Scc476

far

USa3

sea, sak, seg2, sek2

USa4

No virulence genes

SaGIm [14] SaPI3 [25] SaPIm1 [14] SaPI2 [25] SaPIm2 [14] SaPIm3 [14]

Gene symbols: bsa, bacteriocin gene cluster; ccrA,B, cassette chromosome recombinase A,B; far, fusidic acid resistance; geh, lipase;

lpl, lipoproteins; lukDE, components of toxin leukocidin DE; lukS-PV, lukF-PV,

Panton-Valentine leukocidin components S and F; mecA, penicillin binding protein 2a; sak, staphylococcal kinase; set, staphylococcal exotoxins;

splA-F, serine protease; sea, seb, sec3, sec4, seg, seg2, sek2, sel, sem, sen, seo, sep, staphylococcal enterotoxins; tst, toxic shock syndrome toxin 1.

190 | 9 Pathogenic Staphylococci: Lessons from Comparative Genomics 9.2.3.2 Staphylococcal Cassette Chromosome

A unique type of mobile genomic island in staphylococci is represented by the staphylococcal cassette chromosome (SCC) element which is associated with methi-cillin resistance in staphylococci. Although this genomic island does not contain any phage-related genes, mobility has been demonstrated due to the activity of two recombinases of the invertase/resolvase family, designated chromosome cassette recombinases A and B (ccrA and ccrB). Integration occurs site-specifically into the chromosome near the origin of replication in a conserved ORF of unknown function named orfX that contains a 15-bp attachment site (attBscc). In the case of integration of SCC into the genome the attBscc is found at both chro-mosome-SCC junctions. SCC elements were originally described to be associated with methicillin resistance, but in recent studies SCC elements were also described in methicillin-susceptible isolates [15, 66].

Methicillin resistance in staphylococci is related to the presence of the mecA gene encoding an additional penicillin-binding protein, PBP2a, with reduced affinity to b-lactam antibiotics. PBP2a can substitute the essential functions of b-lac-tam-susceptible PBPs in crosslinking the peptidoglycan of the bacterial cell wall [67]. The mecA determinant is located within SCC elements which therefore has been termed staphylococcal cassette chromosome mec (SCCmec) [68, 69]. SCCmec has been shown to be transferable among staphylococcal species. Five major SCCmec types, ranging in size from 21kbp to 67kbp, have been identified [70, 71]. While types I-III are found in hospital-acquired MRSA (HA-MRSA or H-MRSA), strains of types IV and V are restricted to community-acquired MRSA isolates (CA-MRSA, C-MRSA or Co-MRSA). The SCCmec type is defined by the combination of the type of cassette chromosome recombinase (ccr), which confers mobility, and the mec gene complex [70]. There is no equivalent region in methicillin-susceptible staphylococcal strains, suggesting that the mec fragment was acquired by horizontal gene transfer. Moreover, the mosaic structure of the different SCCmec types indicates that several recombination events between the ccr and mec gene complexes along with integration and deletion of DNA sequences has driven the evolution of methicillin resistance in staphylococci. The core sequence of SCCmec consists of class A, B, C, or Dmec and one of five allotypes of ccr gene complex. A recently identified new SCCmec type lacks the ccrAB genes and contains instead of ccrAB a single copy of a recombinase designated ccrC [71]. Class A and Bmec have been found in clinical MRSA, class Cmec is mainly distributed in S. haemolyticus, and class Dmec mainly in S. hominis. Class Amec consists of mecA, a copy of the insertion sequence element IS431mec (an IS257-like element), and the regulatory sequences mecRl and mecl. In class Bmec mecl and the 3'-region of mecRI are deleted and a truncated copy of IS 1272 is inserted (Fig. 9.1). The ccr types are defined by the combination of different ccrA and ccrB homologues. Moreover, transposon Tn554 is detectable in SCCmec of types II and III. Finally, genes encoding resistance genes or whole plasmids (pUB110 or pT181) are found in the 3'-region of mecA (Fig. 9.1).The different types of SCCmec reflect the evolution of methicillin resistance in staphylococci. First MRSA clones such as the pre-MRSA isolate N315 carry intact regulatory genes mecRI and mecl which do not

