Funktionelle Charakterisierung neuer Virulenzfaktoren von Staphylococcus aureus [Elektronische Ressource] = Funktionelle Charakterisierung neuer Virulenzfaktoren von Staphylococcus aureus / vorgelegt von Dorothee Grumann

De
Funktionelle Charakterisierung neuer Virulenzfakteonr von Staphylococcus aureus (Functional chracterization of new virulence sf actorof Staphylococcus aureus) I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer.nat.) an der Mathematisch-Naturwissenschaftlichen Fakutl täder Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Dorothee Grumann geboren am 03.01.1980 in Fürth Greifswald, 11.03.2010                     Dekan:    Prof. Dr. Klaus Fesser  1. Gutachter:    Prof. Dr. Barbara M. Bröker 2. Gutachter:    Prof. Dr. Alex F. van Belkum  Tag der Promotion:  09. Juli 2010   CONTENTS PART I NTIRODUCTION Chapter 1 Staphylococcus aureus in disease and in health………………………….........1. ..........1.1 S. aureus – human pathogen and harmless commensal………………………2 1.2 S. aureus comparative genomics……………………………………………………….4 1.3 What determines staphylococcal virulence?............................................6 1.4 Staphylococcal superantigens…………………………………………………………..7 1.5 Outline of the thesis……………………………………………………………………… .14 PART II ESURLTS Chapter 2 Clonal distribution of superantigen ginen ecslinicSatla phylococcus aureus isolates. S. Holtfreter, D. Grumann, M. Schmudde,u yHe.n , TP.. TE.i chNlegr, B. Strommenger, K. Kopron, J. Kolata, S. Gbiead,r ysI-K.a leSmteinmetz, W. Witte, and B. M. Bröker. 2007. J Clin2 66M9i-c2r6o8bi0o…l …4…5…:…....
Publié le : vendredi 1 janvier 2010
Lecture(s) : 27
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Source : D-NB.INFO/1010483617/34
Nombre de pages : 156
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Funktionelle Charakterisierung neuer Virulenzfakteonr
von Staphylococcus aureus

(Functional chracterization of new virulence sf actor
of Staphylococcus aureus)



I n a u g u r a l d i s s e r t a t i o n
zur
Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer.nat.)
an der Mathematisch-Naturwissenschaftlichen Fakutl tä
der
Ernst-Moritz-Arndt-Universität Greifswald

vorgelegt von
Dorothee Grumann
geboren am 03.01.1980
in Fürth

Greifswald, 11.03.2010
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dekan:    Prof. Dr. Klaus Fesser 
 
1. Gutachter:    Prof. Dr. Barbara M. Bröker 
2. Gutachter:    Prof. Dr. Alex F. van Belkum 
 
Tag der Promotion:  09. Juli 2010 
 
CONTENTS

PART I NTIRODUCTION
Chapter 1 Staphylococcus aureus in disease and in health………………………….........1. ..........
1.1 S. aureus – human pathogen and harmless commensal………………………2
1.2 S. aureus comparative genomics……………………………………………………….4
1.3 What determines staphylococcal virulence?............................................6
1.4 Staphylococcal superantigens…………………………………………………………..7
1.5 Outline of the thesis……………………………………………………………………… .14

PART II ESURLTS
Chapter 2 Clonal distribution of superantigen ginen ecslinicSatla phylococcus
aureus isolates.
S. Holtfreter, D. Grumann, M. Schmudde,u yHe.n , TP.. TE.i chNlegr, B.
Strommenger, K. Kopron, J. Kolata, S. Gbiead,r ysI-K.a leSmteinmetz, W.
Witte, and B. M. Bröker. 2007. J Clin2 66M9i-c2r6o8bi0o…l …4…5…:…....24

