Methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen that causes severe infection with a high mortality rate. Resistance to methicillin in MRSA is mediated by the acquisition of the penicillin-binding protein 2a (PBP2a) encoded by the methicillin resistance gene mecA, which is located on the staphylococcal cassette chromosome mec (SCCmec). Based on the SCCmec elements, MRSA can be divided into 11 different types, among which types Ⅳ are the major strains commonly found ^[1]. Healthcare-associated MRSA (HA-MRSA) infections are generally caused by SCCmec type Ⅰ, Ⅱ, and Ⅲ strains, while community-associated MRSA (CA-MRSA) are mainly SCCmec types Ⅳ and Ⅴ^{[2, 3]}. However, according to recent studies^{[4, 5]}, CA-MRSA strains of SCCmec types Ⅳ and Ⅴ are spreading in hospitals and are replacing the traditional HA-MRSA strains.

In China, the incidence of MRSA infection is high, and the hospitals are exposed to a growing risk of MRSA infection. While SCCmec type Ⅲ HA-MRSA strains remain dominant in the Chinese population ^[6], the epidemiology of SCCmec type Ⅳ and Ⅴ strains in the healthcare setting has not been well documented in China. In this study, we aimed to investigate the occurrence of SCCmec type Ⅳ and Ⅴ strains in a teaching hospital in China, and compare the molecular epidemiological features, drug resistance profiles and the homologies between SCCmec types Ⅳ/Ⅴ and SCCmec types Ⅰ/ Ⅱ/Ⅲ strains.

The study was approved by the Ethics Committee of the Xiangya Hospital of Central South University. The need for informed consent was waived by the Ethics Committee because the study was a retrospective study using a fully anonymized database.

From January to December 2012, a total of 71 nonrepetitive HA-MRSA isolates were collected from Xiangya Hospital, a general teaching hospital affiliated to Central South University (Changsha, Hunan Province, China) that holds 3500 beds with a daily 7000 to 8500 patient visits. HA-MRSA was defined as the isolates from the patients who (ⅰ) had a positive culture for MRSA more than 48 h after hospital admission; (ⅱ) had a history of hospitalization, dialysis, surgery or residence in a long-term healthcare facility in the year prior to the MRSA-positive culture date; or (ⅲ) had permanent indwelling percutaneous catheters or medical devices at the time of culture^[7]. The isolates were identified using the Vitek-2 compact system (bioMérieux, Marcy l' Etoile, France). Strains were confirmed as MRSA by detecting their resistance to methicillin and the presence of mecA gene using polymerase chain reaction (PCR) ^[8].

Antimicrobial susceptibility testing was performed using the broth microdilution method for 14 antimicrobial agents: oxacillin, penicillin, gentamicin, ciprofloxacin, levofloxacin, rifampicin, linezolid, nitrofurantoin, erythromycin, clindamycin, trimethoprim-sulfamethoxazole, vancomycin, tetracycline, and tigecycline. The results were interpreted according to the recommended breakpoints of the CLSI ^[9].

SCCmec types were determined using multiplex PCR ^[10]. The PVL genes were detected by PCR as described previously ^[11].

The isolates were subjected to PFGE analysis according to the PulseNet protocol developed by the U.S. Centers for Disease Control and Prevention ^[12]. The genomic DNA of the bacteria was digested with Sma Ⅰ restriction enzyme. The Salmonella serotype Braenderup (H9812) was selected as a molecular reference marker. PFGE profiles were analyzed with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) using the Dice similarity coefficient. The dendrogram was constructed according to the unweighted pair group method with arithmetic averages (UPGMA). The isolates with a Dice similarity index of 85% or more were thought to belong to the same PFGE group.

The quantitative variables were presented as Mean±SD. Comparisons of the quantitative variables were made using Student's t tests, and comparisons of the proportions were made using Fisher's exact test. A P value less than 0.05 was considered to indicate a statistically significant difference.

The mean age of the 71 patients was 43.8±20.9 years, and most of the patients (53, 74.6%) were male. The HA-MRSA isolates most frequently carried SCCmec Ⅲ (n=49, 69.0%), followed by SCCmec Ⅳ (n=10, 14.1%), SCCmec Ⅴ (n=3, 4.2%) and SCCmec Ⅱ (n=3, 4.2%). SCCmec type Ⅰ was not found in any of the isolates. The remaining 6 isolates (8.5%) were not typeable by multiplex PCR and were categorized into "other SCCmec types" (Fig. 1). Thus, of the 71 HA-MRSA isolates, 13 (18.3%) were HA-SCCmec Ⅳ/Ⅴ MRSA and 52 (73.2%) were HA-SCCmec Ⅱ/Ⅲ MRSA. The patients with SCCmec Ⅳ/Ⅴ MRSA infections had a significantly younger age than those with SCCmec Ⅰ/Ⅱ/Ⅲ MRSA infections (27.5 ± 22.9 vs 48.3 ± 18.8 years, P=0.001), while there were no differences in duration of hospital stay before the first MRSA-positive culture (6.2±4.1 vs 7.7±3.3 days, P=0.185) or the total hospital stay (11.0± 4.8 vs 11.5 ± 3.7 days, P=0.662). Seven (9.9%) isolates positive for the PVL genes were all identified as HA-SCCmec Ⅳ/Ⅴ MRSA (Tab. 1).

