Barbara C. KahlInstitut für Medizinische

MikrobiologieUniversitätsklinikum 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

osteomyelitis endocarditis

pneumoniasepsis

Skin-

and soft tissue infections

carbuncle bursitis

furuncle

Life-threatening infections

S. aureus

colonization and infections

adhesinsearly log phase

secreted proteinslate log/stationary phase

enterotoxinTSST-1-toxin

coagulase

proteinA

fibronectin-bindingprotein

collagen-bindingprotein

clumping factor

elastin-bindingprotein

cell membranepeptidoglycan (cell wall)

micro capsule

Virulence factors of S. aureusWa c hst umsk ur v e

0

2

4

6

8

10

0 1 2 3 4 5 6 7

Zei t i n h

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. aureusSCV

normalS. 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

F0F1ATPaseHemin

FADH 2

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

Antibioticsusceptibility

Pigmented colonies

Menadione

Thiamine PP

Shikimate

Isoprenoid lipid

Menadione biosynthetic enzymes

Hemin biosynthetic enzymes

Negative regulators for toxin production

Intracellular persistence of 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

mar

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6/19

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1996

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CV

6/19

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97

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V5/

1998

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CV

10/1

999

SC

V1/

2000

S

CV

mar

k er

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

Schaedleragar

CO2

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

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

less virulent phenotypespecialized for persistence

agr

spa

sarA

hla

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

P2P3

P1

low thymidine high thymidinelow 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 density2.

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/lPus

nd

18,19 µg/lUrine

540 µg/l 1,818 µg/lLiquor

nd

375 µg/l*Besier S et al J Clin Microbiol 2008;46:3829

thymidine

dTMP

thyA

e.g. component of DNA

extracellular thymidinenupC

N5, N10-

Methylen-THF

DHF

THFPABA +DHpteroate DP

extracellularintracellular

destroyed cells

and pus

from respiratory secretionsin 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 SCVNormal S. aureus

Thymidine-dependent SCV expressing thy exhibits normal phenotype

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

pCX19thyA4906 bps

PstIStuI

Cla I

ScaIIScaINde

RVEcoICla

HIBamINde

RVEc o

ISma

cat

xylR

thyA

'lip

Increased transcription ofthyA 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

Rel

ativ

e G

ene

Expr

essio

n

(EL) (LL) (SP)

normal TD-SCV TD-SCVexpressing thyA

thyA

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

0.02

0.04

0.06

0.08

0.10

Rel

ativ

e G

ene

Exp

ress

ion

(EL) (LL) (SP)B. *

*

normal TD-SCV TD-SCVexpressing 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.

AcknowledgementsMünsterCathrin BaumAndre KriegeskorteMarco KelkenbergClaudia NeumannSimone BrüningBarbara RitzerfeldSusanne DeiwickKatrin WardeckiMarion WallsteinNadine TheimannKarsten BeckerGeorg Peters

HomburgMathias HerrmannIndranil Chatterjee

TübingenChristiane WolzChristiane Görke

FrankfurtSilke BesierThomas Wichelhaus

UlmBarbara SpellerbergNele Wellinghausen

DänemarkHenrik WesthKit Boye

USARichard ProctorAmbrose CheungJean LeeEvgeni SokurenkoBo Shopsin

BelgiumFrancoise van Bambeke

Greetings from Münster