- Email: [email protected]
Staphylococcus aureus small colony variants: a challenge to microbiologists and clinicians
International Journal of Antimicrobial Agents 31 (2008) 507–510
Review
Staphylococcus aureus small colony variants: a challenge to microbiologists and clinicians Christof von Eiff ∗ Institute of Medical Microbiology, University of M¨unster, Domagkstr. 10, 48149 M¨unster, Germany
Abstract The pathogen Staphylococcus aureus may use various strategies to resist antibiotic therapy. One of these strategies is the formation of small colony variants (SCVs), a naturally occurring, slow-growing subpopulation with distinctive phenotypic characteristics and pathogenic traits. SCVs are defined by mostly non-pigmented and non-haemolytic colonies ca. 10 times smaller than the parent strain. In the past decade, many reports and prospective studies have supported a pathogenic role for these variants in patients with persistent and/or recurrent infections. The tiny size of clinical and experimentally derived SCVs on solid agar is often due to auxotrophy for hemin and/or menadione, two compounds involved in the biosynthesis of electron transport chain components. The morphological and physiological features of SCVs present a challenge to clinical microbiologists in terms of recovery of organisms, their identification and susceptibility testing. Based on the knowledge that SCVs may persist intracellularly, treatment including antimicrobial agents with intracellular antistaphylococcal activity appears appropriate. SCVs potentially use the upregulated arginine deiminase pathway to produce ATP or, through ammonia production, to counteract the acidic environment that prevails intracellularly, as shown using a site-directed mutant with SCV phenotype in transcriptomic studies. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Small colony variants; Staphylococcus aureus; Persistent infections; Recurrent infections; Electron transport chain; Intracellular persistence; Arginine deiminase pathway; Oxidative phosphorylation; hemB mutant; menD mutant
1. Small colony variants as a cause of persistent and recurrent infections Small colony variants (SCVs), formerly designated as ‘G’ (gonidial) variants or dwarf colonies, constitute a naturally occurring, slow-growing subpopulation of bacteria with distinctive phenotypic characteristics and pathogenic traits. The recovery of SCVs of Staphylococcus aureus from clinical specimens was first described around 100 years ago. However, the connection of this phenotype to persistent and recurrent infections has only been appreciated in recent years [1]. Five patients were described with unusually persistent and/or antibiotic-resistant infections due to S. aureus SCVs. Since then, many reports and prospective studies have supported a pathogenic role for SCVs in patients with chronic and/or persistent infections such as chronic osteomyelitis and persistent skin and soft-tissue infection [2–4]. The results ∗
Tel.: +49 251 83 52353; fax: +49 251 83 55350. E-mail address: [email protected].
of a 6-year prospective study analysing the prevalence and persistence of S. aureus in patients with cystic fibrosis demonstrated that the airways of >70% patients were persistently colonised/infected by normal and/or SCV S. aureus, with a median persistence of 37 months (range 6–70 months) [5]. Some patients who initially harboured both normal and S. aureus SCVs subsequently lost the normal strain, whilst SCVs persisted for extended periods. The longer persistence of the SCV phenotype indicated a survival advantage of SCVs compared with the normal phenotype in the hostile milieu of the airways, possibly due to optimised adaptation of the SCVs.
2. Intracellular persistence of SCVs S. aureus has various strategies for resisting therapy that extend beyond classic mechanisms. Such strategies include the potential for evading the effect of a given antibiotic even though it tested susceptible by production of diffusion
0924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.10.026
508
C. von Eiff / International Journal of Antimicrobial Agents 31 (2008) 507–510
barriers, e.g. biofilm production, or by withdrawal into the intracellular milieu. The latter mechanism has been documented for SCVs. Indeed, in several assays using various non-professional phagocytes such as endothelial or epithelial cells, these variants were able to persist intracellularly [4,6–8]. To identify the intracellular location of SCVs, primary human endothelial cells were infected with various strain pairs displaying either the normal or the SCV phenotype [9]. Subsequently, maturation of phagosomes using live cell imaging was visualised. Within 1 h, all internalised staphylococci accumulated in lysosomal organelles and remained there for up to 5 days. Whilst an effective bactericidal activity of human endothelial cell lysosomes towards staphylococci was observed, these studies provided evidence that SCVs of selected strains are able to withstand this activity.
