From July 2012 to December 2013, a total of 80 sequential CA-MRSA isolates, which represent all the non-duplicate strains isolated during the study period, were collected from pediatric patients with local or systemic infections in a university hospital in Shanghai, China (Shanghai Children's Medical Center, affiliated with Shanghai Jiao Tong University; CLSI, 2012). Shanghai Children's Medical Center is one of the largest pediatric hospitals in China with 800 beds and ~3000 hospital admissions per day. MRSA isolates were confirmed by classic microbiological methods: Gram stain and catalase and coagulase activity on rabbit plasma. They were further identified by biochemical characterization using the Api-Staph test (bioMérieux, Lyon, France).

CA-MRSA was defined as an MRSA isolate that was obtained either from an outpatient or an inpatient within 48 h of hospitalization, and without the patient having medical history of MRSA infection or colonization, admission to a healthcare facility, dialysis, surgery or insertion of indwelling devices in the past year.

These isolates were recovered from several clinical sources, including the respiratory tract (sputum, pharynx swabs, and bronchial alveolar lavage fluid), skin and soft tissue (cutaneous abscess and wound secretion), cerebrospinal fluid, blood and urine (Klevens et al., 2007). All strains were stored at −70°C and grown overnight on sheep blood agar plates at 37°C.

The Ethics Committee of Shanghai Children's Medical Center exempted this study from review because the present study focused on bacteria.

The antibiotic susceptibility of all isolates in this study was performed using the bioMe'rieux VITEK2 system following manufacturer's instructions. Results were interpreted in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2012). The following 17 drugs were tested: cefazolin (CFZ), linezolid (LZD), ciprofloxacin (CIP), clindamycin (DA), erythromycin (E), trimethoprim-sulfamethoxazole (SXT), moxifloxacin (MOF), nitrofurantoin (FD), vancomycin (V), tetracycline (TET), penicillin (P), rifampicin (RF), levofloxacin (LVX), ampicillin (AMP), gentamicin (GM), quinupristin/dalfopristin (Q/D), and tigecycline (TGC). S. aureus ATCC 29213 was used as a quality control.

Isolates were screened according to the protocol described (Enright and Spratt, 1999) on the S. aureus MLST website (http://saureus.mlst.net) to detect the following seven housekeeping genes (Aanensen and Spratt, 2005): carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glp), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyl coenzyme A acetyltransferase (yqiL). PCR amplicons of seven S. aureus housekeeping genes were obtained from chromosomal DNA. The sequences of the PCR products were compared with the existing alleles available from the MLST website, and the allelic number (sequence type, ST) was determined for each sequence. Clustering of related STs, which were defined as clonal complexes (CCs), was determined using eBURST (based on related STs).

The MRSA isolates were subjected to SCCmec typing as described by Kondo et al. (2007), which was based on a set of multiplex PCRs (M-PCRs) with 14 primers. SCCmec types I–V were assigned according to the combination of the cassette chromosome recombinase (ccr) type and mec class. MRSA isolates that could not be assigned to any expected type were defined as nontypable (NT).

In S. aureus, the polymorphic X region of staphylococcal protein A (spa) gene consists of a variable number of 24 bp repeat units (Shopsin et al., 1999) that allow isolates to be distinguished from one another. The spa typing was based on variations of the repeat units. Amplification and sequencing of the X region were performed as described previously by Koreen et al. (2004). The spa typing was assigned by submitting the data to the S. aureus spa type database (http://spaserver.ridom.de).

All MRSA isolates were screened for the following 22 staphylococcal virulence genes: the staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, seq, sek), the toxic shock syndrome toxin (tsst), the arginine catabolic mobile gene (arcA), the exfoliative toxin genes (eta, etb), the PVL genes (lukF/S-PV; Lina et al., 1999), the hemolysin gene (hla, hlb, hlg), and the adhesin genes (clfA, icaA, sdrC, and sdrE) as previously described (Arvidson and Tegmark, 2001; Peacock et al., 2002; Bubeck Wardenburg et al., 2007).

Statistical analyses were performed using Stata software (version 10.1/SE, Stata Corp., College Station, TX, USA). We used the χ² and Fisher's exact tests, as appropriate for analysis of categorical data. Statistical significance was set at P ≤ 0.05.

Eighty MRSA isolates were obtained from patients 10 days to 11 years old with local or systemic infection and all the isolates were positive for the mecA genes. Among them, 52 patients (65%) were male. 61.3% (49/80) of the children were <1 year old and 18 were neonates (22.5%). From the clinical medical records, respiratory infection was the most frequently determined infection type caused by CA-MRSA; 82.5% (66/80) of the isolates were from the respiratory tract, and 5% (4/80) of the isolates were associated with bloodstream infection (BSI).

