Université Libre de Bruxelles Faculté de Médecine

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Université Libre de Bruxelles

Faculté de Médecine

Laboratoire de Bactériologie, Service de Microbiologie Hôpital Erasme

Contribution to the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA)

in Belgian populations

Docteur Olivier Denis

Dissertation présentée en vue de l’obtention du Grade académique de Docteur en Sciences Médicales

Promoteur : Professeur Marc Struelens

Année académique 2009 – 2010

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Composition du jury de thèse

Prof P.A. Gevenois – Service de Radiologie, Hôpital Erasme, Université Libre de Bruxelles (Président)

Prof M.J. Struelens – Service de Microbiologie, Hôpital Erasme, Université Libre de Bruxelles (Secrétaire)

Prof. A. Allaoui – Laboratoire de Bactériologie Moléculaire, Faculté de Médecine, Université Libre de Bruxelles

Prof. B. Byl - Clinique d'Epidémiologie et d'Hygiène Hospitalière, Hôpital Erasme, Université Libre de Bruxelles

Prof. O. Vandenberg – Service de Microbiologie, Centre Universitaire Saint-Pierre, Université Libre de Bruxelles

Experts extérieurs

Prof. Y. Glupczynski – Service de Microbiologie, Clinique Universitaire de Mont-Godinne, UCL

Prof. P. Tulkens - Unité de Pharmacologie Cellulaire et Moléculaire & Louvain Drug Research Institute, Université Catholique de Louvain

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Table of contents

Composition du jury de thèse 2

Remerciements 7

Résumé 8

List of abbreviations 10

Chapter 1: Introduction 11

1.1. Microbiology of Staphylococcus aureus 12

1.1.1. Taxonomy 12

1.1.2. Genome composition 12

1.1.2.1. Core genome 12

1.1.2.2. Accessory genome 13

1.1.2.2.1. Bacteriophages 13

1.1.2.2.2. Staphylococcal aureus pathogenicity islands (SaPIs) 13

1.1.2.2.3. Plasmids 13

1.1.2.2.4. Transposons 14

1.1.2.2.5. Staphylococcal cassette chromosome (SCC) 14

1.1.2.2.6. Genomic islands 14

1.1.3. Virulence factors 15

1.1.3.1. Capsule 15

1.1.3.2. Surface proteins 15

1.1.3.3. Enzymes 15

1.1.3.4. Exotoxins 15

1.1.3.4.1. Panton-Valentine Leucocidin 15

1.1.3.4.2. Exfoliative toxins 16

1.1.3.4.3. Superantigens 16

1.1.3.4.4. Other membrane active agents 17 1.1.3.5. Arginine catabolic mobile element 17 1.1.3.6. Regulation of staphylococcal virulence 17

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1.1.3.6.1. Regulators of virulence 17

1.1.3.6.2. The agr system 17

1.1.4. Antimicrobial resistance determinants 18

1.1.4.1. Beta-lactams 18

1.1.4.1.1. Penicillinase 18

1.1.4.1.2. Penicillin binding protein 2a 18

1.1.4.2. Glycopeptides 18

1.1.4.2.1. Vancomycin resistant S. aureus (VRSA) 19 1.1.4.2.2. Vancomycin intermediate S. aureus (VISA) 19

1.1.4.3. Fluoroquinolones 20

1.1.4.3.1. Target modification 20

1.1.4.3.2. Active efflux 20

1.1.4.4. Macrolides, lincosamides and streptogramins 21

1.1.4.4.1. Target modification 21

1.1.4.4.2. Active efflux 22

1.1.4.4.3. Inactivating enzymes 22

1.1.4.5. Aminoglycosides 22

1.1.4.6. Tetracyclines 23

1.1.4.6.1. Efflux systems 23

1.1.4.6.2. Ribosomal protection 23

1.1.4.7. Mupirocin 23

1.1.4.8. Rifampicin 24

1.1.4.9. Fusidic acid 24

1.1.4.10. Oxazolidinones 24

1.1.4.11. Sulfonamides and trimethoprim 25

1.1.4.12. Daptomycin 25

1.1.5. Natural habitat 26

1.1.6. Culture, identification and antibiotic susceptibility testing 26

1.1.7. Molecular detection 27

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1.1.8. Typing methods 27

1.1.8.1. Pulsed-field gel electrophoresis 27

1.1.8.2. Multi-locus sequence typing 28

1.1.8.3. spa typing 28

1.1.8.4. PCR analysis of Inter-IS256 spacer length polymorphisms 29

1.1.8.5. SCCmec typing 29

1.2. Clinical manifestations of S. aureus infections 30

1.2.1. Pyogenic infections 30

1.2.2. Toxin mediated diseases 31

1.3. Epidemiology and infection control 31

1.3.1. Healthcare-associated MRSA (HA-MRSA) infections 31

1.3.2. Community-associated MRSA infections 33

1.3.3. Livestock-associated (LA-) MRSA infections 34

1.3.4. Control of MRSA transmission 34

1.4. Antimicrobial therapy of MRSA infections 35

Chapter 2: General objectives 36

Chapter 3: Material and methods 38

3.1. Study populations 39

3.1.1. Hospital care sector 39

3.1.2. Long term care sector 39

3.1.3. Outpatients 39

3.1.4. Swine farmers 39

3.2. Laboratory methods 40

3.2.1. S. aureus screening 40

3.2.2. S. aureus identification 40

3.2.3. Molecular typing 40

3.2.4. Antimicrobial susceptibility testing 40

3.2.5. Toxin gene detection 40

Chapter 4: Emergence and spread of gentamicin-susceptible strains of methicillin-

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resistant Staphylococcus aureus in Belgian hospitals 41 Chapter 5: Molecular epidemiology of resistance to macrolides-lincosamides-

streptogramins in methicillin-resistant Staphylococcus aureus (MRSA) causing bloodstream infections in patients admitted to Belgian hospitals 53 Chapter 6: National surveillance of methicillin-resistant Staphylococcus aureus (MRSA) in Belgian hospitals indicates rapid diversification of epidemic clones 57 Chapter 7: Low prevalence of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in Belgian hospitals 63 Chapter 8: In-vitro activity of ceftobiprole, tigecycline, daptomycin and 19 other antimicrobials against methicillin-resistant Staphylococcus aureus (MRSA) strains

from a national survey of Belgian hospitals. 71

Chapter 9: Genetic relatedness between methicillin-susceptible and methicillin- resistant Staphylococcus aureus: results of a national survey. 78 Chapter 10: Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA)

among residents of nursing homes in Belgium 87

Chapter 11: Polyclonal emergence and importation of community-acquired

methicillin-resistant Staphylococcus aureus strains harbouring Panton-Valentine

leukocidin genes in Belgium 96

Chapter 12: Methicillin-resistant Staphylococcus aureus ST398 in swine farm

personnel, Belgium 101

Chapter 13: Discussion 106

Chapter 14: Conclusions and perspectives 111

References 115

Web-resources 139

Appendix 142

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Remerciements

Ce travail est le fruit de plus de 10 années passées dans le laboratoire de microbiologie de l’hôpital Erasme.

Mes remerciements vont tout particulièrement à ceux qui m’ont aidé à l’aboutissement de cette thèse :

A mon promoteur, Marc Struelens, pour m’avoir permis de mener à bien ce travail, m’avoir suivi toutes ces années, m’avoir conseillé dans la rédaction des manuscrits, les avoir lus et re-lus. Je lui souhaite énormément de plaisir, de joie et de sjömansbiff à Stockholm ;

Aux membres du jury, Abdelmounaaim Allaoui, Baudouin Byl, Pierre-Alain Gevenois Youri Glupczynski, Paul Tulkens et Olivier Vandenberg, pour leur collaboration critique et constructive durant l’examen de ce travail ;

Ce travail n’aurait pas été possible sans Claire Nonhoff et Ariane Deplano, ou Ariane Deplano et Claire Nonhoff, qui m’ont accueilli, aidé, soutenu et supporté dès mon premier jour au laboratoire. Elles m’ont initié l’une au secret du typage et l’autre à celui de l’antibiogramme.

