Abstract
Objective
To investigate the staphylococcal cassette chromosome mec (SCCmec) genotype and molecular epidemiological characteristics of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) in a large teaching hospital in China.
Methods
From January 2012 to December 2012, a total of 71 nonduplicate HA-MRSA were collected in a teaching hospital in Changsha, China. SCCmec types were determined by multiplex PCR, and Panton-Valentine leukocidin (PVL) gene was detected by PCR. The homology among the tested isolates was determined using pulsed-field gel electrophoresis (PFGE).
Results
Of the 71 HA-MRSA isolates, 49 (69.0%) carried SCCmec Ⅲ, 10 (14.1%) carried SCCmec Ⅳ, 3 (4.2%) carried SCCmec Ⅴ and 3 (4.2%) carried SCCmec Ⅱ; the remaining 6 isolates were not typeable by PCR. Compared with patients having SCCmec Ⅰ/Ⅱ/Ⅲ MRSA infections, those with SCCmec Ⅳ/Ⅴ MRSA infections had a significantly younger age and a similar duration of hospital stay before the first MRSA-positive culture and total hospital stay. PVL genes were strongly associated with SCCmec type Ⅳ/Ⅴ MRSA infections. HA-SCCmec Ⅳ/Ⅴ MRSA strains showed a greater susceptibility to rifampicin, gentamicin, levofloxacin, ciprofloxacin, and tetracycline than HA-SCCmec Ⅰ/Ⅱ/Ⅲ MRSA strains. The 13 HA-SCCmec Ⅳ/Ⅴ MRSA isolates formed one large group at the 55% similarity level. Three PFGE clusters with a similarity index of 85% or more were identified, and unique PFGE profiles were observed in 4 isolates.
Conclusions
This is the first report of HA-MRSA isolates carrying SCCmec Ⅴ in Chinese hospitals. SCCmec types Ⅳ and Ⅴ MRSA clones have emerged in Chinese hospitals, which urges more rigorous surveillance of their spread in healthcare facilities in China.
Keywords: methicillin-resistant Staphylococcus aureus, nosocomial infection, SCCmec, Panton-Valentine leukocidin genes, pulsed field gel electrophoresis
Abstract
目的
研究某院临床分离的医院获得性耐甲氧西林金黄色葡萄球菌（HA-MRSA）的葡萄球菌染色体mec盒（SCCmec）分型及分子流行病学特征。
方法
收集中南大学湘雅医院2012年1月~2012年12月临床分离的71株HA-MRSA，采用多重PCR进行SCCmec分型，PCR检测PVL毒素基因，并用脉冲场凝胶电泳（PFGE）分析菌株间的同源性。
结果
71株HA-MRSA以SCCmec Ⅲ型为主，占69.0%（49/71），其次为SCCmec Ⅳ型、SCCmec Ⅴ型和SCCmecⅡ型，分别占14.1%（10/71）、4.2%（3/71）和4.2%（3/71），另有6株（8.5%）菌株未能分型。HA-SCCmec Ⅳ/Ⅴ MRSA感染者年龄显著低于HA-SCCmec Ⅰ/Ⅱ/Ⅲ MRSA感染者，携带PVL基因阳性率显著高于HA-SCCmecⅠ/Ⅱ/Ⅲ MRSA感染者，而两者入院至检出菌株的时间及住院天数均未见明显差异。HA-SCCmec Ⅳ/Ⅴ MRSA对左旋氧氟沙星、环丙沙星、利福平、庆大霉素、四环素等的耐药率均显著低于HA-SCCmecⅠ/Ⅱ/Ⅲ MRSA（P < 0.05）。13株HA-SCCmec Ⅳ/Ⅴ MRSA菌株在55%的相似度水平形成一个大的组群。按照≥85%的相似度，这些菌株共形成3个PFGE簇以及4个单一菌株的PFGE型。
结论
在国内首次发现携带SCCmec Ⅴ型的HA-MRSA菌株，HA-SCCmec Ⅳ/Ⅴ MRSA已有在医疗机构传播的趋势，并成为医院内感染的重要来源。
Keywords: 耐甲氧西林金黄色葡萄球菌, 医院感染, 葡萄球菌染色体mec基因盒, PVL基因, 脉冲场凝胶电泳
INTRODUCTION
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.
METHODS
Institutional review board approval
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.
Bacterial strains
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
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 typing and detection of the Panton-Valentine leukocidin (PVL) genes
SCCmec types were determined using multiplex PCR [10]. The PVL genes were detected by PCR as described previously [11].
Pulsed-field gel electrophoresis (PFGE)
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.
Statistical analysis
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.
RESULTS
SCCmec typing and detection of the PVL gene
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).
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).
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 | - |
Antimicrobial resistance
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).
PFGE analysis
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.
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.
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.
DISCUSSION
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.
Acknowledgment
We thank XIA Xin of Hunan Provincial Center for Disease Control and Prevention and LUO Yun of Zhejiang Provincial Center for Disease Control and Prevention for their technical assistance.
Biography
钟一鸣, 硕士, 技师, E-mail: zhongymxy@163.com
Funding Statement
Supported by National Natural Science Foundation of China (81672066) and a grant from the Development and Reform Commission of Hunan Province ([2014]658)
国家自然科学基金（81672066）；湖南省发改委基金项目（[2014]658）
Contributor Information
(钟 一鸣)Yiming ZHONG, Email: zhongymxy@163.com.
(刘 文恩)Wenen LIU, Email: wenenliu@163.com.
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