The evolutionary and genetic diversity of MRSA isolates within individual patients was analyzed by MLST (Table 1). There were 16 distinct STs identified within the 80 isolates, among which the most frequently represented was ST59 (45.0%, 36/80). It accounted for nearly one half of all MRSA isolates, followed by ST398 (11.3%, 9/80), ST88 (8.8%, 7/80), ST5 (7.5%, 6/80), ST630 (7.5%, 6/80), ST7 (3.8%, 3/80), ST338 (2.5%, 2/80), ST45 (2.5%, 2/80), ST9 (2.5%, 2/80). Among the remaining 7 STs (ST30, ST72, ST6, ST20, ST950, ST1507, ST121), each ST just had one isolate. This methodology revealed that the strains clustered by eBURST into 9 CCs (CC59, CC398, CC88, CC5, CC8, CC7, CC72, CC45, CC9) and 4 singletons (Figure 1), among which were CC59 with 38 isolates, CC398 with 9 isolates, CC88 with 7 isolates, CC5 with 7 isolates, CC8 with 6 isolates, CC7 with 3 isolates, CC72 with 2 isolates, CC45 with 2 isolates, and CC9 with 2 isolates. Sixteen isolates harboring pvl were distributed among 7 different STs, including ST59 (n = 9) and ST338 (n = 2), as well as ST398, ST88, ST30, ST20, and ST121 (one isolate each).

By SCCmec typing, only two types (types IV and V) were found among 80 MRSA isolates. The most common was type IV, which was found in 48 isolates (60%, 48/80), while type V was found in 32 isolates (40%, 32/80).

The spa typing discriminated MRSA isolates into 30 spa types. Among them, spa t437 was the most predominant type (23.8%, 19/80), followed by t441 (8.8%, 7/80), t034 (7.5%, 6/80), t4549 (6.3%, 5/80), t172 (5%, 4/80), t316 (5%, 4/80), and t2310 (5%, 4/80). Each of the remaining spa types was represented in <3 isolates.

There was a strong association observed between specific ST and spa types. The ST59 genotype was associated primarily with spa t437 (47.2%, 17/36) and spa t441 (19.4%, 7/36), and less frequently with six types: t316, t172, t163, t3523, t8886, t1751. The ST398 genotype was associated mainly with spa t034 (6/9) and spa t011 (2/9).

The antimicrobial resistance profiles of 80 MRSA isolates according to MLST are listed in Table 2. All the strains were resistant to cefazolin, penicillin, and ampicillin, but susceptible to vancomycin, linezolid, nitrofurantoin, quinupristin/dalfopristin, and tigecycline. The majority were resistant to clindamycin (80%) and erythromycin (82.5%), however, they were susceptible to most of the antibiotics tested. The resistance rates to other antibiotics tested were 8.8% to ciprofloxacin, 10% to trimethoprim-sulfamethoxazole, 3.8% to moxifloxacin, 32.5% to tetracycline, 2.5% to rifampicin, 5% to levofloxacin, and 3.8% to gentamicin. The resistance profiles of MRSA isolates differed by their STs. For the ST59 strains, the most prevalent antibiotic group contains clindamycin, erythromycin, and tetracycline, while ST9 strains were more resistant to more antibiotics including ciprofloxacin, clindamycin, erythromycin, trimethoprim-sulfamethoxazole and tetracycline.

The distribution of 19 putative virulence genes differed among the 80 MRSA strains according to MLST (Table 3). All virulence genes except eta, etb, and arcA genes were identified within multiple isolates, and all isolates exhibited carriage of at least 4 virulence genes. Thirty-four (42.5%, 34/80) isolates harbored ≥10 tested virulence genes, among which were 1 isolate with 13 genes, 3 isolates with 12 genes, 14 isolates with 11 genes, and 16 isolates with 10 genes.

Adhesion genes were present in most of the MRSA isolates; 100% carried the icaA and clfA genes, 95% harbored sdrC, 63.8% carried sdrE. The most prevalent toxin genes detected were hla (100%), hlg (87.5%), hlb (60%), seb (46.3%), sek (41.3%), and seq (41.3%). The pvl gene was detected in 16 strains, which represented seven different STs, with ST59 being the most common.

In this study, ST59 (45.0%, 36/80) was found to be the most prevalent clone, which was associated primarily with spa t437 (44.7%, 17/36) and spa t441 (18.4%, 7/36). ST59 strains were more resistant to erythromycin (P = 0.002) and clindamycin (P = 0.004) but more susceptible to ciprofloxacin (P = 0.015) and trimethoprim-sulfamethoxazole (P = 0.007) than other STs (Table 4). In addition, non-ST59 strains were more often associated with multiple antibiotic-resistance profiles. They were also resistant to moxifloxacin, rifampicin, levofloxacin, and gentamicin, while ST59 did not have these profiles.

All isolates exhibited icaA, clfA, and hla genes. The frequency of carriage for hlb, sea, seb, sek, seq, or sdrE among ST59 isolates was significantly higher than that for non-ST59 isolates (P < 0.05; Table 5). Twenty-eight (77.8%) of 36 ST59 isolates harbored ≥10 tested virulence genes, which was significantly higher than that among non-ST59 isolates (13.6%, 6/44) (P < 0.05). However, there were no significant differences on the positive rate of pvl between ST59 and non-ST59 strains.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.