3. SCVs: a challenge in terms of recovery, identification and susceptibility testing SCVs are defined by mostly non-pigmented, nonhaemolytic colonies ca. 10 times smaller than the parent strain (hence their name). This tiny size of clinical and experimentally derived SCVs on solid agar is often due to auxotrophy for menadione, hemin or thymidine [1–4,6]. When the medium is supplemented with these compounds, SCVs grow as rapidly as the parent strains. Menadione and hemin are required for the biosynthesis of electron transport chain components, as menadione is isoprenylated to form menaquinone, the acceptor of electrons from nicotinamide adenine dinucleotide (NADH)/flavin adenine dinucleotide (FADH2) in the electron transport chain, and hemin is required for the biosynthesis of cytochromes, which accept electrons from menaquinone. In addition to the atypical colonial morphology, absent or reduced biochemical reactions (e.g. mannitol salt agarnegative using the Api Staph system) are typical features [10]. Most S. aureus SCVs are coagulase-positive by the tube test only after incubation for >18 h. Thus, these uncommon morphological and physiological features of SCVs present a challenge to clinical microbiologists in terms of recovery and identification [2]. A prerequisite for the isolation of this subpopulation is the application of extended conventional culture and identification techniques [10]. Recently, we showed that the most accurate method to detect both the species S. aureus and the SCV phenotype is to inoculate specimens both on Columbia blood agar and on a new chromogenic agar (S. aureus ID agar) [11]. However, SCVs are easily overgrown and missed when the normal S. aureus is present because SCVs divide approximately nine times slower than S. aureus with normal phenotype [2]. S. aureus isolates suspected of being SCVs, which may give a false-negative coagulase test, should be confirmed as S. aureus by testing the species-specific nuc and coa genes. Another diagnostic approach was shown in a patient with a brain abscess in which S. aureus SCVs were
detected in the brain tissue using a 16S rRNA-directed in situ hybridisation technique [12]. Since clinical SCVs often exhibit an unstable phenotype, Fourier-transform infrared spectroscopy has been used to investigate the phase variation from SCV phenotype into the normal phenotype and vice versa [13]. Indeed, this non-invasive technique offered a rapid, reliable and nondestructive approach to trace directly the process of reversion from the normal phenotype into the SCV phenotype and vice versa. Based on spectral information in three different spectral ranges, clustering resulted in dendrograms showing a clear discrimination between the normal and SCV phenotype. For several reasons, SCVs also present a challenge with regard to susceptibility testing. First, SCVs are often present in mixed populations with the normal phenotype (see above), thus even a small percentage of normally growing organisms will rapidly replace the SCVs in liquid medium in an overnight culture, thereby making susceptibility testing of the SCVs difficult [2]. Second, delayed growth makes standardisation of testing difficult because slow growth alters diffusion tests and the times for measuring susceptibility [14]. Third, errors may occur when these variants are resistant to oxacillin when tested by disk diffusion test, Etest, microdilution test and automated susceptibility testing systems as well as antiPBP2a slide latex agglutination tests [15]. As a consequence, detection of the mecA gene by molecular methods or the use of an anti-PBP2a slide latex agglutination test using a drastically increased inoculum (approximately a loopful with 100–200 SCV colonies) should be used for the reliable diagnosis or validation of methicillin-resistant S. aureus (MRSA) SCVs.
4. Treatment of patients infected with S. aureus SCVs Interruption of electron transport reduces the electrochemical gradient across the bacterial membrane, resulting in a decreased uptake of antimicrobial agents that require a charge differential to be active. Therefore, substances such as gentamicin or other aminoglycosides should not be used for the therapy of infections caused by S. aureus SCVs, although single strains with SCV phenotype might be susceptible to aminoglycosides [16]. So far, prospective studies on the treatment of patients infected with S. aureus SCVs are not available, thus optimal therapy has not yet been defined. However, based on the knowledge that SCVs may persist intracellularly, treatment including antimicrobial agents with intracellular antistaphylococcal activity such as rifampicin appears appropriate. As monotherapy with rifampicin is not recommended owing to rapid development of resistance, a combination regimen with either -lactam antibiotics such as oxacillin or a second-generation cephalosporin for methicillin-susceptible S. aureus SCVs, or vancomycin for MRSA SCVs, is necessary [17]. In the past it was found that trimethoprim/sulfamethoxazole combined with rifampicin was the most active therapeutic regimen in a tissue culture system
C. von Eiff / International Journal of Antimicrobial Agents 31 (2008) 507–510
with SCVs inside endothelial cells [18], however more studies are necessary to define this therapy. A combination of rifampicin and a fluoroquinolone, as recently proposed by Sendi et al. [19], appears inadequate at least for treating methicillin-resistant staphylococci, which are often resistant to fluoroquinolones [20]. Another therapeutic approach might be, if possible, to control when staphylococci grow rapidly and when they form SCVs, as development of the SCV phenotype may not always be a clinically disadvantageous situation. Because of their low exotoxin production, SCVs cause less tissue damage than rapidly growing staphylococci. The use of drugs that interfere with electron transport might be particularly valuable as a short-term measure if such drugs could rapidly turn off toxin production [21]. Investigation of the effect of electron transport inhibitors on S. aureus may demonstrate their potential for therapy of staphylococcal infections. Conversely, reversing the phenotype, e.g. by adding menadione in patients infected with menadione-auxotrophic SCVs, could enhance susceptibility to antibiotics and thus decrease the ability of the organisms to reside within cells [14]. Adding vitamin K, the isoprenylated form of menadione, to a standard antibiotic regimen might therefore reduce the rate of development of SCVs or prevent the establishment of SCVs as chronic intracellular pathogens. However, prospective studies are necessary before such strategies can be used for the treatment of patients infected with such variants.
