Staphylococcus aureus and

Barbara C. Kahl Institut für Medizinische

Mikrobiologie Universitätsklinikum Münster

Staphylococcus aureus

and cystic fibrosis

ESCMID Online Lecture Library

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v  most common hereditary disease in the caucasian population

v  1 in 2.900 newborns, app. 8.000 patients in Germany

v  mutation in the cystic fibrosis transmembrane regulator gene (cftr)

v  more than 1.500 mutations known; more than 80% ∆F508

v  CFTR is expressed in epithelial cells of the respiratory, digestive

and reproductive tract

v  multiple organs affected: pancreas, liver, bones, ductus deferens

v  most clinical sequelae: chronic suppurative airway infections, lung-

insufficiency and early death

Cystic fibrosis


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Development of CF lung disease

Clunes, MT and Boucher, RC. Drug Discov Today Dis Mech. 2007

Ø  thick and sticky mucus and impaired mucociliary clearance

Ø  frequent lung infection with specific CF-related pathogens

v  reduced Cl- permeability

v  reduced Cl- secretion

v  reduced water entering the airways

v  ENaC activity

v  inappropriate absorption of salt

v  mucus layer dehydrated

v  arrest of mucociliary clearance


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Age specific prevalence of pathogens

CF Patient Registry Annual Data Report- 2012, USA Data of more than 27.000 CF patients


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Prevalence of pathogens in the airways of CF-patients

CF Patient Registry Annual Data Report- 2013, USA Data of more than 28.000 CF patients


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S. aureus in CF patients- European data

Data from the European Cystic Fibrosis Society, Patient Registry 2013, published 2016 Zolin A, Mkone EF, van Rens J et al.


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CF patients with persistent S. aureus

Persistent S. aureus infection reported from 22 European countries from 21.848 patients 38% (mean) of CF patients Ranging from 53% Switzerland (/642 patients) 50% Russian Federation (/1818 patients) 39% The Netherlands (/1289 patients) 17% United Kingdom (/8801 patients)

u Persistent S. aureus infection as a prerequisite for the emergence of SCVs

Data from the European Cystic Fibrosis Society Patient Registry 2013, published 2016 Zolin A, Mkone EF, van Rens J et al.


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•  Observation period: 151 months •  Prevalence: 132/177 (75%) patients S. aureus •  Persistence: 64 m (median), range 8 – 147 m

associated with small colony variants

High prevalence and persistence of S. aureus

in airways of CF patients

Kahl, B. C. et al., J Infect Dis 1998; J. Clin. Microbiol. 2003, 41:4424-4427 and unpublished data

Gram staining of a sputum from a CF patient

PMNs

S. aureus

mar

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10/1

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1995

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a 12

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5 n

10/1

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10/1

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9/19

97 n

9/

1997

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What are small colony variants? •  Identified in many species

–  P. aeruginosa, B. cepacia, E. coli, Salmonella, Enterococci...

•  Phenotypic description of smaller bacterial colonies cultured on culture plates

SCV normal

S. aureus SCV-characteristics

Ø  Smaller size Ø  Less pigmentation Ø  Less hemolysis Ø  Can revert to normal Ø  Persist intracellularly

v. Eiff et al., Clin. Infect. Dis. 2001;32:1643-7

S. aureus SCV

normal

S. aureus

30 min 48 h


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SCVs on primary plates - easily overlooked

modified from: Kahl BC et al. Clin Microb Rev 2016

laboratories should be aware of SCVs and should actively look for them ESCMID Online Lectu

re Library

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SCVs – different types from clinical infections

•  menadione and hemin-auxotroph S. aureus SCVs electrontransport-deficient SCVs, selected by gentamicin (Proctor et al., Nat. Microb. Med. 2006) –  in vitro enhancedsurvival during co-infection with P. aeruginosa induced

by secreted products of P. aeruginosa (Hoffman et al., PNAS 2006; Biswas et al, Appl. Environ. Microb. 2009)

–  Mostly in patients with chronic osteomyelitis (v. Eiff et al. Clin Inf Dis 97)

•  thymidine-dependent SCVs only survive if extracellular thymidine available, induced by trimethoprim-sulfamethoxazol (TMP/SMX) (Gilligan J Clin Microbiol 1987; Kahl et al. J Infect Dis 1998)) –  CF-patients with persistent S. aureus airway infection

•  CO2-dependent SCVs small colonies on culture plates under normal conditions, normal seized colonies under CO2 (Hale JH Brit J Exp Pathol 1951) –  different infections


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How to determine the underlying mechanism of SCVs?

CDM Agar

normal S. aureus thymidine-dependent SCV

Columbia blood agar

Schaedler agar

thymidine-dependent SCVs under CO2

revertants 27 thymidine 122 hemin 27 thymidin/hemin 26 menadione 1 unknown 36 239

Auxotrophism


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Studies identifying SCVs in CF

all 2,441 291 (12%)

# Investigated patients

#patients with

SCVs (%)

Study period (months)

Auxotrophism

Reference

1 14 7 (50) 7 thy Sparham et al., 1978, UK 2 200 20 (10) 12 thy Gilligan et al., 1987, USA 3 78 26 (33) 34 thy, hem, men

thy+hem Kahl et al., 1998, Ger

4 627 25 (4) 7 n.d.6 Vergison et al., 2007, Belgium 5 252 20 (8) 12 thy, thy+men

not ident. Besier et al., 2007, Ger

6 98 8 (8) 3 n.d. Schneider et al., 2008, Switz. 7 260 17 (6) 6 n.d. Green et al., 2011, USA 8 248 20 (8) 11 thy, thy+hem

not ident. Yagci et al., 2013, Turkey

9 100 24 (24) 24 thy, hem, men,CO2 Wolter et al., 2013, USA 10 222 28 (13) no. inf. thy Morelli et al., 2015, Italy 11 12

