A challenge for the researcher, the clinical microbiologist, and - - PowerPoint PPT Presentation

Barbara C. Kahl Institut für Medizinische Mikrobiologie Universitätsklinikum Münster

Small colony variants of Staphylococcus aureus: A challenge for the researcher, the clinical microbiologist, and the clinician

 healthy nasal carriers  community and nosocomial acquired infections

• steomyelitis

endocarditis pneumonia sepsis Skin- and soft tissue infections

carbuncle bursitis

furuncle Life-threatening infections

• S. aureus

colonization and infections

adhesins early log phase secreted proteins late log/stationary phase

enterotoxin TSST-1 -toxin coagulase proteinA fibronectin- binding protein collagen- binding protein clumping factor elastin-binding protein cell membrane peptidoglycan (cell wall) micro capsule

Virulence factors of S. aureus

Wa chst umskur v e

Acute versus chronic disease

von Eiff et al., Z. Orthop. 136:268-71 (1998)

endocarditis normal S. aureus femoral abscess small colony variant (SCV)

• 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)
• persist intracellularly in in vitro studies
• v. Eiff et al., Clin. Infect. Dis. 2001;32:1643-7
• S. aureus

SCV normal

• S. aureus

30 min 48 h

Proctor RA et al. Nat Rev Microbiol 2006; 4:295-305 SCV normal S. aureus

Various and undetected mechanisms for SCVs occurence

mechanisms:

• hemin- or menadione-dependent (Proctor, von Eiff, Becker,

McNamara, Peters, Lannergard AAC2008; Malouin J Bacteriol, 2006)

NADH Menaquinone Cytochromes F0F1ATPase Hemin FADH2

Cell wall biosynthesis; Amino acid transport/ protein synthesis

Rapid growth, large colonies

P450 Carotenoid biosynthesis Produces an electro- chemical gradient Aminoglycoside and cationic peptide transport

Antibiotic susceptibility Pigmented colonies

Menadione

Thiamine PP

Shikimate Isoprenoid lipid

Menadione biosynthetic enzymes Hemin biosynthetic enzymes Negative regulators for toxin production

Intracellular persistence

• f staphylococci

ATP

Impaired electron transport

• CO2
• 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

Various and undetected mechanisms for SCVs occurence

mechanisms:

• hemin- or menadione-dependent (Proctor, von Eiff, Becker,

McNamara, Peters, Lannergard AAC2008; Malouin J Bacteriol, 2006)

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

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 m a r k e r 6 / 1 9 9 4 n

• r

m a l 9 / 1 9 9 5 S C V 9 / 1 9 9 5 n

• r

m a l 6 / 1 9 9 6 S C V 6 / 1 9 9 6 n

• r

m a l 1 / 1 9 9 7 n

• r

m a l 1 / 1 9 9 7 S C V 5 / 1 9 9 8 S C V 1 / 1 9 9 9 S C V 1 / 2 S C V m a r k e r

65 months persistence

Longterm persistence of 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  survive only in the presence of thymidine  in many patients present even when no normal S. aureus was cultured  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 Schaedler agar CO2

Decreased tanscription of agr and hla in clinical thymidine-dependent (TD) SCVs

Kahl BC et al, 2005, Infect Immun 73:4119-26

 less virulent phenotype specialized for persistence

agr spa sarA hla

Normal S. aureus SCV SCV + thymidine EL LL SP EL LL SP EL LL SP

P2 P3 P1

low thymidine high thymidine low thymidine

 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

TD-SCVs occur not only in CF, but also in other infections and in other species

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

thymidine

dTMP

thyA e.g. component of DNA extracellular thymidine nupC

N5, N10- Methylen- THF DHF THF PABA + DHpteroate DP extracellular intracellular

destroyed cells and pus from respiratory secretions in CF lung with

Model for thymidine-dependency of S. aureus SCVs

dUMP

X

TMP

X

SMX

thymidylate synthase nucleosid transporter

SCV expressing thy Cured mutant Thymidine-dependent SCV Normal S. aureus

Thymidine-dependent SCV expressing thy exhibits normal phenotype

Chatterjee I, J Bacteriol 2008; 190:834-42

pCX19thyA

4906 bps

PstI StuI ClaI ScaI I Sca I Nde RV Eco I Cla HI Bam I Nde RV Ec

• I

Sma cat xylR thyA 'lip

Increased transcription

• fthyA and nupC in TD-SCVs

Chatterjee I, J Bacteriol 2008; 190:834-42

• unexpected: increased transcription of thyA
• expected: increased transcription of nupC

normal TD-SCV TD-SCVpCX19thyA TD-SCVpCX19thyA+Xyl

0.05 0.10 0.15 0.20 0.25 0.30 0.35

Relative Gene Expression

(EL) (LL) (SP)

normal TD-SCV TD-SCV expressing thyA

thyA

normal TD-SCV TD-SCVpCX19thyA TD-SCVpCX19thyA+Xyl

0.02 0.04 0.06 0.08 0.10

Relative Gene Expression

(EL) (LL) (SP)

B.

* *

normal TD-SCV TD-SCV expressing thyA

nupC

* *

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.

Acknowledgements

Münster Cathrin Baum Andre Kriegeskorte Marco Kelkenberg Claudia Neumann Simone Brüning Barbara Ritzerfeld Susanne Deiwick Katrin Wardecki Marion Wallstein Nadine Theimann Karsten Becker Georg Peters Homburg Mathias Herrmann Indranil Chatterjee Tübingen Christiane Wolz Christiane Görke Frankfurt Silke Besier Thomas Wichelhaus Ulm Barbara Spellerberg Nele Wellinghausen Dänemark Henrik Westh Kit Boye USA Richard Proctor Ambrose Cheung Jean Lee Evgeni Sokurenko Bo Shopsin Belgium Francoise van Bambeke

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