Evolution and diversity of community-associated methicillin-resistant Staphylococcus aureus in a geographical region

Background Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in remote regions of Western Australia and is now the predominant MRSA isolated in the state. The objective of this study is to determine the genetic relatedness of Western Australian CA-MRSA clones within different multilocus sequence type (MLST) clonal clusters providing an insight into the frequency of S. aureus SCCmec acquisition within a region. Results The CA-MRSA population in Western Australia is genetically diverse consisting of 83 unique pulsed-field gel electrophoresis strains from which 46 MLSTs have been characterised. Forty five of these sequence types are from 18 MLST clonal clusters and two singletons. While SCCmec IV and V are the predominant SCCmec elements, SCCmec VIII and several novel and composite SCCmec elements are present. The emergence of MRSA in diverse S. aureus clonal clusters suggests horizontal transmission of the SCCmec element has occurred on multiple occasions. Furthermore DNA microarray and spa typing suggests horizontal transfer of SCCmec elements has also occurred within the same CC. For many single and double locus variant CA-MRSA clones only a few isolates have been detected. Conclusions Although multiple CA-MRSA clones have evolved in the Western Australian community only three clones have successfully adapted to the Western Australian community environment. These data suggest the successful evolution of a CA-MRSA clone may not only depend on the mobility of the SCCmec element but also on other genetic determinants.


Background
Based on phenotypic and genotypic typing methods, community onset methicillin-resistant Staphylococcus aureus infections are caused by healthcare-associated MRSA (HA-MRSA) strains, which appear to have been transferred from hospitals or healthcare facilities into the community by patients or healthcare workers [1], or by community-associated MRSA (CA-MRSA) strains, which have been isolated from people who have had little or no contact with healthcare facilities or healthcare workers [2]. This distinction between community and healthcare facility however has become blurred with the replacement of HA-MRSA with CA-MRSA in hospitals [3,4].
In contrast to HA-MRSA, CA-MRSA strains are generally more susceptible to non beta-lactam antibiotics, grow significantly faster, have different clonal backgrounds, carry smaller staphylococcal cassette chromosome mec (SCCmec) elements (most commonly SCCmec type IV or type V), have enhanced virulence properties and frequently harbor genes expressing Panton-Valentine leukocidin (PVL) [5][6][7][8]. Rather than a worldwide spread of a single clone multiple CA-MRSA clones have emerged from diverse genetic backgrounds. Several well characterized CA-MRSA clones predominate in different regions: Sequence type (ST) 8-IV [2B] (USA300) and ST1-IV [2B] (USA400) in North America [9,10]; ST80-IV [2B] (European clone) in Europe [8], North Africa [11] and the Middle East [12]; ST59-V [5C2&5] (Taiwan clone) in Taiwan [13]; ST93-IV [2B] (Queensland clone) in Australia [14], ST30-IV [2B] (South West Pacific [SWP] CA-MRSA) in the Western Pacific [15,16], and ST772-V [5C2] (Bengal Bay clone) in India and Bangladesh [17]. Transmission of these clones into other regions has occurred [18,19]. This occurrence of concurrent epidemics of CA-MRSA in many countries by different clones has been striking. Equally noteworthy are a number of common features of these epidemics, prominent among them the ability to cause severe infections in young otherwise healthy people and the carriage of the lukF-PV/lukS-PV PVL encoding genes by the organism.
The earliest report of CA-MRSA infections involved indigenous people living in remote communities in the sparsely populated Kimberley region of Western Australia (WA) [20]. Approximately 50% of the people in this region are indigenous, many of whom live in poor socioeconomic conditions. Infected skin lesions and staphylococcal sepsis occur frequently and empirical antistaphylococcal therapy is often prescribed. Colloquially known as "WA-MRSA", the early isolates have a similar pulsed-field gel electrophoresis (PFGE) pattern and have subsequently been characterized as a single clone; PVL-negative WA5 (ST8-IV/spa t008) [21]. By 2006 22 CA-MRSA clones were identified in WA, with PVL-negative WA 1 (ST1-IV [2B]/t127) replacing WA5 as the predominant clone [22]. At this time CA-MRSA from indigenous people living in remote areas outside of WA were reported in the Northern Territory [23], Queensland [24] and Central Australia [25]. As may be expected in a geographically large country with relatively few dense concentrations of population, often separated by large areas of desert, different CA-MRSA clones evolved in these communities.
In 1982 colonization or infection with MRSA became a notifiable condition in WA. For infection control purposes all MRSA isolated in the state since 1997 have been referred to the Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research where based on molecular markers they are characterized as either HA-MRSA or CA-MRSA [26]. Although a state-wide policy of screening all patients and healthcare workers who have lived outside the state for MRSA has prevented HA-MRSA from becoming endemic in Western Australian hospitals, it has not prevented CA-MRSA from becoming established in the community. In WA the public health system is divided into two metropolitan health regions and seven country health regions. The state encompasses an area of 1.02 million square miles and has a population of approximately 2.24 million people. In 1983, the overall rate of MRSA notifications was 10 per 100,000 persons in the rural country health regions and 7/100,000 in the metropolitan regions [27]. By 2006 notifications rates throughout the state had increased to 179/100,000 persons of which 144/100,000 were CA-MRSA. In the metropolitan health regions the CA-MRSA notification rate was 134/100,000 whilst in the Kimberley health region the CA-MRSA notification rate had increased 40fold to 391/100,000 [18].
CA-MRSA is thought to emerge when a locally prevalent strain of methicillin susceptible S. aureus (MSSA) acquires a SCCmec element and utilizes mobile genetic elements and single nucleotide polymorphisms to establish local and geographic niches [28]. As WA is a remote region in which all MRSA isolates are referred to a central typing laboratory it is an ideal environment to study the emergence and evolution of CA-MRSA. MLST, SCCmec, spa typing and microarray DNA is performed on all isolates with a unique PFGE pulsotype. The aim of this study is to determine the genetic relatedness of WA CA-MRSA clones within different MLST clonal clusters (CC) providing an insight into the frequency of S. aureus SCCmec acquisition within a region. The genetic profile of these clones may also offer an explanation why only a few WA CA-MRSA clones have successfully adapted to the community environment.

