耐甲氧西林金黄色葡萄球菌分子流行病学及临床感染特征的研究

发布时间：2018-05-03 01:39

本文选题：耐甲氧西林金黄色葡萄球菌 + 耐药机制　；参考：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：[背景]耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)是医院内感染最常见的致病菌之一,由其引起的社区感染也正日益增多。金黄色葡萄球菌能引起包括皮肤与软组织感染、菌血症、骨髓炎、感染性心内膜炎及坏死性肺炎在内的一系列疾病。自1960年以来,耐甲氧西林金黄色葡萄球菌在全世界范围内的许多医院和卫生机构流行性传播开来,其高耐药率成为了临床医生面临的棘手问题,威胁着患者的健康和生命。正因如此,对耐甲氧西林金黄色葡萄球菌进行流行病学监测,为预防和治疗耐甲氧西林金黄色葡萄球菌提供有力依据,具有重要的临床意义和社会效益。分子分型技术通常用来研究金黄色葡萄球菌的进化和流行病学特征,目前,国际上最常用的金黄色葡萄球菌分子分型方法包括:葡萄球菌蛋白A分型(staphylococcal protein A,spa typing)、葡萄球菌染色体盒mec分型(staphylococcal cassette chromosome mec typing,SCCmec typing)、多位点序列分型(multilocus sequence typing,MLST)、脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)和多位点可变串联重复序列分型(multiple-locus variable-number tandem repeat analysis,MLVA)。金黄色葡萄球菌能产生大量毒素,这些毒素在细菌致病过程中具有重要的作用。大约20%的金黄色葡萄球菌分离株具有编码毒性休克综合征毒素-1(toxic shock syndrome toxin 1,TSST-1)的基因,毒性休克综合征毒素-1的结构与外毒素结构相似,可引起毒素综合征。Panton-Valentine杀白细胞素(Panton-Valentine leukocidin,PVL)是白细胞溶解毒素,流行病学上与严重的皮肤感染有关。在我国,耐甲氧西林金黄色葡萄球菌SCCmec III-ST239菌株分布范围非常广,某些全国性的研究结果显示,该型菌株占所有耐甲氧西林金黄色葡萄球菌的75%以上。耐甲氧西林金黄色葡萄球菌的流行病学一直在不断变化,这导致了不同大陆、不同国家和不同地区之间会出现不同的耐药谱,具体到局部地区,耐甲氧西林金黄色葡萄球菌是否具有自身独特的流行趋势和临床感染特征是一个具有现实指导意义的科学问题。基于以上研究背景的调研,本研究对收集自2012年02月至2013年05苏州市一家三甲医院的150株耐甲氧西林金黄色葡萄球菌进行了分子流行病学研究,同时收集样本对应的患者资料,对其临床感染特征进行了分析。[目的](1)探讨本地区耐甲氧西林金黄色葡萄球菌的耐药情况。(2)探寻本地区耐甲氧西林金黄色葡萄球菌的分子流行病学现状,从分子水平阐释耐药机制。(3)探讨本地区耐甲氧西林金黄色葡萄球菌的临床感染特征,为临床防治耐甲氧西林金黄色葡萄球菌提供实验依据。[方法](1)利用自动化肉汤微量稀释法及mec A基因扩增分析本地区耐甲氧西林金黄色葡萄球菌的耐药情况。(2)综合运用spa分型技术、SCCmec分型技术、MLST分型技术、PFGE分型技术和MLVA分型技术研究本地区耐甲氧西林金黄色葡萄球菌的分子流行病学现状。(3)通过扩增pvl和tst-1基因,研究本地区耐甲氧西林金黄色葡萄球菌携带毒力基因的现状。(4)收集感染耐甲氧西林金黄色葡萄球菌患者的临床信息,利用多因素Logistic回归分析研究本地区耐甲氧西林金黄色葡萄球菌的临床感染特征。[结果](1)150株耐甲氧西林金黄色葡萄球菌均扩增出mec A基因。所有菌株均对万古霉素和利奈唑胺敏感。150株实验菌株均对青霉素和苯唑西林耐药,对头孢西丁、哌拉西林-他唑巴坦、氨苄西林-舒巴坦、环丙沙星、呋喃妥因、红霉素、克林霉素、复方新诺明和利福平的耐药率分别为94.0%(141/150),98.0%(147/150),66.0%(99/150),64.0%(96/150),60.0%(90/150),74.0%(111/150),50.0%(75/150),72.0%(108/150)和12.7%(19/150)。(2)分子分型结果显示,150株耐甲氧西林金黄色葡萄球菌共有11个克隆型和28个spa型别,其中CC5-spa t002(29.3%)和CC239-spa t037(14.7%)最为盛行。SCCmec II,III,IV和V型的比例分别占所有菌株的33.3%,21.3%,23.3%和21.3%。PVL基因(luk F/S-PV)在所有150株耐甲氧西林金黄色葡萄球中的检出率为11.3%,分属于6个不同的克隆群(5,8,59,88,239,398)。TSST-1基因(tst)的总体检出率为18.0%,主要为CC5克隆群(96.3%)。本研究中,所有tst-1阳性CC5克隆群的耐甲氧西林金黄色葡萄球菌菌株均为spa t002型。(3)本研究中的150株耐甲氧西林金黄色葡萄球菌,其中71株分离自内科病房(71/150,47.3%),48株分离自外科病房(48/150,32.0%),31株分离自重症监护病房(31/150,20.7%)。本研究中的菌株均分离自住院患者的标本,60%分离自60岁以上患者,平均年龄为63岁,并且74%(111/150)为男性患者。本研究中的菌株大部分分离自痰样本(90/150,60.0%),其次为引流液样本(17/150,11.3%),伤口样本(12/150,8.0%),另外,4株分离自血液样本,5株分离自尿液样本,22株分离自其他部位样本。(4)本研究中,18位患者在住院30天内死亡,病死率为12.0%(18/150)。本研究在三个主要的克隆群内(CC5、CC239和CC59)比较了患者的预后、临床特征和耐甲氧西林金黄色葡萄球菌分离株的基因分型特征。与感染CC239或CC59克隆群菌株的患者相比,感染CC5克隆群菌株的患者30天住院病死率明显较高(P0.05)。另外,CC5克隆群菌株具有更高的机率为SCCmec II型菌株和携带tst-1基因(P0.01),并且具有更高的机率为分离自重症监护病房的患者(P0.05)。本研究也比较了预后不同的患者(生存vs.死亡)所感染的耐甲氧西林金黄色葡萄球菌的临床和分子特征。在单因素分析中,以下因素与患者30天住院病死率相关:年龄、肿瘤、红霉素耐受、感染菌株属于CC5克隆群、分子分型为SCCmec II spa t002以及菌株携带tst-1基因(P0.05)。另外,收入重症监护病房这个因素经统计学分析,与患者30天住院病死率相关性位于临界线(P=0.06)。值得注意的是,本研究观察到感染了tst-1阳性耐甲氧西林金黄色葡萄球菌的患者30天住院病死率明显高于感染tst-1阴性耐甲氧西林金黄色葡萄球菌的患者(51.9%vs.3.3%,P0.001)。然而,感染了PVL阳性耐甲氧西林金黄色葡萄球菌的患者与感染PVL阴性耐甲氧西林金黄色葡萄球菌的患者30天住院病死率无明显差异(0.0%vs.13.5%,P=0.13)。多因素回归分析发现,与患者30天住院病死率相关的独立危险因素包括患者年龄大于60岁(odds ratio[OR]=7.2,95%confidence interval[95%CI]=1.26-41.6,P=0.026)、患有肿瘤基础疾病(OR=9.6,95%CI=1.4-65.7,P=0.02)以及感染tst-1阳性耐甲氧西林金黄色葡萄球菌(OR=62.5,95%CI=12.0-325.2,P0.001)。[结论](1)所研究地区耐甲氧西林金黄色葡萄球菌整体耐药率较高,但尚未出现对万古霉素和利奈唑胺耐药的菌株。(2)本研究的研究结果揭示了,与我国其他地区相比较,所研究地区具有独特的耐甲氧西林金黄色葡萄球菌的流行谱。首先,CC5克隆群是所研究地区耐甲氧西林金黄色葡萄球菌主要的流行克隆群,主要的基因型为SCCmec II-ST002而非SCCmec III-ST239。另外,spa t037型在所研究地区流行程度胜于spat030型,这一流行趋势与之前的其他研究结果提示的spa t037型已被spa t030型取代也不一样。因此,本研究的研究结果提示了中国耐甲氧西林金黄色葡萄球菌在不同医院、不同地区之间的流行具有地域差异。。(3)所研究地区耐甲氧西林金黄色葡萄球菌CC5分离株为优势菌株,其中tst-1阳性的CC5-MRSA-II,spa t002菌株的比例很高。多因素分析结果显示,耐甲氧西林金黄色葡萄球菌tst-1阳性CC5克隆群菌株感染是患者30天住院死亡的独立危险因素,警示医院需要采取严格的感染防控措施阻止这类细菌该地区进一步扩散。