Chapter 3 Diversity of prophages in domStianpahnytl ococcus aureus clonal lineages.
C. Goerke, R. Pantucek, S. Holtfreter, MB.. ZSinchku,l teD,. Grumann, B. M.
Bröker, J. Doskar, and C. Wolz. 2009.9 1:J3 4B6a2c-3te4r6io8l …1…………39

Chapter 4 Recurrent furunculosis - associatiohn s Pwaniton-Valentine leukocidin
and the genetic backgroundSt aopfh ylococcus aureus .
H. Masiuk, K. Kopron, D. Grumann, C. aGtoa,er kJe., JJu.r saK-oKlulesza, S.
Giedrys-Kalemba, B. M. Bröker, and S. H0o1lt0fr.e teirn. p2ress……………47

Chapter 5 Anti-staphylococcal humoral immune rsees poin persistent nasal carriers
and noncarriers Sotfa phylococcus aureus.
iii
N. J. Verkaik, C. P. de Vogel, H. uAm. anBno,e leTn.s, HDo.o geGnrboezem,
C. Vink, H. Hooijkaas, T. J. Foster, H,. AA.. vVaenr bBrueglkhum, and W. J.
van Wamel. 2009. J I n1f9e9c:t6 2D5i-s632…………………………………………..69

Chapter 6 Neutralizing antibody responsSet apthoy lococcus aureus superantigens in
bacteremic patients.
D. Grumann, E. Ruotsalainen, J. Kolata, AP. JKäuruvsinelean,, V. P.
Kontinen, B. M. Bröker, and S. Holtfretbemr.it te2d0…1…0….… …su............78

Chapter 7 Immune cell activation by enterotoex incl usgtenr eg(c)-encoded and
non-egc superantigens froSmta phylococcus aureus.
D. Grumann, S. S. Scharf, S. Holtfrete r,L . C.S teKilo,h leSr., Engelmann, M.
Hecker, U. Völker, and B. M. Bröker. 20l 1081.: 50J 54I-m50m6u1n…o…96

PART III UMSMARY
Chapter 8 Summary and discussion…………………………………………………………...116
8.1 What determines staphylococcal virulence?u laMr-eopleidcemiology
of colonizing and invasSi.v ea ureus isolates……………………………….…117
8.2 Anti-staphylococcal antibody response inz atciolno niand
bacteremia………………………………………………………………………………..…122
8.3 Comparison egocf and noeng-c superantigens – What is the reason
for the lack of eagnc ti-antibodies?....................................................................125

PART IV PAPENDIX
Summary…………………………………………………………………………………………..135
Zusammenfassung……………………………………………………………………………..138
Abbreviations……………………………………………………………………………………142
Publications………………………………………………………………………………………145
iv
Conferences……………………………………………………………………………………....146
Acknowledgments/ Danksagung………………………………………………….…….418
Eidesstattliche Erklärung…………………………………………………………………..150
Curriculum vitae……………………………………………………………………………..…15 1


v

Chapter 1

Staphylococcus aureus in disease and in health


1 STAPHYLOCOCCUS AUREUS IN DISEASE AND IN HEAL TH

Staphylococcus aureus in disease and in health
Staphylococcus (S.) aureu is an opportunistic pathogen and a leading cause o f bacterial
infections worldwide. The spread of antibiotic resi stant strains in hospitals as well as in
the healthy population is of growing concern. Moreo ver, up until now, no anti- S. aureus
vaccine has been approved for medical practice (1).

