Introduction

Staphylococcus aureus is one of the most ubiquitous bacteria among human. It is resistant to many unfavorable environmental conditions such as drying or high concentration of sodium chloride, which enables it to temporary or permanent skin and nasal mucosa colonization as well as makes it one of the major human pathogen (Matouskova and Janout 2008). The rate of S. aureus colonization in the population is about 37.2%. Probably 1% of these are methicillin-resistant S. aureus colonized (Kuehnert et al. 2006).

The first antibiotic, introduced to medical use in 1940, was penicillin. In short time after its introduction, the first penicillin-resistant S. aureus strains were observed. In 1961, 2 years after introduction of methicillin, a penicillinase-resistant penicillin, the first methicillin-resistant S. aureus (MRSA) strain was reported in Great Britain (Jevons 1961). In the second half of the 1960s, multi-drug-resistant MRSA strains were described. Over the next years, S. aureus strains resistant to streptomycin, tetracycline, erythromycin and vancomycin were observed (Schwalbe et al. 1987; Grundmann et al. 2006). For over 40 years, MRSA strains are considered to be the main cause of the majority nosocomial infections worldwide, causing over 50% of nosocomial infections in the United States. (NNIS 2002). However, MRSA infections among people who previously did not have prolonged contact with health services appear to increase (Fridkin et al. 2005).

First, cases of community-associated methicillin-resistant S. aureus (CA-MRSA) infections in drug-addicted patients, who had not been hospitalized earlier, were reported in 1982 in the United States (Saravolatz et al. 1982). Then in 1993, the presence of CA-MRSA strains in populations living in Western Australia was described (Udo et al. 1993; Riley and Rouse 1995). Afterward, in 1999, the death of four children has been reported, caused by CA-MRSA infection (CDC 1999). Recently, more and more cases of CA-MRSA infections have been reported from all over the world (Kowalski et al. 2005).

Community-associated MRSA

With the year 1993, when new CA-MRSA clone was observed, the species were provided to enable differentiation between CA-MRSA and other MRSA strains (Udo et al. 1993). There are different clinical and epidemiological features as well as molecular markers of MRSA types. According to the definition proposed by Center for Disease Control and Prevention, CA-MRSA are strains isolated in an outpatient setting or from patient within 48 h after admission to the hospital. Furthermore, the patient’s medical history cannot contain MRSA infection or colonization, hospitalization or residence in a long-term care facility (nursing home, hospice etc.), dialysis and surgery. Moreover, there cannot be risk factors for acquisition of MRSA such as indwelling catheters and medical devices that pass through the skin into the body. In majority of cases, clinical manifestation presents with skin and soft tissue infections. However, certain cases can progress to invasive tissue infections, bacteremia and death (http://www.cdc.gov).

The main molecular markers of CA-MRSA are presence of lukF-lukS genes, staphylococcal cassette chromosome mec (SCCmec) type IV, V or VII (Tristan et al. 2007; Takano et al. 2008) and accessory gene regulator genotype I or III (Naimi et al. 2003; Tenover et al. 2006). In addition, CA-MRSA strains are resistant to many β-lactam agents (Kowalski et al. 2005). The differences between healthcare-associated MRSA and community-associated MRSA strains are specified. They refer to clinical manifestation, epidemiology, antibiotic resistance and molecular markers (Fey et al. 2003; Huang et al. 2006; Robinson et al. 2008; Chavez and Decker 2008). The comparison of HA-MRSA and CA-MRSA is presented in Table 1.

Table 1 The comparison of CA- and HA-MRSA
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Resistance to antimicrobial agents

CA-MRSA, in comparison with HA-MRSA strains, apart from their resistance to β-lactams, are characterized by high sensitivity toward the remaining antibiotics, mostly above 90%, whereas their resistance toward lincosamides and macrolides is observed at a various degree (Dufour et al. 2002; Naimi et al. 2003; Ellis et al. 2004; Kowalski et al. 2005; Lorber 2006). According to different authors, the sensitivity toward erythromycin and clindamycin was 44 and 83% (Kowalski et al. 2005), 18 and 87% (Lorber 2006), 44 and 83% (Naimi et al. 2003) or 20 and 95.6%, respectively. As reported by Fey et al. among CA-MRSA type IV isolates, 81% were resistant only to β-lactams (Fey et al. 2003). In other study, drug resistance was clone dependent, where clone ST30-IV(USA 1100) displayed high sensitivity toward antibiotics and clone ST8-IVa (USA 300) displayed the highest resistance and 89.4% were resistant to erythromycin, 11.7% toward clindamycin and 62.8 and 24.5% toward ciprofloxacin and tetracycline, respectively (Diep et al. 2006a, b). However, apart from the more frequent presence of the PVL gene, no differences, comparing with HA-MRSA in the distribution of genes encoding virulence factors such as adhesins or toxins, were found (Fey et al. 2003; Graham et al. 2006; Diep et al. 2006a, b; Huang et al. 2007).