Results
The 83 unique PFGE strains isolated in Western Australia from 1989 to 2010 were nuc and mecA gene positive by PCR. The DNA microarray S. aureus species markers gapA (glyceraldehyde 3-phosphate dehydrogenase) and rrn STAU (S. aureus ribosomal marker) were detected in all strains. The array's linear primer elongation method detected the katA (catalase A), coA (coagulase), nuc, spa (protein A) and sbi (IgG-binding protein) S. aureus species markers in 78 strains. These markers were either not detected or detected only by random amplification in five strains (WA8, WA47, WA72, WA76 and WA79).
Forty six STs were identified by MLST. Using the MLST website's eBURST V3 algorithm 45 STs were grouped into 18 CCs and two singletons ( Figure 1) and WA45 harbor a ccrA-1 and ccB-1 gene complex and Q6GD50 (fusidic acid resistance marker) indicating the presence of the mobile fusidic acid SCC element SCCfur. WA1 is known to carry multiple plasmids such as a 2-kb plasmid encoding resistance to erythromycin [29] and this presumably accounts for the differences in the antibiogram and resistance genotype for WA1, WA45 and WA57. In addition to enterotoxin genes the three strains harbor a type D immune evasion cluster [IEC] (seA+sak+scn) [30]. Group   carries several enterotoxin genes including the enterotoxin egc cluster [seG+seI+seM+seN+seO+seU/Y]). Unlike WA1, WA45 and WA57, WA10 does not carry the type D IEC, the pathogenicity island harboring the leukocidin D/E component, the protease splA gene and the hsdS gene. The ssl/set genes and cell surface adhesions encoding genes of WA10 are closely related to the Bengal Bay clone.

Clonal Complex 5
CC5 contains 27 strains. Based on the agr/capsule type the isolates are divided into two groups which are further divided into subgroups based on the SCCmec type.

Clonal Complex 398
Although PVL negative ST398-V [5C2&5]/t034 is frequently associated with livestock, the strain is increasingly isolated from human patients [34]. Rarely identified in Australia, the DNA microarray profile of this isolate is homogeneous with the European livestock-associated ST398 strain and is therefore not considered a WA CA-MRSA.

Queensland Clone (Singleton)
PVL positive ST93-IVa [2B]/t202 is agr group III/capsule type 8 and harbors a type B IEC. The DNA microarray profile is homogeneous with the Queensland clone. Due to its origin and widespread distribution outside WA the Queensland clone is not considered a WA CA-MRSA.