1.1S . AUREUS – HUMAN PATHOGEN AND HARMLESS COMMENS AL
Clinical impact of S. aureus
S. aureus is a major human pathogen capable of causing a wid e spectrum of infections,
from relatively mild skin infections such as follic ulitis and furunculosis to life-
threatening diseases, including sepsis, pneumonia,os teomyelitis, and infective
endocarditis (2). Infections caused by this pathogne have increased over the past 25
years (2, 3S).. a ureus (methicillin-sensitive as well as methicillin-resistant S. aureus;
MSSA and MRSA, respectively) ranks as the most comm on cause of nosocomial
bloodstream infections and leads to increased morbi dity, mortality, length of hospital
stay, and costs (4).
The treatment of such infections is complicated by the ability of this species to become
resistant to antibiotics (5). MRSA strains are wide spread in nosocomial environments
(hospital-associated MRSA; HA-MRSA), and account for > 60% ofS . aureus isolates in US
intensive care units (6). Of growing concern is the emergence and spread of highly
pathogenic MRSA strains in the community outside th e hospital setting (community-
associated MRSA; CA-MRSA), especially the clone USA300 (7, 8). These strains cause
serious infections in otherwise healthy individuals (7, 9). Vancomycin is the drug of
choice for therapy of infections due to MRSA, but i ncrease in vancomycin use has led to
the emergence of vancomycin intermediate-resistent (VISA) or resistant strains (VRSA)
(10). As a consequence of increasing antibiotic resistance non antimicrobial approaches
to control S. aureus are needed. However, due to the complex interactio n between
S. aureus and the immune system of its host, vaccine develop ment is still a challenging
task.

2 STAPHYLOCOCCUS AUREUS IN DISEASE AND IN HEAL TH

S. aureus nasal carriage patterns
In apparent contrast with its infectious potential, S. aureus is also a frequent commensal
that colonizes the skin and mucosal surfaces of hum ans and several animal species (11).
The primary ecological niche of S. aureus are the anterior nares (11, 12). However,
multiple body sites can be colonized including the skin, perineum, pharynx, and less
frequently the gastrointestinal tract, vagina, and axillae (13). Longitudinal studies
distinguish three nasal carriage patterns in health y individuals: about 20% (range 12 -
30%) are persistent carriers, approximately 30% (rnage 16 - 70%) are intermittent
carriers, and about 50% (range 16 - 69%) are noncraierrs (13). While persistent
carriers are usually colonized by a single strain o f S. aureus, intermittent carriers
commonly carry different strains over time (14, 15). Cross-sectional studies yield a
prevalence of ~ 35% carriers in the healthy population, which is ac tually a mix of
persistent and intermittent carriers at the time of investigation (16, 17).
It is clinically relevant to distinguish between pe rsistent and intermittent carriers.
Persistent carriers have higher S. aureus loads, resulting in increased dispersal and a
higher risk of infection in comparison to intermitt ent carriers and noncarriers (13, 18-
21). Intermittent carriers and noncarriers show similar infection risks.
Recently van Belkum et al. suggested a reclassifica tion of nasal carriage types (21). An
artificial colonization study where volunteers were inoculated with a mixture of
S. aureus strains showed that intermittent carriers and nonc arriers quickly eliminated
the inoculated S. aureus strains, while persistent carriers preferentially r eselected their
original resident strain from the inoculum mixture (21, 22). Furthermore, the antibody
levels against 17 S. aureus antigens were equal in intermittent carriers and n oncarriers
but higher in persistent carriers. Along with the p reviously described low risk of
infection of intermittent carriers and noncarriers, the authors suggest to distinguish just
two types of nasal carriers: persistent carriers an d others.

What determines S. aureus nasal carriage?
Mechanisms leading to S. aureus nasal carriage appear to be multifactorial and are still
not fully understood. Bacterial factors (e.g. stapyhlococcal toxins and cell wall-
associated proteins) (2, 23), environmental factor (es.g. hospitalization and crowding)
(24, 25) as well as host susceptibility factors (ge.. immune suppression or other serious
3 STAPHYLOCOCCUS AUREUS IN DISEASE AND IN HEAL TH

underlying diseases) play important roles (11, 24 T).he results of an artificial
colonization study (see above) indicated that host factors are probably the major
determinants of the S. aureus carrier status and an optimal fit between host and bacteria
seems to be essential for long-term colonization (2 2).