Evolution

According to the most probable CA-MRSA evolution hypothesis, staphylococcal cassette chromosome mec was independently introduced to genomes of many different methicillin-susceptible S. aureus (MSSA) strains in various geographical regions. Two theories have been suggested to describe the origin of SCCmec in CA-MRSA strains. According to the first theory, the SCCmec element has been incorporated into the genome of MSSA in an outpatient setting. While the second theory describes the origin of CA-MRSA from HA-MRSA (Deurenberg et al. 2007; Deurenberg and Stobberingh 2008).

The first theory, proposed by Okuma et al. (2002), assumes that IV type of SCCmec was incorporated into the genome of PVL-producing MSSA strains. The Japanese studies confirmed the presence of PVL-producing MSSA strains in the 1960 s, which become then ST30-MRSA-IV strain (Ma et al. 2006; Taneike et al. 2006). Moreover, this hypothesis is confirmed by studies of Monecke et al. (2007), who suggested that CA-MRSA emerged from the various S. aureus isolates by integrating SCCmec into the genome of PVL-producing S. aureus (Deurenberg et al. 2007).

According to the second theory, the evolution of CA-MRSA occurred in a hospital setting; therefore, both CA-MRSA and HA-MRSA have a common ancestor (Aires de Sousa and de Lencastre 2003). Perhaps it was a penicillin-resistant strain, which appeared worldwide both in outpatient and in hospital setting in the 1950 s. As a result of the introduction of β-lactam antibiotics in the 1960 s, the strain disappeared, to reappear after its incorporation of genes encoding PVL and type IV or V SCCmec through horizontal gene transfer by phiSLT phage, giving a rise of CA-MRSA strain, ST30-MRSA-IV (Robinson et al. 2005; Deurenberg and Stobberingh 2008).

Epidemiology

Multiple international investigations have reported the presence of CA-MRSA in many countries and in different populations.

United States

In last few years, the number of infections caused by CA-MRSA in United States has increased. Salgado et al. (2003) reported that CA-MRSA strains are approximately 0.24% of all isolated MRSA strains. However, the research carried out in three American hospitals by Fridkin et al. (2005) showed that the incidence of CA-MRSA infections varied between 18.0 and 25.7 per 100,000 population, while 8–20% of all MRSA isolates were classified as CA-MRSA strains.

Europe

The examination of the data collected from European hospitals participating in European Antimicrobial Resistance Surveillance System (EARSS) from January 1999 through December 2002 showed the prevalence of MRSA and CA-MRSA. MRSA isolates varied between 0.5 and 44.4%. Furthermore, CA-MRSA rate remained low with a prevalence of 0.03–1.5% of all isolates (Tiemersma et al. 2004).

Isolated populations

An interesting phenomenon is the high level of colonization of CA-MRSA strains in isolated ethnic populations. Among the indigenous inhabitants of Australia, Aborigines, this level is 76% (Maguire et al. 1996), while the populations of American Indians 62% (Groom et al. 2001).

The problem of hospital infections caused by methicillin-resistant S. aureus (MRSA) is known and associated with hospital treatment from the 1960s (Hryniewicz 1999). The frequency of bacteremia of this etiology, according to research carried out in the years 1999–2000 in Europe, depending on the country ranged from 44.4% (Greece) to 0.6% (Denmark and Holland) (Tiemersma et al. 2004). According to the research of SENTRY carried out in the years 1997–1999 in USA, Canada, Latin America and Western Pacific, the percentage of infection in these areas was 25.3; 19.2; 20.6; and 21.6, respectively (Diekema et al. 2001). To determine strain differentiation, the method previously used was PFGE (Pulsed Field Gel Electrophoresis). During PFGE, bacterial DNA is immobilized in agarose blocks, in which it is digested with restriction enzymes. Next during electrophoresis, DNA is separated to fragments of different length. Electrophoresis is run in an alternating electrical fields, which allows the separation of DNA fragments in the range of 10–800 Kb (Olive and Bean 1999). The whole genetic material in the cell is analyzed. The additional enzyme digestion of the genome with enzymes, such as SmaI, results in the formation of DNA fragments of different length and number (Murchan et al. 2003). Another technique used in epidemiological studies is RFLP (Restriction Fragments Length Polymorphism) in which differentiation is based on the analysis of restriction patterns obtained through the digestion of the genetic material with restriction enzymes. In the case of S. aureus, the amplified product of the coagulase gene (coa) or the polymorphic X region of the A protein (spa) is digested with restriction enzymes AluI or RsaI, respectively (Frenay et al. 1996; Hookey et al. 1998; Kurlenda et al. 2009). Currently, the spa-typing is thought to be one of the most useful and powerful typing methods (Karynski et al. 2008). One of the most simple PCR-based methods used for differentiation is RAPD (Randomly Amplified Polymorphic DNA). This technique is based on the use of primers consisting of 9–11 nucleotides, which begin the amplification of DNA fragments in many regions of the genome simultaneously. The products are separated in an agarose gel. This method shows sufficient level of differentiation for epidemic investigations (Kurlenda et al. 2007).