图 1(Figure 1)

Figure 1 Electrophoretogram of the PCR amplified products of the SCCmec type. M: DNA ladder marker (100-2000 bp); Lane 1: Standard strain NCTC10442 (SCCmec Ⅰ); Lane 2: Standard strain MRSA N315 (SCCmec Ⅱ); Lane 3: Standard strain MRSA 85/2082 (SCCmec Ⅲ); Lane 4: Standard strain MRSA JCSC4744 (SCCmec Ⅳ); Lane 5: Standard strain SCCmec Ⅴ; Lanes 6-10: Clinical strains (6: SCCmec Ⅲ; 7: Not typeable; 8: SCCmec Ⅳ; 9: SCCmec Ⅱ; 10: SCCmec Ⅴ).

HA-SCCmec Ⅳ/Ⅴ MRSA isolates showed a greater susceptibility than HA-SCCmec Ⅰ/Ⅱ/Ⅲ MRSA isolates to rifampicin, gentamicin, levofloxacin, ciprofloxacin, and tetracycline. None of the isolates were resistant to vancomycin, linezolid, or nitrofurantoin (Tab. 1).

表 1(Table 1)

Table 1 Characteristics of patients with HA-MRSA infection and antimicrobial resistance profiles of HA-MRSA according to SCCmec type Characteristics HA-SCCmec Ⅳ/Ⅴ MRSA (n=13) HA-SCCmec Ⅰ/Ⅱ/Ⅲ MRSA (n=52) P Age (year) 27.5±22.9 48.3±18.8 0.001 Gender (n, %)  Male 10 (76.9%) 40 (76.9%) 1.000  Female 3 (23.1%) 12 (23.1%) Hospital stay before culture (day) 6.2±4.1 7.7±3.3 0.185 Total hospital stay (day) 11.0±4.8 11.5±3.7 0.662 PVL gene (n, %)  Positive 7 (53.8%) 0 < 0.001  Negative 6 (46.2%) 52 (100%) Antimicrobial resistance (n, %)  Penicillin 13 (100%) 52 (100%) -  Erythromycin 13 (100%) 46 (88.5%) 0.335  Clindamycin 13 (100%) 48 (92.3%) 0.576  Rifampin 2 (15.4) 46 (88.5%) < 0.001  Linezolid 0 0 -  Gentamicin 2 (15.4%) 46 (88.5%) < 0.001  Levofloxacin 1 (7.7%) 50 (96.2%) < 0.001  Ciprofloxacin 1 (7.7%) 49 (94.2%) < 0.001  Tetracycline 9 (69.2%) 52 (100%) 0.001  Trimethoprim/sulfamethoxazole 3 (23.1%) 5 (9.6%) 0.192  Tigecycline 0 0 -  Vancomycin 0 0 -  Nitrofurantoin 0 0 -

Table 1 Characteristics of patients with HA-MRSA infection and antimicrobial resistance profiles of HA-MRSA according to SCCmec type

Fig. 2 and Fig. 3 show PFGE analysis results for the 13 HA-SCCmec Ⅳ/Ⅴ MRSA isolates. These MRSA isolates constituted one large group at the 55% similarity level (Fig. 3). Three PFGE clusters with a similarity index of 85% or more were identified. Unique PFGE profiles were observed for 4 isolates.

图 2(Figure 2)

Figure 2 Electrophoretogram of the PCR amplified products of the 13 HA-SCCmec Ⅳ/Ⅴ MRSA isolates (part). M: Marker (Salmonella serotype Braenderup H9812); Lanes 1-10: SCCmec Ⅳ MRSA isolates; Lane 11: SCCmec Ⅴ MRSA isolate.

