A total of 112 non-duplicate S. aureus isolates were collected from eight hospitals in four cities from July 2017 to February 2018 (see Table 1). Isolates were identified as S. aureus using conventional microbiological methods including Gram staining, catalase and coagulase tests. They were further identified by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) System (BD, USA). An antimicrobial susceptibility test was first carried out using a VITEK 2 Compact system and VITEK 2 AST-GP67 Test Kit (bioMerieux, Inc., Durham, NC, USA). Methicillin resistance was confirmed using a cefoxitin disk (30μg, Oxoid) and oxacillin disk (1μg, Oxoid,) in Zhongnan Hospital, Wuhan university. All the isolates were stored at −80°C for further experiments. The Ethics Committee of Zhongnan Hospital, Wuhan University approved this study. Because this was a retrospective study and all patients were anonymized, informed consent was waived.

All S. aureus isolates were grown on blood agar at 37°C overnight, then a single colony was transferred into 5mL Tryptic Soy Broth (TSB) medium to culture for 16h at a rotation speed of 200rpm/min. S. aureus pellets were used to extract chromosomal DNA with TIANamp Bacteria DNA kit (Tiangen, China) supplemented with 1mg/mL lysostaphin (Sigma, China) according to the manufacturer's instructions. All the extracted chromosomal DNAs were stored at −20°C for further tests.

The polymorphic X region of spa consists of a variable number of 24bp repeat units allowing isolates to be distinguished from one another. In the present study, the polymorphic X region of spa was amplified from the extracted chromosomal DNAs using primers spa-1113f and spa-1514r11 (see Table 2). The PCR mixture contained 2μL spa-1113f (10μM), 2μL spa-1514r (10μM), 1μL chromosomal DNA template, 25μL 2×Taq Master Mix (Tiangen, China), and 20μL double distilled water. The PCR conditions were as follow: initial denaturation at 94°C for 5min, followed by 30 cycles of denaturation at 94°C for 45s, annealing at 53°C for 60s and extension at 72°C for 90s, and a final extension at 72°C for 10min. The amplicons were sequenced (Tianyihuiyuan, China) and analyzed using the Ridom web server (http://spaserver.ridom.de).

MLST was carried out as described previously.12 Seven S. aureus housekeeping genes (i.e. arcC, aroE, glpF, gmk, pta, tpi and yqil) were amplified by seven PCR assays followed by sequencing. The sequences of amplicons were compared to the known alleles stored in the MLST database (http://saureus.mlst.net) to identify the sequence type. Clustering of related STs, which were defined as clonal complexes (CCs), was determined using eBURST.

Several PCR assays were used to detect the following 12 staphylococcal virulence genes including the pvl genes (lukF/S-PV), the staphylococcal enterotoxin genes (sea, seb, sec), the exfoliative toxin genes (eta, etb), the hemolysin genes (hla, hlb), the adhesion factor genes (fnbA, fnbB, clfa), and the toxic shock syndrome toxin (tst). All primers are listed in Table 2.13–15 The PCR mixture contained 2μL of a couple of primers (10μM), 1μL DNA template, 12.5μL Taq Master Mix (2×), and double distilled water (9.5μL). The PCR conditions were as follow: initial denaturation at 94°C for 5min, followed by 30 cycles of denaturation at 94°C for 30s, 30s with the respective annealing temperature, extension at 72°C for respective extension time, and a final extension at 72°C for 10min. The annealing temperatures and extension times for different primers are shown in Table 2. All PCR products were analyzed by electrophoresis with 2% agarose gels.

Statistical analyses were performed using SPSS Statistics 24.0 for Windows. The chi-square test was used to compare the distributions of virulence genes between MRSA and MSSA isolates, ST5-t2460 and ST188-t189. The correlations between the prevalence of ST239, ST5 clones and the proportion of MRSA among all S. aureus isolates were analyzed using Spearman rank correlation. p<0.05 was considered to be statistically significant.