Another described and used typing method is multiplex PCR called originally multiple-locus variable-number of tandem repeats analysis (MLVA) and presently multiple-locus VNTR fingerprinting (MLVF). It analzse the variation in number of repeats in seven individual genes (sspA, spa, clfA, clfB, sdrC, sdrD and sdrE). The discriminatory power of MLVF method is comparable with that obtained with PFGE analysis (Sabat et al. 2006; Karynski et al. 2008). Another method used for strain typing is MLST (multi-locus sequence typing). In the year 2000, Enright et al. (2000) described the use of the MLST technique in epidemiological studies regarding both MRSA and methicillin-sensitive MSSA strains for the first time. MLST is the genotyping version of MLEE (multi-locus enzyme electrophoresis), an electrophoretic analysis of sequenced variable region, fragments of 0.5 kb in size and of seven housekeeping genes of S. aureus: arcC, aroE, glif, gmk, pta, tpi and yqiL encoding bacterial reference enzymes (Deurenberg et al. 2007). The accepted nomenclature is based on defining clones with the letters ST combined with the numbers of the SCCmec cassettes, for e.g. the Brazilian/Hungarian clone that was present in countries of Europe, in the USA and South America is defined as ST239-III (Deurenberg et al. 2007). At present, PFGE and MLST are regarded as the most sensitive methods for strain typing (Oliveira et al. 2002); however, as they are technically demanding and laborious, for routine purposes, regarding limited territorial ranges, more simple and quick techniques such as RAPD are used (Kurlenda et al. 2007). Currently, the most widely used method for differentiation is clone determination with the use of MLST. It allows to determine the spreading of strains, maintenance on a particular region and the appearance of new strains (Gosbell 2005; Deurenberg et al. 2007). Introducing universal nomenclature for the description of clones allows to compare centers and also to evaluate maintenance of clones in time and their exchange. Conceicao et al. (2007) presented the results of a 10-year observation where during the years 1994–1998 in Hungary the type ST239-III clone dominated and then totally vanished in the year 2003 replaced by a Southern German clone ST228-I which was present only in Europe (Deurenberg et al. 2007). In USA, in the years 1996–2004, in the region of San Francisco, the clone ST5-II was present among hospital strains. This clone was present also in Europe, Australia, Japan and South America (Diep et al. 2006a, b). In 2005, in the USA, among dialyzed patients, 92% of the isolated MRSA strains from infections belonged to the clone ST5-II (Collins 2007). Two of the biggest clones, Iberian ST247-I and Brazilian/Hungarian ST-239-III, have spread into countries of Europe and the USA, Australia, Asia and South America (Gosbell 2005; Deurenberg et al. 2007).

From the 1990s in many countries (Fey et al. 2003; Faria et al. 2005), in the outpatients infections caused by MRSA strains defined as CA-MRSA (community-associated MRSA) has been observed. According to different authors, it can reach 75% (Naimi et al. 2003; Wannet et al. 2004), 77% (Lorber 2006) and 84% (Faria et al. 2005).

CA-MRSA quickly spread in the outpatient population with the distinct domination of some clones in different periods, countries or continents. The most frequently determined clone ST8-IV(USA 300) was identified both in European countries and in the USA, South Africa and Asia (Pan et al. 2005; Maree et al. 2007; Manzur et al. 2007), whereas in Europe the ST80-IV clone was mainly present (Dufour et al. 2002; Witte et al. 2004). As some authors point out, in recent years, a polyclonal tendency has appeared and so have new clones (Tristan et al. 2007; Hedin and Fang 2007). These strains have widespread so much that they are more frequently isolated from hospitalized patients (Saiman et al. 2003; Maree et al. 2007). This has caused an increase in MRSA infections in hospitals, in Scandinavian countries and Holland (Kaiser et al. 2004; Skov and Westh 2005), where in the 1980 s strict MRSA monitoring was introduced, which allowed to maintain infections on a low level <1%. Since 2001, the situation has changed due to two factors, either the enrollment of patients with CA-MRSA infections (Kaiser et al. 2004; Faria et al. 2005; Lorber 2006) or the infections that developed in a hospital have been introduced by previously enrolled carriers of CA-MRSA (Saiman et al. 2003; Collins and Hampton 2005). A new source of CA-MRSA widespreading can in the future be pig farms and people employed there. During an international conference in Denmark, 272 participants associated with the agriculture industry, among which 12.5% were CA-MRSA carriers from different countries, were analyzed (Wagenaar et al. 2007). Twenty-four cases of MRSA infections in horses hospitalized in the years 2003–2005 in veterinary clinics were also reported where clone CA-MRSA ST254-IV was found in five animals (Cuny et al. 2006).

An important issue, from the therapeutic and epidemiologic point of view, is the identification of infection and carriers of HA-MRSA and CA-MRSA. Identifying carriers is critical for the control of the widespread of multi-resistant strains and reducing the risk of infection development because S. aureus nasal carriage occurs quite frequently, approximately in 20–60% of a normal population (Toshkova et al. 2001; Graham et al. 2006). Kampf et al. (2003) reported that S. aureus carriage among hospital staff was approximately 33.8% of which 1.6% were MRSA strains (Kampf et al. 2003). The example of Holland and Scandinavian countries shows the importance of carriage monitoring where after introducing strict monitoring of enrolled patients the percentage of MRSA incidents was low and maintained on a level <1% (Kaiser et al. 2004; Skov and Westh 2005). Quick evaluation of the etiology of infection is also important in order to apply the proper treatment, as in the case of MRSA it is critical in empiric treatment not to begin treatment with β-lactam antibiotics. Introducing molecular biology methods into diagnostics reduces the time required to obtain information regarding the etiology of the infection and improves the identification of carriers (Nilsson et al. 2005; Holfelder et al. 2006; Ornskov et al. 2008).