A Marie, pour toutes les discussions, les conseils, et les remplacements ; A Ricardo, pour ses conseils techniques et scientifiques ;

A Raf, pour ses bases de données et ses gels magnifiques ;

A Sylvianne, Emilie, Sébastien, Marie-Fabrice, Aram et « aux filles des ana », pour leur précieuse aide technique ;

A Carl Suetens, Béa Jans, Erik Hendrickx et, plus récemment, Boudewijn Catry pour les discussions, les analyses statistiques et les collaborations passées et certainement futures ;

A Ann Douglas, pour avoir lu et corrigé une partie de mes misspelling ; A Muruvet et Mady, pour leur aide ;

A Françoise, pour m’avoir supporté ces dernières semaines dans le bureau ;

A mes collègues, Yves et puis Hector ; A mes plus jeunes collègues Sandrine, Nour, Alina et Jonathan ;

A Véronique, à Nicole et à toute l’équipe du laboratoire de bactériologie ;

A mes collègues, en particulier les microbiologistes, infectiologues et hygiènistes, qui ont participé aux différentes enquêtes ;

A mes parents, pour m’avoir donné envie de faire la médecine ; A Anne, Théo et Garance, pour leur patience et leur présence.

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Résumé

Staphylococcus aureus est une bactérie commensale et pathogène qui s’est rapidement adaptée à la pression sélective des antibiotiques entraînant la diffusion de souches résistantes à la méticilline (MRSA). En Belgique, des souches appelées Healthcare- associated (HA)-MRSA sont devenues endémiques dans les hôpitaux, causant de nombreuses épidémies durant les années 90. Parallèlement, des cas d’infections communautaires par des souches appelées Community-associated (CA)-MRSA produisant la leucocidine de Panton-Valentine (PVL) ont été rapportées en Europe, en Australie et aux USA, chez des patients jeunes sans contact avec les institutions de soins. Depuis 2005, une prévalence élevée de portage de MRSA a été observée chez les porcs, les éleveurs et les vétérinaires aux Pays-Bas et dans les pays voisins. Ces souches dites Livestock-associated (LA)-MRSA montrent une origine génétique distincte des souches humaines précédemment décrites.

Nos travaux de recherche ont porté sur l’épidémiologie et la caractérisation moléculaire de la colonisation et l’infection par S. aureus dans diverses populations humaines en Belgique afin d’élucider : 1) l’évolution de la distribution spatio-temporelle des clones épidémiques de MRSA parmi les patients hospitalisés au cours de la période de 1995 à 2003 ; 2) les mécanismes moléculaires de résistance aux antibiotiques associés à ces clones ; 3) les relations phylogénétiques entre souches de S. aureus sensibles et résistantes à la méticilline parmi les patients hospitalisés afin d’identifier l’origine probable des clones épidémiques ; 4) la prévalence et les facteurs de risque de portage de MRSA parmi les résidents des maisons de repos et l’origine de ces souches ; 5) l’origine des souches de CA-MRSA et les profils cliniques des infections associées ; 6) la prévalence et les facteurs de risque associés au portage de MRSA et la diffusion des souches de MRSA dans les fermes d’élevage porcin.

Des enquêtes multicentriques nous ont permis de caractériser les souches de S. aureus résistantes et sensibles à la méticilline affectant les patients hospitalisés (4 enquêtes, 1995 -2003), les patients ambulants (1 enquête, 2002-2004), les résidents dans des maisons de repos et de soins (1 enquête en 2005) et les habitants des fermes d’élevage porcin (1 enquête en 2007). Ces souches ont été caractérisées par détermination du type de cassette mec (SCCmec typing), séquençage d’un gène hypervariable (spa typing) et de 7 gènes de ménage (multi-locus sequence typing, MLST), combinées à l’analyse par électrophorèse en champs pulsé (PFGE) du profil de macrorestriction génomique. Les gènes codant pour trois classes d’antibiotiques et les toxines, PVL, TSST-1 et exfoliatines, ont été recherchés par PCR.

L’étude des souches de MRSA a montré une diversification importante des clones dans les hôpitaux, avec le remplacement d’un clone multi-résistant par des clones plus sensibles aux antibiotiques. La distribution des gènes de résistance ainsi que du gène TSST-1 était fortement liée au génotype. Les S. aureus sensibles à la méticilline (MSSA) montraient une plus grande diversité génotypique que les MRSA. La majorité des MRSA épidémiques appartient à des génotypes endémiques de MSSA, suggérant la possibilité d’émergence locale de MRSA par acquisition récente de l’élément mobile SCCmec.

D’autres souches de génotypes pandémiques pourraient avoir été importées en Belgique.

L’enquête dans les maisons de repos et de soins a montré une prévalence élevée de résidents porteurs de MRSA. L’exposition aux antibiotiques, les lésions cutanées, la perte de mobilité, l’absence de formulaire thérapeutique et de procédures d’hygiène pour le contrôle du MRSA, associée à un nombre élevé de médecins, augmentaient significativement le risque de portage. La présence des mêmes génotypes dans les

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hôpitaux et les maisons de repos d’une même province suggère des transferts locaux de MRSA entre patients résidant dans et circulant entre ces secteurs de soins.

Les souches de CA-MRSA productrices de toxine PVL importées ou acquises en Belgique appartiennent à divers génotypes. La présence de MRSA de même lignée clonale mais présentant des profils de virulence différents clonale dans les institutions de soins et dans la population générale suggère que ces souches ont évolué de manière divergente dans des environnements différents.

Nous avons documenté une prévalence très élevée de porteurs de MRSA de génotype ST398 chez les éleveurs de porcs. Le réservoir de ce clone est très probablement d’origine animale, la transmission à l’homme ayant lieu par contact avec des animaux d’élevage ou de compagnie et potentiellement, par voie alimentaire.

En conclusion, S. aureus est un pathogène capable de s’adapter dans des environnements très différents. Les souches épidémiques résistantes aux antibiotiques, qu’elles soient d’origine hospitalière, communautaire ou animale, appartiennent à un nombre limité de génotypes bien établis dans chaque écosystème au niveau local ou régional. L’étude approfondie de la dynamique de transmission des MRSA, et de leur profil de résistance dans la communauté, les secteurs des soins aigus et chroniques et l’élevage, est indispensable pour définir les stratégies cliniques et de santé publique pour adapter les schémas thérapeutiques et endiguer l’endémie d’infections à MRSA.

