with rifampicin or fosfomycin to combat infections associated with biofilm formation. Managing staphylococcal infections is challenging due to antibacterial resistance, biofilms, and S. aureus's ability to invade and replicate within host cells. Intracellular invasion shields the bacteria from antibacterial agents and the immune system, often leading to incomplete bacterial clearance and chronic infections. Additionally, S. aureus can assume a dormant phenotype, known as the small colony variant (SCV), further complicating eradication and promoting persistence. This study investigated the impact of antibiotic combinations on the persistence of S. aureus 6850 and its stable small colony variant (SCV strain JB1) focusing on intracellular survival and biofilm formation. The results from the wild-type strain 6850 demonstrate that β-lactams combined with RIF effectively eliminated biofilms and intracellular bacteria but tend to select for SCVs in planktonic culture and host cells. Higher antibiotic concentrations were associated with an increase in the zeta potential of S. aureus, suggesting reduced membrane permeability to antimicrobials. When using the stable SCV mutant strain JB1, antibiotic combinations with rifampicin successfully cleared planktonic bacteria and biofilms but failed to eradicate intracellular bacteria. Given these findings, it is reasonable to report that β-lactams combined with rifampicin represent the optimal treatment for MSSA bacteremia. However, caution is warranted when employing this treatment over an extended period, as it may elevate the risk of selecting for small colony variants (SCVs) and, consequently, promoting bacterial persistence.

Combinatorial efficacy of Manuka honey and antibiotics in the in vitro control of staphylococci and their small colony variants

IntroductionStaphylococci are among the list of problematic bacteria contributing to the global antibiotic resistance (ABR) crisis. Their ability to adopt the small colony variant (SCV) phenotype, induced by prolonged antibiotic chemotherapy, complicates staphylococcal infection control options. Novel and alternative approaches are needed to tackle staphylococcal infections and curb ABR. Manuka honey (MH), a non-antibiotic alternative is recognized for its unique antibacterial activity based on its methylglyoxal (MGO) component.MethodsIn this study, MH (MGO 830+) was tested in combination with gentamicin (GEN), rifampicin (RIF), or vancomycin (VA) against staphylococcal wildtype (WT) and SCVs. To our knowledge, there are no current studies in the literature documenting the effects of MH on staphylococcal SCVs. While Staphylococcus aureus is well-studied for its international ABR burden, limited data exists demonstrating the effects of MH on S. epidermidis and S. lugdunensis whose pathogenic relevance and contribution to ABR is also rising.Results & discussionThe three staphylococci were most susceptible to RIF (0.06-0.24 μg/ml), then GEN (0.12-0.49 μg/ml), and lastly VA (0.49-0.96 μg/ml). The MICs of MH were 7%, 7-8%, and 6-7% (w/v), respectively. Fractional inhibitory concentration (FIC) evaluations showed that the combined MH + antibiotic effect was either additive (FICI 1-2), or partially synergistic (FICI >0.5-1). While all three antibiotics induced SCVs in vitro, stable SCVs were observed in GEN treatments only. The addition of MH to these GEN-SCV-induction analyses resulted in complete suppression of SCVs (p<0.001) in all three staphylococci, suggesting that MH’s antibacterial properties interfered with GEN’s SCV induction mechanisms. Moreover, the addition of MH to growth cultures of recovered stable SCVs resulted in the inhibition of SCV growth by at least 99%, indicating MH’s ability to prevent subsequent SCV growth. These in vitro analyses demonstrated MH’s broad-spectrum capabilities not only in improving WT staphylococci susceptibility to the three antibiotics, but also mitigated the development and subsequent growth of their SCV phenotypes. MH in combination with antibiotics has the potential to not only resensitize staphylococci to antibiotics and consequently require less antibiotic usage, but in instances where prolonged chemotherapy is employed, the development and growth of SCVs would be hampered, providing a better clinical outcome, all of which mitigate ABR.