Direct/ inverted repeats

SCCmectype I

mecA recombinases

DirectJ inverted repeats pis s. aureus col

SCCmec type II

Tl>554

tnsw hsdR IS431 te ft

S. aureus N315

Tn 554

SCCmectype III

SCCmec type IV

S. aureus 85 2085

5. aureus MUV2

10 kb

Fig. 9.1 Structure of type I, II, III, and IV staphylococcal cassette chromosome mec (SCCmec) elements (adapted from [70]). SCCmec is characterized by two common gene complexes, the ccr (cassette chromosome recombinase) gene complex (gray) and the mec gene complex (blue). Integrated IS43 7, Tn554, and plasmid pT181 are also indicated. Gene symbols: cad, cadmium resistance; ccr, cassette chromosome recombinase; ermA, erythromycin resistance; hsdR, host specificity determinant; kdp, two-component system regulating potassium transport; mec, penicillin binding protein 2a, methicillin resistance; mer, mercury resistance; tetK, tetracycline resistance. (This figure also appears with the color plates.)

respond well to b-lactam antibiotics including methicillin. Now, epidemic clones are homogeneously resistant to high methicillin concentrations and in addition to many other antibiotics including tobramycin, bleomycin, and tetracycline. Importantly, such resistance determinants inserted into SCCmec by recombination across IS431 insertion elements.

Specific types of SCCmec elements have been described in CA-MRSA. In contrast to HA-MRSA, which was first described in 1961, CA-MRSA was detected quite recently among healthy individuals with no recognizable risk factors [72, 73]. The expression of the Panton-Valentine leukotoxin (PVL) by CA-MRSA represents a major virulence factor of these strains as PVL is implicated in life-threatening necrotizing pneumonia [74]. Interestingly, PVL is carried by different bacteriophages [75]. Obviously, CA-MRSA strains are epidemiologically and clonally unrelated to hospital-acquired isolates. Most CA-MRSA isolates worldwide carry the type IV SCCmec, which was presumably acquired by community clones showing a high fitness. The type IV SCCmec element (21-24kbp) is shorter than SCCmec elements found in HA-MRSA and carries no further resistance determinants [16]. Recently, a fifth SCCmec type was discovered in a CA-MRSA isolate from Australia [71]. The 28-kb type V SCCmec carries a C2 mec gene complex, a new ccr recombi-

nase designated ccrC, and in addition, a complete set of a type I restriction modification system composed of hsdR, hsdS, and hsdM. The restriction modification systems seem to be important for the stability of a given region of the genome. Interestingly, hsdR and hsdM are also present on the two genomic islands vSaa and vSayb found in all S. aureus genomes sequenced so far (see above). As several CA-MRSA lineages with substantial genetic diversity are found in different parts of the world, in all probability new SCCmec types will be described in the future. The origin of SCCmec is still obscure and a number of speculations have been proposed. Probably, the evolutionary origin of the precursor mecA is a coagulase-neg-ative staphylococcal species [67].

MLST studies revealed that methicillin resistance in nosocomial strains due to the presence of SCCmec is restricted to approximately five clonal complexes (CC5, CC8, CC22, CC30, CC45) [28]. It has therefore been supposed that despite mobility of SCCmec, a host restriction barrier ensures stable integration and maintenance of mecA resistance [76, 77]. The reason why some clones are able to pick up SCCmec and not others is still unknown. Obviously, mecA expression is connected with loss of fitness which has to be counterbalanced by an adaptive process including regulation by mec/bla regulatory elements [78].

Recent work reveals that SCC elements are also present in methicillin-sensitive strains. For example, strain MSSA476 carries a unique 22.6-kb genetic element with a high similarity to SCCmec. The SCC476 element has the same left and right boundaries (attL and atlR), similar inverted repeats, and is integrated at the same site on the chromosome as SCCmec elements but carries a putative fusidic acid resistance determinant instead of mecA [15]. In addition, SCC-specific sequences were identified in several clinical MSSA isolates by DNA microarray analysis, indicating extensive integration and deletion events within this region [22].

