Güler Ismail, Kılıç Hüseyin, Atalay Mustafa Altay, Perçin Duygu, Erçal Barış Derya
Erciyes University Faculty of Medicine, Department of Medical Microbiology, Kayseri, Turkey.
Mikrobiyol Bul. 2011 Oct;45(4):581-91.
Methicillin-resistant Staphylococcus aureus (MRSA) infections are associated with increased cost, mortality and length of hospital stay compared with the other infections. Therefore, controlling the spread of this pathogen by screening patients, personnel and the environment remains as a high priority in infection control programs. The aim of this study was to detect the clonal relationship between nosocomial MRSA strains by using repetitive-sequence-based polymerase chain reaction (rep-PCR) method which has several advantages owing to its speed and ease of use. A total of 100 MRSA stock strains that had been isolated from various clinical samples of hospitalized patients in Erciyes University Medical Faculty Hospitals between September 2008-October 2009, were included in the study. Methicillin resistance of the strains were determined by cefoxitin disc diffusion test according to CLSI guidelines. Rep-PCR (Diversilab, bioMerieux, France) method was performed in the following four steps in order to determine genetic proximity of MRSA strains: (1) Manual DNA extraction (UltraClean Microbial DNA Isolation Kit; MoBio Laboratories, USA), (2) Rep-PCR by using fingerprinting kits in the thermocycler (Diversilab DNA Fingerprinting Kit), (3) Automated microfluidic electrophoresis by bioanalyzer (Diversilab DNA LabChip kit), (4) Analysis and rapid evaluation with the use of web-based DiversiLab software (version 2.1.66). Rep-PCR analysis have shown the presence of a total 11 clones, including 3 major clones [A (4 subtypes), B (2 subtypes) and C (2 subtypes)] and 8 unique clones (DK). Clone A was found to be the dominant type. Seventy-eight percent of the 100 MRSA isolates belonged to clone A (63 were A1; 9 were A2; 4 were A3, 2 were A4), 11% belonged to clone B (10 were B1, 1 was B2), 3% belonged to clone C (2 were C1, 1 was C2), and one of each belonged to the other clones (D, E, F, G, H, I, J, K). Clone A was isolated from 93.3% (14/15) of the samples sent from internal diseases intensive care unit (ICU), from 66.6% (10/15) of the samples sent from infectious diseases ward and 91% (10/11) of hematology-oncology ward samples. All MRSA strains isolated from anesthesiology and newborn ICU were of clone A. The isolation dates of these strains were in proximity. In conclusion, MRSA strains showed clonal dissemination in our hospital, clone A being the predominant one during the study period. Rep-PCR which is a rapid and reliable method, can easily be applied for molecular epidemiological purposes and aid to infection control measures.
与其他感染相比，耐甲氧西林金黄色葡萄球菌（MRSA）感染会导致成本增加、死亡率上升以及住院时间延长。因此，通过筛查患者、医护人员和环境来控制这种病原体的传播仍是感染控制项目中的重中之重。本研究的目的是使用基于重复序列的聚合酶链反应（rep-PCR）方法检测医院内MRSA菌株之间的克隆关系，该方法因其速度快且易于使用而具有诸多优势。本研究纳入了2008年9月至2009年10月间从埃尔西耶斯大学医学院附属医院住院患者的各种临床样本中分离出的100株MRSA储备菌株。根据CLSI指南，通过头孢西丁纸片扩散试验测定菌株的耐甲氧西林情况。为了确定MRSA菌株的遗传亲缘关系，rep-PCR（法国生物梅里埃公司的Diversilab）方法按以下四个步骤进行：（1）手工DNA提取（美国MoBio实验室的UltraClean微生物DNA分离试剂盒），（2）在热循环仪中使用指纹识别试剂盒进行rep-PCR（Diversilab DNA指纹识别试剂盒），（3）通过生物分析仪进行自动微流控电泳（Diversilab DNA LabChip试剂盒），（4）使用基于网络的DiversiLab软件（版本2.1.66）进行分析和快速评估。rep-PCR分析显示共存在11个克隆，包括3个主要克隆[A（4个亚型）、B（2个亚型）和C（2个亚型）]和8个独特克隆（DK）。发现克隆A是主要类型。100株MRSA分离株中，78%属于克隆A（63株为A1；9株为A2；4株为A3，2株为A4），11%属于克隆B（10株为B1，1株为B2），3%属于克隆C（2株为C1，1株为C2），其他克隆（D、E、F、G、H、I、J、K）各有1株。克隆A从内科重症监护病房（ICU）送检样本的93.3%（14/15）、传染病病房送检样本的66.6%（10/15）以及血液肿瘤病房样本的91%（10/11）中分离得到。从麻醉科和新生儿ICU分离出的所有MRSA菌株均为克隆A。这些菌株的分离日期相近。总之，MRSA菌株在我院呈克隆性传播，在研究期间克隆A是主要类型。rep-PCR是一种快速可靠的方法，可轻松应用于分子流行病学研究，并有助于采取感染控制措施。
