山东省食品中单核细胞增生性李斯特氏菌耐药性与分子分型研究

发布时间：2018-12-27 13:08

【摘要】：[研究背景] 单核细胞增生性李斯特氏菌(Listeria monocytogenes,以下简称单增李斯特氏菌),为革兰氏阳性无芽孢杆菌,是一种重要的食源性人畜共患病原菌。据报道,单增李斯特氏菌在欧洲及美国的发病率为0.2-0.8/100000,但其病死率却高达20-70%,国际各卫生研究机构已经对此给予了高度关注。单增李斯特氏菌在自然界中分布广泛,人类主要通过接触即时食品和冷冻食品发生感染。人类感染后的临床症状主要表现为败血症,脑膜炎,单核细胞增多及自发性晚期流产等,孕妇、新生儿、老人及免疫缺陷者为易感人群。因单增李斯特氏菌污染而引起的食源性疾病暴发在全球范围内屡有报道,加之其较高的死亡率,为公共卫生健康造成了巨大的危害。美国马萨诸塞州于1983年因食用该菌污染的牛奶导致49人患病,14人死亡；加利福尼亚州于1985年因食用污染的奶酪造成142人发病,其中29人死亡；2002年多个州又因单增李斯特氏菌污染的火鸡导致46人患病,7人死亡,3人流产。1992年因食用该菌污染的猪舌头造成法国279人患病。我国从1964-2003年因感染单增李斯特氏菌的病例为150例,死亡39例。从1988年首次报道单增李斯特氏菌对四环素产生耐药以来,从不同环境、食品及临床病例中分离出的菌株对各种抗生素的耐药及多重耐药现象愈发严重。随着单增李斯特氏菌耐药性的日益加重,临床药物选择压力也逐渐加重,这进一步增加了疾病的死亡率。单增李斯特氏菌已成为中国严重的公共卫生问题。近年来虽有不少学者开展了食品中单增李斯特氏菌的研究,但多数局限于对该菌污染率的主动性监测。因此,本研究对山东省2003-2010年食品中单增李斯特氏菌分别运用耐药性试验、血清学分型和脉冲场凝胶电泳分型(PFGE)三种方法进行综合的流行病学研究,探讨不同菌株之间的遗传相关性及不同分型方法之间的联系,为有效地控制和预防食源性疾病提供科学依据。 [研究目的] 1.研究山东省不同食品中单增李斯特氏菌的耐药性,分析山东省单增李斯特氏菌的耐药趋势,为抗生素耐药的危险性评估和管理提供理论基础。 2.研究不同食品中单增李斯特氏菌的血清学型别,分析优势血清型菌株的分布情况。 3.研究单增李斯特氏菌PFGE型别的分布特点,分析不同菌株之间的遗传相关性,建立山东省单增李斯特氏菌的数据库,为单增李斯特氏菌的分布特点、菌株关联及分子流行病学研究提供平台。 4.比较耐药性试验、血清学分型和脉冲场凝胶电泳分型三种方法的优劣,并分析不同分型方法之间的关系。 [研究方法] 1.采用CLSI推荐的微量肉汤稀释法进行耐药性试验。 2.采用玻片凝集法、试管凝集法对单增李斯特氏菌进行血清学分型。 3.按照PulseNet标准方法进行脉冲场凝胶电泳分型。 [研究结果] 1.252株单增李斯特氏菌对15种抗生素的平均耐药率为15.87%,可分为21种耐药型别。其中,对亚胺培南的耐药率最高(3.97%),其次为四环素和环丙沙星(均为1.59%),对庆大霉素、氨苄西林和青霉素尚未出现耐药菌株。多重耐药率为4.37%。不同样品、采样年份和采样地点中分别以生猪肉中(22.09%),2007年(38.89%),临沂(29.41%)的菌株耐药率最高。 2.252株单增李斯特氏菌分为6种血清型,以1/2c型菌株最多,出现了致病力最强的4b型菌株,且4b型菌株的耐药率最高(22.74%)。不同样品、采样年份和采样地点中分别以生鸡肉中(优势血清型为1/2a型,59.15%),2005年(优势血清型为1/2c型,40.54%),济南地区(优势血清型为1/2c型,38.71%)的菌株血清型种类最多,分别包括6种血清型。 3. PFGE分型将252株单增李斯特氏菌分为73个型别,同源性在50.43-100.00%之间,优势型别为24型(15.48%),不同食品来源、采样年份和采样地点的优势型别分别为33型(23.94%)、24型(22.97%)及24型(15.48%)。 4.三种方法的辛普森分辨指数分别为：耐药性试验D=0.7048,血清学分型D=0.7023, PFGE分型D=0.9371,可见PFGE分型方法具有较好的分辨能力。PFGE分型结果与血清学分型结果存在较为密切的联系,与耐药谱分型结果无明显联系。 [结论] 1.山东省不同种类食品中存在单增李斯特氏菌的污染,应当加强对食品卫生的监管力度。 2.首次运用耐药性试验、血清学分型和PFGE分型相结合的方法对山东省食品中单增李斯特氏菌进行研究。不同菌株存在一定的耐药性,但尚未出现对临床一线药物耐药的菌株。山东省单增李斯特氏菌的血清学型别分布较广,应加强对致病型菌株的监测,尤其应警惕4b型菌株的扩散和新型耐药型别的出现。山东省食品中单增李斯特氏菌来源于不同的克隆株,但部分菌株存在不同程度的亲缘关系。PFGE型别与菌株的样品来源不存在明显相关性。PFGE型别与血清型别联系密切,与耐药谱无明显联系。 3.通过三种方法之间的比较,PFGE分型是分辨力最高的分型方法(辛普森分辨指数D=0.9371)。在进行食源性致病菌的流行病学研究时,建立以PFGE型别为主,结合血清型别和耐药谱的数据库,可以为掌握食源性致病菌流行趋势、调查食源性疾病暴发及追踪传染源提供依据。
[Abstract]:[Study Background] Listeria monocytogenes (hereinafter referred to as Listeria monocytogenes), which is a Gram-positive bacillus-free bacterium, is an important food-derived human animal It is reported that the incidence of Listeria monocytogenes in Europe and the United States is 0.2-0.8/ 100000, but the case fatality rate is high by 20-70%. Note: Listeria monocytogenes are widely distributed in nature, human being mainly through contact with instant food and frozen food The clinical symptoms after human infection are mainly characterized by septicemia, meningitis, increased mononucleosis and spontaneous late-stage abortion. The outbreak of foodborne disease caused by the contamination of Listeria monocytogenes has been reported