The distribution of MLST and spa types of bloodborne S. aureus isolates is presented in Table 3. Among the 112 S. aureus isolates, 25 STs were identified. ST5 was the most predominant (21.4%), followed by ST188 (12.5%), ST59 (8.9%), ST398 (8.0%), ST1 (6.3%), ST630 (4.5%), and ST121 (4.5%). Other STs accounted for 33.9% of the 112 S. aureus isolates. In addition, one isolate could not be identified for ST. 95.8% of the ST5 isolates were collected from Wuhan, whereas 57.1% of the ST188 isolates were isolated from Haikou, which indicates that the distribution of STs of S. aureus have regional differences. Sixteen clone complexes (CCs) and three singletons were identified by eBURST (Fig. 1), CC5 (22.3%) was the most prevalent clonal complex, followed by CC8 (10.7%) and CC59 (9.8%). Thirty-two (28.6%) S. aureus isolates were MRSA in this study. The most major epidemic ST in MRSA isolates was ST5 (50.0%), followed by ST59 (15.6%), ST239 (12.5%), and ST45 (9.4%). In contrast, the most prevalent ST in MSSA isolates was ST188 (17.5%) followed by ST398 (10.0%) and ST5 (10.0%). In last ten years in China, five studies about molecular characteristics of S. aureus isolates causing bloodstream infection were carried out.6,8,9,16,17 ST239 was found to be the most prevalent clone except in the studies by Yu and Li where ST239 was the most common clone among the MRSA isolates, suggesting that the prevalence of ST239 clone may be closely associated with MRSA infections. Twelve previous studies about molecular characteristic profiles of S. aureus isolates in China6,8,9,16–24 and our present study analyzed the correlation between the proportion of MRSA among all S. aureus isolates and the prevalence of ST239 and ST5 clone. All S. aureus isolates both MRSA and MSSA in the above studies were collected from patients rather than volunteers. The information of these S. aureus isolates is listed in Table 4. The rates of MRSA to total S. aureus isolates and the prevalence of ST239 and ST5 clone are shown in Fig. 2. Spearman rank correlation analysis showed that the proportion of MRSA among all S. aureus isolates correlated closely with the prevalence of ST239 clone (r=0.8, p<0.001) rather than ST5 clone (r=0.2, p=0.5), and the prevalence of ST239 clone decreased as the proportion of MRSA among all S. aureus isolates decreased, indicating that molecular characteristics of S. aureus isolates change as the proportion of MRSA among all S. aureus isolates fall. These studies also indicated that the implementation of infection control contributed to preventing the spread of ST239 S. aureus isolates.

Fig. 1.

Distribution of STs in the clonal complexes. The diagram generated by eBURST with the default group definition based on the MLST data of this study, representing the relationships of 111 S. aureus isolates. Each number implies an MLST ST, STs that are linked by a line belong to the same cluster and the dot area indicates the prevalence of the ST in the MLST data of this study.

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Fig. 2.

The proportions of MRSA among all S. aureus isolates and the prevalence of ST239 or ST5 clone.

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Among the 112 S. aureus isolates, 48 spa types were found. The most prevalent spa type was t2460 (14.3%) followed by t189 (10.7%), t437 (8.9%), t377 (5.4%), t127 (5.4%), t002 (3.6%), and t030 (3.6%). In addition, two isolates could not be identified for spa. All the t2460 S. aureus isolates were collected from Wuhan except one isolate from Beijing, whereas the t189 S. aureus isolates were collected in Haikou (58.3%), Wuhan (25.0%), Jingzhou (8.3%) and Beijing (8.3%). In 2007, t2460 clone was first found in South Korea,25 whereas in China it was not be found in large numbers until May 2015.26 These data may supported the view of the temporary outbreak of t2460 in Wuhan. To date, t091 and t030 were the most common spa types in bloodstream infection in China,6–9 whereas t437 and t127 were found to be the most common spa types in other infections.27–29