Risk factors

Although the epidemiology of CA-MRSA has not been fully explained, several important risk groups have been described. The higher prevalence of CA-MRSA has been observed in various population groups such as military personnel, soldiers (Ellis et al. 2004), intravenous and intranasal drug users, people belonging to a lower socioeconomic status, homeless persons (Charlebois et al. 2002), athletic and competitive sport participants (CDC 2003), a homosexual mans (Sztramko et al. 2007), newborns (Saunders et al. 2007), children, young people (Herold et al. 1998), prison inmates (CDC 2001), ethically closed or semi-closed communities such as Pacific Islanders (CDC 2004), rural American Indian and rural southwestern Alaska population (Groom et al. 2001) remote Aboriginal Community (Maguire et al. 1996).

Clinical manifestation

Clinical manifestation of CA-MRSA infections manifest most commonly as skin and soft tissue infection (STTI) such as furuncles, boils and abscess (Eady and Cove 2003). However, it may cause more severe symptoms as folliculitis, cellulitis, impetigo, septic arthritis, sepsis, wound infections, pneumonia (Fridkin et al. 2005), necrotizing pneumonia (Roberts et al. 2002), meningitis (von Specht et al. 2006) or endocarditis (Millar et al. 2008).

Genetic characterization of CA-MRSA strains

Accessory gene regulator (agr)

The accessory gene regulator, (agr), also termed agrBCDA, is the S. aureus global regulatory system (Cotter and Miller 1998). It consists of five genes (hld, agrB, agrD, agrC and agrA), whose products establish a quorum-sensing regulatory mechanism and modulate the expression of virulence factors. Moreover, it serves a crucial role in S. aureus virulence and reduced susceptibility to glycopeptides antibiotics.

The agr polymorphism

Polymorphism at the locus across two genes, agrD and agrC, defines four S. aureus agr groups (I–IV). The distribution of S. aureus agr groups varies with geographical region (Novick 2000; Gomes et al. 2005). The differences between agr groups concern the production of biofilms (Boles and Horswill 2008), expression of virulence factors and resistance to antibiotics (Campoccia et al. 2008). CA-MRSA strains correlate to agr group I and III (Naimi et al. 2003; Tenover et al. 2006). There is a strong correlation between agr group and the pathogenesis of the infection (Jarraud et al. 2002). Seventy-one percent of all MRSA strains correlate to agr group I what makes it the most prevalent genotype. Agr II group genotype is characteristic to isolates from patients with endocarditis, and it is related to reduced susceptibility to vancomycin. Furthermore, toxic shock syndrome toxin-1 (TSST)-producing isolates belong to agr group III, while most exfoliatin-producing strains responsible for staphylococcal scaled-skin syndrome (SSSS) correlate to agr group IV (Jarraud et al. 2000). Campoccia et al. (2008) compared the prevalence of antibiotic resistance in different agr polymorphism groups. Isolates correlated to agr I and II group were resistant to many antimicrobial agents including oxacillin, penicillin, gentamicin, erythromycin, clindamycin, chloramphenicol and ciprofloxacin. However, the agr type III strains showed the lowest prevalence of antibiotic resistance with high susceptibility to oxacillin, clindamycin, chloramphenicol and sulfamethoxazole with trimethoprim.

Biofilm production

The activity of the agr system and its polymorphism influence different biofilm production in S. aureus. Among these bacteria, there are four types of agr quorum-sensing systems. Each of agr types (agr I through agr IV) recognizes a unique auto-inducing peptide (AIP) structure (AIP-I through AIP-IV). AIP-I and AIP-IV differ by only one amino acid for that reason there are three cross-inhibitory groups: agr I/IV, agr II and agr III. The addition of indicated AIP (type I agr strain was treated with AIP-I, type II agr strain was treated with AIP-II, and type III agr strain was treated with AIP-III) to each biofilm, resulted in its detachment over a period of 2 days (Boles and Horswill 2008). In addition, mutations in agr quorum-sensing system resulted in increased biofilm production (Beenken et al. 2003).

Geographic dominance

The observation of Gomes et al. (2005) showed that the isolates with different agr groups are most frequently recovered in distinct geographic regions. Strains of agr I group are mainly isolated in Europe and South America. Type II agr is characteristic to isolates from Japan and North America. Furthermore, strains of agr III group are predominant in European countries. Different geographic distribution of strains of distinct agr groups can be an evidence of bacterial interference mediated by agr operon.