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List of abbreviations

ACME Arginine catabolic mobile element agr Accessory gene regulator

AME Aminoglycoside modifying enzyme BICS Belgian Infection Control Society CA-MRSA Community-associated MRSA

CC Clonal complex

CDC Centers for Disease Control and Prevention

Clf Clumping factor

CLSI Clinical and Laboratory Standards Institute CNS Coagulase negative staphylococci

DHFR Dihydrofolate reductase

DHPS Dihydropteroate synthetase

EARSS European Antimicrobial Resistance Surveillance System EFSA European Food Safety Authority

ET Exfoliative toxin

EUCAST European Committee on Antimicrobial Susceptibility Testing FEM Factors essential for methicillin resistance

Fnb Fibrinogen binding protein

GIs Genomic islands

GISA Glycopeptide intermediate Staphylococcus aureus HA-MRSA Hospital-associated MRSA

HL Haemolysin J region Junkyard region

LA-MRSA Livestock-associated MRSA MGEs Mobile genetic elements

MLS Macrolides – lincosamides - streptogramins MLST Multilocus sequence typing

MRSA Methicillin-resistant Staphylococcus aureus

MSCRAMM Microbial surface components recognizing adhesive matrix molecules

NH Nursing home

PFGE Pulsed-field gel electrophoresis PBP Penicillin binding protein

PVL Panton-Valentine Leucocidin

Sag Superantigen

SaPIs Staphylococcus aureus pathogenicity islands SCCmec Staphylococcal cassette chromosome mec

SE Staphylococcal enterotoxin

SFP Staphylococcal food poisoning SNPs Single nucleotide polymorphisms spa Staphylococcus aureus protein A SSSS Staphylococcal scaled skin syndrome

ST Sequence type

TSS Toxic shock syndrome

TSST-1 Toxic shock syndrome toxin – 1

VISA Vancomycin intermediate Staphylococcus aureus VNTRs Variable tandem of number repeats

VRSA Vancomycin resistant S. aureus

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Chapter 1: Introduction

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Figure 1. Representation of the MRSA252 genome with each gene colored according to whether it is a core gene or core variable gene or whether it is found on a mobile genetic element. The outer circle represents genes on the forward coding strand, and the inner circle represents genes on the complementary strand. (From Lindsay et al. 2006)

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1.1. Microbiology of Staphylococcus aureus

1.1.1. Taxonomy

Staphylococci are Gram-positive cocci (0.5 to 1.5 µm in diameter) that are associated in pairs or in clusters and classified into the Staphylococcaceae family, which includes the genera Staphylococcus, Macrococcus, Salinicoccus and Jeotgalicoccus (Bergeys online). Staphylococci are non-motile, catalase positive and mostly facultative anaerobic bacteria.

The genus Staphylococcus comprises 41 species, 16 of which are found in humans (LPSN online). The majority of these are coagulase negative species which are not pathogenic for humans except in the presence of predisposing host conditions like immunosuppression or a foreign body. Staphylococcus aureus is the most virulent species and can cause a wide spectrum of diseases from benign skin and soft tissue infections to life-threatening infections including bacteraemia, endocarditis and pneumonia (Lowy 1998).

1.1.2. Genome composition

The genome of S. aureus consists of a circular chromosome of approximately 2700 to 2900 Mb with low G+C content (32.8%) and up to 3 extra-chromosomal plasmids, (Baba et al. 2008). It contains between 2600 and 2750 genes. The annoted genome sequence of 14 S. aureus strains is available (Genomes Pages online). The overall structure of the S. aureus genome is well conserved and is composed of a core genome and accessory genome (Lindsay et al. 2006a) (Figure 1).

1.1.2.1. Core genome

A large proportion of genes that is present in nearly all (> 95%) S. aureus strains constitute the core genome. These genes are encoding functions that are essential for growth and survival of the bacterium. They include genes associated with metabolism and other housekeeping functions but also contain virulence genes such as surface binding proteins, toxins, exoenzymes and capsule biosynthesis genes. The sequence divergence in the core genome (variable core genome) is due to single nucleotide polymorphisms (SNPs), large variation from few nucleotides to several kb, and insertion of repeat sequences (Lindsay et al.

2006a). The SNPs within coding regions can be phenotypically silent (synonymous substitution) or can alter positively or negatively the expression or the function of genes (non-synonymous substitution). SNPs affecting housekeeping genes have been used by multi-locus sequence typing (MLST) schemes to investigate the phylogenetic relationship of strains within bacterial species that display a clonal population structure such as S. aureus. Homologous recombination results in larger areas of variations of the core genome, including complete or partial gene sequence, in operons like the capsule cluster and accessory gene regulator (agr) loci. Repeat sequences are widespread in S.

aureus and can vary in size from a single nucleotide to several hundreds. Many surface associated protein genes contain large variable repeats including genes encoding fibrinogen-binding proteins (FnbA, FnbB) and collagen binding protein (Cna) (Lindsay et al. 2006a). Variation in the repeat regions may represent a means by which pathogenic bacteria can evade the host immune system by modulating the expression or antigen composition of its surface proteins.

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Figure 2. Circular representation of S aureus chromosome (N315) and Mu50-specific genetic elements. Outer circle=distribution of mobile genetic elements (yellow, SCCmec;

light blue, Tn554; red, phages and pathogenic islands; brown, IS1181). Red boxes outside circle=locations of Mu50-specific elements. Second circle=every 100th open reading frame.

Third circle=pathogenicity (red) and antibioticresistance (blue) determinants. Fourth and fifth circles=predicted protein-coding regions on the plus and minus strand, respectively. Sixth circle=taxonomic distribution of BLAST best-hit entries (blue, Bacillus/Clostridium group;

green, firmicutes; pink, viruses/insertion sequences/transposons; orange, Archaea/eubacteria/eukaryota; white, no hit or ribosomal and transfer RNAs). Seventh circle=GC content at third codon positions (GC3) and synonymous codon-usage bias of each open reading frame (green, highly expressed; red, putative alien; orange, possible alien based on GC3 skew, blue, other). Size of coloured bar corresponds to deviation of GC3 value of each open reading frame from average. Eighth circle=distribution of GC content with window size of 1 kb and shift increment of 1 kb. Red arrowhead=rRNA and its orientation.

Black bars=locations of tRNAs. Blue bars=locations of staphylococcal repeat sequences.

Ninth circle=GC skew (dark blue, positive value; light blue; negative value). Tenth circle=nucleotide position in Mb.

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1.1.2.2. Accessory genome

Mobile genetic elements (MGEs) are segments of DNA that can replicate autonomously or possess specific mechanisms to insert into or out of the chromosome or plasmids (Lindsay 2008) (Figure 2). MGEs quantitatively represent an important component (10 to 20%) of the S. aureus genome and form the main part of its subspecies diversity. Many MGEs carry virulence or antibiotic resistance genes. They have a G+C content equivalent to chromosomal DNA, suggesting that horizontal transfer predominant occur between isolates of S.

aureus or other closely related bacteria with similar G+C content.

1.1.2.2.1. Bacteriophages

The majority of S. aureus strains carry one to four prophages (approximately 45 kb in size) integrated into the chromosome at specific sites (Firth et al. 2006;

Lindsay 2008). Prophages can be classified into five families on the basis of integrase gene that specify their insertion site. Notably, S. aureus strains contain no more than one phage of each family type. Prophage carry genes encoding virulence factors and toxins like the staphylococcal enterotoxin A (SEA), exfoliative toxin A (ETA) and Panton-Valentine leucocidin (PVL).

1.1.2.2.2. Staphylococcal aureus pathogenicity islands (SaPIs)

The S. aureus pathogenicity islands (SaPIs) are DNA sequences of 15 to 27-kb which are located at specific sites in the chromosome (Novick et al. 2007; Lindsay et al. 2006a; 2008). Their transfer between strains depends on specific interactions with a helper bacteriophage. The SaPIs have a highly conserved set of core genes including the integrase, the rep (initiator protein) and the terminase.

Many SaPIs encode two or more superantigen toxins, i.e. toxic shock syndrome toxin (TSST-1), staphylococcal enterotoxin B (SEB) and C (SEC) and antibiotic resistance determinants like fusidic acid resistance island, i.e. SARIfusB (O’Neill et al. 2007a). All but one of the sequenced S. aureus strains contain one or more SaPIs (Novick et al. 2007).