5. Physiology and pathogenic potential of defined mutants with SCV phenotype To study the physiological characteristics of SCVs, stable mutants in electron transport were generated by interrupting hemin (hemB) or menadione (menD) biosynthetic genes in S. aureus [8,22,23]. In fact, these mutants mimicked the characteristic features of SCVs recovered from clinical specimens. The SCV concept, in particular their significance in chronic and persistent infections, has been investigated in a comprehensive approach comprising genomic, transcriptomic, proteomic and metabolic studies as well as in animal models. A full-genome DNA microarray was used to compare the transcriptome of a clinical S. aureus strain with normal phenotype with its hemB mutant displaying the SCV phenotype [24]. Genes of enzymes involved in glycolytic and fermentative pathways were particularly found to be upregulated in the mutant. Profound differences were identified in purine biosynthesis as well as in arginine and proline metabolism. The hemB mutant potentially uses the upregulated arginine deiminase pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. Studies using a high-resolution two-dimensional protein gel electrophoresis technique combined with matrix-assisted laser desorption ionisation-time of flight mass spectrom-
509
etry (MALDI-TOF-MS) indicated that the hemB mutant generates ATP from glucose or fructose only by substrate phosphorylation [25]. Most of the known virulence factors expressed during the late exponential phase were not found in the mutant or were present at low levels. Both the menD and the hemB mutant were also subjected to Phenotype MicroArray Analysis of over 1500 phenotypes [26]. In this metabolomic approach, the hemB mutant was shown to be defective in utilising a variety of carbon sources including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than seen with the hemB mutant. Hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth of both mutants. To assess the pathogenic potential of SCVs, both mutants displaying the SCV phenotype were studied in various animal models. In a murine model of septic arthritis, mice challenged with the hemB mutant displayed a higher frequency and a significantly higher severity of arthritis than mice inoculated with the parent strain [27]. Mice inoculated with the hemB mutant also showed a reduced bacterial burden in their kidneys and joints compared with mice exposed to the normal phenotype strain. It was suggested that SCVs might be more virulent on a per-organism basis than their parental isolates owing to the ability of SCVs to produce high amounts of destructive proteases. In another study, the S. aureus hemB and menD mutants were compared with the parental strains and complemented mutants in a rabbit endocarditis model [22]. The hemB mutant was found to be equally virulent to wild-type and hemB-complemented strains as measured by vegetation bacterial densities, dissemination to the liver and spleen, and sensitivity to oxacillin therapy. In contrast, the menD mutant revealed reduced colonisation levels in the liver and spleen, and disseminated foci of infection were less responsive to oxacillin therapy than wild-type and menD-complemented strains. The differences between these mutants were thought to be related to the fact that each organ is probably replete with hemin derived from embolic infarcts that occur during the course of experimental endocarditis. Thus, hemin might circumvent the hemB mutation-induced defect in the cytochrome system. Recently, these mutants, as well as SCVs recovered from clinical specimens, were studied using the Caenorhabditis elegans infection model [28]. Indeed, hemB and menD mutants as well as clinical SCVs that were auxotrophic for hemin or menadione were found to be less virulent than isogenic parental and complemented strains. Of particular interest, reduced virulence of the strains with SCV phenotype was not the result of an impaired ability to colonise the nematode digestive tract. Consequently, it was assumed that reduced production of ␣-haemolysin and perhaps other virulence products in the SCV strains owing to the loss of oxidative phosphorylation leads to reduced virulence in nematodes and that inhibition of bacterial respiration as a virulence-inhibiting mecha-
510
C. von Eiff / International Journal of Antimicrobial Agents 31 (2008) 507–510
nism might be an interesting future therapeutic approach [28]. Funding: Deutsche Forschungsgemeinschaft EI 247/7-1. Competing interests: None declared. Ethical approval: Not required.
