Characterization of Staphylococcus aureus Isolated from Non-Native Patients with Skin and Soft Tissue Infections in Shanghai

Background Staphylococcus aureus is one predominant cause of skin and soft-tissue infections (SSTIs), but little information exists regarding the characterization of S. aureus from non-native patients with SSTIs in China. Methods In this study, we enrolled 52 non-native patients with S. aureus SSTIs, and 65 native control patients with S. aureus SSTIs in Shanghai. 52 and 65 S. aureus isolates were collected from both groups, respectively. S. aureus isolates were characterized by antimicrobial susceptibility testing, toxin gene detection, and molecular typing with sequence type, spa type, agr group and SCCmec type. Results Methicillin-resistant S. aureus (MRSA) was detected in 8 non-native patients and 14 native patients with SSTIs. Overall, antimicrobial susceptibilities of S. aureus isolated from non-native patients were found higher than those from native patients. CC59 (ST338 and ST59) was found in a total of 14 isolates (4 from non-native patients; 10 from native patients), 9 of which were carrying lukS/F-PV (3 from non-native patients; 6 from native patients). ST7 was found in 12 isolates and all 12 isolates were found in native patients. The livestock-associated clone ST398 was found in 11 isolates (6 from non-native patients; 5 from native patients), and 5 ST398 lukS/F-PV-positive methicillin-susceptible S. aureus (MSSA) were all discovered among non-native patients. The molecular epidemiology of S. aureus isolated from non-native patients was quite different from those from native patients. lukS/F-PV was more frequent in isolates originating from non-native patients with SSTIs compared to native patients (31 vs. 7, P <0.0001). Conclusions CC59 was the most common clonal complex among patients with SSTIs in Shanghai. The other most common sequence types were ST7 and Livestock ST398. The molecular epidemiology of S. aureus isolated from non-native patients was quite different from those from native patients. S. aureus isolated from non-native patients was more likely to carry lukS/F-PV.


Introduction
Skin and soft tissue infections(SSTIs) are common and range in severity from minor, self-limiting, and superficial infections to life-threatening diseases requiring all resources of modern medicine [1].Staphylococcus aureus currently is the leading cause of SSTIs across all continents [2,3].MRSA was first discovered in the 1960s, and quickly became a critical pathogen in hospitals globally, leading to the emergence of healthcare-associated MRSA (HA-MRSA). Community-associated MRSA (CA-MRSA) subsequently appeared in Chicago, among other part of North America, in the late 1980s and early 1990s [4].Infection of the skin and soft tissues is by far the most frequent disease manifestation associated with CA-MRSA, accounting for at least 90% of all CA-MRSA infections [5].
S. aureus contains a wide variety of enzymes and toxins including the Panton-Valentine leukocidin(PVL). PVL is one toxin produced by S. aureus, and consists of two components with subunits LukS-PV and LukF-PV which are secreted individually by the bacteria before forming a heptamer that induces pores in polymorphonuclear leukocytes [6].PVL may well be the key toxin in SSTIs, but there is some debate about whether it is the key pathogenic factor in necrotic SSTIs and necrotic haemorrhagic pneumonia, or remains just as a marker of such diseasecausing potential. Other virulence factors playing a role in SSTIs include coagulase, protein A, toxic shock syndrome toxin (TSST1) and scalded skin syndrome toxin (SSST) [7].
In China, ST59-MRSAIV/V-t437 was the most common clone among CA-MRSA isolates reported among children with SSTIs [8,9].One recent study in Beijing reported the livestock ST398 clone as having a high prevalence in S. aureus SSTIs, where 64.3% of ST398 strains were harboring the lukS/F-PV gene [10]. In Japan, the most common clone among SSTI-associated MRSA was MLST-CC8/spa-CC008-SCCmec-IV [11].CC8 is the most common type in S. aureus SSTIs, especially among PVL-positive S. aureus as reported in New York, the United States [12].
Shanghai is a large, dynamic city in China where about 200,000 foreigners reside, many of whom lead different lifestyles and receive much different medical care in comparison to the native Chinese population. To our knowledge, there exists no information characterizing the differences, if any, of S. aureus in SSTIs between non-native patients and native patients which respect to antimicrobial resistance, toxin production and gene typing. Our study aims to discover any significant differences in antimicrobial susceptibility, toxin gene detection and molecular typing of S. aureus between non-native and native patients with SSTIs.