The mechanism of agr regulation

The agr system consists of 3-kb locus and contains divergent transcription units regulated by promoters P2 and P3. The 120-bp region between promoters P2 and P3 contains sites of activation of these two promoters (Novick 2003). When bacteria reach a critical density, a 7–9-amino acid peptide (auto-inducing peptide, AIP) derived from agrD is secreted into the culture supernatant. For agr I/IV group strains, it is octapeptide, agr II type – nonapeptide and agr III type – heptapeptide. The structure of all auto-inducing peptides is rather similar with conserved structural feature as a gradient of increasing hydrophobicity from N- to C-termi ending with two hydrophobic residues (phenylalanine, leucine, valine, tyrosine, occasionally methionine). In addition, AIPs form a thiolactone ring with the conserved essential cysteine in central position (Ji et al. 1997; Novick 2003). AgrD-encoded AIP is produced in form of propeptide. Then, it is processed and secreted by agrB-encoded transmembrane protein AgrB (Zhang et al. 2002). Mature AIP binds to AgrC histidine protein kinase what results in phosphorylation of AgrA transcription factor. Consequently, it leads to transcriptional activation of two promoters, P2 and P3. The agr system is regulated via autoinduction mechanism where P2 promoter transcribes agr operon itself. The P3 promoter regulates expression of RNAIII transcript (Cotter and Miller 1998). RNAIII is a 0.5-kb effector of agr regulon. (Novick et al. 1993). Its intracellular concentration is high as well as it has a long half-time of ≈ 15 min (Janzon and Ardvidson 1990). In the complex secondary structure of RNAIII can be distinguished the sequence complementary to the Shine–Dalgarno sequence, by which RNAIII can block translation and non-overlapping 5′ and 3′ regions, which are active in the regulation of hemolysin-encoding gene (hla) transcription (Novick 2003). RNAIII regulates transcription of many genes. By binding to individual transcription factors, it can cause their allosteric modifications and change their ability to bind to target sequences. Furthermore, RNAIII could influence stability of gene transcripts (Arvidson and Tegmark 2001). RNA III inhibits the expression of genes encoding for protein A, fibronectin-binding proteins (FBP), coagulase and other surface proteins that can be important in the early stages of infection and expressed during exponential growth phase. In contrast, RNAIII serves as a transcription factor turning on expression of secreted virulence factors as hemolysins, toxic shock syndrome toxins and exotoxins (Cotter and Miller 1998).

Bacterial interference

S. aureus strains of different agr groups produce structurally different auto-inducing peptides. AIPs secreted by one strain can inhibit virulence gene expression in another. For that reason, colonization of competing strains can be repressed. This phenomenon is termed bacterial interference (Cotter and Miller 1998). The results obtained by Boles and Horswill (2008), who examined AIP-mediated biofilm detachment, proved the existence of this S. aureus phenomenon. In this research, AIP-I addition to 2-day-old biofilm of agr I type strain resulted in its detachment. In contrast, addition of interfering AIP-II did not cause biofilm dispersal.

The agr operon as a virulence factor

The agr operon is an important virulence factor of CA-MRSA strains. McDougal et al. (2003) obtained that agr system plays a crucial role in pathogenesis of staphylococcal soft tissue infections. Moreover, the virulence of agr-mutated stains was suppressed. Wright et al. (2005) reported a rapid growth of bacteria within first 3 h of infection to activate agr system. During this time, agr-negative strains were eradicated and abscess formation continued in the presence of agr-positive bacteria. In this study, inhibition of agr system in early stage of infection was sufficient to block abscess formation. Furthermore, agr operon increases expression of Panton–Valentine leukocidin and δ-hemolysins, a virulence factors associated with CA-MRSA strains. Ninety-seven percent of CA-MRSA strains produce δ-hemolysin, while PVL is considered to be the major virulence factor of these strains (Said-Salim et al. 2003; Sakoulas 2006).

The agr operon and resistance to glycopeptide antibiotics

The agr system may serve a crucial role in reduced susceptibility to glycopeptide antibiotics in S. aureus. The analysis of glycopeptide intermediate-resistant S. aureus (GISA) strains showed the loss of δ-hemolysin, α-hemolysin and RNAIII production, what is associated to loss of agr system expression. Sakoulas et al. (2002) reported that exposition of bacteria to glycopeptide antibiotics such as vancomycin and teicoplanin resulted in decreased RNAIII production. Furthermore, the exposition to vancomycin of agr knockout strain resulted in increased heteroresistance compared with the wild type. The electron microscopy analysis of cell wall of agr-mutated strain showed its thickening in comparison with non-mutated strain (Sakoulas 2006).

Staphylococcal cassette chromosome mec (SCCmec)

In S. aureus, staphylococcal cassette chromosome mec (SCCmec) causes resistance to methicillin and other β-lactam antibiotics. It consists of three main regions.

The mec gene complex

The mec gene complex consists of following elements: insertion sequence IS431mec, mecA and regulatory genes - mecR1 and mecI. The mecA gene, which is 2.1 kb in length, encodes for 78-kDa penicillin-binding protein (PBP2′ or PBP2a). It is responsible for resistance to methicillin and other β-lactam antibiotics (Ito et al. 2003).

The cassette chromosome recombinases (ccr) gene complex

The ccr genes, which are of invertase/resolvase class, are responsible for the mobility of SCCmec element. Integration and excision occurs at the specific site that is SCCmec attachment site (attBscc) at the 3′ of the open reading frame (orfX) of unknown function (Ito et al. 1999).