[14]
References
[16]
[1] Proctor RA, van Langevelde P, Kristjansson M, Maslow JN, Arbeit RD. Persistent and relapsing infections associated with smallcolony variants of Staphylococcus aureus. Clin Infect Dis 1995;20: 95–102. [2] Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, Herrmann M, et al. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 2006;4:295–305. [3] von Eiff C, Bettin D, Proctor RA, Rolauffs B, Lindner N, Winkelmann W, et al. Recovery of small colony variants of Staphylococcus aureus following gentamicin bead placement for osteomyelitis. Clin Infect Dis 1997;25:1250–1. [4] von Eiff C, Becker K, Metze D, Lubritz G, Hockmann J, Schwarz T, et al. Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier’s disease. Clin Infect Dis 2001;32:1643–7. [5] Kahl BC, Duebbers A, Lubritz G, Haeberle J, Koch HG, Ritzerfeld B, et al. Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study. J Clin Microbiol 2003;41:4424–7. [6] Kahl B, Herrmann M, Everding AS, Koch HG, Becker K, Harms E, et al. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis 1998;177:1023–9. [7] Vaudaux P, Francois P, Bisognano C, Kelley WL, Lew DP, Schrenzel J, et al. Increased expression of clumping factor and fibronectin-binding proteins by hemB mutants of Staphylococcus aureus expressing small colony variant phenotypes. Infect Immun 2002;70:5428–37. [8] von Eiff C, Heilmann C, Proctor RA, Woltz C, Peters G, G¨otz F. A sitedirected Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly. J Bacteriol 1997;179:4706–12. [9] Schroder A, Kland R, Peschel A, von Eiff C, Aepfelbacher M. Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes. Med Microbiol Immunol 2006;195:185–94. [10] Becker K, Harmsen D, Mellmann A, Meier C, Schumann P, Peters G, et al. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol 2004;42:4988–95. [11] Kipp F, Kahl BC, Becker K, Baron EJ, Proctor RA, Peters G, et al. Evaluation of two chromogenic agar media for recovery and identification of Staphylococcus aureus small-colony variants. J Clin Microbiol 2005;43:1956–9. [12] Kipp F, Ziebuhr W, Becker K, Krimmer V, Hobeta N, Peters G, et al. Detection of Staphylococcus aureus by 16S rRNA directed in situ hybridisation in a patient with a brain abscess caused by small colony variants. J Neurol Neurosurg Psychiatry 2003;74:1000–2. [13] Becker K, Laham NA, Fegeler W, Proctor RA, Peters G, von Eiff C. Fourier-transform infrared spectroscopic analysis is a powerful tool for
[15]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
studying the dynamic changes in Staphylococcus aureus small-colony variants. J Clin Microbiol 2006;44:3274–8. Proctor RA, Kahl B, von EC, Vaudaux PE, Lew DP, Peters G. Staphylococcal small colony variants have novel mechanisms for antibiotic resistance. Clin Infect Dis 1998;27(Suppl 1):S68–74. Kipp F, Becker K, Peters G, von Eiff C. Evaluation of different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureus. J Clin Microbiol 2004;42:1277–9. Baumert N, von Eiff C, Schaaff F, Peters G, Proctor RA, Sahl HG. Physiology and antibiotic susceptibility of Staphylococcus aureus small colony variants. Microb Drug Resist 2002;8:253–60. von Eiff C, Friedrich AW, Becker K, Peters G. Comparative in vitro activity of ceftobiprole against staphylococci displaying normal and small-colony variant phenotypes. Antimicrob Agents Chemother 2005;49:4372–4. Proctor RA, Peters G. Small colony variants in staphylococcal infections: diagnostic and therapeutic implications. Clin Infect Dis 1998;27:419–22. Sendi P, Rohrbach M, Graber P, Frei R, Ochsner PE, Zimmerli W. Staphylococcus aureus small colony variants in prosthetic joint infection. Clin Infect Dis 2006;43:961–7. Schmitz FJ, von Eiff C, Gondolf M, Fluit AC, Verhoef J, Peters G, et al. Staphylococcus aureus small colony variants: rate of selection and MIC values compared to wild-type strains, using ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin. Clin Microbiol Infect 1999;5:376–8. Proctor RA, Dalal SC, Kahl B, Brar D, Peters G, Nichols WW. Two diarylurea electron transport inhibitors reduce Staphylococcus aureus hemolytic activity and protect cultured endothelial cells from lysis. Antimicrob Agents Chemother 2002;46:2333–6. Bates DM, von Eiff C, McNamara PJ, Peters G, Yeaman MR, Bayer AS, et al. Staphylococcus aureus menD and hemB mutants are as infective as the parent strains, but the menadione biosynthetic mutant persists within the kidney. J Infect Dis 2003;187:1654–61. Senn MM, Bischoff M, von Eiff C, Berger-B¨achi B. sigmaB activity in a Staphylococcus aureus hemB mutant. J Bacteriol 2005;187:7397– 406. Seggewiß J, Becker K, Kotte O, Eisenacher M, Yazdi MR, Fischer A, et al. 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. J Bacteriol 2006;188:7765–77. Kohler C, von Eiff C, Peters G, Proctor RA, Hecker M, Engelmann S. Physiological characterization of a heme-deficient mutant of Staphylococcus aureus by a proteomic approach. J Bacteriol 2003;185: 6928–37. von Eiff C, McNamara P, Becker K, Bates D, Lei XH, Ziman M, et al. Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype. J Bacteriol 2006;188:687–93. Jonsson IM, von Eiff C, Proctor RA, Peters G, Ryden C, Tarkowski A. Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis. Microb Pathog 2003;34:73–9. Sifri CD, Baresch-Bernal A, Calderwood SB, von Eiff C. Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model. Infect Immun 2006;74:1091–6.
