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Molecular Epidemiology of Methicillin-Resistant

Staphylococcus aureus

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Örebro Studies in Medicine 20

Carolina Berglund

Molecular Epidemiology of Methicillin-Resistant

Staphylococcus aureus

Epidemiological aspects of MRSA and the dissemination

in the community and in hospitals

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© Carolina Berglund, 2008

Title: Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals.

Publisher: Örebro University 2008

www.publications.oru.se

Editor: Maria Alsbjer

maria.alsbjer@oru.se

Printer: Intellecta DocuSys, V Frölunda 08/2008

issn 1652-4063 isbn 978-91-7668-611-9

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ABSTRACT

Carolina Berglund (2008): Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals. Örebro Studies in Medicine 20, 126 pp.

Methicillin-resistant S. aureus (MRSA) arise by the acquisition of the mecA gene located on the staphylococcal cassette chromosome mec (SCCmec) that occur in at least six different variants, type I, II, III, IV, V and VI. MRSA has shown a unique capability to adapt to almost every situation, as demonstrated by the ability to accumulate resistance to basically all groups of antibiotics. Furthermore, the recent changing epidemiology and increase of MRSA in the healthy population outside the hospitals may be a serious future threat, especially since these community-acquired MRSA (CA-MRSA) may possess virulence genes, such as the Panton Valentine leukocidin (PVL), that confers a higher pathogenic po-tential in comparison with the nosocomial MRSA or methicillin-susceptible S. aureus.

This thesis describes the molecular epidemiology of MRSA with focus on community-acquired isolates that represent more than half of the MRSA cases in Örebro County. Investigation of the long-term epidemiology revealed that SCCmec was integrated at several occasions into unrelated clones of S. aureus and it was clear that the CA-MRSA had arisen independently from the nosocomial MRSA by the acquisition of predominantly type IV and V SCCmec. In addition, the birth of a new clone of MRSA at the neonatal intensive care unit in Örebro University Hospital was observed, where a variant of a type V SCCmec had most likely been horizontally transferred from a methicillin-resistant Staphylococcus

haemolyticus (MRSH) into the chromosome of a susceptible S. aureus (MSSA).

Many of the MRSA from Örebro County, and elsewhere, were shown to carry SCCmec other than type I, II, II, IV, V or VI, or subtypes other than IVa, IVb, IVc or IVd and these were mainly community-acquired. In this thesis, two new subtypes of the type IV SCCmec were described with common J1-regions shared by other types of SCCmec (type I and II), which indicated a close relationship and also that J1-regions had occurred as precursor SCC. In addition, the type IV SCCmec was detected in heterogeneous clones of MRSA in Sweden. Furthermore, a novel SCCmec was identified in a CA-MRSA, which was a unique composite element consisting of a SCCmec and a chromosome cassette, that were independently excised from the S. aureus chromosome. The nucleotide sequence of the novel SCCmec was nearly identical to the element SCCmercury and indicated that

ccrC driven SCC existed as a primordial element into which the mec complex or mercury

resistance determinants have been integrated independently and it is capable of transfer-ring resistance traits among staphylococci.

More than 60 % of the CA-MRSA carried the genes for PVL and further investigation indicated that both PVL positive MRSA and MSSA are genetically diverse, but in contrast, the PVL genes were well conserved in these different clones of S. aureus. In addition, a non-synonymous mutation detected in the lukS-PV had no consequence for the biological activity of the toxin.

In conclusion, nosocomial MRSA disseminate throughout the whole world by colonized or infected persons following health care contact, while in contrast, the CA-MRSA appear to arise spontaneously by horizontal acquisition of SCCmec into the chromosome of a pre-viously susceptible S. aureus from a donor, possibly methicillin-resistant CoNS. This thesis contributed to an insight on the evolutionary origin of MRSA and the understanding of the epidemiology of community-acquired MRSA. Furthermore, it has provided important information that will help to improve the identification and diagnosis as well as prevention

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of MRSA. This highlights the significance of emergence of new effective clones of MRSA in the community, with potential to cause serious infections that are difficult to treat, but also to disseminate among the healthy population.

Keywords: methicillin-resistant Staphylococcus aureus (MRSA), community-acquired

MRSA (CA-MRSA), staphylococcal cassette chromosome mec (SCCmec), multilocus sequence typing (MLST), horizontal transfer, Panton Valentine leukocidin (PVL).

Carolina Berglund, Department of Clinical Microbiology, Örebro University Hospital, SE-701 85 Örebro, Sweden. EMAIL: carolina.berglund@orebroll.se

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POPULÄRVETENSKAPLIG SAMMANFATTNING

Methicillin-resistenta Staphylococcus aureus (MRSA) som bär på genen mecA, har förekommit och spridit sig över hela världen, främst i sjukhusmiljö, och orsakat utbrott av vårdrelaterade (så kallade nosokomiala) infektioner. Dessa infektioner kan inte behandlas med stafylokock-penicilliner och MRSA-bakterierna är ofta resistenta även mot flera andra grupper av antibiotika vilket medför att infektionerna är påtagligt svårbehandlade. Under senare år har emellertid allt fler fall beskrivits av samhällsförvärvad MRSA infektion, det vill säga uppträdande av MRSA hos personer som tidigare ej har haft kontakt med sjukhusvård eller behandlats med antibiotika. Det har länge varit oklart om samhällsförvärvad MRSA (community-acquired [CA-MRSA]) representerar spridning av bakterier från sjukhusmiljön ut till samhället eller om dessa MRSA är spontant uppträdande. Många av dessa stammar har dessutom visat sig bära på sjukdomsrelaterade faktorer som vanligen inte återfinns hos S. aureus, t.ex. Panton Valentine leukocidin (PVL) som associeras med hudinfektioner och allvarlig lunginflammation med hög dödlighet hos unga och annars friska individer.

Denna avhandling beskriver den molekylära epidemiologin hos MRSA med fokus på de samhällsförvärvade isolaten som utgjorde mer än hälften av samtliga fall av MRSA i Örebro län och som dessutom ofta producerade PVL, vars funktion vidare analyserades i detalj. Undersökning av ursprung och släktskap hos samtliga MRSA som isolerats i Örebro län, samt karaktärisering av det gensegment som kallas staphylococcal cassette chromosome mec (SCCmec) vilket innehåller genen mecA och ibland även andra resistensgener, visade att CA-MRSA inte är relaterade till de nosokomiala MRSA, och att dessa uppstått oberoende av varandra. Flertalet MRSA visade sig dessutom bära på SCCmec som tidigare inte beskrivits. Troligen har dessa MRSA uppstått genom ett genetiskt utbyte av SCCmec mellan methicillin-resistenta koagulas-negativa stafylokocker, som utgör huvudparten av normalfloran på huden, och methicillin-känsliga S. aureus som därvid erhåller genen mecA och resistensmekanismer mot samtliga stafylokockantibiotika. I den här avhandlingen framläggs bevis för att ett sådant genetiskt utbyte kan ha skett på Barnkliniken på Universitetssjukhuset i Örebro i slutet på 1990-talet, vilket resulterade i uppkomsten av en ny klon av MRSA som orsakade ett allvarligt utbrott. Kartläggning av DNA-sekvensen hos flertalet unika SCCmec från svenska MRSA gav dessutom en bättre förståelse för hur resistens uppkommer och sprider sig, samt mekanismerna bakom detta. Dessa nya kunskaper kan bidra till en förbättrad diagnostik av MRSA. Detta är framför allt av stor betydelse eftersom nya och effektiva kloner av MRSA verkar kunna uppstå ute i samhället med potential att orsaka svårbehandlade infektioner och som dessutom kan sprida sig bland den friska befolkningen.