Patients and Bacterial isolates
A total of 117 patients, including 52 non-native patients and 65 native patients, from three hospitals (Hospital A, B and C) with SSTIs were enrolled in this study. All 117 S. aureus isolates were collected from these patients during the period of January 2011 to November 2013. Hospital A is a joint venture hospital mainly serving foreigners in Shanghai, while Hospital B and C are tertiary public hospitals in Shanghai that mainly serve native patients. Native patients in our study were defined as patients who were born in Mainland China, live permanently in Shanghai and carry publicly provided medical insurance. Non-native patients were defined as those who originated from other countries or regions, and short-term or long-term stay in Shanghai for employment or travel. Non-native patients do not carry publicly provided medical insurance, and instead generally seek medical care from specific hospitals (like Hospital A in our study) that accept health plans offered by private international insurance carriers. Four patients from Hong Kong SAR of PRC in Hospital A in this study conform to the definition of non-native patients and carried private international insurance. They were enrolled in the non-native group.
Fifty-two S. aureus isolates from 52 non-native patients with SSTIs in Hospital A were collected. Sixty-five S. aureus isolates collected from 65 native patients with SSTIs were from Hospitals B and C, and served the controls in this study. The SSTIs we studied were considered superficial, uncomplicated infections, including impetigo, erysipelas and cellulitis. All 117 S. aureus isolates enrolled in this study were identified by a combination of phenotypic tests as previously described [13].
CA-MRSA is defined as MRSA isolated from an outpatient or within 2 days of a patient's hospitalization. Exclusion criteria included a history of hospitalization for illness, surgery, or dialysis in the previous year or the presence of indwelling catheters or other medical devices. HA-MRSA was defined by isolation >2 days after hospitalization or presence of any of the aforementioned healthcare risks [14].
This study was approved by the Ruijin Hospital Ethics Committee (Shanghai Jiao Tong University School of Medicine).The Review Board exempted the need for informed consent for this retrospective study.

Detection of toxin genes
A variety of clinically significant toxin genes were detected by PCR, including lukS/F-PV (encoding Panton-Valentine leukocidin); tst (encoding toxic shock syndrome toxin 1); eta and etb (encoding exfoliative toxin A and B); sea-see and seg-sej (encoding staphylococcal enterotoxins SEA-SEE and SEG-SEJ) [16]; and sasX (encoded in a mobile genetic element) which also acts as a virulence determinant and plays a key role in MRSA colonization and pathogenesis as recently reported by Li et al. [17].

Detection of molecular types
DNA was extracted by the simplified alkaline-lysis method [13]. All S. aureus isolates were investigated by multilocus sequence typing (MLST), spa typing and accessory gene regulator (agr) typing as previously described [18]. MRSA isolates were confirmed by the mecA gene, and SCCmec types of MRSA were determined by a method previously described [19].

Statistical analysis
The chi-square or Fisher's exact test were used where appropriate using the software package SAS 8.2 (SAS Institute Inc., Cary, NC, USA). A two-sided P value of < 0.05 was considered statistically significant.

Clinical data
The median age of non-native patients and native patients was 30 years (range: 2-56 years) and 48 years (range: 8-84 years) respectively, while the sex distribution (male/ female) was 32/20 (61.5%/38.5%) and 35/30 (53.8%/46.2%) respectively. Among the non-native patients, 51.9% were outpatients, whereas as 43.1% of the native patients were considered outpatients. Abscesses were the most common infection type in both groups.

Antimicrobial susceptibility testing
Ninety-five isolates (44 from non-native patients; 51 from native patients) were MSSA, and 22 isolates (8 from non-native patients; 14 from native patients) were confirmed as MRSA. All 117 isolates were susceptible to quinupristin-dalfopristin, linezolid and vancomycin. Only one MRSA isolated from a native patient showed intermediate susceptibility to teicoplanin. Sixteen isolates (13.7%) were susceptible to penicillin with β-lactamase negative. Seven isolates (4 MRSA and 3 MSSA from native patients) were observed showing high-level mupirocin resistance and 5 (4 MRSA and 1 MSSA) of which all belong to ST764. A total of 25 isolates (1 MRSA and 9 MSSA from non-native patients, 4 MRSA and 11 MSSA from native patients) were inducibly resistant to clindamycin based on D-test results. Antimicrobial susceptibilities of S. aureus isolated from non-native patients were mostly higher than those from native patients. These findings are summarized in Table 1.