[Abstract]:[background] methicillin resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus, MRSA) is one of the most common pathogenic bacteria in hospital infection, and the community infection is increasing. Staphylococcus aureus can cause skin and soft tissue infection, bacteremia, osteomyelitis, infective endocarditis and necrosis. A series of diseases, including pneumonitis. Since 1960, methicillin resistant Staphylococcus aureus has spread widely in many hospitals and health institutions around the world. The high drug resistance rate has become a difficult problem for clinicians, threatening the health and life of the patients. The epidemiological monitoring of cocci provides a powerful basis for the prevention and treatment of methicillin resistant Staphylococcus aureus. It has important clinical significance and social benefits. Molecular typing is usually used to study the evolutionary and epidemiological characteristics of Staphylococcus aureus. At present, the most commonly used molecular typing side of Staphylococcus aureus at the international level The methods include: staphylococcal protein A typing (staphylococcal protein A, spa typing), staphylococcal chromosome box MEC typing (staphylococcal cassette chromosome mec typing), multiple point sequence typing, pulsed field gel electrophoresis, and multiple sites Multiple-locus variable-number tandem repeat analysis, MLVA). Staphylococcus aureus can produce a large number of toxins, which play an important role in the bacterial pathogenesis. About 20% of the Staphylococcus aureus isolates have toxic shock syndrome toxin -1 (toxic shock syndrome toxin). 1, TSST-1) gene, the structure of the toxic shock syndrome toxin -1 is similar to the structure of the exotoxin, which causes the toxin syndrome,.Panton-Valentine, Panton-Valentine leukocidin, PVL, is leucocyte lysis toxin, and is closely related to severe skin infection. In China, methicillin resistant Staphylococcus aureus SCCmec III The distribution of -ST239 strains is very wide. Some national studies show that the strain accounts for more than 75% of all methicillin resistant Staphylococcus aureus. The epidemiology of methicillin resistant Staphylococcus aureus has been constantly changing, which leads to different resistance spectrum between different continents, different countries and different regions. In specific areas, whether or not methicillin resistant Staphylococcus aureus has its own unique epidemic trend and clinical infection characteristics is a practical scientific problem. Based on the research background above, this study collected 150 methicillin resistant strains of methicillin in a three a hospital in Suzhou from 02 to 2013 2012. The molecular epidemiology study of Staphylococcus aureus was carried out, and the corresponding patient data were collected and the clinical infection characteristics were analyzed. [Objective] (1) to explore the resistance of methicillin resistant Staphylococcus aureus in the local area. (2) to explore the molecular epidemiology of Staphylococcus aureus in local methicillin resistant Staphylococcus aureus, and