1.1.2.2.3. Plasmids

Plasmids harboured by S. aureus belong to three families that are defined based on size and ability to conjugate (Firth et al. 2006; Lindsay et al. 2006a; 2008).

Class I, which comprises the smallest (< 5 kb in size) plasmids, are found at multiple copy number and encode one or two resistance genes, such as pT181 encoding the tetK gene for tetracycline resistance. These plasmids can integrate into the chromosome. Class II plasmids are larger (up to 40 kb in size) and carry multiple genes encoding resistance to antibiotics, heavy metals and antiseptics.

Resistance genes are located on transposons that have secondarily integrated into the plasmid, like Tn4001 which confers resistance to gentamicin. Class III plasmids are large (up to 60 kb in size), similar to class II plasmids but possess transfer (tra) genes enabling their self-conjugative transfer. There is high variation in the distribution of plasmids in S. aureus. Some strains lack free plasmids while others have up to three. Many plasmids are integrated in high copy number into the chromosome, particularly within the SCCmec elements.

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Figure 3. Schematic representation of the molecular structure of SCCmec type I to VIII of S. aureus.

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1.1.2.2.4. Transposons

Transposons are small pieces of DNA (3-60 kb in size) which encode a transposase gene for excision and replication (Firth et al. 2006; Lindsay et al., 2006; 2008). Their transfer between S. aureus strains involves integration into another mobile element like a plasmid. They are integrated randomly into the chromosome, sometimes in multiple copies. S. aureus transposons may carry antibiotic resistance genes, such as Tn554 encoding resistance to erythromycin and Tn4001 to gentamicin.

1.1.2.2.5. Staphylococcal cassette chromosome (SCC)

Staphylococcal cassette chromosome (SCC) elements are single copy elements inserted into a specific region of the S. aureus genome, the attBSCC site located at the 3’ end of the orfX gene, near the origin of replication. They carry recombinase (ccr) genes that catalyze excision and integration of the element. The mechanism of horizontal transfer of SCC elements between staphylococci is unknown. The SCC elements contain antibiotic resistance genes such as the SCCmec and SCCfar for methicillin and fusidic acid resistance, respectively (Ito et al. 2001;

Holden et al. 2004).

The SCCmec elements have been subdivided into types I to VIII which range in size from 20.9 kb to 66.9 kb (Ito et al. 2001; 2004; Ma et al. 2004; Daum et al.

2002; Oliveira et al. 2006; Berglund et al. 2008; Zhang et al. 2009) (Figure 3).

They are classified by the combination of ccr genes and mec complex that they carry (Figure 3). Five major mec complexes (A to E) have been described in staphylococcus, of which three (A to C) in S. aureus (Hanssen et al. 2006). The mec complexes differ by integration of IS1272 and IS431 elements and by deletion of mecI and a part of mecR genes. The ccr genes are classified into five allotypes designated ccrAB1, ccrAB2, ccrAB3, ccrAB4 and ccrC. The SCCmec type III prototype is a composite element that consists of the recombination of two SCC elements, i.e. SCCmec type III and SCCmercury (Chongtrakool et al. 2006).

SCCmec elements often contain integrated plasmids, e.g. pUB110, pI258 and pT181, and transposons, e.g. Tn554 and ΨTn554.

SCCmec elements also comprise three junkyard (J) regions. The J1 region is located between the right junction and the ccr complex. The region between the ccr complex and the mec complex is called J2. The J3 region extends from the mec complex to the orfX. SCCmec elements, particularly SCCmec II and IV, show many variants in their J regions (Chongtrakool et al. 2006; Kondo et al. 2007).

These variations in the J regions of SCCmec elements with the same mec and ccr combination define the SCCmec subtypes. To date, more than 46 structural- differences in SCCmec elements have been identified (Stephens et al. 2007). An international nomenclature has been published recently for SCCmec type and subtype classification (SCCmec Home online) (International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) 2009).

1.1.2.2.6. Genomic islands (GIs)

The term GI refers to non-phage and non-SCC genomic islands (20-30 kb in size) that are exclusively found in S. aureus (Lindsay 2008). These islands frequently, encode virulence determinants, are inserted at a specific locus in chromosome, and are associated with either intact or remnant DNA recombinase (Baba et al.

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Figure 4. Structure of S. aureus. Panel A shows the surface and secreted proteins.

The synthesis of many of these proteins is dependent on the growth phase, as shown by the graph, and is controlled by regulatory genes such as agr. Panels B and C show cross sections of the cell envelope. Many of the surface proteins have a structural organization similar to that of clumping factor, including repeated segments of amino acids (Panel C). TSST-1 denotes toxic shock syndrome toxin 1. (From Lowy 1998)

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2002). The GIs encode multiple exotoxins, lipoproteins (lpl) and serine protease (spl).

1.1.3. Virulence factors 1.1.3.1. Capsule

S. aureus isolates produce a polysaccharidic capsule which is thin (< 0.05 µm) and usually not visible by microscopic examination (Figure 4) (Wright III et al.

2003; Fournier 2008). Among 11 capsular serotypes described, serotypes 5 and 8 account for 75 to 85% of S. aureus strains isolated from human infections. The capsule increases resistance to phagocytosis by neutrophils and macrophages. In several animal models, the capsule increases virulence. The capsule antigens are among the candidate components for S. aureus vaccine currently in development, although trials with conjugated polysaccharide vaccines alone were not successful (Creech II et al. 2009).

1.1.3.2. Surface proteins

S. aureus produces several proteins which bind extracellular matrix (Wright III et al. 2003; Fournier 2008). These proteins have been designated as microbial surface components recognizing adhesive matrix molecules (MSCRAMM). Most of them are covalently anchored to the cell wall peptidoglycan. The most important surface proteins are FnbA and FnbB, clumping factors ClfA and ClfB, collagen binding protein (Cna) and S. aureus protein A (Spa) (Figure 4). These proteins are encoded by genes of the core chromosome. They have been associated with invasive capacity in experimental endocarditis and osteoarthritis.

1.1.3.3. Enzymes

S. aureus produces a wide variety of extracellular enzymes including proteases, lipases, staphylokinase and coagulase (Figure 4). Most of them are involved in the pathogenesis of staphylococcal infections (Wright III et al. 2003; Fournier 2008).

Coagulase (Coa) forms a complex with prothrombin, known as staphylothrombin, which converts fibrinogen to fibrin. Its role in staphylococcal infections remains unclear. Coagulase could generate a protective layer of fibrin around the bacterium and inhibit immune cell recruitments. Staphylokinase (Sak), encoded by a gene located on prophage, is a potent thrombotic agent that acts by converting plasminogen to plasmin.

1.1.3.4. Exotoxins

1.1.3.4.1. Panton-Valentine Leucocidin

The Panton-Valentine leucocidin (PVL) is a bi-component cytotoxin encoded by two co-transcribed genes, lukS-PV and lukF-PV (Bohach 2006). PVL genes are located on prophages, including ϕ108PVL, ϕPVL, ϕSLT and ϕSa2mw, which are integrated in the S. aureus chromosome (Ma et al., 2006; Feng et al. 2008). The two components of the PVL target cells of the immune system, e.g.

polymorphonuclear neutrophils, monocytes and macrophages, by binding to their cell membrane to form pores. In vitro, PVL causes cell deaths of human and rabbit polymorphonuclear neutrophils, whereas the intradermal injection in rabbits induces skin erythema followed by necrosis. However, there is still debate about

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Figure 5. T-Cell activation by superantigens. In the conventional response to bacterial antigens (left), the antigen is presented by the antigen-presenting cell (APC) in association with MHC II. Superantigens (right) bypass this process by binding between MHC II and the Vβ domain of the T lymphocyte receptor. (From Lynn 2004)

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the role of PVL in the virulence of CA-MRSA. A mouse pneumonia model has shown that PVL alone was sufficient to cause pneumonia but also that the toxin induced expression changes of genes encoding secreted and cell wall-anchored staphylococcal proteins (Labandeira-Rey et al. 2007). PVL was detected in samples from patients with PVL producing S. aureus skin infections at concentrations that were toxic for rabbit skin (Badiou et al. 2008). In contrast, no significant differences between USA300 and USA400 wild-type and isogenic PVL- deficient strains were detected in mouse and rat models of abscess, pneumonia and bacteraemia (Voyich et al. 2006; Diep et al. 2008a).