The J regions

The J (junkyard) regions contain different genes or pseudogenes whose function does not appear essential for the bacteria. The exceptions are J regions encoding for non-β-lactam antibiotic and heavy metal-resistance genes (Ito et al. 2003).

SCCmec polymorphism

At the moment, eight main types of SCCmec (I to VIII) are distinguished. The differences concern the presence of non-β-lactam antibiotic-resistance genes, integrated plasmids, transposons as well as insertion sequences (Ito et al. 2003; Takano et al. 2008). While all SCCmec types contain mecA gene, which causes resistance to methicillin and other β-lactam agents, SCCmec type II and III consist of additional integrated plasmids and transposons causing multi-drug and heavy metals resistance. Integrated plasmid pUB110 harbors the antt (4′) gene encoding for aminoglycosides resistance. The resistance to penicillin is determined by the presence of integrated plasmid pI258, while pT181a harbors tetracycline-resistance genes. The constitutive as well as inducible resistance to macrolide, linkosamide and streptogramin is encoded by Tn554 transposon, which harbors the ermA gene. In addition, the presence of ΨTn554 transposon causes resistance to cadmium, while integrated plasmid pI258 encodes for resistance to mercury (Ito et al. 2003; Oliveira et al. 2006; Takano et al. 2008). The comparison of different SCCmec types is showed in the Table 2. CA-MRSA strains harbor SCCmec type IV, V or VII, which do not contain additional integrated plasmids and transposons. Consequently, they are not resistant to multiple classes of antibiotics as well as heavy metals (Deurenberg and Stobberingh 2008).

Table 2 The characteristic of distinct staphylococcal cassette chromosome mec types
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The mechanism of resistance to β-lactam agents

β-lactam antibiotics inhibit the final stages of peptidoglycan biosynthesis, which are the components of bacterial cell wall (Giesbrecht et al. 1998). The cell wall of methicillin-sensitive S. aureus (MSSA) contains native penicillin-binding protein (PBP). β-lactams bind to transpeptidase domain of PBP, disrupting the synthesis of peptidoglycan layer. Whereas methicillin-resistant S. aureus strains acquired the mecA gene, which codes for low-affinity penicillin-binding protein (PBP2′ or PBP2a). Consequently, β-lactams do not inhibit cell wall biosynthesis, resulting in the growth of MRSA (Berger-Bachi and Rohrer 2002). The transcription of mecA gene is regulated by two regulatory elements—the MecI repressor and the transmembrane receptor MecR1, which contains a β-lactam-induced extracellular sensor domain. The two proteins are transcribed separately. In the absence of β-lactam agents, MecI represses both transcription of the mecA and mecR1-mecI. On the other hand, in the presence of β-lactams, MecR1 is cleaved, activating its metalloprotease domain, which in turn cleaves the MecI repressor. In consequence, the transcription of mecA gene and PBP2 production occur (Berger-Bachi and Rohrer 2002). CA-MRSA strains, due to the presence of mecA gene, are resistant to β-lactam agents. However, they are susceptible to many antibiotics, comparing to HA-MRSA strains (Kilic et al. 2006; Kim et al. 2006). Kilic et al. (2006) compared antibiotic susceptibility patterns of strains harboring SCCmec type II (HA-MRSA) and IV (CA-MRSA). The results showed that MRSA strains of SCCmec type II were greater resistant that SCCmec-IV MRSA strains to clindamycin, erythromycin, levofloxacin, gentamicin, rifampicin, minocycline and sulfamethoxazole-trimethoprim.

The association between SCCmec type and virulence factors production

Kim et al. (2006) reported an association between SCCmec type of MRSA strains and toxin gene profiles. The toxin gene carriage differed in different SCCmec types (95.9% in SCCmec II, 74.4% in SCCmec III and 68.8% in SCCmec IV). Furthermore, Kilic et al. (2006) obtained that the strains of SCCmec type IV more frequently harbored lukS-PV and lukF-PV genes, coding for PVL than isolates of SCCmec type II. The PVL genes were detected in 93.6% of SCCmec-IV isolates, comparing to 0.2% of SCCmec-II isolates. In addition, higher occurrence of PVL was notified in isolates of SCCmec-IV in children (98.1%) than in adults (89.1%).

Panton–Valentine leukocidin

Panton–Valentine leukocidin was discovered by Van deVelde in 1894 due to its ability to lyse leukocytes. In 1932, Panton and Valentine first associated the leukotoxin with skin and soft tissue infections (Panton and Valentine 1932; Boyle-Vavra and Daum 2007). PVL shows cytolytic activity with high specificity to leukocytes. Furthermore, it is one of the molecular markers of CA-MRSA strains (Tomita and Kamio 1997; Tristan et al. 2007; Takano et al. 2008).