Extracellular DNA released by glycine-auxotrophic Staphylococcus epidermidis small colony variant facilitates catheter-related infections

Though a definitive link between small colony variants (SCVs) and implant-related staphylococcal infections has been well-established, the specific underlying mechanism remains an ill-explored field. The present study analyzes the role SCVs play in catheter infection by performing genomic and metabolic analyses, as well as analyzing biofilm formation and impacts of glycine on growth and peptidoglycan-linking rate, on a clinically typical Staphylococcus epidermidis case harboring stable SCV, normal counterpart (NC) and nonstable SCV. Our findings reveal that S. epidermidis stable SCV carries mutations involved in various metabolic processes. Metabolome analyses demonstrate that two biosynthetic pathways are apparently disturbed in SCV. One is glycine biosynthesis, which contributes to remarkable glycine shortage, and supplementation of glycine restores growth and peptidoglycan-linking rate of SCV. The other is overflow of pyruvic acid and acetyl-CoA, leading to excessive acetate. SCV demonstrates higher biofilm-forming ability due to rapid autolysis and subsequent eDNA release. Despite a remarkable decline in cell viability, SCV can facilitate in vitro biofilm formation and in vivo survival of NC when co-infected with its normal counterparts. This work illustrates an intriguing strategy utilized by a glycine-auxotrophic clinical S. epidermidis SCV isolate to facilitate biofilm-related infections, and casts a new light on the role of SCV in persistent infections.

Spontaneously Occurring Small-Colony Variants of Staphylococcus aureus Show Enhanced Clearance by THP-1 Macrophages.

Staphylococcus aureus is a common cause of chronic and relapsing infection, especially when the ability of the immune system to sterilize a focus of infection is compromised (e.g., because of a foreign body or in the cystic fibrosis lung). Chronic infections are associated with slow-growing colony phenotypes of S. aureus on solid media termed small-colony variants (SCVs). Stable SCVs show characteristic mutations in the electron transport chain that convey resistance to antibiotics, particularly aminoglycosides. This can be used to identify SCVs from within mixed-colony phenotype populations of S. aureus. More recently, populations of SCVs that rapidly revert to a “wild-type” (WT) colony phenotype, in the absence of selection pressure, have also been described. In laboratory studies, SCVs accumulate through prolonged infection of non-professional phagocytes and may represent an adaptation to the intracellular environment. However, data from phagocytic cells are lacking. In this study, we mapped SCV and WT colony populations in axenic growth of multiple well-characterized methicillin-sensitive and methicillin-resistant S. aureus strains. We identified SCVs populations on solid media both in the presence and absence of gentamicin. We generated stable SCVs from Newman strain S. aureus, and infected human macrophages with WT S. aureus (Newman, 8325-4) and their SCV counterparts (SCV3, I10) to examine intracellular formation and survival of SCVs. We show that SCVs arise spontaneously during axenic growth, and that the ratio of SCV:WT morphology differs between strains. Exposure to the intracellular environment of human macrophages did not increase formation of SCVs over 5 days and macrophages were able to clear stable SCV bacteria more effectively than their WT counterparts.

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.

Novel Research Models for Staphylococcus aureus Small Colony Variants (SCV) Development: Co-pathogenesis and Growth Rate.

Staphylococcus aureus remains a great burden on the healthcare system. Despite prescribed treatments often seemingly to be successful, S. aureus can survive and cause a relapsing infection which cannot be cleared. These infections are in part due to quasi-dormant sub-population which is tolerant to antibiotics and able to evade the host immune response. These include Small Colony Variants (SCVs). Because SCVs readily revert to non-SCV cell types under laboratory conditions, the characterization of SCVs has been problematic. This mini-review covers the phenotypic and genetic changes in stable SCVs including the selection of SCVs by and interactions with other bacterial species.

Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections.