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The bamboo that bends is stronger than the oak that resists (Japanese proverb)

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ABBREVIATIONS

ACME arginine catabolic mobile element

agr accessory gene regulator

BLAST Basic Local Alignment Search Tool

bp basepair

CA-MRSA community-acquired MRSA

CC clonal complex

ccr cassette chromosome recombinase CLSI Clinical and Laboratory Standards Institute

coa coagulase

CoNS coagulase-negative staphylococci

dcs downstream constant region (of SCCmec) ddNTP dideoxynucleoside triphosphate

DNA deoxyribonucleic acid

dNTP deoxynucleoside triphosphate

e.g. exempli gratia (for example)

E-MRSA epidemic MRSA

et al et alibi (and others)

ET exfoliative toxin

fem factors essential for methicillin-resistance HA-MRSA hospital-acquired MRSA

hsd host specificity determinant (restriction-modification system)

i.e. id est (that is)

ICU intensive care unit

IS insertion sequence

ISS integration site sequence

IWG-SCC the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements

J junkyard region (of SCCmec)

MIC minimum inhibitory concentration MLST multilocus sequence typing

MRSA methicillin-resistant Staphylococcus aureus

MRSH methicillin-resistant Staphylococcus haemolyticus

MSCRAMM microbial cell surface components recognizing adhesive matrix molecules MSSA methicillin-susceptible Staphylococcus aureus

NCBI National Center for Biotechnology Information NICU neonatal intensive care unit

NT not typable

orf open reading frame

p plasmid

PBP penicillin-binding protein

PCR polymerase chain reaction PFGE pulsed-field gel electrophoresis

PHP phene plate system

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PSM phenol-soluble modulin PVL Panton Valentine leukocidin

ROX 6-carboxy-x-rhodamine

S Staphylococcus

SC Staphylocoagulase

SCCmec staphylococcal cassette chromosome mec

SE staphylococcal enterotoxin

SmaI restriction enzyme (from Serratia marcescens)

Spa staphylococcal protein A

SRGA the Swedish Reference Group for Antibiotics SSSS staphylococcal scalded skin syndrome

ST sequence type

TAMRA 6-carboxytetramethylrhodamine

Tm melting temperature of DNA

Tn transposon

TSST toxic shock syndrome toxin

UK United Kingdom

US United States (of America)

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ORIGINAL PAPERS

Paper I Carolina Berglund, Paula Mölling, Lennart Sjöberg, Bo Söderquist. 2005.

Predominance of staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA) in a Swedish county and presence of unknown SCCmec types with Panton-Valentine leukocidin genes. Clin. Microbiol. Infect. 11:447-56.

Paper II Carolina Berglund, Paula Mölling, Lennart Sjöberg, Bo Söderquist. 2005.

Multilocus sequence typing of methicillin-resistant Staphylococcus aureus from an area of low endemicity by real-time PCR. J. Clin. Microbiol. 43:4448-54.

Paper III Carolina Berglund, Bo Söderquist. 2008. The origin of a

methicillin-resistant Staphylococcus aureus (MRSA) at a neonatal ward in Sweden - possible horizontal transfer of a Staphylococcal Cassette Chromosome mec between methicillin-resistant Staphylococcus haemolyticus and

Staphylococcus aureus. Accepted for publication in Clin. Microbiol. Infect.

Paper IV Carolina Berglund, Xiao Xue Ma, Megumi Ikeda, Shinya Watanabe, Bo

Söderquist, Teruyo Ito and Keiichi Hiramatsu. 2008. Genetic diversity of Methicillin-Resistant Staphylococcus aureus carrying the type IV SCCmec in Sweden. Provisionally accepted in J. Antimicrob. Chemother.

Paper V Carolina Berglund, Teruyo Ito, Megumi Ikeda, Xiao Xue Ma, Bo

Söderquist, and Keiichi Hiramatsu. 2008. Novel type of Staphylococcal Cassette Chromosome mec in a Methicillin-Resistant Staphylococcus

aureus isolated in Sweden. Antimicrob. Agents. Chemother. 52: In press.

Available online 1 August 2008.

Paper VI Carolina Berglund, Gilles Prévost, Benoît-Joseph Laventie, Daniel Keller,

Bo Söderquist. 2008. The genes for Panton Valentine leukocidin (PVL) are conserved in diverse lines of resistant and

methicillin-susceptible Staphylococcus aureus. Microb. Inf. 10:878-884. Available online 7 May 2008.

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CONTENTS

INTRODUCTION ... 15

Staphylococci... 15

S. aureus chromosome ... 16

S. aureus enzymes and toxins... 17

Staphylococcal enzymes... 17

Toxic shock syndrome toxin... 18

Enterotoxins... 18

Exfoliative toxins... 19

Hemolysins... 19

Leukocidins... 19

Antibiotic treatment of Staphylococcal infections... 20

Penicillin... 21

Multiresistance... 22

Methicillin-resistance... 23

Mechanisms of resistance... 23

Staphylococcal cassette chromosome mec ... 24

SCCmec types... 26

SCC without the mecA gene ... 31

Evolution of MRSA... 31 Epidemiology... 33 Epidemic MRSA... 34 Community-acquired MRSA... 34 Typing of MRSA... 36 Antibiogram... 37

Pulsed field gel electrophoresis... 37

Polymerase chain reaction... 38

Real time PCR... 39

Nucleotide sequencing... 41

DNA Cloning... 42

Multilocus sequence typing (MLST)... 43

SCCmec typing ... 45

AIMS ... 47

MATERIALS AND METHODS ... 49

Bacterial isolates... 49

Culturing... 51

Antibiotic susceptibility testing... 51

DNA preparation... 52

Detection of nuc and mecA ... 52

PFGE... 53

Detection of the PVL locus, ETA, ETB, and TSST-1... 53

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Purification... 56

Sequencing... 56

MLST... 57

Spa typing... 59

Partial sequencing of the class C mec complex ... 59

Long range PCR in SCCmec carrying the class C mec complex ... 60

Staphylocoagulase typing... 61

Nucleotide sequencing of novel staphylocoagulase genes... 61

Nucleotide sequencing of novel type IV SCCmec... 61

Cloning of MRSA JCSC6082... 63

PCR amplification and sequencing of lukS-PV and lukF-PV... 65

Detection of the LukS-PV, LukF-PV, LukE and LukD... 65

Purification of the LukS-PV and LukF-PV... 65

Human PMNs and flow cytometry measurements... 66

Competition experiments of the LukS-PV... 66

Evaluation of pre-pore formation and Ca2+ entry... 67

Ethidium entry... 67

Nucleotide accession numbers... 68

RESULTS AND DISCUSSION... 69

Molecular epidemiology of MRSA (Paper I and II)... 69

The birth of a new MRSA in Örebro (Paper III)... 75

Novel subtypes of type IV SCCmec in Swedish MRSA (Paper IV)... 79

A novel type VII SCCmec in a Swedish CA-MRSA (Paper V) ... 86

Panton Valentine leukocidin in S. aureus (Paper VI)... 93

FURTHER PERSPECTIVES ... 99

CONCLUSIONS ... 103

ACKNOWLEDGEMENTS ... 105

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INTRODUCTION

Staphylococci

The staphylococci are members of the Micrococcaceae bacterial family and consist of more than 40 species and subspecies (http://www.bacterio.cict.fr/). Staphylococcus

aureus (S. aureus) is the foremost important and most studied human pathogen of the

staphylococcal genus and it is capable of causing a broad variety of infections ranging from minor skin infections to serious conditions such as osteomyelitis, bacteremia and infective endocarditis. However, S. aureus is usually a harmless coloniser of about one third of healthy humans without causing infections and it is most likely found in the nares [102, 133]. Accordingly, nasal carriage of S. aureus is associated with an increased risk for being infected by the own strain [118] and such infections typically arise when the skin and mucous barriers are damaged, after surgery or if the immune system is compromised. Moreover, S. aureus is capable of producing a wide range of enzymes and toxins that are associated with specific clinical conditions, such as food poisoning or the toxic shock syndrome [103].