147 195

12 (8%) 84 (43%)

36 21

thy, hem thy

Masoud-Landgraf et al. 2016, Öst. Junge et al., 2016, under review

modified from Kahl BC et al. Clin Microb Rev 2016

thymidine-dependent SCVs most common in CF patients


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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

TMP SMX

thymidylate synthase

nucleosid transporter


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Characteristics of patients with SCVs

•  Patients with S. aureus SCVs

–  Older –  More advanced lung disease –  More often treated with antibiotics –  More often co-infected with P. aeruginosa

Besier et al. J Clin Microbiol 2007 Schneider et al. 2008 Wolter et al. Clin. Infect. Dis. 2014


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Observational prospective study

•  prospective (21 months) multicenter observational study –  195 CF patients from –  16 centers in Germany and 1 center in Austria

with persistent S. aureus infection

Junge et al., under review


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Patients with SCVs

–  84 (43%) patients with SCVs

• older (p=0.0066) • lower FEV1% predicted at

baseline (p=0.0133) and during the study period (p=0.0337)

• were more likely treated with TMP/SMX (p=0.0078)

Blue circles: never SCVs Red squares: ever SCVs Generalized linear mixed model

Junge et al., under review


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Susceptibility testing of SCVs

•  no growth on Mueller-Hinton agar •  testing on blood agar

Kriegeskorte et al. mBio 2014

Optimized susceptibility testing on MH-agar supplemented with thymidine Precit M et al. J A Chem 2016

ref.strain on MH ref. strain on MH suppl TD-SCV on MH suppl


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Strain Alteration in nucleotide sequence

Predicted result

Normal-12

SCV-12 Δ3 bp deletion AAT 247-249 In frame deletion; unclear function of the protein

Normal-22

SCV-22 Δ3 bp deletion AAT 247-249 In frame deletion; unclear function of the protein

Normal-31 T76C Cys⇒Arg (aa26)

SCV-31 C139T; G337A; A493G; ΔA565 Leu⇒Por (aa47); Gly⇒Ser (aa113); Asn⇒Asp (aa165); 1 bp deletion⇒stop-mutation (aa188)

Normal-42 T730G; G835A Ser⇒Ala (aa244); Gly⇒Ser (aa279)

SCV-42 T82A; Δ11bp deletion CTTCCGCCTTG

11 bp deletion⇒frameshift mutation⇒nonsense protein

Normal-51 G308A; C838T Gly⇒Asp (aa103); Leu⇒Phe (aa280)

SCV-5 Δ51 1 bp deletion⇒frameshift mutation⇒stop mutation at aa19

Normal-61 C383T; A482G; G886A Ser⇒Phe (aa128); Asp⇒Gly (aa161); Val⇒Ile (aa296)

SCV-61 G359A; ΔG743 Arg⇒Gln (aa120); 1 bp deletion frameshift mutation leading to stop mutation at aa251

SCV-7-11 T7A; C521T; G806A; ΔA893 Tyr⇒Asn (aa3); Tyr⇒Ile (aa174); Gly⇒Asp (269); 1 bp deletion⇒frameshift mutation ⇒stop mutation at aa311

SCV-7-21 T7A; T616C Tyr⇒Asn (aa3); pointmutation⇒ stop mutation at aa206

SCV-8-1 ΔT121 1 bp deletion⇒frameshift mutation⇒ stop mutation at aa50

SCV-8-21 Δ7bp deletion ACATCGT 7 bp deletion⇒frameshift mutation⇒stop mutation at aa212

Mutations of thyA in 10 clinical TD-SCVs and S. aureus WT

Mutations # SCVs

in frame del 2

deletions resulting in stop

mutation

6

pointmutations resulting in stop

mutation

1

Deletion resulting in frameshift mutation

1

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

Mutations # WT

none 2

SNPs 4

1 SNP 1

2 SNPs 2

3 SNPs 1


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thyA-SCV

on blood agar

With SXT

Gram staining

A B C

1 2 3

WT complemented mutant

A thyA deletion mutant exhibits a SCV phenotype

Kriegeskorte et al, mBio 2014


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Competition of WT and mutant in a chronic mouse pneumonia model

WT M WT M

untreated treated

untreated treated

Kriegeskorte et al. JAC2015


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Summary

•  S. aureus –  is highly prevalent in the airways of CF patients –  is long-term persisting there

•  thymidine-dependent SCVs –  are often recovered from the airways of CF patients

–  display an attenuated phenotype –  are isolated from older patients with more advanced disease –  are induced and selected by TMP/SMX –  are caused by mutations in thyA –  a thyA-deletion mutant displays the phenotype of clinical TD-

SCVs –  the mutant is less effected by TMP/SMX treatment in a chronic

mouse pneumonia model than the WT


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Acknowledgements •  Janina Treffon •  Desiree Block •  Nina Hirschhausen •  Andre Kriegeskorte •  Angelika Dübbers •  Peter Küster •  Jörg Große-Onnebrink •  Holger Schültingkemper •  Susanne Deiwick •  Barbara Ritzerfeld •  Karsten Becker •  Georg Peters

Cooperations with •  Susanne Engelmann •  Alessandra Bragonzi, Milano, It •  Francoise van Bambeke, Brussels, BE •  Pavel Drevinek, Prague, CZ •  Willem van Wamel, NL •  Martijn den Reijer, NL


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Staphylococcus aureus and

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