Virulence factors
As presented in Table 2, seg and sei were the most frequently found of the enterotoxin genes that we detected, occurring in 42 and 43 isolates respectively. lukS/F-PV was found in 38 isolates, of which 31 isolates (8 MRSA and 23 MSSA) were from non-native patients and7 isolates (4 MRSA and 3 MSSA) were from native patients. No see-positive isolates were detected in non-native patients or native patients. S. aureus isolates from non-native patients were more likely to carry lukS/F-PV, seg and sei (P<0.0001, P = 0.0045 and P = 0.0023 respectively), while those from native patients tended to carry sea (P = 0.0409).

Molecular epidemiologic characteristics
Among the 117 S. aureus isolates, 31 sequence types (STs) and 59 spa types were identified (S1 Table). We detected 8 SCCmec type I, 4 SCCmec type II, 2 SCCmec type III, 3 SCCmec type IV, and 4 SCCmec type V isolates. One MRSA isolate could not be SCCmec typed. As seen in Table 3, MRSA and MSSA isolates from both non-native patients and native patients expressed a great diversity in molecular epidemiologic characteristics. Only one specific clone, ST338-SCCmecV-t437 was present in MRSA isolates from both non-native patients and native patients. As shown in Table 3, we discovered a few characteristics to be similar among clones from S. aureus isolates from non-native patients and native patients. The most common ST was ST7 (12/117, 10.3%), followed by ST338 (11/117, 9.4%), ST398 (11/117, 9.4%) and ST217 (8/117, 6.8%). All 12 ST7 isolates were sourced from native patients, and all 8 ST217 isolates were isolated from non-native patients. ST338 was discovered in 3 isolates from non-native patients and 8 isolates from native patients, and ST398 was found in 6 isolates from non-native patients and 5 isolates from native patients.ST338 was the most common ST in lukS/F-PV-positive isolates, including 3 isolates (2 MRSA and 1 MSSA) from non-native patient and 5 isolates  agrI was the most frequent agr group found in isolates from both non-native patients and native patients (27/52, 51.9%; 47/65, 72.3%respectively), but was found more prevalent in native patients (P = 0.0231). In contrast, agrIII and agrIV were found more likely to occur in non-native patients (11 vs. 5, P = 0.0352; 6 vs. 1, P = 0.0234, respectively). agrII was detected in 8isolates from non-native patients and 12 isolates from native patients (P = 0.6604).