S. aureus is primarily separated from the majority of the other staphylococci by its

production of the enzyme coagulase and the subsequent ability to coagulate plasma. Remaining staphylococci are hence referred to as coagulase-negative staphylococci (CoNS) and many of these are found as natural colonisers of the human skin and mucous membranes, whereas other CoNS are exclusively identified in animals [89]. The S. epidermidis is the most important representative of the CoNS group, its entire genome has been determined [48] and it is associated with infections in patients with implanted medical devices or in immunocompromised patients [173], while S.

saprophyticus is a common cause of urinary tract infections in women [167]. S. haemolyticus is a frequently found CoNS in human blood cultures and it may cause

clinical relevant infections such as prosthetic joint infections, septicemia, peritonitis, otitis, and urinary tract infections, and this species has also been thoroughly studied by whole genome sequencing [154]. In addition, CoNS are the foremost cause of infections occurring in neonatal intensive care units (NICUs) where they may endemically persist for many years [88]. In Örebro County (approximately 280 000 inhabitants), Sweden, the CoNS accounted for 10.8 % to 15.7 % of the true bacteremias detected in blood cultures at the Department of Clinical Microbiology at Örebro University Hospital, between the years 2000 and 2005, while S. aureus accounted for 12.2 % to 15.5 % during the same time period (unpublished data).

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S. aureus chromosome

The circular chromosome of S. aureus is approximately 2.85 Mb and it is composed of ancestral DNA and genomic islands (GI) that have been acquired from other bacteria. Genes that have housekeeping functions, i.e. are essential for growth, metabolism and survival, comprise the ancestral DNA. In contrast, genes that are involved in specific functions, such as virulence or resistance, mostly consist of mobile genetic elements (bacteriophages, plasmids, insertion sequences, transposons and chromosomal cassettes) that have been horizontally transferred between bacteria. There are eight GIs identified on the S. aureus chromosome and they convey pathogenicity or antibiotic resistance but is unknown whether these GIs are self-transmissible [72, 101]. Horizontal gene transfer in bacteria may occur by three different mechanisms; conjugation, transformation or transduction, as detailed in figure 1.

Figure 1. The three main mechanisms of horizontal transfer by which bacteria acquire antibiotic resistance genes are; a) Transformation, which involves acquisition of DNA from the extracellular environment, for example when DNA is released on lysis of an organism and subsequently picked up by another organism. The acquired gene can be integrated into the chromosome or into a plasmid of the recipient cell. b) Transduction involves acquisition of foreign DNA by means of bacteriophages. c) Conjugation occurs by direct contact between two bacteria and the formation of a mating bridge across the bacteria in which the DNA is exchanged. (Reproduced with permission, Nature reviews Microbiology).

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Substantial information on the pathogenicity as well as mechanisms of antibiotic resistance has been revealed after the determination of the entire genome of several S.

aureus isolates displaying different origins and characteristics. Information about the

genetic background as well as about additional genes that have been acquired horizontally contribute to the understanding of the unique versatility and characteristics of S. aureus that may help to predict the evolution and outcome of the staphylococcal infections.

S. aureus enzymes and toxins

S. aureus produce numerous of cell wall anchored proteins and extracellular products,

mainly enzymes and toxins, that possibly will enhance the virulence and subsequently contribute to infections [103]. Several cell surface proteins have been suggested to play an important role in the host interaction and pathogenicity of staphylococci, such as the staphylococcal protein A (spa), the adhesion proteins denominated microbial cell surface components recognizing adhesive matrix molecules (MSCRAMMs), and the capsule polysaccharides that protects the bacteria from being phagocytised [29, 165]. The various staphylococcal enzymes and toxins could act together or alone and the expression is coordinated by global regulatory systems, such as the agr (accessory gene regulator), and induces the production of various proteins during different bacterial growth phases. The staphylococci are hence capable to sense the surrounding environment and adjust the production of virulence factors suitable for colonization, dissemination and for causing infection [52]. For example, the combination of various toxins has been shown to cause specific syndromes, such as antibiotic-associated diarrhea and the simultaneous production of enterotoxin A and leukocidins lukE-lukD [53].

Several of the staphylococcal toxins are carried on bacteriophages, for example the enterotoxin A, exfoliative toxin A and Panton Valentine leukocidin (PVL). The bacteriophages are divided into five families and it has been shown that a S. aureus could only carry one bacteriophage from the same family simultaneously, which has been explained by phage immunity or competition for insertion sites. Consequently, the acquisition of related bacteriophages is not possible and may influence the evolution and success of a specific S. aureus isolate [67, 101].

Staphylococcal enzymes

The function of the staphylococcal enzymes is primarily to produce nutrients that are necessary for the cell growth. However, some enzymes have also been proposed to play an important role in pathogenicity. For example, the proteases may be involved in the

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inactivation of host defence peptides and perhaps also blocking of antibodies. Hyaluronidase is an enzyme produced by approximately 90 % of S. aureus and promotes bacterial spread by digesting hyaluronic acid in connective tissue and thereby degrading the tissue. Lipases may have an effect on the host immune response by inactivating the fatty acids that are intended to disrupt the bacteria [29]. The enzyme coagulase may protect the bacteria from the host defence by clotting fibrin around a focal infection, although its exact role in staphylococcal pathogenesis is unclear [46].

Toxic shock syndrome toxin

The toxic shock syndrome (TSS) is a rare condition associated with infections in children related with wounds or burn damage, as well as in women using tampons during menstruation and these infections are characterized by a rapid onset with high fever and multiorgan failure [101]. These serious conditions, as well as staphylococcal scarlet fever, is caused by S. aureus that are producing the toxic shock syndrome toxin-1 (TSST-toxin-1), which belong to the group of staphylococcal superantigens that are capable of inducing a massive activation of T-cells [29]. TSST-1 has been identified in as many as 20 % of S. aureus strains in the UK as well as in Sweden [81, 101], however, the TSST-1 producing strains have been reported to consist of a limited amount of clones and to be rarely or never found in the remaining S. aureus clones [37, 134, 157]. The gene encoding TSST-1 (tst) is located on the pathogenicity island designated SaPI 1 that is suggested to be a less transmissible mobile element, which would explain presence of

tst in only a few restricted clones [134]. Enterotoxins

The staphylococcal enterotoxins also belong to the group of staphylococcal superantigens and consist of several toxins of which some have been suggested to be involved in an increased virulence that have been reported in strains of nosocomial S.

aureus [67, 157]. There were initially five serotypes of staphylococcal enterotoxins

(SEA to SEE) reported, however, numerous additional (SEG to SEU) enterotoxins have continuously been identified [10]. The SEA has been associated with more severe infections, such as staphylococcal food poisoning and septic shock, and induces a strong proinflammatory response in comparison with other enterotoxins. Staphylococcal food poisoning is actually not an infection but an intoxication caused by the toxins that were produced by the bacteria prior to ingestion [29]. In contrast, a defined group of enterotoxins, the enterotoxin gene cluster (SEG, SEI, SELM, SELN, SELO), is correlating only with colonization and is not associated with infection [30].

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Most of the enterotoxins are carried by phages, plasmids, pathogenicity islands or other mobile genetic elements [67].