The emergence of MRSA strains has dramatically changed the diversity of clones causing diseases in humans. A small number of clonal types are now responsible for the vast majority of nosocomial S. aureus infections worldwide. Moreover, CA-MRSA strains seem to be widely distributed in the community, and there is substantial fear that PVL-positive strains with increased fitness may enter the hospital environment, where they would become multiresistant. In consequence, new dangerous pathotypes might emerge which are more aggressive than those presently found and would be extremely difficult to treat.

9.2.3.3 Bacteriophages

Most ofthe naturally occurring S. aureus strains are polylysogenic. Based on virus morphology, staphylococcal bacteriophages are members of two groups of tailed phages: Myoviridae and Siphoviridae. Bacteriophages of S. aureus belong to seven serological groups, designated A, B, C, D, F, G, L. Alternatively, staphylococcal prophages can be classified into five families based on integrase gene homology or, depending on genome sizes, into three classes: class I (<20kbp), class II (approx. 40kbp), and class III (>125kbp) [29, 79]. More than 40 staphylococcal phage genomes have been determined including phages carrying important viru lence determinants such enterotoxin A, exfoliative toxin A, and Panton-Valentine leukocidin [80-82]. The sequence data revealed some interesting structural features of staphylococcal phages. Although the genomes of the phages show a G+C content which is similar to that of the host, most phages contain a large set of genes of unknown function and no homology to bacterial sequences [79]. The coding regions are tightly packed with more than 90% coding capacity. The majority of genes are transcribed from one strand. Some large phages contain a second DNA replication mode which is associated with lytic functions encoding amidase and holin genes. These functions may be important for a broad host range of this class of phages as they infect both coagulase-positive and coagulase-negative sta-phylococci. Interestingly, phage genomes possess a mosaic structure, suggesting that recombination events between different phages are common. All these characteristics highlight the importance of phages for structural flexibility of staphylo-coccal genomes.

The best characterized staphylococcal phage is U11 with a 43.6-kb genome [83]. U11 is a member of the serogroup B and possesses int and xis genes, as does UL54a. Phage U11 is a general transducing phage capable of packaging up to 45 kbp of DNA. The phage is able to package plasmids and chromosomal genes of the host at low frequency, which in turn can be injected into other S. aureus cells. Since these plasmids can replicate and chromosomal genes might be integrated into the chromosome of the new host, transducing phages are important vehicles involved in horizontal gene transfer. For example, tst carrying staphylococcal pathogenicity islands (see above) can be transferred by generalized transduction between different strains of S. aureus. It is tempting to speculate that other gene clusters, too, such as biofilm converting genes or other toxin loci, or even resistance determinants such as mecA, may become subject to transduction by bacteriophages. For many phages, including U12, U13, UPVL, UETA, USLT, and UMu50A, no intrinsic xis function has been identified [83]. Instead these phages carry an ORF of unknown function named OrfC which might perform the xis function, but this has yet to be shown.

Phages have a great impact on the evolution of new pathotypes of staphylococci, mediating both positive and negative lysogenic conversion. For example, the gene for the superantigenic staphylococcal enterotoxin A (sea) that causes food poisoning and toxic shock syndrome is carried by a polymorphic family of related temperate bacteriophages found, e.g., in the genomes of strains Mu50, MW2, MSSA476, and MRSA252 [14-16, 84]. Interestingly, the phage integrates into the genome using an attachment site located within the determinant encoding the b-toxin (hlb) [85]. By a similar mechanism the structural gene of the lipase (geh) is inactivated by bacteriophage L54a or an L54-like phage (UCOL), which att site is composed of an 18-bp core sequence within the reading frame for lipase [17, 86]. Moreover, double- or triple-converting bacteriophages have been described. U13 negatively affects the expression of b-toxin but simultaneously confers the ability to produce staphylokinase, and a serotype F bacteriophage of S. aureus has been found mediating the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase, and b-toxin [87, 88].