worldwide, and the higher mortality rate has caused great danger for public health and health in 1983, 49 people were sick and 14 died in Massachusetts, Massachusetts, in 1983; in 1985, 142 people were killed by eating tainted cheese, of which 29 were dead. in many states in 2002, 46 people were affected by a single increase of Listeria monocytogenes. Seven people were killed and three were miscarriages. The pig's tongue, which was contaminated with the bacteria in 1992, caused 279 people in France. The number of Listeria monocytogenes in China from 1964 to 2003 is 150, and death 39 Example: Since the first report of the drug resistance of Listeria monocytogenes to tetracycline in 1988, the strains isolated from different environment, food and clinical cases have become more resistant to the drug resistance and multiple drug resistance of various antibiotics. Heavy. As the drug resistance of Listeria monocytogenes is increasing, the pressure of clinical drug selection is also gradually increased, which further increases the death of the disease. The rate of Listeria monocytogenes has become a serious public health guard in China In recent years, many scholars have carried out the study of Listeria monocytogenes in food, but most of them are limited to the activity of the bacteria pollution rate. In this study, the drug resistance test, serological typing and pulse-field gel electrophoresis (PFGE) method were used to make a comprehensive epidemic of Listeria monocytogenes in the food of Shandong Province from 2003 to 2010. To study the relationship between different strains and the relationship between different types of different strains, and to provide the effective control and prevention of foodborne diseases. The basis of the study.[Study Objective] 1. To study the drug resistance of Listeria monocytogenes in different foods in Shandong Province, and to analyze the drug resistance of Listeria monocytogenes in Shandong Province. To provide a theoretical basis. 2. To study the serological type of Listeria monocytogenes in different foods and to analyze the dominant blood. Distribution of the Qing-type strain. 3. The distribution of PFGE-type of Listeria monocytogenes was studied. The genetic relationship among different strains was analyzed, and the database of Listeria monocytogenes was established. Sub-epidemiological studies provide the platform. 4. Compare the advantages and disadvantages of drug resistance test, serological typing and pulse-field gel electrophoresis. Analysis of different types The relationship between the methods.[Study method] 1. CLSI is used The drug resistance test was carried out by the recommended microbroth dilution method. Serological typing of Listeria monocytogenes by tube agglutination test. lseNet Standard method for pulse-field gel electrophoresis typing.[Results] 1. 