Exfoliative toxins

There are four serological isoforms of exfoliative toxins (ETA, ETB, ETC and ETD) of

S. aureus, which may cause exfoliation of the skin epidermis followed by secondary

infections. The exfoliative toxins A and B are the two most important isoforms in humans and those are associated with staphylococcal bullous impetigo and staphylococcal scalded skin syndrome (SSSS) that predominantly affects neonates and children [29]. The ETD has been associated with wound infections, while the ETC has not yet been associated with human disease [136]. The eta gene (encoding exfoliative toxin A) is chromosomal, while the etb (encoding exfoliative toxin B) is located on a plasmid. The exfoliative toxin genes are infrequently found in S. aureus, however major geographical differences in both prevalence and presence of particular toxins have been reported [52, 97].

Hemolysins

The staphylococcal D-toxin (D-hemolysin) is encoded by the hla gene and has been

extensively studied and shown to be associated with an increased virulence of S.

aureus. The D-toxin creates pores in cell membranes and is capable of lysing

erythrocytes and it may also stimulate apoptosis in lymphocytes. In addition, the

D-toxin is dermonecrotic and neurotoxic. Interestingly, E-hemolysin (hlb) displays the opposite effect by acting on the same sites of the erythrocyte membrane and thereby

preventing D-toxin to attach and create a pore. Yet, the haemolytic activity of

E-hemolysin is enhanced if the erythrocytes are first refrigerated prior to the toxin treatment. The action and importance of the G-toxin in staphylococcal virulence has not been fully investigated, but it is suggested to also display pore-forming activity [29].

Leukocidins

The J-hemolysins and PVL are staphylococcal bi-component toxins that display a

leukocytolytic activity. These leukocidins are referred to as synergohymenotropic toxins and the cytolytic activity requires the action of two components, the S and the F

subunits. The leukocidins are associated with a total of five genes; the J-hemolysins are

encoded by three orfs, hlgA, hlgB and hlgC, whereas PVL is encoded by two co-transcribed orfs, the lukS-PV and lukF-PV. The hlgA, hlgC and lukS-PV are

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functioning as S components, whereas hlgB and lukF-PV are functioning as F components. There is a complex and not yet fully understood interplay between these subunits that will form six combinations with various leukocytolytic effect [112, 138].

The J-hemolysins are produced by 99 % of S. aureus isolates and therefore, its

role in the staphylococcal pathogenesis has been difficult to infer [138]. In contrast, the PVL toxin has been specifically associated with community-acquired primary skin and soft tissue infections, but also severe necrotizing pneumonia in young and otherwise healthy individuals [49, 100]. The prevalence of the PVL locus is reported to be below 2 % in the general S. aureus population, but with a much higher prevalence in community-acquired infections [12, 49, 142]. A recent study presented a low prevalence of the PVL locus in a consecutive material of isolates obtained from cutaneous infections (2 %), pneumonia (8 %) and bacteremia (1 %) in Örebro County in Sweden [77]. The PVL has toxic effect on the polymorphonuclear cells (PMNs), monocytes and macrophages, but not on human erythrocytes. The PMNs are involved in the inflammatory response and they play a major role in the protection against infection. Depending on the concentration of the toxin, the PVL will act upon these cells by either creating a pore that will eventually lyse the cells, or it will cause apoptosis, probably involving pore formation of the mitochondrial membrane [47]. In addition, the PVL will induce calcium influx in the PMNs and trigger a cascade of signal transduction that will initiate the production of cytokines and other inflammatory mediators [139, 166].

Antibiotic treatment of Staphylococcal infections

The staphylococci cause a wide range of infections. The choice of antibiotic treatment should assess the location and severity of the infection. In addition, the mechanism of the antimicrobial agent should be validated, thus it could be either bacteriostatic (inhibiting) or bactericidal (lethal) as well as the type of infection is important in determining which kind of drug to use. Furthermore, the treatment could be complicated, for example in case of infections associated with foreign bodies when the staphylococci often are adhering to the foreign material and embedded in biofilm and thus protected from the blood stream and consequently also to the antibiotics. In addition, difficult to treat infections include localized or focal infections such as abscesses or osteomyelitis in which penetration of antibiotics is poor. The duration of treatment is also related to the specific type and localization of the infection and varies from days to months or even life long suppressive treatment in some selected cases [42, 78].

There is a wide range of antibiotics available for the treatment of staphylococcal infections, thus isoaxazolyl-penicillin is generally the most effective and appropriate

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choice for treatment of infections caused by susceptible staphylococci. Other antibiotics, such as vancomycin, clindamycin and fusidic acid are used predominantly for treatment of resistant staphylococci or in case of allergy [42, 78].

Penicillin

Many of the formerly serious infections caused by S. aureus were possible to successfully cure after the introduction of penicillin in 1941 [1]. However, only four years after the introduction of penicillin in clinical practise, penicillin-resistant staphylococci were identified [87]. The prevalence of penicillin-resistant S. aureus within hospitals began to increase after World War II because of the increased availability and perhaps inappropriate use of penicillin, which positively selected the resistant bacteria and allowed these to spread. Initially, penicillin-resistant strains were solely a nosocomial problem in the 1950s, whereas the S. aureus circulating in the community remained susceptible [23]. However, penicillin was considered as an effective agent for treatment of staphylococcal infections until the early 1970s when a Danish study reported a prevalence of penicillin resistance of 85% to 90% for nosocomial isolates of S. aureus, and 65 % to 70 % also in the community [23, 74].

Resistance to penicillin is mediated by the enzyme E-lactamase (penicillinase) that

inactivates the penicillin molecule by hydrolyzing the E-lactam ring before it has

reached its target on the bacterial cell. E-lactamase is encoded by the blaZ gene, which is carried on a large plasmid. The expression of blaZ is under tight regulation of two adjacent regulatory genes, the blaR1 (transmembrane signal transducer) and blaI (repressor). When the penicillin binds the BlaR1 on the surface of the cell membrane an intracellular signalling pathway will be initiated and lead to the cleavage of the BlaI repressor on the operator region, which will allow transcription of the blaZ gene and hence production of E-lactamase [26]. An illustration of the induction of E-lactamase synthesis and regulation is shown in figure 2.

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Figure 2. The induction of E-lactamase synthesis in the presence of staphylococcal penicillins; A) In the absence of penicillin, the BlaI binds to the operator region and thus repress the RNA transcription from

blaZ as well as blaR1 and blaI and the E-lactamase is expressed only at low levels. B) Once the penicillin bind to the transmembrane sensor-transducer BlaR1 it will lead to BlaR1 autocatalytic activation. C-D) The active BlaR1 will cleave and thus inactivate the BlaI, either directly or via a second BlaR2 protein, and allow transcription of blaZ, blaR1 and blaI. E) The E-lactamase, which is encoded by blaZ, hydrolyze the E-lactam ring and thereby inactivates the penicillin (F-G).

Multiresistance

The rapid spread of resistance to penicillin is an example of the unique adaptability of

S. aureus to quickly respond to new environments and antibiotics. Several new

antibiotics were introduced in order to treat the E-lactamase-producing staphylococci,

however, until today, there is not one single group of antibiotics that have not been overcome by S. aureus, which begun with the inactivation of penicillin, followed by erythromycin, tetracycline and aminoglycosides, and more recently new agents such as linezolid and daptomycin [18, 102, 130]. The glycopeptide vancomycin has been considered as the antibiotic of “last resort” available for treatment of multidrug resistant staphylococcal infections, however there are now several reports of S. aureus showing intermediate or high level of resistance also to this drug [63, 147]. There is a wide range of mechanisms altering in bacterial resistance to different groups of antibiotics, including enzymatic inactivation for example of penicillin caused by the action of E-lactamase. Additional mechanisms include efflux pumps located in the cell membrane that transports the antibiotic outside the bacteria, alteration of the target

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site of the antibiotics, decreased cell permeability or capturing of the drug in a thickened cell wall. The mechanisms of vancomycin resistance are not fully understood but are suggested to be multifactorial and to vary in different strains [7, 147]. Acquisition of bacterial resistance could be mediated either by natural mutations in the chromosomal genes or by acquisition of additional genes by genetic exchange and the resistant bacteria are positively selected as the surrounding concentration of antibiotics increase [7, 72].