The presence of PVL genes in clinical isolates is associated with furonculosis, severe skin and soft tissue infections including pyomyositis and necrotizing fasciitis, severe necrotizing pneumonia and osteomyelitis in previously healthy children and young adults (Lina et al. 1999a; Gillet et al. 2002; Bocchini et al.

2006). The PVL genes are infrequently detected in hospital S. aureus isolates (<5%) but are present in the majority of CA-MRSA strains (Prévost et al. 1995;

Vandenesch et al. 2003; Tristan et al. 2007).

1.1.3.4.2. Exfoliative toxins

Exfoliative toxins (ETs) are serine proteases that attack the stratum granulosa cells causing intra-epidermal skin cleavage. ETs cleave the desmoglein-1 at the glutamic residue at position 381, which results in a loss of function and cell separation, leading to blister formation (Bohach 2006).

Four ETs have been reported in S. aureus designated as ETA, ETB, ETC and ETA. The eta gene is chromosomally located on a phage, whereas the etb and etd are located on plasmids and pathogenicity island, respectively.

ETs are responsible for staphylococcal scalded-skin syndrome (SSSS) which is characterized by the formation of bullae or skin blisters (Moreillon et al. 2005).

SSSS occurs predominantly in infants and children. Outbreaks of SSSS have been described in nurseries.

1.1.3.4.3. Superantigens

S. aureus produces a large number of pyrogenic exotoxins known as superantigens (SAgs), which are responsible for toxic shock syndrome and food poisoning (Moreillon et al. 2005). The Sags are proteins which link non-specifically major histocompatibility complex class II (MCH II) of the antigen presentation cells to the Vβ domain of the T lymphocytes (Bohach 2006). They activate polyclonal populations of T cells leading to their massive proliferation and uncontrolled release of proinflammatory cytokines (Figure 5). The mechanism of action of SE at surface the intestinal mucosa remains unclear (Lina et al. 2004). Food poisoning may result of specific interaction with parasympathetic nerve in the gastro-intestinal tract (Wright III et al. 2003).

The pyrogenic exotoxins include the TSST-1, staphylococcal enterotoxins (SEs) A to G, and SE-like toxins with unconfirmed biological activity, types H to R and U (Nomenclature Committee for Staphylococcal Superantigens) (Lina et al. 2004).

Seven SEC minor variants have been described. The genes encoding pyrogenic enterotoxins are harboured on various mobile genetic elements (Table 1) including plasmids (sed, selj and selr), phages (sea, sed and sep) and pathogenicity islands (seb, sec, selk, sell, selq and tst) (Omoe et al. 2005). The large operon termed the

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Table 1. Genes and activity of staphylococcal pyrogenic toxins

Gene Location Protein Activity

sea Phage φSa3 Enterotoxin A Food poisoning, TSS

seb Pathogenicity island SaPI1, SaPI3, Enterotoxin B Food poisoning, TSS sec* Pathogenicity island SaPI2, SaPI4 Enterotoxin C Food poisoning, TSS sed Plasmid or phage Enterotoxin D Food poisoning, TSS see Pathogenicity island SaPI1 Enterotoxin E Food poisoning seg Enterotoxin gene cluster (egc), phage φSa3, SaPI1 Enterotoxin G Food poisoning

selh Phage φSa3 Enterotoxin-like H Unknown

sel Enterotoxin gene cluster (egc), SaPI2 Enterotoxin i Food poisoning

selj Plasmids Enterotoxin-like J Unknown

selk Pathogenicity island SaPI1, SaPIbov, phage φSa3 Enterotoxin-like K Unknown

sell Pathogenicity island SaPI2, SaPI3 Enterotoxin-like L Unknown

selm Enterotoxin gene cluster (egc) Enterotoxin-like M Unknown seln Enterotoxin gene cluster (egc) Enterotoxin-like N Unknown selo Enterotoxin gene cluster (egc) Enterotoxin-like O Unknown

selp Phage φSa3 Enterotoxin-like P Unknown

selq Pathogenicity island SaPI1 Enterotoxin-like Q Unknown

selr Plasmids Enterotoxin-like R Unknown

selu Enterotoxin gene cluster (egc) Enterotoxin-like U Unknown tst Pathogenicity island SaPI1 SaPI2, bov1 TSST-1 TSS

* Seven variants; TSS, toxic shock syndrome

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enterotoxin gene cluster (egc) encodes seg, sei, selm, seln, selo, selu (Collery et al. 2009). Enterotoxin genes regulated by the agr and sar loci (Fournier 2008).

The distribution of genes encoding Sags is strongly linked to the genetic background of the strains (Durand et al. 2006; Diep et al. 2006a; Holtfreter et al.

2007).

1.1.3.4.4. Other membrane active agents

Several other pore-forming cytolytic toxins are produced by S. aureus including the alpha haemolysin (Hla), beta haemolysin (Hlb), delta haemolysin (Hld) and gamma haemolysin (Hlg) (Bohach 2006). These toxins can cause the destruction of a wide range of host cells, including epithelial cells, erythrocytes, fibroblasts and monocytes.

1.1.3.5. Arginine catabolic mobile element

Recently, the data from whole genome sequencing of the USA300 MRSA strain revealed a novel genetic element named the arginine catabolic element (ACME) inserted into the orfX gene adjacent to the SCCmec IV element (Diep et al.

2006b). This DNA element contains two putative virulence factors, a cluster of arginine catabolism (arc) genes that encode an additional arginine deiminase pathway and opp3, which encodes an oligopeptide permease. The deletion of ACME has been shown to decrease the fitness of USA300 in an animal bacteraemia model (Diep et al. 2008b).

This genetic feature is characteristic of the USA300 MRSA ST8-SCCmec IV clone but has also been found occasionally in strains of MSSA ST8, MRSA USA100 ST5-SCCmec II and MRSA ST97-SCCmec V (Goering et al. 2007; Ellington et al.

2007). The presence of ACME in strains of diverse genetic background and SCCmec types suggests that this DNA element may be highly mobile (Ellington et al. 2007).

1.1.3.6. Regulation of staphylococcal virulence

1.1.3.6.1. Regulators of virulence

The pathogenicity of S. aureus is a complex process involving coordinated expression of extracellular and cell wall components during the different phases of growth. Global regulators control the expression of these unlinked virulence genes. S. aureus possesses several of the global regulatory systems including agr system, sae, sarA and sigmaB (Fournier 2008).

1.1.3.6.2. The agr system

The accessory gene regulator (agr) modulates the expression of virulence factors including toxins, enzymes and cell wall proteins (Fournier 2008). The agr locus encodes of two divergent transcripts under the control of promoters P2 and P3 (Figure 6). The P2 transcript RNA II contains four genes of which agrA and agrC represent a two-component signal transduction system, and agrB and agrD encode an auto-inducing peptide (AIP) that activates the quorum sensing system.