Review
Staphylococcus aureus small colony variants: a challenge to microbiologists and clinicians Christof von Eiff ∗ Institute of Medical Microbiology, University of M¨unster, Domagkstr. 10, 48149 M¨unster, Germany
Abstract The pathogen Staphylococcus aureus may use various strategies to resist antibiotic therapy. One of these strategies is the formation of small colony variants (SCVs), a naturally occurring, slow-growing subpopulation with distinctive phenotypic characteristics and pathogenic traits. SCVs are defined by mostly non-pigmented and non-haemolytic colonies ca. 10 times smaller than the parent strain. In the past decade, many reports and prospective studies have supported a pathogenic role for these variants in patients with persistent and/or recurrent infections. The tiny size of clinical and experimentally derived SCVs on solid agar is often due to auxotrophy for hemin and/or menadione, two compounds involved in the biosynthesis of electron transport chain components. The morphological and physiological features of SCVs present a challenge to clinical microbiologists in terms of recovery of organisms, their identification and susceptibility testing. Based on the knowledge that SCVs may persist intracellularly, treatment including antimicrobial agents with intracellular antistaphylococcal activity appears appropriate. SCVs potentially use the upregulated arginine deiminase pathway to produce ATP or, through ammonia production, to counteract the acidic environment that prevails intracellularly, as shown using a site-directed mutant with SCV phenotype in transcriptomic studies. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Small colony variants; Staphylococcus aureus; Persistent infections; Recurrent infections; Electron transport chain; Intracellular persistence; Arginine deiminase pathway; Oxidative phosphorylation; hemB mutant; menD mutant
1. Small colony variants as a cause of persistent and recurrent infections Small colony variants (SCVs), formerly designated as ‘G’ (gonidial) variants or dwarf colonies, constitute a naturally occurring, slow-growing subpopulation of bacteria with distinctive phenotypic characteristics and pathogenic traits. The recovery of SCVs of Staphylococcus aureus from clinical specimens was first described around 100 years ago. However, the connection of this phenotype to persistent and recurrent infections has only been appreciated in recent years [1]. Five patients were described with unusually persistent and/or antibiotic-resistant infections due to S. aureus SCVs. Since then, many reports and prospective studies have supported a pathogenic role for SCVs in patients with chronic and/or persistent infections such as chronic osteomyelitis and persistent skin and soft-tissue infection [2–4]. The results ∗
Tel.: +49 251 83 52353; fax: +49 251 83 55350. E-mail address: [email protected].
of a 6-year prospective study analysing the prevalence and persistence of S. aureus in patients with cystic fibrosis demonstrated that the airways of >70% patients were persistently colonised/infected by normal and/or SCV S. aureus, with a median persistence of 37 months (range 6–70 months) [5]. Some patients who initially harboured both normal and S. aureus SCVs subsequently lost the normal strain, whilst SCVs persisted for extended periods. The longer persistence of the SCV phenotype indicated a survival advantage of SCVs compared with the normal phenotype in the hostile milieu of the airways, possibly due to optimised adaptation of the SCVs.
2. Intracellular persistence of SCVs S. aureus has various strategies for resisting therapy that extend beyond classic mechanisms. Such strategies include the potential for evading the effect of a given antibiotic even though it tested susceptible by production of diffusion
0924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.10.026
508
C. von Eiff / International Journal of Antimicrobial Agents 31 (2008) 507–510
barriers, e.g. biofilm production, or by withdrawal into the intracellular milieu. The latter mechanism has been documented for SCVs. Indeed, in several assays using various non-professional phagocytes such as endothelial or epithelial cells, these variants were able to persist intracellularly [4,6–8]. To identify the intracellular location of SCVs, primary human endothelial cells were infected with various strain pairs displaying either the normal or the SCV phenotype [9]. Subsequently, maturation of phagosomes using live cell imaging was visualised. Within 1 h, all internalised staphylococci accumulated in lysosomal organelles and remained there for up to 5 days. Whilst an effective bactericidal activity of human endothelial cell lysosomes towards staphylococci was observed, these studies provided evidence that SCVs of selected strains are able to withstand this activity.