We might face a worrisome future similar to the period prior to the penicillins because resistance to new agents arise and spread quickly and since it is both time-consuming and expensive to develop new and effective drugs.

Methicillin-resistance

The S. aureus infections are often successfully treated with the E-lactamase stabile antibiotics, i.e. the isoxazolyl-penicillins. Methicillin, originally called celbenin, is a semisynthetic penicillin that was introduced in 1960 in order to treat infections caused by the E-lactamase-producing S. aureus. However, resistance to methicillin was observed within one year [75] and since then, the methicillin-resistant S. aureus (MRSA) has rapidly emerged and disseminated and has become a major nosocomial problem worldwide. These infections are not available for treatment with the staphylococcal penicillins and the MRSA isolates are often in addition resistant to various other groups of antibiotics resulting in difficulties treating such infections [130].

Mechanisms of resistance

Methicillin-resistance is characterized by the acquisition of the mecA gene, which encodes an alternative penicillin-binding protein (PBP) that is referred to as PBP2´ or PBP2a. The PBP2´ confers resistance to all clinically in use E-lactam antibiotics, i.e. the penicillins, cephalosporins, carbapenems and monobactams. The only exception is the E-lactam agents ceftobiprole and ceftaroline, which belong to the next generation‚ broad spectrum‚ cephalosporin antibiotics and remain active despite the presence of the PBP2´ [16, 120]. Actually, methicillin has been replaced by other isoxazolyl penicillins and is no longer used in clinical practise today, although the definition of MRSA has remained.

Penicillin-binding proteins are transpeptidases that catalyze the cell wall assembly and since PBP2´ has low affinity for the E-lactam antibiotics, the peptidoglycan synthesis continues despite the presence of the antibiotics. There are four different PBP

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described in staphylococci and the 78 kDa PBP2´ takes over the function of the ordinary PBPs once the MRSA is exposed to the E-lactam antibiotics [60, 135].

The expression of the mecA gene is regulated by the adjacent genes mecR1 and

mecI (which are shown in figure 3). The regulation of the mecA transcription is not

completely understood but it involves the binding of the E-lactam antibiotics to an extracellular sensor domain and subsequent cleavage of a sensor transducer [8] in a

similar course of action to that described for E-lactamase (as shown in figure 2). This

leads to the activation of at least one intracellular domain that will cleave the repressor and enable transcription of the mecA gene. The induction of the PBP2´ is a slow process in presence of methicillin and oxacillin and the MRSA may appear susceptible in the laboratory. For this reason, cefoxitin is used for laboratory susceptibility testing of S. aureus since this substrate causes stronger induction [45, 145]. Additional genes might influence the expression of mecA, such as the bla regulatory genes (blaR1 and

blaI) and the factors essential for methicillin-resistance (fem) genes that encode

peptidoglycan-modifying enzymes. However, the repressors (mecI and blaI) can repress either mecA or blaZ, whereas the inducers (mecR1 and blaR1) are specific for their respective repressor [8].

MRSA that contain functionally intact mec regulator genes will remain a susceptible phenotype since the transcription of the mecA gene is strongly repressed. Such strains are infrequent and referred to as pre-MRSA [70]. In pre-MRSA,

methicillin-resistance will not be induced by the presence of E-lactam antibiotics.

However, when exposed to antibiotic, the mecI repressor will eventually be deleted or mutations will occur in the mecI or the promoter region where the mecI would bind, allowing production of the PBP2´ and transformation of pre-MRSA into MRSA. The phenotypically susceptible profile is commonly observed in methicillin-resistant CoNS (MR-CoNS) but is probably regulated by additional unknown systems since mutations in the mecI and mecR1 does not seem to be required for the expression of mecA [163].

Staphylococcal cassette chromosome mec

The mecA gene, encoding the alternative PBP2’, is carried on a mobile genetic element, the staphylococcal cassette chromosome mec, SCCmec. Historically, MRSA was found to possess more than 30 kb extra DNA in comparison to methicillin-susceptible S.

aureus (MSSA), and this phenomenon was referred to as additional DNA, or mecDNA

[6]. The mecDNA surrounding the mecA gene was thoroughly investigated and subsequently described as the specific structure responsible for methicillin-resistance and was designated SCCmec [70, 84].

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The SCCmec is integrated at a specific site in the staphylococcal chromosome, the

attBscc, located on the open reading frame X (orfX) with unknown function. The

location on the genome is close to the origin of replication, a position that has been suggested as an advantage for the utilization of the resistance genes [72, 94]. The SCCmec is precisely excised from the chromosome by the action of the cassette chromosome recombinases, ccr. SCCmec is demarcated by integration site sequences (ISS) consisting of direct and inverted repeat sequences that are recognized by the recombinases for excision and integration of the SCCmec from and into the staphylococcal chromosome. The Ccr proteins belong to the invertase/resolvase family and carry characteristic catalytic motifs at the N-terminal domain. There are five known types of ccr in MRSA and these provide mobility of the SCCmec. Type 1, 2, 3 and 4 ccr is composed of two recombinase genes, ccrA and ccrB, and surrounding orfs [72, 129]. Type 5 ccr composes only one recombinase gene, the ccrC, and surrounding

orfs [71]. In addition, several variants of the ccrC have been identified in both MRSA

and MR-CoNS [57, 62]. It is notable that only type 2 and type 5 ccr is shown to be functionally intact. It has been suggested that alterations of ccr would stabilize the SCCmec on the chromosome and that would be an advantage for MRSA exposed to the antibiotic pressure in the hospitals [123]. Also, the characteristic nucleotide sequences located at the extremities of the SCCmec elements are suggested to influence the efficiency of excision in SCCmec since different Ccr proteins recognize specific nucleotides associated with different SCCmec [71]. The ccr is one of the essential components of the SCCmec, however, recent studies have shown the presence of ccr also on other locations of the chromosome, outside the SCCmec [57].

The mecA gene is always carried by the SCCmec and it is located on a structure

referred to as a mec complex. The mec complex is composed of an insertion sequence (IS) IS431, followed by mecA and the regulatory genes mecR1 and mecI, which could be intact or mutated. There are four major classes of mec complex, class A, B, C and D [72], identified in staphylococci, as well as a few minor variants [144]. The class D mec complex have solely been identified in S. caprae [83]. The genetic structures of the four

mec classes are shown in figure 3.

Figure 3. Structural comparison of the four variants of mec gene complex that have been identified in staphylococci.

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The mec complex and the ccr comprise the two essential components of the

SCCmec. Other regions of the SCCmec are designated junkyard (J) regions and contain other genes, mainly open reading frames with unknown or non-essential function, but also other antibiotic resistance genes, besides mecA, that have been acquired by plasmids or transposons.