Currently, the phage carrying the PVL gene associated with CA-MRSA strains has serious clinical implications. CA-MRSA strains are being increasingly recognized in the community where they colonize persons without any specific risk factors, frequently causing skin and soft tissue infections [73]. Most alarming, however, these strains carry a bacteriophage encoding the PVL toxin, which has been shown to be the major virulence determinant in necrotizing pneumonia [74]. Several recent cases of necrotizing pneumonia in children and young adults were associated with a very high mortality, around 40% [74]. Fortunately, CA-MRSA pneumonia has been rarely reported so far; however, the recent spread of PVL-ex-pressing CA-MRSA strains increases the probability that new, even more aggressive lineages will evolve, and that such strains will reach the hospitals, where they may become resistant to many antibiotics. The short-term evolution of PVL-carry-ing CA-MRSA strains impressively illustrates the importance of bacteriophages for particular virulent forms of S. aureus and the genome flexibility of this important human pathogen.

9.2.3.4 Plasmids

Most naturally occurring staphylococcal strains contain plasmids. These plasmids can be grouped into three major classes. Class I consists of small (1-5 kbp) multicopy plasmids that are either cryptic or carry a single resistance determinant [89]. Plasmids of this class mainly contribute to the development of resistance against oxytetracycline, chloramphenicol, and to macrolides-lincosamides-streptogra-mines (MLS) [89, 90].

Plasmids of class II are 15-30 kbp in size, have low copy numbers (4-6/cell), and mostly carry several resistance determinants: for b-lactams, often associated with transposon Tn552, for mercury resistance, for resistance to cadmium and lead, and also for MLS resistance of the ermB type due to insertion of Tn551 [89, 91,92].

Class III plasmids are conjugative multiresistance plasmids and are therefore of special epidemiological interest. They are comparatively large (30-60 kbp), and besides determinants for conjugative transfer, they carry a number of different resistance determinants: for aminoglycoside resistance on Tn4001, for trimethoprim resistance on Tn4003, for resistance to quaternary ammonium compounds, and in some cases also for b-lactams on a transposon related to Tn552 [93-95].

The association of resistance determinants in S. aureus with transposable elements such as Tn4001 has led to a clustering of resistance determinants on plasmids [93, 96, 97]. There is evidence that certain multiresistance plasmids have evolved by sequential acquisition of resistance determinants based on cointegra-tion of target molecules. This process is mediated by IS257, which can already exist on the captured DNA sequence. Most concerningly, a plasmid carrying the full vanA vancomycin resistance transposon Tn 1546 has recently emerged in S. aureus [13]. The vanA determinant was received from an Enterococcus faecalis strain coinfecting a patient in a hospital. The conjugative enterococcal plasmid was transferred to S. aureus, in which it cannot replicate, but the vanA-containing transposon Tn!546 jumped into a naturally S. aureus-conjugative plasmid. This event impressively shows how resistance determinants can cross the species border, accelerating the development of new resistance types and jeopardizing anti-staphylococcal therapy. Since vancomycin is used as an antibiotic of last resort, the spread of vancomycin-resistant staphylococci, either vertically or by transfer of vanA plasmids horizontally, would have dramatic consequences for the management of infections.

Whereas plasmids carry many resistance determinants, they carry only a few virulence genes. For example, the superantigenic enterotoxins SED and SEJ are located on a 27.6-kb penicillinase plasmid designated pIB485 [98, 99]. The exfoliative toxin B (ETB) gene is carried by a large plasmid of 38.2 kbp which shows similarity to certain conjugative resistance plasmids of the pSK family [100]. Interestingly, pETB contains another virulence-related gene encoding the ADP-ribosyltransferase EDIN-C which modifies Rho GTPases. In addition, a cadmium resistance operon and a lantibiotic-producing region are encoded by this plasmid. Three copies of IS257 divide the pETB genome into three functional regions, suggesting the integration of particular parts of the plasmid by homologous recombination between insertion elements [100].

Some staphylococcal plasmids carry bacteriocin-like genes. The approximately 40-kb plasmid pACK1 of S.simulans biovar staphylolyticus ATCC1362 carries the lysostaphin gene (lss), which codes for an extracellular glycylglycine endopepti-dase, and the lysostaphin immunity factor gene (lif), which leads to an increase of the serine/glycine ratio of the interpeptide bridges of peptidoglycan [101]. Interestingly, lss and lif are flanked by insertion sequences such as IS257and IS2293, indicating that S. simulans biovar staphylolyticus received lif and lss by horizontal gene transfer.

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