The lukS-PV and lukF-PV genes

In the S. aureus genome, the cotranscribed lukS-PV and lukF-PV genes are separated by only one thymine. The ORF of lukS-PV is 939 nucleotides long, whereas the lukF-PV ORF is 978 nucleotides long. The G + C contents of lukS-PV and lukF-PV are 31.5 and 30.3%, respectively. Two identical putative ribosome-binding sites (GAAAGGA) are located 10 bp upstream from the ATG codons of the two genes. The ribosome-binding site for the lukF-PV is included within the ORF of lukS-PV. Two hypothetical transcription signals are located 80 bp upstream from lukS-PV, whereas the putative transcription terminator is supposed to be located 67 bp downstream from lukF-PV. The unique mRNA of cotranscribed lukS-PV and lukF-PV is 2.1 kbp long. The first transcribed gene of the operon, coding for 312-amino acid protein, is lukS-PV. However, the cleavage between 28 and 29 position of amino acid results in 284-amino acid mature protein. The second transcribed gene is lukF-PV, which encodes a protein of 325 amino acids. The N-terminal end of the mature protein starts at 25 amino acid position. Consequently, the mature protein is of 301 amino acids (Prevost et al. 1995).

PVL is bicomponent, pore-forming leukotoxin, composed of two components—LukS and LukF. LukS and LukF consist of three domains—β-sandwich, rim and stem.

The structure of LukS-PV and LukF-PV

The three-dimensional structure of LukS (33 kDa) is composed of 19 strands that participate to four antiparallel β-sheets and three shorts segments of either 310 or α helices (Guillet et al. 2004). In contrast, the three-dimensional structure of LukF compound (34 kDa) consists of 22 β-strands and three short segments of either 310 or α helices. β-sandwich domain is composed of two antiparallel, six-stranded β-sheet domains, facing with an angle of 30°. The rim domain forms an antiparallel four-stranded structure of open-face sandwich. The stem domain consists of three antiparallel β-strands linked by one β-turn (Pedeacq et al. 1999). The two components, LukF-PV and LukS-PV, have a similar conformation. The structure of β-sandwich and stem domain of LukS-PV is more similar to their counterparts in LukF-PV, comparing to rim domain. The four peptide segments in rim domain are less conserved (Guillet et al. 2004). The two components of PVL leukocidin, the LukS-PV and LukF-PV, are secreted by S. aureus to the environment before they form a heptamer on polymorphonuclear leukocytes membranes. First, the LukS-PV binds to an unidentified receptor on PMN leukocyte where it dimerizes with LukF-PV. The two components bind together until the heptamer is assembled (Kaneko and Kamio 2004). Depending on the concentration of PVL, it can cause either lysis (necrosis) of the leukocyte or apoptosis. High concentrations of PVL cause the lysis of the leukocyte due to the assembly of many octameric pores on the PMNs membrane, leading to an influx of extracellular substances as ethidium ions. Furthermore, LukS-PV by binding to PMNs membrane may be phosphorylated by A or C kinase, leading to the stimulation of calcium channels of PMNs, causing influx of extracellular ions. This contributes to the production of interleukins and inflammatory mediators (Finck-Barbançon et al. 1993; Staali et al. 1998; Genestier et al. 2005). On the other hand, the low PVL concentration causes PMNs apoptosis via pathway that involves PVL-mediated pore assembly on the mitochondrial membrane. Consequently, cytochrome c is released, inducting apoptotic proteins—caspases 3 and 9 that lead to DNA fragmentation and PMNs apoptosis (Genestier et al. 2005).

The contribution of PVL in CA-MRSA pathogenesis

The role of PVL in CA-MRSA pathogenesis is controversial. On the one hand, mouse infection models used by Voyich et al. (2006) showed that PVL is not the major virulence determinant of CA-MRSA. On the other hand, the epidemiological studies as well as rabbit bacteremia model developed by Diep et al. (2008a, b) and studies carried out on neutrophils isolated from different species by Loeffler et al. (2010) confirm its major role for the pathogenesis of CA-MRSA infections.

In a PubMed database, there are more than 300 articles, published in 2002–2007, suggesting an association between PVL and CA-MRSA infections. The most frequently cited conditions are furuncles, ulcers, severe necrotizing skin infections, leucopenia and hemoptysis. Moreover, the respiratory tract infections, extensive necrosis of respiratory epithelium and necrotizing pneumonia are often mentioned (Gillet et al. 2002). Only 2% of strains isolated from patients with necrotizing pneumonia contain genes coding for PVL leukotoxin. This is small percentage; however, the infections caused by PVL-positive strains are more severe as well as the lethality rate in such infections is increased (Lopez-Aguilar et al. 2007).