252 strains of Listeria monocytogenes were used for 15 antigenics The average drug-resistance rate was 15.87%, which could be divided into 21 drug-resistant types, among which, the drug-resistance rate of imipenem was the highest (3.97%), followed by tetracycline and ciprofloxacin (both 1.59 and 1.59).%), for gentamicin, aminophene There were no drug-resistant strains in the forest and the penicillins. The multiple drug-resistance rate was 4.37%. In different samples, the sampling year and the sampling location were in raw pork (22.09%), 2007 (3 The strains of Listeria monocytogenes (8.89%) and Linyi (29. 41%) were the highest. The resistance rate of the 4b-type strain was the highest (22.74%). In different samples, in the sample year and the sampling place, in the raw chicken (the dominant serotype is 1/ 2a, 55.9%), in 2005 (the dominant serotype is 1/ 2c, 40. 54%), the Jinan region (the dominant serotype is 1/ 2c type, The type of strain of the strain of 38. 71%) was the most, including six serotypes. 3. PGE type, 252 strains of Listeria monocytogenes were divided into 73 types, with the homology of 50. 43-100. 00%, the dominant type was 24 (15.48%), and the advantages of different food sources, sampling year and sampling place were 33 (23 (94%), 24-type (22. 97%) and 24 (15.48%). 4. The Simpson-resolved indexes of the three methods were: drug resistance test D = 0.7048, serologic type D = 0.7023, PFG E-type D = 0. 9371, it can be seen that the PFGE classification method has better resolving power. PFGE typing results and serology There was a close relationship between the typing results and there was no significant association with the results of the drug resistance spectrum typing.[Conclusion] 1. Shandong The contamination of Listeria monocytogenes in different kinds of food in the province should strengthen the supervision of food hygiene. The method of combination of serological typing and PFGE's typing in the food of Shandong province is Listeria monocytogenes. The strains of Listeria monocytogenes have not yet been resistant to the first-line drug. The serological type of Listeria monocytogenes in Shandong Province has a wide distribution. The monitoring of the pathogenic strains should be strengthened, especially the spread of the 4b-type strain and the emergence of a new drug-resistant type. Listeria monocytogenes in the food of the province are derived from different clones, but the part There is a different degree of genetic relationship among the strains. PFGE-type is not the same as that of the strain. There was no significant correlation between the source of the product. The PFGE type was not in close contact with the serotype, and there was no significant association with the resistance spectrum. The type of PFGE was the highest-resolution method (Simpson-resolved index, D = 0.97371). In the case of the epidemiological study of food-borne pathogenic bacteria, PFGE-type was the main type, which was combined with serotype and drug-resistance spectrum.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5

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