The pathogenicity of S. aureus is closely related to presence of various virulence genes.30 The frequencies of virulence encoding genes identified in the 112 isolates are listed in Table 5. Our data showed that all S. aureus isolates carried at least two virulence genes and almost all the isolates contained clfa (100%, 112/112) and hla (99.1%, 111/112) genes. These findings are comparable to those reported in many hospitals of south of China,31,32 which indicated that clfa and hla were widely present and may play a pivotal role in the pathogenicity of S. aureus. Enterotoxins, including sea, seb and sec were found to cause food poisoning primarily. In our study, the most common enterotoxin gene was seb (66.1%), which is significantly different from those reported in foodborne S. aureus, where the most common enterotoxin gene is sea.33,34 Our study showed that 55.4% (62/112) isolates harbored six or more virulence genes, of which two isolates contained nine virulence genes, 15 isolates harbored eight virulence genes, 15 isolates carried eight virulence genes, 18 isolates had seven virulence genes, 27 isolates harbored six virulence genes. Furthermore, six or more virulence genes were present in 81.3% of MRSA isolates and 45% of MSSA isolates and MRSA isolates harbored more virulence genes than the MSSA isolates (χ2=12.154, p<0.01). The above results suggested that S. aureus isolates with different genetic background have different ability to acquire mobile genetic elements such as plasmids, phages and pathogenicity islands.

MLST typing and spa typing were performed to analyze the molecular characteristics of S. aureus isolates. There was a strong association observed between ST and spa types: the ST5 type was primarily associated with t2460 (66.7%, 16/24), the ST188 type with t189 (85.7%, 12/14), the ST59 type with t437 (90%, 9/10), the ST1 type with t127 (85.7%, 6/7) and the ST630 type with t377 (100%, 5/5). ST5-t2460 (16/112, 14.3%) were found to be the most common in this study, followed by ST188-t189 (12/112, 10.7%). Molecular characteristics of ST5-t2460 and ST188-t189 are summarized in Table 6. 93.8% of ST5-t2460 were collected from four hospitals in Wuhan, whereas the distribution of ST188-t189 was more dispersed including in Haikou (58.3%), Wuhan (25%), Beijing (8.3%) and Jingzhou (8.3%). In 2007, ST5-t2460 became the main type of MRSA bloodstream infections in Korea which had never been seen before.25 In China, extremely few cases of ST5-t2460 were reported in previous studies.16,35 Therefore, the prevalence of ST5-t2460 in our research may be consequence of a temporary outbreak of ST5-t2460 in Wuhan. In our present study, all the ST5-t2460 were MRSA, whereas all the ST188-t189 were MSSA. The prevalence of strains with six or more virulence genes was significantly higher in ST5-t2460 strains (93.8%) compared with ST188-t189 strains (8.3%) (p<0.001). In addition, of the tested 12 virulence genes, pvl, fnbA, hlb, sec, and tst were found to be more frequent in ST5-t2460 than in ST188-t189. These results indicated that ST5-t2460 acquire mecA and virulence genes easily, which supported the above conclusion that S. aureus with different genetic background have different ability to acquire mobile genetic elements. In addition to genetic background, ST5-t2460 often lead to persistent and recurrent MRSA bloodstream infections,36 which also contribute to promoting S. aureus isolates to acquire virulence genes easily.

In conclusion, ST5-t2460 was the most common clone in S. aureus causing bloodstream infection, followed by ST188-t189, which had never been reported in China before. All the ST5-t2460 were MRSA, whereas all the ST188-t189 were MSSA. Moreover, ST5-t2460 harbored more virulence genes than ST188-t189, which indicates that ST5-t2460 acquire mecA and virulence genes more easily than ST188-t189. In addition, the prevalence of ST239 clone decreased with the decrease of the proportion of MRSA among all S. aureus isolates.