Small colony variants of Staphylococcus aureus: A challenge for the researcher, the clinical microbiologist, and the clinician Barbara C. Kahl Institut für Medizinische Mikrobiologie Universitätsklinikum Münster
S. aureus colonization and infections healthy nasal carriers community and nosocomial acquired infections Skin- and soft tissue infections Life-threatening infections osteomyelitis endocarditis furuncle sepsis pneumonia carbuncle bursitis
Virulence factors of S. aureus Wa chst umskur v e 10 8 adhesins secreted proteins 6 4 2 early log phase late log/stationary phase 0 0 1 2 3 4 5 6 7 Zei t i n h elastin-binding protein clumping factor enterotoxin TSST-1 collagen- binding -toxin protein fibronectin- cell membrane binding protein peptidoglycan (cell wall) micro capsule proteinA coagulase
Acute versus chronic disease femoral abscess endocarditis small colony normal S. aureus variant (SCV) S. aureus von Eiff et al., Z. Orthop. 136:268-71 (1998)
SCV normal S. aureus Small colony variants (SCVs) • subpopulation of S. aureus • emerge after longterm antibiotic therapy • associated with persistent, recurrent infections, difficult treatable infections • osteomyelitis, device-related infections, cystic fibrosis • more resistant to antibiotics (aminoglykosides, TMP/SMX, ß-lactams) 48 h 30 min S. aureus • persist intracellularly in in vitro studies SCV normal S. aureus Proctor RA et al. Nat Rev Microbiol 2006; 4:295-305 v. Eiff et al., Clin. Infect. Dis. 2001;32:1643-7
Various and undetected mechanisms for SCVs occurence mechanisms: • hemin - or menadione -dependent (Proctor, von Eiff, Becker, McNamara, Peters, Lannergard AAC2008; Malouin J Bacteriol, 2006)
Impaired electron transport Negative regulators Shikimate NADH for toxin production Menadione Thiamine PP Intracellular persistence biosynthetic FADH2 enzymes of staphylococci Menadione Menaquinone Hemin biosynthetic enzymes Cytochromes Isoprenoid Hemin lipid F 0 F 1 ATPase ATP Produces an electro- chemical gradient P450 Cell wall biosynthesis; Amino acid transport/ Aminoglycoside and protein synthesis cationic peptide Carotenoid transport biosynthesis Rapid growth, Pigmented Antibiotic large colonies colonies susceptibility
Various and undetected mechanisms for SCVs occurence mechanisms: • hemin - or menadione -dependent (Proctor, von Eiff, Becker, McNamara, Peters, Lannergard AAC2008; Malouin J Bacteriol, 2006) • CO 2 -dependent (Gomez-Gonzalez, J Clin Microbiol 2010) • mutations in stringent stress response genes (Gao et al. Plos Pathogen 2010) • thymidine -dependent (Gilligan JCM1987, Besier I&I2008, Kahl, JID1998) • many SCVs with so far unknown underlying mechanism SCVs: • can revert to the normal phenotype within short periods
Intra/extracellular phenotypic switching Tuchscherr et al. EMBO MolMed 2010
Primary cultures from clinical specimens Tuchscherr et al. EMBO MolMed 2011
Altered bacterial gene expression and host cell response Tuchscherr et al. EMBO MolMed 2011
Chronic infection in mice Tuchscherr et al. EMBO MolMed 2011
First conclusions • Bacterial phenotype switching is an integral part of the infection process, which enable the bacteria to hide inside the host thereby providing a reservoir for chronic infection.