Studies on animal infections models provide contradictory conclusions about the contribution of PVL in CA-MRSA pathogenesis. On the one hand, Voyich et al. (2006), using mouse bacteremia model to compare the virulence of PVL-positive and PVL-negative strains, showed that PVL is not a major virulence determinant of CA-MRSA. Strains that did not produce leukotoxin were as virulent as those containing PVL. The clinical manifestation of infections caused by PVL-positive as well as PVL-negative strains was similar and manifested as sepsis, abscess and skin disease. Furthermore, lysis of human PMN leukocytes was the same with PVL-positive and PVL-negative strains. On the other hand, Diep et al. (2008a, b) reported a modest and transient contribution of PVL in CA-MRSA infection. In contrast to Voyich et al. (2006), Diep et al. (2008a, b) developed a rabbit bacteremia model because rabbit’s granulocytes are more sensitive to the leukolytic activity of PVL, compared with mouse (Szmigielski et al. 1999). At 24 and 48 h after infection, PVL plays a modest, but measurable role in pathogenesis. It is important in early stages of bacteremic seeding of the kidney. However, after 72 h postinfection, the PVL activity is lost. This is consistent with clinical manifestation of rapid and acute onset of CA-MRSA infections (Gonzalez et al. 2005). It is still unclear why PVL shows a transient effect. Perhaps the leukotoxin production in the end stages of the infection could lead to priming the innate immune response, resulting in limited survival of bacteria. Another reason may be the changes in bacterial metabolism to produce other virulence factors, important in maintaining persistent infection (Diep et al. (2008a, b). The most recent studies by Loeffler et al. (2010) showed the effect of PVL on polymorphonuclear cells from different species including humans, mice, rabbits and monkeys. These studies demonstrated that PVL induces rapid activation and cell death in human and rabbit PMN leukocytes, but not in murine or simian cells. The reason for the differential sensitivity of cells isolated from various species is unknown. It has been suggested that animal models using mice and non-human primates do not correctly replicate S. aureus diseases in human. Taken together, PVL is a strong cytotoxic factor for human PMN leukocytes and their premature death may be extremely relevant in the virulence of CA-MRSA.

Arginine catabolic mobile element

A 30.9-kb genetic island—the arginine catabolic mobile element (ACME), present in the genome of CA-MRSA strain USA 300 which contain the SCCmec type IVa element, has been associated with emerging dominance of its background. It contains loci that encode at least 33 genes. One of these loci, arcABCD, encodes a complete arginine deiminase pathway that converts l-arginine to carbon dioxide, ATP and ammonia (Ito et al. 2001; Mongkolrattanothai et al. 2004; Diep et al. 2006a, b, 2008a, b). In addition, ACME contains an operon, opp-3, which comprises five genes that, together, encode a putative oligopeptide permease (Opp). It could be involved in the regulation of virulence factor expression (Wang et al. 2005).

The linkage between ACME and SCCmec

ACME could enhance growth, survival and dissemination of USA300, thus allowing for the genetic “hitchhiking” of SCCmec. SCCmec protects against exposure to β-lactams, further enhancing rapid dissemination of USA300 with antibiotic use (Diep et al. 2008a, b). Furthermore, ACME integrates into orfX on the USA300 chromosome, using the same attachment site as SCCmec element, and is flanked by repeat sequences characteristic of SCCmec elements. SCCmec provides the ccrAB recombinases that could catalyze mobilization and integration of ACME into the chromosome (Katayama et al. 2000).

The contribution of ACME in virulence of CA-MRSA strains

The studies of Diep et al. (2008a, b) consisting of simultaneous inoculation of rabbits with a wild-type USA300 parent strain and an isogenic ACME deletion mutant. The parent strain was recovered in greater numbers than the deletion mutant from the lungs, spleen and blood. They found that the deletion of ACME significantly attenuated the pathogenicity or fitness of USA300. The most recent studies by Montgomery et al. (2009) compared USA300 CA-MRSA clinical isolates differing in the presence or absence of ACME and a USA300 wild-type/ACME deletion mutant isogenic pair in two animal models of S. aureus infection. They showed that presence of ACME was not associated with increased virulence in a rat model of necrotizing pneumonia, as assessed by mortality, in vivo bacterial survival and severity of lung pathology. Furthermore, the presence of ACME was not associated with increased dermonecrosis in a model of skin infection. They concluded that ACME is not necessary for virulence in rodent models of CA-MRSA USA300 pneumonia or skin infection.

Conclusion

The studies on the genetic characterization of CA-MRSA play a crucial role in the identification of CA-MRSA strains, the monitoring of infections, improvement of treatment, working on new antimicrobial agents as well as understanding the evolution of these pathogens. First of all, the classical microbiological methods such as microscopy, culturing and biochemical identification are not sufficient for precise identification of the strain. The molecular methods, in contrast, that use genetic analysis of the nucleic acids are much more precise and are used for rapid identification, what plays an important role in monitoring of the pathogen spread and rapid identification of the bacteremia (Matouskova and Janout 2008). Then, the information about genetic elements of the strain is important in the treatment of CA-MRSA infection. It plays a significant role in clinical decision making. For instance, PVL-positive strains seem to be dangerous for pulmonary patients, so when the presence of PVL-encoding genes is confirmed, the reinforced antibiotic treatment and patient’s isolation are recommended (Lopez-Aguilar et al. 2007). Another treatment of PVL-positive strains infections uses anti-PVL leukotoxin antibodies (Gauduchon et al. 2004). Furthermore, analysis of the genetic elements responsible for virulence and antibiotic resistance as well as understanding their mechanisms of action enables the development of new anti-CA-MRSA antimicrobial agents (Deurenberg and Stobberingh 2008). For example, bacterial interference, the phenomenon of producing structurally different auto-inducing peptides by S. aureus strains of different agr groups, may have potential application in the treatment of infections associated with biofilm production (Boles and Horswill 2008). In addition, genetic analysis of different CA-MRSA strains enables to study the evolution of these pathogens, both in hospital setting, as well as in outpatient. This contributes to a better understanding of the mechanisms of spread and acquisition of virulence factors (Deurenberg and Stobberingh 2008).