Additional structures could be located in the J-regions of the SCCmec and these structures presumably convey resistance to other groups of antibiotics. The type III SCCmec carries a copy of the widespread plasmid pT181 with either tetK or tetM that confers tetracycline resistance. The composite type III SCCmec also carries the mer operon and transposon Tn554, that is either carrying resistance determinants for macrolides, lincosamides and streptogramines (MLS) and spectinomycin, Tn554(MLS) or cadmium resistance, Tn554(cad) [72]. Resistance to aminoglycosides could be introduced either by the plasmid pUB110 or the transposon Tn4001, which in turn is carried on a large plasmid. The insertion sequence IS431, which is a component of the

mec gene complex, may be present in more than one copy on the same SCCmec or the

same mec complex as in the case of the class C mec. IS are mobile genetic elements that can inactivate a gene by direct integration into the gene, or it could activate an adjacent gene by providing a promoter, yet IS do not contain additional genes. IS in staphylococci may therefore contribute to the ability of acquiring antibiotic resistance by either activating or inactivating genes that directly mediate or regulate resistance [154]. The IS431 is regarded to be involved in the integration of exogenous material by homologous recombination between the SCCmec and either plasmids or transposons [8].

Other genes, except those encoding resistance, could be found on SCCmec. The type I SCCmec sometimes carries the pls (plasmin-sensitive surface protein) gene that is encoding a surface adhesion protein and which presence have been shown to decrease the adherence of the MRSA to host proteins, such as fibrinogen, but also to decrease the cellular invasiveness of MRSA [164]. In contrast, the kdp operon in type II SCCmec is regarded to improve the bacterial colonization and invasion of the host. The kdp is a potassium transport system that is suggested to be responsible for the osmolarity resistance in staphylococci that makes the organism tolerant to high concentrations of sodium chloride [94, 130].

SCCmec types

The SCCmec are classified according to the combination of ccr and mec complex contained. There are six different structural types of SCCmec that are designated according to the content of ccr and mec class, and there are two approaches for describing SCCmec. The first is by roman numerals (I, II, III, IV, V and VI) that are

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given to the SCCmec in the order of appearance and it has been the generally most used nomenclature [72]. As an increasing number of MRSA has been investigated, the SCCmec have shown to be extensively diversified and complex, and for that reason a new nomenclature has been suggested for description of the SCCmec [24]. According to the latter, the SCCmec are defined by the ccr type (indicated by a number) and mec class (indicated by an uppercase letter). The six SCCmec types are; type I (1B), type II (2A), type III (3A), type IV (2B), type V (5C), and type VI (4B). The type I SCCmec is defined as carrying the type 1 ccr and class B mec complex. The class B mec complex is partially deleted from the upstream region of mecA, leaving only a 5’-part of mecR1 followed by the insertion of a truncated copy of IS1272. Type II SCCmec carries a type 2 ccr and a class A mec complex with intact regulatory genes mecI and mecR1. Type III SCCmec carries the type 3 ccr and in common with type II SCCmec, a class A mec gene complex. Type IV SCCmec carries a type 2 ccr gene complex and a class B mec gene complex. The type V SCCmec is characterized by the class C mec complex, in which the mecA is bracketed by IS431, and it contains a single copy of a recombinase gene,

ccrC (type 5 ccr). The final type VI SCCmec resembles the type IV SCCmec yet it

carries the type 4 ccr instead of type 2 ccr. A genomic illustration of the six SCCmec types is shown in figure 4.

SCCmec is further classified into subtypes due to differences in the J-regions. Structural variations in the J-regions are the primary explanation for the size differences of the SCCmec types. The size of the type IV SCCmec of MRSA is more variable and the composition more diverse than among the other SCCmec types and there has been most focus on the description of subtypes of this specific element. There are until today six known subtypes of type IV SCCmec based on structural differences in the J1-region located between the ccr complex and the right chromosomal region of SCCmec. The type IVa was first described and frequently found in MRSA from the US and Australia [106, 127], and has then been identified all over the world [68, 98]. Type IVb has been scarcely found since it was first described in the US [86, 127] which is in contrast to the subtype IVc that has spread all over Europe and is one of the dominating clones of MRSA [36, 158, 160]. The type IVc and IVd were frequently found in MRSA isolated in Japan in the early 80s [105] and the latter has recently also been identified in MRSA isolated from horses in Ireland [107]. The subtype IVg was found in MRSA isolated from bovine milk in Korea [95]. The most recently described subtype, IVh, was specific for EMRSA-15 from Portugal [113]. By contrast, the described subtypes IVe and IVf have variations in the J3-regions [24]; type IVe SCCmec has an identical J1-region as the type IVc SCCmec, yet it differs due to the presence or absence of transposon Tn4001 in the J3-region. The type IVf SCCmec carries the same

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J1-region as the type IVb SCCmec and differs because of the presence or absence of the downstream constant region (dcs) in the J3-region [144].

However, variants and subtypes have recently been reported for other types of SCCmec as well [13, 65, 90, 144]. A uniform nomenclature for the various types of SCCmec is presented in table 1. Difficulties in designation of SCCmec may arise when more than one J-region is investigated, since the different regions should be separately described in order to be compared with extant types.

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Figure 4. Schematic illustration and comparison of the six extant types of SCC

mec

described in MRSA. The various

types s

h

are two esse

ntial ge netic components; the ccr gene c o

mplex (type 1, 2, 3, 4 or 5) and the

mec gene com plex (class A, B or C) . The non-essenti al par ts of SCC mec

are divided into three junkyard re

gions (J1 -J3 ) an d m ay co ntain ad dition al antibi otic resistance genes. Integr atio n site seq uences are ind icated as red flags.

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Table 1. A uniform nomenc

lature

for the extant

SCC

me

c elements and description of the major difference

s

between these (reproduced with permission, T.

Ito). J1 re gion J2 and J3 re gions I 1 B.1.1 ( I.1.1 ) 1 B pl s Ia 1B.1.2 ( I.1.2) 1 B pl s carrie s pUB 1 1 0 IIa 2A.1.1 (II.1.1) 2 A kdp operon carrie s pUB 1 1 0 II varian t 2A.1.2 (II.1.2) 2 A kdp operon d o es no t ca rry pUB110 IIb 2A.2 (II. 2) 2 A IIA 2A.3.1 (II.3.1) 2 A same as type IVb inse rt io n o f IS 1182 IIB 2A.3.2 (II.3.2) 2 A same as type IVb IIC 2A.3.3 (II.3.3) 2 A same as type IVb inse rt ion/de let ion of I S1182 IID 2A.3.4 (II.3.4) 2 A same as type IVb inse rt io n o f IS 1182 , doe s not c arry pUB110 IIE 2A.3.5 (II.3.5) 2 A same as type IVb inse rt ion/de let ion of I S1182 , doe s n ot ca rry pUB110 III 3A.1. 1 (III. 1) 3 A IIIA 3A.1.2 (III.1.2) 3 A d o es no t ca rry pT18 1 o r ip s IIIB 3 A.1 .3 (III. 1 .3 ) 3 A d o es n o t car ry p T 1 8 1 IIIC 3A.1.4 (III.1.4) 3 A carrie s mec le ft e x tr em it y polymorp hism (MLEP) ty pe ii IVa 2B.1 (IV.1) 2 B IVb 2B.2.1 (IV.2 .1) 2 B IVc 2B.3.1 (IV.3 .1) 2 B d o es no t ca rry Tn 4001 IVc 2B.3.2 (IV.3 .2) 2 B IVe 2B.3.3 (IV.3 .3) 2 B

same as type IVc

IVf

2B.2.2 (IV.2

.2)

2

B

same as type IVb

IVd 2B.4 (IV.4) 2 B IVg 2B.5 (IV.5) 2 B IVh 2B.6 (IV.6) 2 B V 5 C.1 (V.1) 5 C VI 4B (VI. 1) 4 B C h arac te ris tic f eat ure s in SCC mec Altern ative names ccr type mec cl ass

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SCC without the mecA gene

The staphylococcal cassette chromosomes (SCC) are mobile genetic elements that are mainly associated with the carriage of the mecA gene, encoding methicillin resistance in staphylococci. However, several SCC elements have been found to carry other sets of genes besides mecA. The S. aureus strain MSSA476 carried a SCC-like element

designated SCC476 that was demarcated by ISS and carried a gene with homology to

far1, encoding resistance to fusidic acid [101]. A similar SCC (SCC12263) was identified

in a S. hominis isolate and carried the type 1 ccr [85]. Also, the SCCcap1 was carrying a

capsule gene cluster on a SCC that was integrated at the orfX, and although it was not carrying ccr genes, it was shown to be precisely excised from the chromosome by complementation of ccrA and ccrB [104]. Obviously, SCC is functioning as a vehicle for genetic exchange of both resistance- and pathogenicity genes.