Thymidine-dependent (TD) SCVs emerge in vivo after treatment with trimethoprim/sulfamethoxazole (TMP/SMX) rely on extracellular thymidine (no growth on Mueller-Hinton Agar) are TMP/SMX resistant Longterm persistence of SCVs survive only in the presence of thymidine l l l l a a a a m m m m V V V V V V r r r r C C C C C C o o o o S S S S S S n n n n in many patients present even when no normal S. aureus was 9 r r 4 5 5 6 6 7 7 8 9 0 e e 9 9 9 9 9 9 9 9 9 0 k k 9 9 9 9 9 9 9 9 1 0 cultured r r a 1 1 1 1 1 1 1 1 / 2 a 0 m / / / / / / / / / m 6 9 6 6 1 9 1 5 1 1 persisted after TMP/SMX therapy was stopped (>4 years) induction of TD-SCVs of S. aureus Newman after in vitro culture in BHI after TMP/SMX challenge Columbia blood agar 65 months persistence Schaedler agar From Kahl B. C. et al. J. Clin. Microbiol. 2003, 41:410-3; Kahl B. C. et al. J. Clin. Microbiol. 2003, 41:4424-7; and unpublished data CO 2
Decreased tanscription of agr and hla in clinical thymidine-dependent (TD) SCVs Normal S. aureus SCV SCV + thymidine EL LL SP EL LL SP EL LL SP P2 sarA P3 P1 agr hla spa low thymidine low thymidine high thymidine less virulent phenotype specialized for persistence Kahl BC et al, 2005, Infect Immun 73:4119-26
TD-SCVs occur not only in CF, but also in other infections and in other species 11% SCVs of 3972 isolates from a CF multicenter study (193 patients from 17 centers) 40% TD-SCVs are reported in other CF-centers in Belgium, US, Germany, Turkey, Czech Republic in other chronic infections: soft tissue infection recurrent abscesses chronic bronchitis septicaemia tympanitis (Besier S J Clin Microbiol 2008; 46:3829; Seifert H, Emerg Infect Dis 1999; 5:450) not only in humans but also in chronic bovine mastitis (Atalla H VetMicro09) can complicate correct diagnosis of MRSA (Cleeve VJ, Hosp Infect 2006) also reported in other species: Salmonella, Escherichia
When to expect TD-SCVs? 1. S. aureus in high density 2. extracellular thymidine 3. treatment with TMP/SMX 4. Due to the rise of CA- and HA-MRSA recommendations of the IDSA to treat with TMP/SMX critical response (Proctor RA, Clin Infect Dis 2008; 46:584) Concentration of thymidine or dTMP in various human specimens* CF sputum 346 µg/l 34,8 µg/l Pus nd 18,19 µg/l Urine 540 µg/l 1,818 µg/l Liquor nd 375 µg/l *Besier S et al J Clin Microbiol 2008;46:3829
Model for thymidine-dependency of S. aureus SCVs thymidylate synthase thyA dUMP nucleosid transporter N 5 , N 10- dTMP Methylen- extracellular thymidine nupC THF DHF from respiratory secretions X TMP in CF lung X SMX with THF PABA + thymidine destroyed cells DHpteroate DP and pus e.g. component of DNA intracellular extracellular
Thymidine-dependent SCV expressing thy exhibits normal phenotype Normal S. aureus Thymidine-dependent SCV Pst I Sma I Stu I Ec RV o 'lip cat thyA Nde I Bam HI Cla I pCX19 thyA Eco RV 4906 bps Cla I xylR Nde I Sca I Sca I SCV expressing thy Cured mutant Chatterjee I, J Bacteriol 2008; 190:834-42
Increased transcription of thyA and nupC in TD-SCVs (EL) B. (EL) * 0.10 0.35 (LL) (LL) (SP) (SP) 0.30 * 0.08 Relative Gene Expression Relative Gene Expression 0.25 * * 0.06 0.20 0.15 0.04 0.10 0.02 0.05 normal TD-SCV TD-SCVpCX19thyA TD-SCVpCX19thyA+Xyl normal TD-SCV TD-SCV normal TD-SCV TD-SCV normal TD-SCV TD-SCVpCX19thyA TD-SCVpCX19thyA+Xyl expressing thyA expressing thyA thyA nupC unexpected: increased transcription of thyA expected: increased transcription of nupC Chatterjee I, J Bacteriol 2008; 190:834-42
Conclusions For the clinical microbiology laboratory: important to know when TD-SCVs are to be expected and how they look like patho-adaptive mechanism lead to a loss of function of thymidylate synthase – an essential protein clinical and in vitro data provide evidence that TD-SCVs are optimized for survival in the hostile environment of the lung TD-SCVs are attenuated in their virulence Intracellular location of bacteria difficult to treat Therefore, the work of defining the cellular pharmacokinetics and – dynamics of antibiotics against these bacteria are of importance.
Münster Frankfurt Cathrin Baum Silke Besier Acknowledgements Andre Kriegeskorte Thomas Wichelhaus Marco Kelkenberg Claudia Neumann Ulm Simone Brüning Barbara Spellerberg Barbara Ritzerfeld Nele Wellinghausen Susanne Deiwick Katrin Wardecki Dänemark Marion Wallstein Henrik Westh Nadine Theimann Kit Boye Karsten Becker Georg Peters USA Richard Proctor Homburg Ambrose Cheung Mathias Herrmann Jean Lee Indranil Chatterjee Evgeni Sokurenko Bo Shopsin Tübingen Christiane Wolz Belgium Christiane Görke Francoise van Bambeke
Greetings from Münster
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