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMIs) that can evolve into difficult-to-treat chronic mastitis. To date, no vaccine formulation has shown high protective efficacy against S. aureus IMI, partly because this bacterium can efficiently evade the immune system. For instance, S. aureus small colony variants (SCVs) have intracellular abilities and can persist without producing invasive infections. As a first step towards the development of a live vaccine, this study describes the elaboration of a novel attenuated mutant of S. aureus taking advantage of the SCV phenotype. A genetically stable SCV was created through the deletion of the hemB gene, impairing its ability to adapt and revert to the invasive phenotype. Further attenuation was obtained through inactivation of gene vraG (SACOL0720) which we previously showed to be important for full virulence during bovine IMIs. After infection of bovine mammary epithelial cells (MAC-T), the double mutant (ΔvraGΔhemB) was less internalized and caused less cell destruction than that seen with ΔhemB and ΔvraG, respectively. In a murine IMI model, the ΔvraGΔhemB mutant was strongly attenuated, with a reduction of viable counts of up to 5-log10 CFU/g of mammary gland when compared to the parental strain. A complete clearance of ΔvraGΔhemB from glands was observed whereas mortality rapidly (48h) occurred with the wild-type strain. Immunization of mice using subcutaneous injections of live ΔvraGΔhemB raised a strong immune response as judged by the high total IgG titers measured against bacterial cell extracts and by the high IgG2a/IgG1 ratio observed against the IsdH protein. Also, ΔvraGΔhemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of mastitis associated spa types. This double mutant could serve as a live-attenuated component in vaccines to improve cell-mediated immune responses against S. aureus IMIs.

A full genomic characterization of the development of a stable Small Colony Variant cell-type by a clinical Staphylococcus aureus strain

A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form.

Prolonged growth of a clinical Staphylococcus aureus strain selects for a stable small-colony-variant cell type.

An undetermined feature of Staphylococcus aureus pathogenesis is its persistence and then relapse of disease. This has been explained by its switch to alternative lifestyles, mainly as biofilm or small-colony variants (SCVs). Studying the native characteristics of SCVs has been problematic due to their reversion to the parental lifestyle. We have observed that for a number of S. aureus strains as they switch to an SCV lifestyle, there is the formation of an extracellular matrix. We focused our analysis on one strain, WCH-SK2. For bacterial survival in the host, the combination of low nutrients and the prolonged time frame forms a stress that selects for a specific cell type from the population. In this context, we used steady-state growth conditions with low nutrients and a controlled low growth rate for a prolonged time and with methylglyoxal. These conditions induced S. aureus WCH-SK2 into a stable SCV cell type; the cells did not revert after subculturing. Analysis revealed these cells possessed a metabolic and surface profile that was different from those of previously described SCVs or biofilm cells. The extracellular matrix was protein and extracellular DNA but not polysaccharide. The SCV cells induced expression of certain surface proteins (such as Ebh) and synthesis of lantibiotics while downregulating factors that stimulate the immune response (leucocidin, capsule, and carotenoid). Our data reveal cell heterogeneity within an S. aureus population and under conditions that resemble long-term survival in the host have identified a previously unnoticed S. aureus cell type with a distinctive metabolic and molecular profile.

Staphylococcus aureus small colony variants are susceptible to light activated antimicrobial agents

BackgroundAntibiotic therapy can select for small colony variants of Staphylococcus aureus that are more resistant to antibiotics and can result in persistent infections, necessitating the development of more effective antimicrobial strategies to combat small colony variant infections. Photodynamic therapy is an alternative treatment approach which utilises light in combination with a light-activated antimicrobial agent to kill bacteria via a non-specific mechanism of action. In this study, we investigated whether the combination of 665 nm laser light and the light-activated antimicrobial agent methylene blue was able to successfully kill S. aureus small colony variants. S. aureus and isogenic stable small colony variant were exposed to varying doses (1.93 to 9.65 J/cm2) of 665 nm laser light in the presence of varying concentrations (1 to 20 μM) of methylene blue.ResultsThe combination of 665 nm laser light and methylene blue was found to be an effective strategy for the killing of small colony variants. At the highest light dose (9.65 J/cm2) and methylene blue concentration (20 μM) tested, the number of viable bacteria decreased by approximately 6.9 log10 for the wild type and approximately 5 log10 for the small colony variant.ConclusionsThese results suggest that photodynamic therapy has potential for use in the treatment of superficial infections caused by small colony variants of S. aureus and supports further research in this field.

Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent.