Discussion
As all MRSA isolated in WA are referred to a central typing laboratory it is possible to investigate the emergence and evolution of CA-MRSA in a remote region.
Prior The emergence of WA1, WA2 and WA3 has been due to the acquisition and insertion of the small and highly mobile type IVa [2B] SCCmec element, presumably harbored by methicillin resistant coagulase negative staphylococci (MRCNS). Several hypotheses to explain the transmission of a SCCmec element from MRCNS to S. aureus have been proposed including the increased use of antimicrobials within a community [35]. Many of the Kimberley indigenous population live in poor socioeconomic conditions. Staphylococcal skin lesions, commonly resulting from scabies infestation, trachoma and venereal diseases such as chlamydia and gonorrhea occur frequently in this population. Consequently empirical therapy using β-lactamase stable penicillins and azithromycin is often prescribed [36]. The frequent use of these antimicrobials may have assisted in the acquisition of the SCCmec element and erm genes into S. aureus. Genetic studies however have shown these newly emerged CA-MRSA clones did not originate in the predominant methicillinsusceptible S. aureus (MSSA) clones found in these communities, suggesting not all clones are able to acquire or retain the SCCmec element [37]. The subsequent dissemination of WA1, WA2 and WA3 into the wider community suggests the acquisition of the SCCmec element and the erm genes has given these clones a selective advantage. WA4 and WA5 however have not been successful in spreading beyond the indigenous communities suggesting the acquisition of the SCCmec element does not provide a universal selective advantage.
Many of the remaining 46 CA-MRSA clones, identified between July 2003 and June 2010, were not isolated in remote WA indigenous communities. The geographical spread of CA-MRSA over long distances and across cultural borders is believed to be a rare event compared to the frequency in which the SCCmec element is acquired by S. aureus [38]. Most of these clones are therefore likely to have evolved in WA. Some clones are slvs and dlvs of pre-existing CA-MRSA, and their SCCmec type, spa type and DNA microarray profile suggests vertical transmission of the SCCmec element has occurred. However the emergence of MRSA in several unrelated S. aureus clonal clusters suggests horizontal transmission of the SCCmec element has also occurred. SCCmec typing and spa typing and DNA microarray results also suggests horizontal transfer of SCCmec elements has occurred into the same CC on more than one occasion.
Although several SCCmec elements have been acquired by multiple S. aureus clones from which many CA-MRSA clones have emerged, only a few clones have successfully adapted to the WA community  [18,31,32]. Patients colonized or infected with the Bengal Bay clone have been observed to be epidemiologically linked to Indian healthcare workers (unpublished data). The USA300, European, Taiwanese and Bengal Bay CA-MRSA clones are not frequently isolated in WA. This may be due, in part, to WA Health Department infection control interventions applied to patients who are colonized or infected with international PVL positive pandemic clones. A seventh pandemic clone has recently been identified. The DNA microarray profile and the SCCmec element of the PVL negative ST398-V [5C2&5] is indistinguishable from the pandemic ST398 clone initially isolated from pigs and pig farmers in the Netherlands [39]. Only one isolate, from a patient with travel outside of Australia, has been identified in WA.
The Queensland clone (ST93-IVa [2B]) first detected on the east coast of Australia in the Caucasian population in 2000 [40], has become one of the most prevalent CA-MRSA isolated in Australia [18] and in 2010 accounted for 18% of CA-MRSA in WA. This suggests the acquisition of the SCCmec element has given this clone a selective advantage. Although the Queensland clone is believed to have been introduced into WA in 2001 [22], PVL positive ST93-MSSA was identified as the most prevalent S. aureus clone in WA's remote indigenous communities in surveys performed in the mid 1990s. Although found in an environment of high β-lactam use a methicillin-resistant variant of ST93-MSSA was not found in WA during these surveys.
WA1, WA2 and WA3 are PVL negative and do not harbor multiple virulence genes (Tables 1). Similarly the successful Queensland clone, although PVL positive, carries almost no other exotoxin genes and no additional resistance genes. Although most other WA CA-MRSA clones are also PVL negative, many of these clones have acquired multiple resistance and/or virulence determinants (Tables 1). For example WA78 (ST188-IVa [2B]/ t315) in addition to mecA and blaZ, harbors aacA-aphD, tetK and cat and is phenotypically resistant to erythromycin, trimethoprim and ciprofloxacin; WA64 (ST5-IVa [2B]/t3778) has acquired seA enterotoxin genes and edinA and lukF-PV lukS-PV virulence genes; and WA62 (ST923[ST8slv]-IVa [2B]/t1635) harbors seD+seJ+seR and seK+seQ enterotoxin genes and lukF-PV lukS-PV. The acquisition of multiple resistance and/or virulence genes may have come at a high fitness cost as none of these clones have established a niche in the WA community.
As WA1, WA2 and WA3 CA-MRSA lack PVL as well as other virulence genes that are found in pandemic international CA-MRSA clones, such ACME in USA300, the epidemiology of CA-MRSA disease in WA is different to other regions. Outside of WA the majority of diseases related to CA-MRSA infection are severe skin and soft tissue infections such as soft tissue abscess, carbuncles and furuncles. Many of these infections have occurred in healthy individuals, especially children and adolescents, usually via skin-to-skin contact [41]. In WA the majority of CA-MRSA related diseases were initially associated with the indigenous population and then other groups normally susceptible to S. aureus infections such as the elderly. As the original WA CA-MRSA are PVL negative, many of these infections were superficial skin infections such as impetigo. However with the introduction of the PVL-positive Queensland CA-MRSA clone more severe skin and soft tissues infections have been observed.
The limitation of this study is that only the initial isolate of each PFGE pulsotype was included in the study. To determine if the successful CA-MRSA clones found in the WA community are evolving the genetic profiles of subsequent isolates need to be investigated.