3. SCVs: a challenge in terms of recovery, identification and susceptibility testing SCVs are defined by mostly non-pigmented, nonhaemolytic colonies ca. 10 times smaller than the parent strain (hence their name). This tiny size of clinical and experimentally derived SCVs on solid agar is often due to auxotrophy for menadione, hemin or thymidine [1–4,6]. When the medium is supplemented with these compounds, SCVs grow as rapidly as the parent strains. Menadione and hemin are required for the biosynthesis of electron transport chain components, as menadione is isoprenylated to form menaquinone, the acceptor of electrons from nicotinamide adenine dinucleotide (NADH)/flavin adenine dinucleotide (FADH2) in the electron transport chain, and hemin is required for the biosynthesis of cytochromes, which accept electrons from menaquinone. In addition to the atypical colonial morphology, absent or reduced biochemical reactions (e.g. mannitol salt agarnegative using the Api Staph system) are typical features [10]. Most S. aureus SCVs are coagulase-positive by the tube test only after incubation for >18 h. Thus, these uncommon morphological and physiological features of SCVs present a challenge to clinical microbiologists in terms of recovery and identification [2]. A prerequisite for the isolation of this subpopulation is the application of extended conventional culture and identification techniques [10]. Recently, we showed that the most accurate method to detect both the species S. aureus and the SCV phenotype is to inoculate specimens both on Columbia blood agar and on a new chromogenic agar (S. aureus ID agar) [11]. However, SCVs are easily overgrown and missed when the normal S. aureus is present because SCVs divide approximately nine times slower than S. aureus with normal phenotype [2]. S. aureus isolates suspected of being SCVs, which may give a false-negative coagulase test, should be confirmed as S. aureus by testing the species-specific nuc and coa genes. Another diagnostic approach was shown in a patient with a brain abscess in which S. aureus SCVs were
detected in the brain tissue using a 16S rRNA-directed in situ hybridisation technique [12]. Since clinical SCVs often exhibit an unstable phenotype, Fourier-transform infrared spectroscopy has been used to investigate the phase variation from SCV phenotype into the normal phenotype and vice versa [13]. Indeed, this non-invasive technique offered a rapid, reliable and nondestructive approach to trace directly the process of reversion from the normal phenotype into the SCV phenotype and vice versa. Based on spectral information in three different spectral ranges, clustering resulted in dendrograms showing a clear discrimination between the normal and SCV phenotype. For several reasons, SCVs also present a challenge with regard to susceptibility testing. First, SCVs are often present in mixed populations with the normal phenotype (see above), thus even a small percentage of normally growing organisms will rapidly replace the SCVs in liquid medium in an overnight culture, thereby making susceptibility testing of the SCVs difficult [2]. Second, delayed growth makes standardisation of testing difficult because slow growth alters diffusion tests and the times for measuring susceptibility [14]. Third, errors may occur when these variants are resistant to oxacillin when tested by disk diffusion test, Etest, microdilution test and automated susceptibility testing systems as well as antiPBP2a slide latex agglutination tests [15]. As a consequence, detection of the mecA gene by molecular methods or the use of an anti-PBP2a slide latex agglutination test using a drastically increased inoculum (approximately a loopful with 100–200 SCV colonies) should be used for the reliable diagnosis or validation of methicillin-resistant S. aureus (MRSA) SCVs.
4. Treatment of patients infected with S. aureus SCVs Interruption of electron transport reduces the electrochemical gradient across the bacterial membrane, resulting in a decreased uptake of antimicrobial agents that require a charge differential to be active. Therefore, substances such as gentamicin or other aminoglycosides should not be used for the therapy of infections caused by S. aureus SCVs, although single strains with SCV phenotype might be susceptible to aminoglycosides [16]. So far, prospective studies on the treatment of patients infected with S. aureus SCVs are not available, thus optimal therapy has not yet been defined. However, based on the knowledge that SCVs may persist intracellularly, treatment including antimicrobial agents with intracellular antistaphylococcal activity such as rifampicin appears appropriate. As monotherapy with rifampicin is not recommended owing to rapid development of resistance, a combination regimen with either -lactam antibiotics such as oxacillin or a second-generation cephalosporin for methicillin-susceptible S. aureus SCVs, or vancomycin for MRSA SCVs, is necessary [17]. In the past it was found that trimethoprim/sulfamethoxazole combined with rifampicin was the most active therapeutic regimen in a tissue culture system
C. von Eiff / International Journal of Antimicrobial Agents 31 (2008) 507–510
with SCVs inside endothelial cells [18], however more studies are necessary to define this therapy. A combination of rifampicin and a fluoroquinolone, as recently proposed by Sendi et al. [19], appears inadequate at least for treating methicillin-resistant staphylococci, which are often resistant to fluoroquinolones [20]. Another therapeutic approach might be, if possible, to control when staphylococci grow rapidly and when they form SCVs, as development of the SCV phenotype may not always be a clinically disadvantageous situation. Because of their low exotoxin production, SCVs cause less tissue damage than rapidly growing staphylococci. The use of drugs that interfere with electron transport might be particularly valuable as a short-term measure if such drugs could rapidly turn off toxin production [21]. Investigation of the effect of electron transport inhibitors on S. aureus may demonstrate their potential for therapy of staphylococcal infections. Conversely, reversing the phenotype, e.g. by adding menadione in patients infected with menadione-auxotrophic SCVs, could enhance susceptibility to antibiotics and thus decrease the ability of the organisms to reside within cells [14]. Adding vitamin K, the isoprenylated form of menadione, to a standard antibiotic regimen might therefore reduce the rate of development of SCVs or prevent the establishment of SCVs as chronic intracellular pathogens. However, prospective studies are necessary before such strategies can be used for the treatment of patients infected with such variants.