to explore the molecular epidemiology of methicillin resistant Staphylococcus aureus. Molecular level interpretation of drug resistance mechanism. (3) to explore the clinical infection characteristics of methicillin resistant Staphylococcus aureus in local area, provide experimental basis for clinical prevention and control of methicillin resistant Staphylococcus aureus. [method] (1) the analysis of methicillin resistant Staphylococcus aureus in local methicillin-resistant region by automated broth microdilution and mec A gene amplification (2) the molecular epidemiology of methicillin-resistant Staphylococcus aureus in the local area was studied by spa typing, SCCmec typing, MLST typing, PFGE typing and MLVA typing. (3) by amplifying PVL and tst-1 genes, the virulence genes in methicillin resistant Staphylococcus aureus in the region were studied. (4) the clinical information of Staphylococcus aureus patients infected with methicillin resistant Staphylococcus aureus was collected and the clinical infection characteristics of methicillin resistant Staphylococcus aureus were analyzed by multiple factor Logistic regression analysis. [results] 150 strains of methicillin resistant Staphylococcus aureus all amplified MEC A gene. All strains were all vancomycin and vancomycin. Linezolid sensitive strain.150 strains were resistant to penicillin and zoxicillin, and the resistance rates for cefoxitin, piperacillin - tazobactam, ampicillin - sulbactam, ciprofloxacin, furadyrin, erythromycin, clindamycin, compound sulfamethamine and rifampin were 94% (141/150), 98% (147/150), 66% (99/150), 64% (96/150), 60%, respectively. (90/150), 74% (111/150), 50% (75/150), 72% (108/150) and 12.7% (19/150). (2) molecular typing results showed that 150 strains of methicillin resistant Staphylococcus aureus had 11 clones and 28 spa types, of which CC5-spa T002 (29.3%) and CC239-spa T037 (14.7%) were most prevalent.SCCmec II, respectively, and accounted for 33.3 of all strains, respectively. The detection rate of%, 21.3%, 23.3% and 21.3%.PVL gene (Luk F/S-PV) in all 150 methicillin resistant golden yellow grapevine balls was 11.3%, and the total physical examination rate of 6 different clones (5,8,59,88239398).TSST-1 gene (TST) was 18%, mainly CC5 clone group (96.3%). In this study, all tst-1 positive CC5 clones were in methicillin resistant group. Staphylococcus aureus strains were spa T002 type. (3) 150 strains of methicillin resistant Staphylococcus aureus, of which 71 were isolated from the internal medicine ward (71/150,47.3%), 48 isolated from the surgical ward (48/150,32.0%), and 31 isolated from the intensive care unit (31/ 150,20.7%). The strains in this study were isolated from the hospitalized patients, 60% The average age of 60 years old was 63 years old and 74% (111/150) was male. The strains in this study were mostly isolated from sputum samples (90/150,60.0%), followed by drainage samples (17/150,11.3%), wound samples (12/150,8.0%), and 4 isolated from blood samples, 5 isolated from urine samples and 22 isolated from other sites. (4) in this study, 18 patients died within 30 days of hospitalization and the fatality rate was 12% (18/150). This study compared patients' prognosis in three major clones (CC5, CC239, and CC59), the clinical features and genotyping of methicillin resistant Staphylococcus aureus isolates. Compared to patients infected with CC239 or CC59 cloned strains, Patients infected with CC5 clones were