Conclusions
In conclusion although the vertical and horizontal transmission of SCCmec elements into S. aureus has occurred on multiple occasions in the WA community only three WA CA-MRSA clones have found an ecological niche. These three PVL negative clones harbor few additional resistance and virulence genes which paradoxically may account for their success.

Isolates
The isolates studied are representative of the 83 CA-MRSA unique PFGE strains identified in WA from 1989 to 2010 (Figure 3). They include five strains isolated  nuc and mecA S. aureus species and methicillin resistance was confirmed by the detection of nuc (thermostable extracellular nuclease) and mecA (methicillin resistance) genes by PCR [43].

PVL
PCR for the detection of PVL determinants was performed as previously described [48].

PFGE
Electrophoresis of chromosomal DNA was performed as previously described [49], using a contour-clamped homogeneous electric field (CHEF) DR III system (Bio-Rad Laboratories Pty Ltd). Chromosomal patterns were examined visually, scanned with a Quantity One device (Bio-Rad Laboratories Pty Ltd), and digitally analyzed using FPQuest (Bio-Rad Laboratories Pty Ltd). S. aureus strain NCTC 8325 was used as a reference strain.

MLST and spa typing
Chromosomal DNA for MLST and spa typing was prepared using a DNeasy tissue kit (Qiagen Pty Ltd).
MLST was performed as previously described [50]. The sequences were submitted to http://www.mlst.net/ where an allelic profile was generated and an ST assigned. Clonal complex (CC) was determined using the eBURST V3 algorithm at the same website. Clones that diverged at no more than one of the seven MLST loci were considered to belong to the same CC. Double locus variants (dlvs) were included if the linking single locus variant (slv) was present in the MLST database.
spa typing, a DNA sequenced-based analysis of the protein A gene variable region was performed as previously described [51] using the nomenclature as described on the Ridom website (http://spa.ridom.de/).

SCCmec typing
The strategy used for SCCmec typing was as previously described [32]. SCCmec nomenclature is used as proposed by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) [52]. Briefly, the structural type is indicated by a Roman numeral, with a lowercase letter indicating the subtype, and the ccr complex and the mec complex are indicated by an Arabic numeral and an uppercase letter respectively in parenthesis. Where there is an extra ccr element, this is indicated by "&" and an Arabic numeral designating the ccr type. When there is an extra ccr element present whose precise location is unknown it is indicated by an "&" and ccr number outside the parentheses.

DNA microarray
Arrays and reagents were obtained from Alere Technologies, Jena Germany. The principle of the assay, related procedures, and a list of targets has been described previously [53,54]. An iterated, linear primer elongation was employed for the simultaneous amplification of all targets. An alternative protocol was used for a few isolates in which amplification and labeling was directed by random primers [55]. This method detects target genes for which the binding sites of the primers used in the first protocol were deleted or changed by nucleotide polymorphisms. Target genes included species markers, markers for accessory gene regulator (agr) alleles and capsule types, virulence factors, resistance genes, staphylococcal superantigen-like/exotoxin-like genes (set/ssl genes) and genes encoding adhesion proteins. Probes for mecA, ugpQ, xylR, and two probes for mecR were used for SCCmec typing. The last two probes allowed detection and discrimination of untruncated mecR and ΔmecR, respectively. Probes for the recombinase genes ccrA1, ccrB1, ccrA2, ccrB2, ccrA3, ccrB3, ccrA4, ccrB4, and ccrC1; the fusidc acid resistance marker Q6GD50; and the J region proteins, dcs, pls-SCC and the kdp-operon were also included.

MRSA Strain Definition
MRSA strains are defined according to their unique PFGE pulsotype

MRSA Clone Definition
MRSA clones are defined by the combination of the multilocus sequence type (ST) and the SCCmec type [56]. For instance ST1-SCCmec IVa [2B] is abbreviated as ST1-IVa [2B].