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 106 to 109 colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 107 or 108 CFUs of the normal phenotype, respectively. A minimum dose of 108 or 109 CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm2 vs. 28 mm2). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.

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

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

Activities of Daptomycin and Comparative Antimicrobials, Singly and in Combination, against Extracellular and Intracellular Staphylococcus aureus and Its Stable Small-Colony Variant in Human Monocyte-Derived Macrophages and in Broth

We investigated the antistaphylococcal activities of daptomycin, gentamicin, and rifampin against two Staphylococcus aureus strains and their stable small-colony variants, singly and in combination, in human monocyte-derived macrophages and in broth. Intracellularly, the three-drug combination and two-drug combinations with rifampin were most effective. Extracellularly, daptomycin, daptomycin plus gentamicin, gentamicin plus rifampin, and the three-drug combination had similar activities.

Reporter Metabolite Analysis of Transcriptional Profiles of a Staphylococcus aureus Strain with Normal Phenotype and Its Isogenic hemB Mutant Displaying the Small-Colony-Variant Phenotype

In this study, full-genome DNA microarrays based on the sequence of Staphylococcus aureus N315 were used to compare the transcriptome of a clinical S. aureus strain with a normal phenotype to that of its isogenic mutant with a stable small-colony-variant (SCV) phenotype (hemB::ermB). In addition to standard statistical analyses, systems biology advances were applied to identify reporter metabolites and to achieve a more detailed survey of genome-wide expression differences between the hemB mutant and its parental strain. Genes of enzymes involved in glycolytic and fermentative pathways were found to be up-regulated in the hemB mutant. Furthermore, our analyses allowed identification of additional differences between the normal-phenotype S. aureus and the SCV, most of which were related to metabolism. Profound differences were identified especially in purine biosynthesis as well as in arginine and proline metabolism. Of particular interest, a hypothetical gene of the Crp/Fnr family (SA2424) that is part of the arginine-deiminase (AD) pathway, whose homologue in Streptococcus suis is assumed to be involved in intracellular persistence, showed significantly increased transcription in the hemB mutant. The hemB mutant potentially uses the up-regulated AD pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. Moreover, genes involved in capsular polysaccharide and cell wall synthesis were found to be significantly up-regulated in the hemB mutant and therefore potentially responsible for the changed cell morphology of SCVs. In conclusion, the identified differences may be responsible for the SCV phenotype and its association with chronic and persistent infections.

The GacS sensor kinase controls phenotypic reversion of small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14

The GacS/GacA two-component regulatory system in pseudomonads regulates genes involved in virulence, secondary metabolism and biofilm formation. Despite these regulatory functions, some Pseudomonas species are prone to spontaneous inactivating mutations in gacA and gacS. A gacS(-) strain of Pseudomonas aeruginosa PA14 was constructed to study the physiological role of this sensor histidine kinase. This loss-of-function mutation was associated with hypermotility, reduced production of acylhomoserine lactones, impaired biofilm maturation, and decreased antimicrobial resistance. Biofilms of the gacS(-) mutant gave rise to phenotypically stable small colony variants (SCVs) with increasing frequency when exposed to silver cations, hydrogen peroxide, human serum, or certain antibiotics (tobramicin, amikacin, azetronam, ceftrioxone, oxacilin, piperacillin or rifampicin). When cultured, the SCV produced thicker biofilms with greater cell density and greater antimicrobial resistance than did the wild-type or parental gacS(-) strains. Similar to other colony morphology variants described in the literature, this SCV was less motile than the wild-type strain and autoaggregated in broth culture. Complementation with gacS in trans restored the ability of the SCV to revert to a normal colony morphotype. These findings indicate that mutation of gacS is associated with the occurrence of stress-resistant SCV cells in P. aeruginosa biofilms and suggests that in some instances GacS may be necessary for reversion of these variants to a wild-type state.

MP073 Host-cell responses in models of persistent C. pneumoniae infection

A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form.

Selection of small-colony variants of Salmonella enterica serovar typhimurium in nonphagocytic eucaryotic cells.