图 3(Figure 3)

Figure 3 SmaI PFGE-based dendrogram for the 13 HA-SCCmec Ⅳ/Ⅴ MRSA isolates. The dendrogram was generated according to the unweighted pair group method based on Dice coefficients. PVL: PVL genes (N, negative; P, positive); PEN: Penicillin; ERY: Erythromycin; CLI: Clindamycin; RIF: Rifampin; GEN: Gentamicin; LEV: Levofloxacin; CIP: Ciprofloxacin; TET: Tetracycline; SXT: Trimethoprim-sulfamethoxazole. The 13 HA-SCCmec Ⅳ/Ⅴ MRSA isolates formed one large group at the 55% similarity level and formed 7 PFGE groups at ≥85% similarity level.

In this study, we examined the presence of HA-MRSA of different SCCmec types in clinical isolates collected from a teaching hospital in China. We found that the isolated HA-MRSA were mainly SCCmec type Ⅲ strains. Surprisingly, however, SCCmec type Ⅳ was the second most prevalent HA-MRSA genotype, which was found in 14.1% of the isolates. To the best of our knowledge, this is the first report of the identification of the SCCmec Ⅴ genotype in a HA-MRSA isolate in China. Moreover, our results showed a high proportion of HA-MRSA isolates of SCCmec Ⅳ/Ⅴ types at our institution, suggesting the high likeliness that these strains with the genetic characteristics of CA-MRSA are replacing the traditional HA-MRSA strains to become important sources of nosocomial infection.

The reason for nosocomial spread of SCCmec type Ⅳ/Ⅴ strains is not clear. Recent studies suggest that their spreading is probably associated with the higher growth rate and genetic fitness of CA-MRSA strains ^{[5, 13, 14]}. Further studies are still required to better understand the mechanisms that facilitate the nosocomial spread of these strains.

We found a prevalence rate of PVL of 9.9% in the isolated HA-MRSA, which was consistent with the results of previous studies^[6]. Our finding suggested a strong association between PVL genes and SCCmec type Ⅳ/Ⅴ (53.8%) to urge careful monitoring and screening of the PVL genes in HA-MRSA strains to prevent and control the spread of nosocomial infection.

Our findings showed that HA-SCCmec types Ⅳ/Ⅴ isolates were more susceptible to multiple antibiotics than HA-SCCmec Ⅱ/Ⅲ isolates. This may be explained by the observation that SCCmec Ⅱ/Ⅲ strains carry mecA and additional antibiotic drug-resistance genes, whereas SCCmec Ⅳ/Ⅴ strains generally carry only mecA gene^[15]. Nevertheless, we found that SCCmec types Ⅳ/Ⅴ isolates were also resistant to erythromycin, clindamycin and tetracycline. We presume that under the nosocomial antibiotic selection pressure, SCCmec Ⅳ/Ⅴ isolates may obtain drug-resistant genetic elements to exhibit multidrug resistance similar to that of HA-SCCmec Ⅱ/Ⅲ isolates. Still, further studies are needed to verify this possibility.

Similar to the results of Dhawan et al^[5], we found that SCCmec types Ⅳ/Ⅴ HA-MRSA infections were significantly associated with a younger age, and SCCmec types Ⅳ/Ⅴ isolates were more likely to carry the PVL genes. The total hospital stay was similar between patients with SCCmec types Ⅳ/Ⅴ MRSA infections and those with SCCmec Ⅰ/Ⅱ/Ⅲ MRSA infections. We postulate that this may be associated with the higher susceptibility of HA-SCCmec Ⅳ/Ⅴ MRSA isolates to multiple antibiotics and the younger age of patients with HA-SCCmec Ⅳ/Ⅴ MRSA infections despite the fact that HA-SCCmec Ⅳ/Ⅴ MRSA isolates are more virulent. Other studies reported that SCCmec elements of types Ⅳ and Ⅴ are smaller and can spread more easily through mobile genetic elements, such as plasmids and phages ^{[4, 5]}. Thus, hospitals should carefully monitor these strains to prevent potential outbreaks of nosocomial infections.

Despite of our new findings, this study also has limitations. As a single-center study, the results may only reflect the local trends and might not be generalisable to other facilities in different geographic areas; in addition, only representative SCCmec Ⅳ/Ⅴ strains were genotyped by PFGE. But still, the findings in our institution indicate a potential high risk of nosocomial spread of SCCmec type Ⅳ and Ⅴ strains in Chinese hospitals and urge more rigorous surveillance of HA-MRSA infections.

In conclusion, we report the first identification of SCCmec Ⅴ in HA-MRSA isolates in China. The high proportions of HA-MRSA strains carrying SCCmec types Ⅳ and Ⅴ, together with the high prevalence of PVL-positive MRSA strains, suggest that CA-MRSA strains have emerged and spread within Chinese hospitals. The strains with the genetic characteristics of CA-MRSA may gradually replace the traditional HA-MRSA strains to become an important source of nosocomial infection, and more rigorous measures should be taken to monitor and screen bacterial infections in Chinese hospitals.