The activation of the agrAC signalling pathway leads to increased transcription from the agr promoters P2 and P3. The P3 transcript RNA III is the effector molecule of the agr regulon which activates the transcription of secreted protein

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Figure 6. The staphylococcal agr quorum-sensing system. The extracellular signal of the agr quorum-sensing system is a posttranslationally modified peptide autoinducer. The membrane located enzyme AgrB is responsible for the maturation and export of the autoinducing peptide. AgrA and AgrC form a twocomponent signal transduction system, which in an auto-regulatory fashion, after binding of the autoinducer to AgrC, is responsible for a rapid increase of agr activity at the onset of stationary growth phase. Target genes are controlled by a regulatory RNA molecule.

The RNAIII region contains a gene (hld) coding for the peptide delta-toxin (PSMg), the expression of which does not affect the quorum-sensing mechanism. (From Kong et al. 2006)

Figure 7. Structure of the glycopeptide: peptidyI-D-Ala-B-Ala complex. Binding of a glycopeptide to the D-Ala-o-Ala extremity of peptidoglycan precursors involves five hydrogen interactions indicated by dashed lines. (From Arthur et al. 1997)

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genes and down-regulates surface protein genes.Although the agr locus is well conserved throughout staphylococci, a polymorphism exists in the region encoding the AIP and its receptor (Fournier 2008). S. aureus can be divided into four major agr allelic groups which are strongly linked to the clonal population structure.

1.1.4. Antimicrobial resistance determinants 1.1.4.1. Beta-lactams

Β-lactams inhibit cell wall biosynthesis by binding irreversibly to the transpeptidase active site of penicillin binding proteins (PBPs) resulting in the formation of a stable ester-linked acyl enzyme which lacks catalityic activity (Pinho 2008; Llarrull et al. 2009). Two mechanisms are involved in staphylococcal resistance to β- lactams: the production of penicillinase and target modification.

1.1.4.1.1. Penicillinase

Resistance to penicillin is conferred by penicillinase, which is encoded by the bla gene carried on a plasmid (Pinho 2008). Penicillinase hydrolyzes penicillin and its derivates into penicilloic acid. Expression of the blaZ gene is regulated by the blaRI and blaI determinants. Penicillin-resistant S. aureus strains emerged shortly after the therapeutic introduction of penicillin in the 1940s. Today, more than 90%

of S. aureus strains are resistant to penicillin.

1.1.4.1.2. Penicillin binding protein 2a

Methicillin resistance is mediated in the majority of strains by acquisition of protein binding protein 2a (PBP2a) encoded by the mecA gene (Pinho 2008). PBP2a confers intrinsic resistance to all β-lactam antibiotics by its very low affinity. New cephalosporins, such as ceftobiprole, have been recently developed showing a broad spectrum activity against gram-negative and gram-positive bacteria, including methicillin-resistant staphylococci. The latter activity of ceftobiprole is dueto its high affinity to PBP2a (Van Bambeke et al. 2008; Barbour et al. 2009).

Transcription of mecA is controlled by the mecI and mecR regulatory elements:

mecI represses mecA transcription while mecR encodes a signal-transduction protein (MecR1) with an extra-cellular penicillin-binding domain that activates its cytoplasmic domain in the presence of β-lactams. The mecA gene and its regulatory system are located on the staphylococcal cassette chromosome mec (SCCmec) which is transferable between staphylococcal species.

Additional genes are necessary for the expression of methicillin resistance. Over 30 of these “factor essential for methicillin resistance” (fem) genes have been identified. They are involved in cell wall metabolism (Pinho 2008). Among them, PBP2 is the only of the four native PBPs having both transpeptidase and trasnglycolase activity in S. aureus. Therefore, cell wall biosynthesis in MRSA is completely dependent on the cooperative function of the PBP2a transpeptidase with the transglycosylase domain of PBP2 (Pinho 2008; Llarrull et al. 2009).

1.1.4.2. Glycopeptides

Glycopeptides inhibit cell wall biosynthesis by binding to the terminal D-alanyl-D-

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Figure 8. Mechanisms of S. aureus resistance to vancomycin: VRSA strains. VRSA strains are resistant to vancomycin because of the acquisition of the vanA operon from an enterococcus that allows synthesis of a cell wall precursor that ends in D-Ala-D-Lac dipeptide rather than D-Ala-D-Ala. The new dipeptide has dramatically reduced affinity for vancomycin. In the presence of vancomycin, the novel cell wall precursor is synthesized, allowing continued peptidoglycan assembly. (From Périchon et al. 2009)

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alanine (D-ala-D-ala) of N-acetylmuramic acid (MurNAc) pentapeptide cell wall precursor (Pinho 2008) (Figure 7). Two mechanisms of resistance to glycopeptides have been described in S. aureus.

1.1.4.2.1. Vancomycin resistant S. aureus (VRSA)

High-level resistance to vancomycin in S. aureus is associated with acquisition of the vanA operon which is located in Tn1546-like transposons (Weigel et al. 2003;

Périchon et al. 2009). Enzymes encoded by the vanA gene cluster synthesize cell wall precursor with terminal D-alanyl-D-lactate (D-ala-D-lac) residues in place of D-ala-D-ala. This residue reduces glycopeptide affinity by a factor 1000 compared with the normal cell wall precursor (Figure 8).

Interspecies transfer of vanA by conjugation from Enterococcus faecalis to S.

aureus was obtained, both in in-vitro and in in-vivo models in 1992 (Noble et al.

1992). In 2002, the first clinical vancomycin-resistant S. aureus (VRSA) strain was isolated from a dialysis patient in the USA (Chang et al. 2003). Since then, eight more cases of VRSA infections have been confirmed by the Centers for Disease Control and Prevention (CDC) in Pennsylvania, New-York and Michigan (Sievert et al. 2008, Finks et al. 2009). VRSA isolates carried the vanA gene and had vancomycin MIC ranging from 32 to 1024 mg/l (Sievert et al. 2008) (NARSA online). Two of these isolates remained susceptible to teicoplanin with MIC of 8 mg/l. The vanA gene was carried on Tn1546-like elements integrated into plasmids ranging in size from 40 to 120 kb. In vitro transfer of vanA gene from VRSA to other strains of S. aureus has reinforced concerns about potential widespread resistance to glycopeptides (Weigel et al. 2003). These VRSA isolates belonged either to the “New-York/Japan clone” ST5-SCCmec II (USA 100) or ST5-SCCmec IV (USA 800) (Sievert et al. 2008). Patients with VRSA infections had a history of a prior MRSA and enterococcal/VRE colonization or infection with multiple underlying conditions including chronic skin ulcers, diabetes, chronic renal failure and obesity (Sievert et al. 2008). All but one patient had received vancomycin during the previous 3 months. No secondary transmission was observed after implementation of infection control measures (Sievert et al. 2008;

Finks et al. 2009).

1.1.4.2.2. Vancomycin intermediate S. aureus (VISA)

Since their first description in 1997 from Japan, VISA isolates have been reported worldwide (Hiramatsu et al. 1997a; Ploy et al. 1998; Smith et al. 1999; Denis et al.

2002a). VISA isolates were in the majority of cases recovered from chronically ill patients failing prolonged glycopeptide therapy of infections associated with an indwelling device or undrained collections of pus. Hetero-VISA strains are borderline susceptible to vancomycin (MIC 2–4 mg/L) with low-level subpopulations (10–6 cells)that are able to grow at vancomycin concentrations of 4–8mg/L (Hiramatsu et al. 1997b). Those strains may represent first-stepmutants that are precursors of VISA strains in patients receiving vancomycin. CLSI and EUCAST decreased vancomycin susceptibility breakpoints for testing S. aureus (CLSI online; EUCAST online). According to these new breakpoints, many Hetero- VISA isolates would now be reclassified as VISA. Reduced susceptibility to vancomycin was found in MRSA strains belonging to different pandemic lineages (Howe et al. 2004).