Salmonella enterica strains are enteropathogenic bacteria that survive and proliferate within vacuolar compartments of epithelial and phagocytic cells. Recently, it has been reported that fibroblast cells are capable of restricting S. enterica serovar Typhimurium intracellular growth. Here, we show that prolonged residence of bacteria in the intracellular environment of fibroblasts results in the appearance of genetically stable small-colony variants (SCV). A total of 103 SCV isolates, obtained from four independent infections, were subjected to phenotypic analysis. The following phenotypes were observed: (i) delta-aminolevulinic acid auxotrophy; (ii) requirement for acetate or succinate for growth in glucose minimal medium; (iii) auxotrophy for aromatic amino acids; and (iv) reduced growth rate under aerobic conditions not linked to nutrient auxotrophy. The exact mutations responsible for the SCV phenotype in three representative isolates were mapped in the lpd, hemL, and aroD genes, which code for dihydrolipoamide dehydrogenase, glutamate-1-semyaldehyde aminotransferase, and 3-dehydroquinate dehydratase, respectively. The lpd, hemL, and aroD mutants had intracellular persistence rates in fibroblasts that were 3 to 4 logs higher than that of the parental strain and decreased susceptibility to aminoglycoside antibiotics. All three of these SCV isolates were attenuated in the BALB/c murine typhoid model. Complementation with lpd(+), hem(+), and aroD(+) genes restored the levels of intracellular persistence and antibiotic susceptibility to levels of the wild-type strain. However, virulence was not exhibited by any of the complemented strains. Altogether, our data demonstrate that similar to what it has been reported for SCV isolates of other pathogens, S. enterica SCV display enhanced intracellular persistence in eucaryotic cells and are impaired in the ability to cause overt disease. In addition, they also suggest that S. enterica SCV may be favored in vivo.

Decreased susceptibility to antibiotic killing of a stable small colony variant of Staphylococcus aureus in fluid phase and on fibronectin-coated surfaces.

The frequency of small colony variants of staphylococci associated with persistent, antibiotic resistant and relapsing infections is probably underestimated. These variants demonstrate decreased metabolism, leading to slow growth, increased resistance to cell-wall-active antibiotics, and decreased uptake of aminoglycoside antibiotics. This altered phenotype arises from defects in menadione and haemin biosynthesis resulting in impaired electron transport and decreased ATP concentrations. The recent acquisition of a stable small colony variant (SCV strain JB1), generated from strain 6850 of Staphylococcus aureus, allowed us to study the susceptibilities to antibiotic killing of parent and variant strains. Because differences in susceptibilities have been found between unattached and surface-adherent organisms, we tested both strains in solid and fluid-phase assays. Suspensions of SCV strain JB1 exposed to 8 x MIC of either oxacillin, vancomycin or fleroxacin, exhibited lower reductions in viable counts than the parent strain 6850, especially when high bacterial concentrations (1-2 x 10(7) cfu/mL) of either strain were tested. Susceptibility to antibiotic killing of bacteria attached to fibronectin-coated coverslips was markedly influenced by their growing or nongrowing state on the surface. In the latter condition, surface-bound SCV organisms were highly resistant to the bactericidal action of oxacillin or vancomycin in contrast to the parental strain which was normally eliminated by each antimicrobial agent. In conclusion, the decreased susceptibility of the stable SCV strain of S. aureus to bactericidal concentrations of antibiotics may help to explain the persistence of such organisms in chronic infections.

Staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu.

Recent studies have reported that Staphylococcus aureus small colony variants (SCVs) can cause highly persistent infections in humans and in cultured endothelial cells. To understand the process by which SCVs of S. aureus appear in subjects who have not received antibiotic treatment, bovine endothelial cells were coincubated with a wild S. aureus strain for 72 h in the presence of lysostaphin. Intracellular bacteria were harvested and screened for stable SCVs. Intracellular bacteria developed the SCV phenotype at a greater rate than control bacteria not exposed to endothelial cells: The intracellular induction rate was approximately 10(-3) versus a spontaneous rate of <10(-7). This observation suggest that SCVs are induced by the intracellular milieu and suggest a possible mechanism for the intriguing pathophysiology of tissue persistence of staphylococci.