S. aureus nasal carriage is a major risk factor for infectio n
Carriage of S. aureus has been identified as a risk factor for the devel opment of infection
in various settings (11, 16, 26-28). Importanostlyt, of m these S. aureus infections are
caused by the patients´ own flora. Von Eiff et ald.e monstrated in a prospective study
that nasal strains and subsequent bacteremic strain s have the same genotype in more
than 80% of the cases (29). Similarly, hemodialysis patients and chronic ambulatory
peritoneal dialysis patients have increased infecti on rates of mostly endogenous origin
(16, 30).
Wertheim and coworkers investigated the incidence o f bacteremia in carriers as well as
noncarriers in a non-surgical patient population (n = 14,008) (31). Nasal carriage
increased the risk of nosocomial infection by a fac tor of three. Surprisingly, the mortality
rate from S. aureus bacteremia was four times higher in noncarriers th an in carriers (8%
vs. 32%P, = 0 .006) (31). An explanation for this observationh as not yet been provided,
although a role for the immune system has been prop osed (32).
Moreover, several studies have demonstrated that er adication of S. aureus from nasal
and other body sites with the anti-staphylococcal d rug mupirocin effectively prevents
S. aureus infections (15, 33-37).

1.2 S. AUREUS COMPARATIVE GENOMIC S
Currently, 14 annotated whole genome sequences of S. aureus are available. A
comparison of these sequences and whole genome micr oarray analyses revealed that
the S. aureus genome consists of a core genome (~ 75%), a core va riable genome
(~ 10%) and mobile genetic elements (MGEs, ~ 15%) (38, 39).
The core genome is highly conserved concerning gene order and gene sequence and
comprises house keeping genes, which are essential for growth and survival (5). Allelic
variations in the core genome are exploited by sequ ence-based genotyping methods, like
4 STAPHYLOCOCCUS AUREUS IN DISEASE AND IN HEAL TH

multilocus sequence typing (MLST) (40) and protein A (spa) genotyping (41). MLST
determines the phylogenetic relationship of bacteri al strains based on the sequence
fragments of seven housekeeping genes. In contrast, spa genotyping compares the
variable repeat region of the protein A gene ( spa), which differs in sequence and number
of spa repeats. As pa type is assigned to each unique repeat pattern, an d closely related
spa types are referred to as clonal complexes (CCs). S everal studies demonstrated that
pulsed-field gel electrophoresis (PFGE), MLST and spa genotyping provide largely
concordant results, however, only sequence-based ty ping methods can be easily
compared between laboratories (41, 42). The genotiynpg analyses revealed that
S. aureus has a highly clonal population structure, dominate d by ten prevalent clonal
lineages (43-45).
The core variable genome includes most surface-asso ciated genes (MSCRAMMs) and
regulator genes. Core variable genes are encoded on the bacterial chromosome and are,
therefore, typically stable and transferred vertica lly (39). Lindsay et al. demonstrated
that each staphylococcal lineage carries a unique c ombination of core variable genes
(39).
MGEs include bacteriophages, pathogenicity islands, plasmids, transposons, and
staphylococcal chromosomal cassettes (SCC) (38, 46.) They mainly encode resistance
and virulence genes (e.g., Panton-Valentine leukdocini (PVL) genes, superantigen (SAg)
genes). MGEs can be distributed either by vertical transmission to daughter cells or by
horizontal transfer by bacteriophages or conjugatio n (38). Microarray analyses revealed
substantial variation in the distribution of MGEs w ithin lineages, suggesting frequent
horizontal transfer or loss of MGEs (47). However, there is evidence that some MGEs are
distributed within certain lineages at higher frequ ency than between lineages,
suggesting the existence of some barriers in horizo ntal transfer of MGEs (39). The
recently described Sau1 type I restriction-modification system, which recognizes and
digests foreign DNA may serve as an example (48). The accumulation of such MGEs may
result in the emergence of “superbugs” that are inc reasingly antibiotic resistant and of
higher virulence (5).
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