5. Physiology and pathogenic potential of defined mutants with SCV phenotype To study the physiological characteristics of SCVs, stable mutants in electron transport were generated by interrupting hemin (hemB) or menadione (menD) biosynthetic genes in S. aureus [8,22,23]. In fact, these mutants mimicked the characteristic features of SCVs recovered from clinical specimens. The SCV concept, in particular their significance in chronic and persistent infections, has been investigated in a comprehensive approach comprising genomic, transcriptomic, proteomic and metabolic studies as well as in animal models. A full-genome DNA microarray was used to compare the transcriptome of a clinical S. aureus strain with normal phenotype with its hemB mutant displaying the SCV phenotype [24]. Genes of enzymes involved in glycolytic and fermentative pathways were particularly found to be upregulated in the mutant. Profound differences were identified in purine biosynthesis as well as in arginine and proline metabolism. The hemB mutant potentially uses the upregulated arginine deiminase pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. Studies using a high-resolution two-dimensional protein gel electrophoresis technique combined with matrix-assisted laser desorption ionisation-time of flight mass spectrom-
509
etry (MALDI-TOF-MS) indicated that the hemB mutant generates ATP from glucose or fructose only by substrate phosphorylation [25]. Most of the known virulence factors expressed during the late exponential phase were not found in the mutant or were present at low levels. Both the menD and the hemB mutant were also subjected to Phenotype MicroArray Analysis of over 1500 phenotypes [26]. In this metabolomic approach, the hemB mutant was shown to be defective in utilising a variety of carbon sources including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than seen with the hemB mutant. Hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth of both mutants. To assess the pathogenic potential of SCVs, both mutants displaying the SCV phenotype were studied in various animal models. In a murine model of septic arthritis, mice challenged with the hemB mutant displayed a higher frequency and a significantly higher severity of arthritis than mice inoculated with the parent strain [27]. Mice inoculated with the hemB mutant also showed a reduced bacterial burden in their kidneys and joints compared with mice exposed to the normal phenotype strain. It was suggested that SCVs might be more virulent on a per-organism basis than their parental isolates owing to the ability of SCVs to produce high amounts of destructive proteases. In another study, the S. aureus hemB and menD mutants were compared with the parental strains and complemented mutants in a rabbit endocarditis model [22]. The hemB mutant was found to be equally virulent to wild-type and hemB-complemented strains as measured by vegetation bacterial densities, dissemination to the liver and spleen, and sensitivity to oxacillin therapy. In contrast, the menD mutant revealed reduced colonisation levels in the liver and spleen, and disseminated foci of infection were less responsive to oxacillin therapy than wild-type and menD-complemented strains. The differences between these mutants were thought to be related to the fact that each organ is probably replete with hemin derived from embolic infarcts that occur during the course of experimental endocarditis. Thus, hemin might circumvent the hemB mutation-induced defect in the cytochrome system. Recently, these mutants, as well as SCVs recovered from clinical specimens, were studied using the Caenorhabditis elegans infection model [28]. Indeed, hemB and menD mutants as well as clinical SCVs that were auxotrophic for hemin or menadione were found to be less virulent than isogenic parental and complemented strains. Of particular interest, reduced virulence of the strains with SCV phenotype was not the result of an impaired ability to colonise the nematode digestive tract. Consequently, it was assumed that reduced production of ␣-haemolysin and perhaps other virulence products in the SCV strains owing to the loss of oxidative phosphorylation leads to reduced virulence in nematodes and that inhibition of bacterial respiration as a virulence-inhibiting mecha-
510
C. von Eiff / International Journal of Antimicrobial Agents 31 (2008) 507–510
nism might be an interesting future therapeutic approach [28]. Funding: Deutsche Forschungsgemeinschaft EI 247/7-1. Competing interests: None declared. Ethical approval: Not required.