Discussion
S. aureus particularly CA-MRSA is the predominant cause of SSTIs worldwide [20].In our study, 22 isolates (18.8%) of 117 isolates were found as MRSA, and it was lower than the occurrences of MRSA among S. aureus SSTIs previously reported [1,2,21].Eight MRSA isolates (3 HA-MRSA and 5 CA-MRSA) were found among non-native patients and 14 MRSA isolates (12 HA-MRSA and 2 CA-MRSA) were found among native patients. We found no significant difference between the occurrences of MRSA in non-native patients compared to native patients. Nevertheless, as shown in Table 1, the isolates from non-native patients showed higher antimicrobial susceptibilities than those from native patients, and high-level mupirocin resistance was detected only in 7 isolates from native patients. High-level mupirocin resistance is mediated by the mupA gene, which is located on plasmids that vary in size, restriction patterns and ability to transfer by conjugation [22]. In this study, mupA was detected in the 7 high-level mupirocin resistance isolates and found positive in 5 isolates. Of 5 mupA-positive isolates, 4  (1) ST338-SCCmecI-t437 59 ST338-SCCmecV-t437 59 ST338-SCCmecIV-t437 59 (2), sei (2) ST88-SCCmecI-t1814 88 isolates were ST764 (3 MRSA and 1 MSSA) and one isolate was ST338 MSSA. These resistance findings among native patients are probably due to overuse of antibiotic therapy which commonly occurs in public hospitals in China. The different occurrences of HA-and CA-MRSA between non-native and native patients may also imply the different routes of MRSA infection among these two groups. It is likely that non-native patients are more likely to contract CA-MRSA SSTIs in comparison to native patients according to the feature of MRSA as previously described, whereas native patients are more frequently infected with HA-MRSA SSTIs.
As common clone lineages in MRSA, ST338-t437was usually associated to SCCmec types IV or V, and ST30-t019 was usually associated to SCCmecIV. However, in this study we have found 4 ST338-t437 MRSA isolates and 1 ST30-t019MRSA isolate that associated to SCCmecI.  Ever since its emergence in 2000 in the United States, prevalence ofST8 (USA300) has led to a high burden of SSTIs globally [23], as witnessed by S. aureus clone outbreaks among SSTIs in Japan, Korea, Singapore and Europe [11,[24][25][26][27].We found one ST8 isolate from a non-native patient in our study as ST8-SCCmecI-t5160 with lukS/F-PV positive. However, CC8 (ST8, ST239, ST630 and ST1821) was found in 10 isolates, of which 2 (1 MRSA and 1MSSA) were from non-native patients and 8 (2 MRSA and 6 MSSA) were from native patients. CC8 might also be an important clonal complex among SSTIs in China especially in native patients. The occurrence of CC8 was significantly higher among native patients in comparison to non-native patients (8vs. 2, P = 0.0230). ST239 is recognized as a common epidemic clone in bloodstream infections in China [18] and ST630 was also recently reported to cause severe infective endocarditis with systemic embolism in China [28].ST7,found in a total of 12 MSSA isolates, was the most common ST in our study. ST7 has also been found to be among the most common genotypes of MSSA in invasive community-acquired S. aureus infection in Chinese children, along with ST88, ST25, ST2155 and ST188 [29]. As shown in Table 3, the clone characteristics of S. aureus isolated from non-native patients and native patients were quite different. The molecular characterization of isolates from non-native patients did not show any centralized tendency or distinguishing features related to the countries (regions) or continents of patients' origin.
A study by Zhao et al. from Beijing reported that the livestock-associated ST398clone in MSSA was the most prevalent PVL-positive clone in SSTIs [10]. Similarly, ST398 in our study was found in 11 MSSA isolates (6 from non-native patients; 5 from native patients), and 5 lukS/F-PV-positive isolates were all from non-native patients.ST398 presented decreased human-to-human transmissibility, and we lack any clinical data on any possible patient contact with animals. Zhao et al. also did not find any association between livestock contact and ST398 infection [10]. However, careful monitoring of human-to-human transmissibility of ST398 of course remains important [30]. Even though there is no statistically significant difference between the ST398 isolates from non-native patients and those from native patients in our study, it is notable that the 5 lukS/F-PV-positive ST398 isolates were only found in non-native patients. We believe the occurrence of lukS/F-PV only in non-native patients is not coincidental when compared to the complete lack of lukS/F-PV in native patients (5/6 vs. 0/5, P = 0.0152).
PVL production by S. aureus is associated with SSTIs, most notably in a recent outbreak of PVL-positive MSSA SSTIs in France [31]. A total of 38 lukS/F-PV-positive isolates were discovered in our study, of which 31 isolates (8 MRSA and 23 MSSA) were from non-native patients and7 isolates (4 MRSA and 3 MSSA) were from native patients. PVL-positive S. aureus isolates were more likely to spread among non-native patients, possibly due to the different lifestyles and medical care between non-native patient and native patients. Further studies are recommended to further explore exactly why non-native patients are more likely to carry lukS/F-PV in comparison to native patients.
Different PVL-positive MRSA clones predominate indifferent regions, e.g., ST8 in the United States, ST30 and ST80 in Europe, and ST59 in Asia [14,32]. As a single locus variant (SLV) of ST59, ST338 (8/38, 21.1%)was the most common lukS/F-PV-positive ST in our study. CC59 (ST338 and ST59) was found in a total of 14 isolates (4 from non-native patients; 10 from native patients, P = 0.0570). Nine of these 14 isolates (3 from non-native patients; 6 from native patients, P = 0.3469) carried lukS/F-PV. Even though ST59 was found in only 3 isolates in this study, CC59 remains the most common clonal complex among the patients with SSTIs in China when ST59 is accounted for together with ST338 (14/117, 12.0%). The other most common lukS/F-PV-positive STs were ST217 (6 isolates), ST398 (5 isolates) and ST88 (5 isolates), all of which were isolated from non-native patients.
Supporting Information S1 Table. Clinical data and molecular characteristics of 117 S. aureus isolates from non-native patients and native patients with skin and soft tissue infections (SSTIs). (XLSX)