The mechanism of heterogeneous vancomycin resistance in VISA/ hetero-VISA isolates is not well understood. Expression of resistance is not linked to acquisition

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Figure 9. Transmission electron micrographs of (a) S. aureus ATCC 29213, (b) VISA strain P1V44, (c) hetero-VISA strains P1V69 and (d) MRSA P39575.

Magnification x60 000. (From Denis et al. 2002a)

Figure 10. Mechanisms of S. aureus resistance to vancomycin: VISA strains.

VISA strains appear to be selected from isolates that are heterogeneously resistant to vancomycin. These VISA strains synthesize additional quantities of peptidoglycan with an increased number of D-Ala-Dala residues that bind vancomycin, preventing the molecule from getting to its bacterial target. (From Lowy 2003)

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of a particular determinant but to accumulation of point mutations that lead to changes in peptidoglycan biosynthesis. Loci associated with decreased vancomycin susceptibility include genes involved in cell wall and membrane biosynthesis and global regulator systems such as agr, vraSR and graSR (Sakoulas et al. 2005; Howden et al. 2008; Cui et al. 2009). The two-component system vraSR and graSR modulate the cell wall biosynthesis (Ciu et al. 2009).

VraSR regulates the expression of enzymes involved in peptidoglycan synthesis including PBP2, PBP1A/1B and MurZ. A recent study suggests that graRS controls at least 248 genes including ABC transporters vraF and vraG which are unregulated in VISA strains (Howden et al. 2008).

VISA strains have common phenotypic characteristics including a thickened cell wall (Figure 9) (Denis et al. 2002a; Cui et al. 2003; Reipert et al. 2003), reduced autolytic activity, increased production of glutamine non-amidated muropeptides (Hanaki et al. 1998), increased number of D-Ala-D-Ala residues and reduced peptidoglycan cross-linking. Accumulation of dipeptides in the cell wall traps the antibiotic away from its target site of cell wall synthesis adjacent to the membrane (Figure 10).

Since their first description in 1997, VISA isolates have remained rare. The reported prevalence of hetero-VISA isolates ranges widely from one study to another, but was similarly low in large surveys conducted in the UK, Italy, France, USA and Korea (Aucken et al. 2000; Marchese et al. 2000; Kim et al. 2002;

Cartolano et al. 2004; Rybak et al. 2005). Most studies have reported a higher prevalence of (hetero-)resistance of S. aureus to teicoplanin than to vancomycin, even in countries, such as the USA, where teicoplanin has not yet been licensed for clinical use (Rybak et al. 2005).

1.1.4.3. Fluoroquinolones

The quinolones inhibit bacterial type II DNA topoisomerases, namely DNA gyrase and topoisomerase IV. Both enzymes are tetramers made up of 2 units, GyrA and GyrB for the DNA gyrase and ParC or ParE for topoisomerase IV (which are known as GrlA and GrlB in S. aureus). Two mechanisms of resistance to quinolones have been described in S. aureus including target modification and hyper-expression of active efflux system.

1.1.4.3.1. Target modification

Mutations in the genes encoding DNA gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB) confer stepwise resistance to fluoroquinolones by decreasing their drug affinity (Tanaka et al. 2000). In vitro studies have shown that mutations in grlA gene occur before gyrA gene in S. aureus under ciprofloxacin exposure.

Mutations in GrlA (Ser80 to Phe or Tyr and Glu84 to Lys) and GyrA (Ser84 to Leu or Lys, Glu88 to Lys or Val and Ap73 to Gly) confer resistance to fluoroquinolones both in laboratory and clinical S. aureus isolates (Deplano et al. 1997a; Schmitz et al. 2000; Guirao et al. 2001). Few mutations in grlB and no mutation in gyrB have been reported in S. aureus.

1.1.4.3.2. Active efflux

Resistance to fluoroquinolones has also been associated with over-expression of the chromosomally encoded efflux system NorA (Tanaka et al. 2000). This non- specific pump extrudes fluoroquinolones and unrelated compounds such as

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Table 2. Mechanisms, genes and phenotypes of resistance to MLS in S. aureus

Mechanism Genes Phenotype designation Resistance profile

14-/ 15-Md 16-Md L SA SB

Ribosomal methylation erm(A) erm(C) erm(B)

MLSB inducible MLSB constitutive

R R

R R

Sa R

S S

S R

Efflux system msrA vgaC

MSB R

S

S S

S R

S S

Enzymatic modification lnu(A)

vatA, vatB, vatC, vgbA, vgbB L S

S S

S S

R S

S R or S

S R or S

Ribosomal mutation rplD, rplV, rrn M, MSB, MLSB, ML R R R or S S R or S

14-Md, 14-membered ring macrolides (clarithromycin, erythromycin and roxithromycin); 15-Md, 15-membered ring macrolide (azythromycin); 16-Md, 16- membered ring macrolide (josamycin and spiramycin); L, Lincosamides; SB streptogramins B (dalfopristin, pristinamycin IA); SA streptogramins A (quinupristin, pristinamycin IIA)

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chloramphenicol, ethidium bromide and cetrimide. Resistance is conferred by mutations in the norA regulatory region. Efflux mediated resistance results in a lower level of resistance than topoisomerase mutations. The over-expression of NorA efflux pump may enhance the emergence of secondary grlA mutation leading to high level resistance to fluoroquinolones.

Shortly after the introduction of fluoroquinolones in clinical use, the proportion of hospital-associated (HA)-MRSA strains resistant to ciprofloxacin agents reached up to 90% (Fluit et al. 2001; Van Bambeke et al. 2008). Community-associated (CA)-MRSA strains and MSSA strains remained more frequently susceptible to the fluoroquinolones than HA-MRSA strains. New fluoroquinolones with more potent activity on Gram-positive bacteria like moxifloxacin show reduced activity against ciprofloxacin-resistant MRSA strains.

1.1.4.4. Macrolides, lincosamides and streptogramins

Macrolides, lincosamides and streptogramins (MLS) are chemically distinct classes of antimicrobials that inhibit protein synthesis by stimulating dissociation of the peptidyl-tRNA molecule from the 50S ribosomal subunit during elongation.

In S. aureus, three mechanisms of resistance to MLS have been described: target modification, active efflux and enzymatic drug modification (Table 2) (Leclercq 2002, Roberts 2008). The nomenclature of MLS resistance determinants is available online (Faculty Washington online).

1.1.4.4.1. Target modification Methylation

The main mechanism of resistance to those agents is methylation of the A2058 residue of peptidyl transferase centre in domain V of 23S rRNA (Leclercq 2002).

The family of enzymes responsible for methylation has been designated as erythromycin resistance methylase (Erm) encoded by the corresponding erm genes. Five erm genes have been reported in S. aureus: ermA, ermC, and, less frequently, ermB, ermF and ermY. These determinants are located on mobile genetic elements. The ermA gene is harboured on transposon Tn554 whereas the ermC gene is found on small plasmids such as pE194 or pE5 (Firth et al. 2006).

Methylation of the A2058 residue confers cross-resistance to macrolides, lincosamides and streptogramins B (MLSB). This resistance profile is known as the MLSB phenotype which can be either inducible or constitutive.

A newly described enzyme encoded by cfr gene methylates the 23S rRNA at the position A2503 (Toh et al. 2007). This enzyme confers cross resistance to lincosamides, streptogramins, chloramphenicol, pleuromutilins and linezolid (Long et al. 2006). The cfr gene is carried on plasmids found both in S. aureus and coagulase negative staphylococci (CNS) (Mendes et al. 2008; Kehrenberg et al.