In addition, the type III SCCmec of isolate 85/2082 has been reported to carry a second SCC-element adjacent to the SCCmec [72] in a composite SCCmec. The additional SCC was carrying the mercury resistance operon (mer) and the ccrC (type 5

ccr) and was demarcated by ISS [24]. The additional SCC was precisely excised from

the chromosome and was designated SCCmercury. A similar element, demarcated by DR, was identified in the MSSA strain ATCC25923 and in MRSA strain 81/108 [69, 72]. Also, a 30 kb genetic element designated arginine catabolic mobile element (ACME) has been identified in the chromosome of MRSA representing the USA300 clone. This genetic element contained the arc gene cluster but no ccr, and it has been suggested to enhance the growth and survival of the successful epidemic USA300 MRSA. The ACME was integrated in the orfX, next to the type IV SCCmec, and carried the same ISS sequences as SCCmec, suggesting that it could be excised and integrated from and into the chromosome by the ccr genes [33].

In contrast, excision of a SCCmec from the chromosome of a MRSA results in a susceptible phenotype. It has been shown that the excision of the SCCmec is not always done precisely and that parts of the SCCmec may remain in the chromosome of the post-MRSA [35, 124].

Evolution of MRSA

The first MRSA originated when a SCCmec was integrated into the chromosome of a MSSA [70, 84]. It has been a matter of debate whether this genetic transfer had occurred at only one occasion, and if this MRSA had spread and generated the present geographical variants, or whether MRSA have arisen at several independent occasions [69, 92]. Thorough investigations of large populations of MRSA, employing several

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typing methods (that are presented in detail below), have provided information that indicates arising of MRSA clones continuously [40, 130]. This is mainly supported by the presence of SCCmec in MRSA that are not related to each other but also the general diversified nature of some MRSA clones [40, 130]. However, because of the global presence of a relatively restricted amount of MRSA clones, the MRSA should primarily have disseminated clonally, by human contact and travelling.

The origin of SCCmec and mecA is not known, however, since the SCCmec is widely spread among the MR-CoNS, it has been suggested that horizontal transfer of SCCmec between these species have occurred [57, 73], although it has not been proven. It has been proposed that as much as 60-80 % of the CoNS from several European countries as well as the US are methicillin-resistant [110] and a high prevalence has been reported also in Sweden [167]. The MR-CoNS may therefore be considered as an important reservoir for SCCmec. There is not as much information available about the MR-CoNS in comparison with MRSA, although an increased interest in MR-CoNS has been seen during recent years, which has led to a better insight on the mechanisms of resistance and presence of SCCmec similar to those carried by MRSA [57, 110, 114]. However, detailed studies have shown a more complex structure of SCCmec in MR-CoNS and presence of more than one cassette on the chromosome and commonly also several copies of the ccr genes [57, 114, 154].

In vitro experiments of MRSA have confirmed precise excision of SCCmec from and into the chromosome by the action of type 2 and type 5 ccr, that would further support such hypothesis of a horizontal transfer between the staphylococcal species [71-73]. However, the exact mechanisms behind such genetic transfer remain unknown. The predominance of type IV and type V SCCmec in genetically distant clones of MRSA may suggest that these smaller SCCmec, or parts of SCCmec, are more easily transferred between bacteria [34, 71, 73, 123]. In contrast, the type I, II and III SCCmec are identified in a restricted amount of MRSA clones, which may be explained by its larger size. An alternative explanation is that the SCCmec becomes stabilized on the chromosome and thereby loses its mobility [123]. The restriction-modification system (hsd) is suggested to protect GIs from deletional loss and it may influence the stability of SCCmec on the chromosome in the same manner [5, 71]. In addition, recent data suggest that the movement of SCCmec could be dependent on an orf that is located outside the recognition sites (ISS) that are necessary for the ccr action. This sequence has been absent or not functional in MRSA strains belonging to ST1, a clonal type of MRSA that is known to have stabilized the SCCmec on the chromosome [123].

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Epidemiology

The prevalence of MRSA in the Nordic countries remain below 1 % and this low frequency is most likely due to the infection control procedures including search and destroy strategy applied in order to track MRSA and to prevent further spread, as well as a restricted use of antibiotics. Nevertheless, a worrying increase of the number of MRSA has been observed during the last decade, which could also be explained by a higher awareness and screening of MRSA that results in more MRSA being detected [Swedish Institute for Infectious Disease Control (www.smittskyddsinstitutet.se)]. As shown in figure 5, the prevalence of MRSA in 2006 was low also in the Netherlands, and this is mainly due to preventive efforts similar to those applied in Sweden. A scenario that is in contrast to many other southern European countries possessing a MRSA prevalence of approximately 50 % [http://www.rivm.nl/earss/database].

Figure 5. Prevalence of MRSA in the European Countries in 2006 (http://www.rivm.nl/earss/).

In Sweden, outbreaks of MRSA have been very rare, mainly due to the thorough epidemiological surveillance and infection control as well as isolation procedures of all patients with suspected or verified MRSA infections including carriers. Despite that strategy some outbreaks have occurred and spreading has taken place in Sweden, predominantly in Gothenburg and Stockholm, but also at the Örebro University Hospital. MRSA has been identified in Örebro County (approximately 280 000

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inhabitants), in Sweden, since 1987 and these are mainly represented by sporadic or imported cases of MRSA. However, one MRSA outbreak occurred at the NICU at Örebro University Hospital at the end of the 90s and involved more than 30 patients and hospital staff.

Epidemic MRSA

Strains of epidemic MRSA (EMRSA) are well adapted to survive, spread, and colonize humans in a hospital environment and have disseminated between healthcare facilities over large geographic areas [40]. This could be exemplified by the clones EMRSA-15 and EMRSA-16 in the UK, which dominates in hospitals and account for more than 95% of MRSA bacteremias [76, 116]. The EMRSA-16 is also one of the predominant MRSA clones in the US (USA200) [66]. Today, there are at least 17 EMRSA clones in the UK reported by the Health Protection Agency [76].

A small number of MRSA clones, referred to as pandemic MRSA, have spread worldwide [38, 140]. Pandemic MRSA are EMRSA that have been characterized both upon the type of SCCmec and the genetic background, as depicted by multilocus sequence typing (described below). These clones are named according their origin or other epidemiological properties and are referred to as the Iberian (ST247-SCCmec type I), Brazilian (ST239-SCCmec type III), New York/Japan (ST5-SCCmec type II), Pediatric (ST5-SCCmec type IV), Clone V (ST8-SCCmec type IV) Berlin (ST45-SCCmec type IV), EMRSA-15 (ST22-(ST45-SCCmec type IV) and EMRSA-16 (ST36-(ST45-SCCmec type II) [2, 40, 130]. It has been shown that five of these major MRSA clones accounted for 70 % of all MRSA from southern and eastern Europe, South America, and the USA [2, 40, 130]. Such restricted amounts of clones circulating in the hospitals indicate that these MRSA are adapted to survive in a clinical environment and this may be due to yet unidentified virulence factors. The pandemic MRSA clones have been identified also in Sweden, however, about one third of the MRSA cases in 2007 were known to be imported from abroad (www.smittskyddsinstitutet.se). MRSA that cause nosocomial infections contribute to a higher morbidity and mortality, longer hospital stays, and consequently higher costs of healthcare, in comparison to corresponding infections caused by MSSA [27].