significantly higher in hospital mortality in 30 days (P0.05). In addition, the CC5 clone strain had a higher probability of SCCmec II strain and tst-1 gene (P0.01), and had a higher probability of being separated from the intensive care unit (P0.05). The study also compared patients with different prognosis (survival vs. death). Clinical and molecular characteristics of methicillin resistant Staphylococcus aureus resistant Staphylococcus aureus. In a single factor analysis, the following factors are related to the mortality of 30 days in patients: age, tumor, erythromycin tolerance, infected strains of CC5 clones, molecular typing SCCmec II spa T002 and tst-1 gene (P0.05). In addition, income severe The factor of the guardianship was statistically analyzed and the correlation with the mortality rate of 30 days of hospitalization was on the critical line (P=0.06). It is worth noting that in this study, patients infected with tst-1 positive methicillin resistant Staphylococcus aureus were significantly higher in hospital mortality than those infected with tst-1 negative methicillin resistant Staphylococcus aureus. 51.9%vs.3.3% (P0.001). However, there was no significant difference in mortality between patients infected with PVL positive methicillin resistant Staphylococcus aureus and patients infected with PVL negative methicillin resistant Staphylococcus aureus (0.0%vs.13.5%, P=0.13) at 30 days. Independent risk factors associated with the mortality rate of 30 days hospitalization in patients with multiple factor regression analysis. The patients were older than 60 years old (odds ratio[OR]=7.2,95%confidence interval[95%CI]=1.26-41.6, P=0.026), with basic tumor disease (OR=9.6,95%CI=1.4-65.7, P=0.02), and tst-1 positive methicillin resistant Staphylococcus aureus (OR=62.5,95%CI=12.0-325.2, P0.001). [Conclusion] (1) the study area of methicillin resistant Staphylococcus aureus The overall resistance rate of Staphylococcus was high, but there was no strain resistant to vancomycin and linezolid. (2) the results of this study revealed that compared with other regions of our country, the study area has a unique epidemic spectrum of methicillin resistant Staphylococcus aureus. First, the CC5 clone is the golden yellow resistance of methicillin resistant region in the study area. The main epidemic clones of Staphylococcus aureus are SCCmec II-ST002 and not SCCmec III-ST239.. The prevalence of spa T037 in the area studied is better than that of the spat030 type. The epidemic trend is not the same as the spa T037 type of the previous research results suggested by spa t030 type. Therefore, the results of this study The prevalence of Staphylococcus aureus in methicillin resistant Staphylococcus aureus in different hospitals in China has regional differences. (3) methicillin resistant Staphylococcus aureus CC5 isolates are the dominant strains in the study area, and the proportion of tst-1 positive CC5-MRSA-II and Spa T002 strains is very high. The results of multifactor analysis show that methoxy resistant strains are resistant to methicillin. The infection of the tst-1 positive CC5 clone strain of Staphylococcus aureus is an independent risk factor for the patient's death in hospital for 30 days, warning the hospital to take strict control measures to prevent the further spread of the bacteria in the area.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.5;R515

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