2007).

Ribosomal mutation

Mutations of ribosomal proteins L22 and L4, encoded by rplV and rplD genes respectively, or in rrn genes at position A2058 and A2059 have been rarely described in S. aureus strains, mostly isolated from cystic fibrosis patients (Prunier et al. 2002; 2005). These mutations also confer cross-resistance to macrolides, lincosamides and streptogramins B.

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Table 3. Genes and resistance phenotypes of aminoglycoside modifying enzymes in S. aureus

Enzyme Gene Resistance profile

Transposon/plasmid (Frith et al. 2006) AAC(6’)-APH(2”) aac(6’)-Ie+aph(2”) Gentamicin, tobramycin, kanamycin Tn4001

ANT(4’)-I ant(4’)-Ia Tobramycin, kanamycin pUB110

APH(3’)-III aph(3’)-IIIa Kanamycin, neomycin Tn5404, Tn5405

ANT(6)-I ant(6)-I Streptomycin Tn3854

ANT(9)-I ant(9)-I Spectinomycin Tn554

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1.1.4.4.2. Active efflux

The msrA gene codes for ATP-dependant efflux pump (ABC) which confers resistance to 14- and 15-membered macrolides and streptogramins B (Leclercq 2002). Other ATP-binding transporters of lincosamides and streptogramins A described in S. aureus are encoded by vgaA and vgaB genes (Roberts 2008).

1.1.4.4.3. Inactivating enzymes

Many enzyme systems that modify macrolides, lincosamides, streptogramins A or streptogramin B have been described in staphylococci including S. aureus (Lina et al. 1999b; Roberts 2008). vatA, vatB and vatC genes encode acetyltransferases which inactivate streptogramin A. vgb gene encodes a lactonase which confers resistance to streptogramin B and ereA/ereB genes encode enzymes that hydrolyze the lactone ring of macrolides. lnu(A) (formerly known as linA or linA’) encodes O-nucleotidyltransferase which inactivates lincomycin and clindamycin (Lüthje et al. 2007). These determinants are frequently found in combination with erm genes (Lina et al. 1999b). These combinations of genes may confer resistance to pristinamycin and quinupristin-dalfopristin.

Shortly after erythromycin was introduced in 1952, the first macrolide resistant staphylococci were described in Europe, USA and Japan. In Europe, MRSA strains showed a high rate of resistance to MLS (Fluit et al. 2001; Van Bambeke et al. 2008). MRSA strains resistant to MLS harbour mainly ermA or ermC genes (Lina et al. 1999b; Denis et al. 2002b). Other mechanisms including efflux pumps and inactivating enzyme have been rarely observed in S. aureus, often in combination with methylase gene. msrA is the most frequently reported gene encoding for efflux pumps, particularly in CA-MRSA strains (Tenover et al. 2006;

Pérez-Vázquez et al. 2008).

1.1.4.5. Aminoglycosides

Aminoglycosides inhibit protein synthesis by binding to the 30S ribosomal subunit.

The major mechanism of resistance to aminoglycosides in staphylococci is drug inactivation by modifying enzymes (Vakulenko et al. 2003). These enzymes include N-acetyltransferases (AAC), O-nucleotidyltransferase (ANT) and O- phosphotransferase (APH) which modify specific amino or hydroxyl groups.

Five aminoglycoside modifying enzyme (AME) genes have been identified in S.

aureus (Table 3) (Vakulenko et al. 2003). The bifunctional aac(6’)-Ie + aph(2’’) gene, carried on transposon Tn4001, confers cross-resistance to gentamicin, tobramycin and kanamycin. High-level resistance to tobramycin is mediated by the ant(4’)-Ia gene located on plasmid pUB110 which can be integrated in different SCCmec types. The aph(3”)-IIIa gene confers resistance to kanamycin and is carried on various transposons located either on chromosome or plasmid. The ant(6)-I and ant(9)-I genes encode resistance to streptomycin and spectinomycin, respectively. These genes are frequently found associated in the same strain.

Surveys conducted in Europe and Japan in the 1980s and 1990s found a high prevalence of MRSA strains resistant to aminoglycosides, harbouring ant(4’)-Ia gene (>50%) and the aac(6’)-Ie + -aph(2”) gene (>60%) in MRSA strains (Wildemauwe et al. 1996; Schmitz et al. 1999; Lelièvre et al. 1999) . The aph(3’)- IIIa gene was found less frequently (<10%) associated with one of the two other AME genes. At the end of the 1990s, the prevalence of MRSA strains carrying the

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aac(6’)-Ie + aph(2”) gene decreased significantly in Europe (Galbart et al. 2000;

Denis et al. 2003; Campanile et al. 2009; Vindel et al. 2009).

1.1.4.6. Tetracyclines

Tetracyclines inhibit protein synthesis by binding to the 30S subunit of the bacterial ribosome and blocking the association of aminoacyl-tRNA with the ribosome. New derivatives of tetracyclines have been recently developed.

Tigecycline, a 9-t-butylglycylamido derivative of minocycline, is the first drug of the new class of antimicrobial agents called glycylcycline.

Two mechanisms of resistance to tetracyclines have been reported in S. aureus:

active efflux and ribosome protection (Trzcinski et al. 2000; Chopra et al. 2001;

Schmitz et al. 2001; Roberts 2005). The nomenclature of tetracycline resistance determinants is available online (Faculty Washington online).

1.1.4.6.1. Efflux systems

The tet(K) and tet(L) genes encode efflux pumps that belong to the major facilitator superfamily (MFS) group 2. These two genes are found on small plasmids, such as pT181 (Firth et al. 2006). They confer resistance to tetracycline and chlortetracycline but neither to minocycline nor doxycycline. Glycylcyclines are not transported by any of these efflux pumps. Recently, a new MATE family efflux pump has been reported that confer reduced susceptibility to tigecycline in a laboratory-mutant S. aureus (McAleese et al. 2005).

1.1.4.6.2. Ribosomal protection

Ribosomal protection proteins have N-terminal regions with close homology to those of elongation factor GTPAses EF-Tu and EF-G. These proteins interact with the ribosome by removing bound tetracycline from the ribosome. They confer cross resistance to tetracycline, doxycycline and minocycline but not to glycylcyclines. Like with efflux proteins, expression of ribosomal protection can be induced by pre-incubation with tetracycline (Trzcinski et al. 2000). Two ribosomal protection protein genes, tet(M) and, less frequently, tet(O), have been described in S. aureus. The tet(M) gene is found on conjugative transposons, such as Tn5801 and Tn916, which are generally located on the chromosome in S. aureus (Firth et al. 2006; de Vries et al. 2009).

Considerable geographical and temporal variations have been reported in the antimicrobial susceptibility of S. aureus to tetracyclines (Fluit et al. 2001; Van Bambeke et al. 2008). Interestingly, high resistance rate are found in livestock- associated (LA)-MRSA strains (Nemati et al. 2008; Lewis et al. 2008; Kadlec et al.

2009; Denis et al. 2009a).

1.1.4.7. Mupirocin

Mupirocin, an analog of isoleucine, inhibits bacterial protein synthesis by binding competitively isoleucyl-tRNA synthetase. Two types of mupirocin resistance have been observed in S. aureus. Low-level resistance (MIC between 8 and 256 mg/l), is attributed to point mutations in the chromosomal gene encoding isoleucyl-tRNA synthetase (Fujimura et al. 2003; Hurdle et al. 2004). High-level resistance (MIC >

256 mg/l) is due to an acquired gene, mupA, which encodes an additional enzyme ileS-2 with no affinity to mupirocin (Udo et al. 2001). The mupA gene has been

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