Community-acquired MRSA

MRSA has mainly been a nosocomial problem and especially in persons displaying risk factors, such as surgery or previous antibiotic treatment. However, during the last decade, cases of MRSA have increasingly been described in healthy individuals that had not had any recent contact with healthcare and did not display any of the typically risk

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factors for MRSA [23]. These MRSA are referred to as community-acquired or community-associated MRSA (CA-MRSA) and was first described among injection drug users in Detroit in 1981 [19]. This was followed by several reports in the 1990s of CA-MRSA as a health problem among Australian aborigines [108], prison inmates [21, 132], people practicing contact sports [93] and other categories of people [23, 32, 61]. CA-MRSA is now described all over the world and in Sweden the CA-MRSA have become more prevalent than the nosocomial MRSA isolates (www.smittskyddsinstitutet.se). However, this increase may be partly explained by an extensive contact tracing and screening of MRSA among for example family members. The application of such MRSA preventive strategies in Sweden have managed to keep the CA-MRSA outside the hospitals, but it may be a question of time before these clones invade the hospitals as well.

A major concern is the increased rate of serious infections caused by the CA-MRSA, including primary skin infections and a highly lethal form of necrotizing pneumonia in young and immunocompetent individuals [49, 100]. These stains have been associated with the PVL toxin that may contribute to the higher virulence and increased capability of CA-MRSA to spread among the healthy population and to compete with the natural bacterial flora. At present, there is a controversy in the discussion of the role of the PVL toxin in necrotizing pneumonia [96, 142, 161, 171]. In addition, surface proteins such as spa have been suggested to act together with the PVL toxin and cause the severe tissue damage and inflammation associated with necrotizing pneumonia, as shown in mouse models, and it is likely that unknown factors are inferring with the regulatory systems that control the gene expression [96].

The isolate MW2 is an example of a PVL producing CA-MRSA that caused fatal infections in four children in the United States in 1998 whom displayed no risk factors associated with healthcare, that attracted worldwide attention of both media and healthcare workers [5, 20]. Further studies on this specific MRSA that is a representative of the epidemic USA400 clone (ST1-SCCmec type IVa) and one additional CA-MRSA (Los Angeles County Clone, LAC) representing the USA300 clone (ST8-SCCmec type IVa or IVb) have identified a range of exoproteins that are

produced in abundance, such as D-haemolysin, collagen adhesion, staphylokinase,

coagulase and enterotoxins C3 and Q. Although it seems to be evident that it is not a single protein that is responsible for the increased dissemination and virulence of CA-MRSA [15]. As much as 20 % of the genomic content of these CA-CA-MRSA was shown to be acquired horizontally, by mobile genetic elements, and had not been identified in representative nosocomial MRSA [34]. In contrast, comparative whole-genome sequencing was performed on several isolates representing the same USA300 clone, yet from different origins, and concluded that MRSA exhibiting significant variations in

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virulence varied by only a limited number of polymorphisms that could explain these dramatic differences in virulence [86].

CA-MRSA strains are associated with the carriage of mainly the type IV SCCmec, and more increasingly also the type V SCCmec [13, 125, 127]. In contrast, a different epidemiology is observed in the nosocomial, or hospital-acquired MRSA (HA-MRSA), which consists mainly of SCCmec type I, II or III [72, 140]. In addition, the CA-MRSA generally exhibits much more diverse origins, they have a higher growth rate, and are more likely to be susceptible to other groups of antibiotics in comparison to HA-MRSA. A hypothesis is that the CA-MRSA originates spontaneously by exchange of SCCmec between MR-CoNS and MSSA and thereby acquires the mecA gene and mechanisms of resistance against all available E-lactam antibiotics [83].

CA-MRSA has been identified in Örebro County since 1995 and with a drastic increase during the early 2000’s. This thesis focuses on the CA-MRSA in order to evaluate the origin, dissemination and characteristics of this potential pathogen. Considering the increasing rates of MRSA that has been reported in many countries, especially in the community, the importance of surveillance and preventive strategies has been emphasized. An unambiguous characterization and typing of MRSA is necessary in order to understand the origin and epidemiology of this pathogen.

Typing of MRSA

Epidemiology is the study of the occurrence and distribution of diseases. Epidemiological surveillance of bacterial pathogens that has an increased virulence, dissemination and antibiotic resistance are depending on effective methods for identification and for tracking their spread. Several typing methods have been developed and used in order to describe the origin and spread of MRSA. Initially, phenotypical methods, such as phage typing and antibiogram, were used for the characterization of MRSA but have subsequently been replaced by the use of mainly molecular techniques, in order to gain enhanced specificity and to save time. This has been described as a new discipline, the molecular epidemiology of MRSA, which has contributed to the insights into the evolutionary history of MRSA [130] but also provided a rapid and unambiguous identification, characterization and tracking of the spread of MRSA at clinical microbiological laboratories. Detection of specific genes and variation in the genome or within genes of a pathogen is used for molecular typing and the choice of method is dependent on the biology of the organism and the epidemiological questions. Short term or local epidemiology is usually of interest concerning the spread of a strain within a hospital, from an individual or in the local community. It is important to find out if the isolates from an outbreak of disease are caused by the same or by different strains. There will be little or no variation among

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the isolates if they are representing the spread of the same strain and short time epidemiology therefore requires a method that is able to monitor genetic variation that accumulates rapidly. Long-term or global epidemiology examines how the strains are causing disease in one geographic area related to those isolated worldwide. Typing methods for long-term epidemiology should therefore investigate genetic variation that accumulates slowly but requires the ability to distinguish a large number of genotypes [41, 109, 151]. The present thesis focuses on the molecular characterization of MRSA in a low endemic area and an introduction to some of the most important tools that are available today for typing of pathogenic organisms is given below.

Antibiogram

The antibiogram has been an epidemiological tool for typing of MRSA in which the antibiotic susceptibility profile is determined using selected sets of antibiotics. The antibiogram is important for the clinical laboratory and gives the first indication of an MRSA. However, it should not be used as the only epidemiological instrument since the resistance determinants are often carried by mobile genetic elements, such as plasmids, and since these are subject to a selective pressure they are likely to undergo genetic rearrangements that might affect the susceptibility profile. The discriminatory power of the antibiogram is considered as poor because genetically unrelated strains may carry the same resistance profile and, in contrast, two related strains may exhibit different phenotypes because of loss or gain of resistance determinants [11].

Pulsed field gel electrophoresis

Pulsed field gel electrophoresis (PFGE) has been regarded the gold standard method for characterization of MRSA outbreaks since it reflects genetic events that occur on a short time basis, but it has also been widely used for national studies and studies of long-term epidemiology [56, 156]. The bacterial DNA is cut by a restriction enzyme and is separated under an electrical field, which results in specific band patterns. Any gain or loss of DNA as well as DNA rearrangements is revealed by differences in the band pattern. PFGE has high discriminatory power and has been one of the most used and important tools for classification of MRSA, as well as other pathogens, but several disadvantages have been discussed, for example that PFGE may blur the long-term epidemiology and genetic relatedness among S. aureus since minor genetic changes may lead to significant differences in the band pattern [40]. Also, the method is time-consuming, the interpretation is relatively subjective and interlaboratory results are difficult to compare because of lack of appropriate standardization [119, 141]. For the purpose to investigate long-term epidemiology, methods that are based on slowly

References

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