[14]
References
[16]
[1] Proctor RA, van Langevelde P, Kristjansson M, Maslow JN, Arbeit RD. Persistent and relapsing infections associated with smallcolony variants of Staphylococcus aureus. Clin Infect Dis 1995;20: 95–102. [2] Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, Herrmann M, et al. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 2006;4:295–305. [3] von Eiff C, Bettin D, Proctor RA, Rolauffs B, Lindner N, Winkelmann W, et al. Recovery of small colony variants of Staphylococcus aureus following gentamicin bead placement for osteomyelitis. Clin Infect Dis 1997;25:1250–1. [4] von Eiff C, Becker K, Metze D, Lubritz G, Hockmann J, Schwarz T, et al. Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier’s disease. Clin Infect Dis 2001;32:1643–7. [5] Kahl BC, Duebbers A, Lubritz G, Haeberle J, Koch HG, Ritzerfeld B, et al. Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study. J Clin Microbiol 2003;41:4424–7. [6] Kahl B, Herrmann M, Everding AS, Koch HG, Becker K, Harms E, et al. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis 1998;177:1023–9. [7] Vaudaux P, Francois P, Bisognano C, Kelley WL, Lew DP, Schrenzel J, et al. Increased expression of clumping factor and fibronectin-binding proteins by hemB mutants of Staphylococcus aureus expressing small colony variant phenotypes. Infect Immun 2002;70:5428–37. [8] von Eiff C, Heilmann C, Proctor RA, Woltz C, Peters G, G¨otz F. A sitedirected Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly. J Bacteriol 1997;179:4706–12. [9] Schroder A, Kland R, Peschel A, von Eiff C, Aepfelbacher M. Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes. Med Microbiol Immunol 2006;195:185–94. [10] Becker K, Harmsen D, Mellmann A, Meier C, Schumann P, Peters G, et al. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol 2004;42:4988–95. [11] Kipp F, Kahl BC, Becker K, Baron EJ, Proctor RA, Peters G, et al. Evaluation of two chromogenic agar media for recovery and identification of Staphylococcus aureus small-colony variants. J Clin Microbiol 2005;43:1956–9. [12] Kipp F, Ziebuhr W, Becker K, Krimmer V, Hobeta N, Peters G, et al. Detection of Staphylococcus aureus by 16S rRNA directed in situ hybridisation in a patient with a brain abscess caused by small colony variants. J Neurol Neurosurg Psychiatry 2003;74:1000–2. [13] Becker K, Laham NA, Fegeler W, Proctor RA, Peters G, von Eiff C. Fourier-transform infrared spectroscopic analysis is a powerful tool for
[15]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
studying the dynamic changes in Staphylococcus aureus small-colony variants. J Clin Microbiol 2006;44:3274–8. Proctor RA, Kahl B, von EC, Vaudaux PE, Lew DP, Peters G. Staphylococcal small colony variants have novel mechanisms for antibiotic resistance. Clin Infect Dis 1998;27(Suppl 1):S68–74. Kipp F, Becker K, Peters G, von Eiff C. Evaluation of different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureus. J Clin Microbiol 2004;42:1277–9. Baumert N, von Eiff C, Schaaff F, Peters G, Proctor RA, Sahl HG. Physiology and antibiotic susceptibility of Staphylococcus aureus small colony variants. Microb Drug Resist 2002;8:253–60. von Eiff C, Friedrich AW, Becker K, Peters G. Comparative in vitro activity of ceftobiprole against staphylococci displaying normal and small-colony variant phenotypes. Antimicrob Agents Chemother 2005;49:4372–4. Proctor RA, Peters G. Small colony variants in staphylococcal infections: diagnostic and therapeutic implications. Clin Infect Dis 1998;27:419–22. Sendi P, Rohrbach M, Graber P, Frei R, Ochsner PE, Zimmerli W. Staphylococcus aureus small colony variants in prosthetic joint infection. Clin Infect Dis 2006;43:961–7. Schmitz FJ, von Eiff C, Gondolf M, Fluit AC, Verhoef J, Peters G, et al. Staphylococcus aureus small colony variants: rate of selection and MIC values compared to wild-type strains, using ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin. Clin Microbiol Infect 1999;5:376–8. Proctor RA, Dalal SC, Kahl B, Brar D, Peters G, Nichols WW. Two diarylurea electron transport inhibitors reduce Staphylococcus aureus hemolytic activity and protect cultured endothelial cells from lysis. Antimicrob Agents Chemother 2002;46:2333–6. Bates DM, von Eiff C, McNamara PJ, Peters G, Yeaman MR, Bayer AS, et al. Staphylococcus aureus menD and hemB mutants are as infective as the parent strains, but the menadione biosynthetic mutant persists within the kidney. J Infect Dis 2003;187:1654–61. Senn MM, Bischoff M, von Eiff C, Berger-B¨achi B. sigmaB activity in a Staphylococcus aureus hemB mutant. J Bacteriol 2005;187:7397– 406. Seggewiß J, Becker K, Kotte O, Eisenacher M, Yazdi MR, Fischer A, et al. 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. J Bacteriol 2006;188:7765–77. Kohler C, von Eiff C, Peters G, Proctor RA, Hecker M, Engelmann S. Physiological characterization of a heme-deficient mutant of Staphylococcus aureus by a proteomic approach. J Bacteriol 2003;185: 6928–37. von Eiff C, McNamara P, Becker K, Bates D, Lei XH, Ziman M, et al. Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype. J Bacteriol 2006;188:687–93. Jonsson IM, von Eiff C, Proctor RA, Peters G, Ryden C, Tarkowski A. Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis. Microb Pathog 2003;34:73–9. Sifri CD, Baresch-Bernal A, Calderwood SB, von Eiff C. Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model. Infect Immun 2006;74:1091–6.