不同来源产志贺毒素大肠埃希菌的分子流行病学研究

发布时间：2018-02-28 14:13

本文关键词： 产志贺毒素大肠埃希菌分子流行病学血清分型耐药性　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景产志贺毒素大肠埃希菌(Shiga toxin-producing Escherichia coli,STEC)是一类能够产1种或多种志贺毒素(Shigatoxin,STX)大肠埃希菌。是能引起一系列人畜共患病的重要食源性病原菌。反刍动物是引起人类发病的STEC主要宿主。自1982年0157:H7血清型STEC在美国首次被确认为食物中毒新型致病菌以来,前后发现了超过400种血清型的STEC致病菌株,血清型的复杂性严重威胁着人类的健康和生命。目前,细菌耐药已成为全球性的公共卫生难题,STEC菌株的耐药现状在我国也较为严峻,因此,及时监测和比较不同来源的STEC的耐药性并分析其分子流行病学特征,有助于分析菌株亲缘关系,掌握其变异规律,并为临床用药提供一定指导意见。研究目的1.通过收集不同来源的STEC菌株,了解STEC的检出及流行情况,获得菌株实验室基础数据,探索全面分析不同来源STEC的技术路线,为其长期监测奠定基础;2.了解STEC的药物敏感性及多重耐药性现状,为指导临床用药提供参考。3.分析STEC的分子分型结果,探究不同来源的菌株间的亲缘关系,并比较分子分型与表型结果间的联系。研究方法1.样本来源:(1)采集山东省济宁微山县及烟台莱州市农村散养家禽家畜新鲜粪便样本;(2)依托于山东省食品安全风险监测工作,收集食品源大肠埃希菌;(3)依托山东省食源性疾病主动监测工作,收集食源性患者源大肠埃希菌。2.STEC菌株分离:(1)对于动物粪便样本,分别采用免疫磁珠捕获法及ECC显色平板培养法初筛O157:H7型及非0157:H7型STEC可疑菌落,飞行质谱鉴定可疑菌落菌种,以PCR扩增法检测毒力基因,携带一种或两种STX毒力基因的大肠埃希菌鉴定为STEC;(2)采用飞行质谱鉴定收集到的食品源及食源性疾病患者源大肠埃希菌的菌种,通过PCR扩增法检测毒力基因,携带毒力基因的大肠埃希菌鉴定为STEC。3.生化特征分析:采用DBI-09致泻大肠埃希菌干制生化鉴定试剂盒对分离到的STEC进行一系列生化特征的鉴定和分析。4.血清型:通过特异性引物PCR扩增测序法结合血清玻板凝集法确认STEC菌株的血清型。5.药物敏感性试验:微量肉汤稀释法对7类、16种常用抗生素进行药敏试验,得到最低抑菌浓度(MIC)值,分析其药物敏感性。6.分子分型:对分离到的STEC菌株进行PFGE分型及MLST分型。结果1.从1022份动物粪便样本中共分离出26株STEC;从157株食品源大肠埃希菌中检测出10株致泻性大肠埃希菌,未检测出STEC阳性菌株;从726株食源性疾病患者源大肠埃希菌中检测出3株STEC;另获得6株2007-2012年分离到的历史动物源STEC。2.全部35株STEC中,23株只携带STX2基因,2株只携带STX1基因;9株同时携带STX2与黏附基因eae;1株同时携带STX1、STX2及eae三种基因3.35株STEC菌株中有8株鉴定为0157:H7血清型菌株,占22.86%。27株非0157:H7型STEC菌株血清型分布分散,无优势血清型。10株食品源致泻性大肠埃希菌的血清型各不相同。4.35株STEC有不同程度的耐药性,对磺胺异恶唑的耐药率最高(65.71%),其次为四环素(60.00%)、复方新诺明(57.14%)和萘啶酸(54.29%)。总体上对β-内酰胺类抗生素耐药率呈现较低水平,对磺胺类抗生素耐药率呈现较高水平。10株食品源致泻大肠埃希菌对磺胺异恶唑耐药率最高(90.00%),对其他抗生素耐药率较低。35株STEC中共24株多重耐药菌株,占68.57%;10株食品源致泻大肠埃希菌中2株为多重耐药菌,占 20.00%。5.35株STEC与10株食品源致泻大肠埃希菌分子分型结果较为复杂,全部45株菌株分属30个基因型别,36个PFGE基因指纹图谱,同源性在57.6%-100%之间,反映了一定基因多态性。SD025和SD028基因型别的菌株最多,均为4株(8.89%);其次为SD011、SD021和SD023,均为3株(6.67%)。全部45株菌株共有24种MLST型别。其中35株STEC有14种MLST型别,ST11型别的菌株最多,包含8株菌(22.86%)。其次为ST5133和ST540,均包含4株菌(11.43%)。10株食品中分离的致泻性大肠埃希菌的MLST型别结果各不相同。结论1.STEC菌株血清型分布分散,无优势血清型,表型结果反映了一定的菌株变异性。2.STEC菌株对β-内酰胺类抗生素耐药率呈现较低水平,对磺胺类抗生素耐药率呈现较高水平;3.分子分型结果呈多态性和复杂性,STEC菌株变异性较高,不同来源STEC菌株间未形成明显的亲缘聚集性。总体上PFGE的分型能力高于MLST,MLST分型结果与血清型相关性较高。
[Abstract]:The research background of Shiga toxin producing Escherichia coli (Shiga toxin-producing Escherichia coli, STEC) is a kind of can produce 1 or more kinds of Shiga toxin (Shigatoxin, STX) of Escherichia coli. Can cause a series of important foodborne pathogens of zoonosis. Ruminant animal STEC is caused by the main host disease in humans. Since 1982 0157:H7 serotype STEC in America was first recognized as a new food poisoning pathogen, before and after STEC was found more than 400 pathogenic strains of serotypes, the serotype complexity is a serious threat to human health and life. At present, bacterial resistance has become a public health problem worldwide, the status of resistance strains in STEC our country is also more severe, so the resistance of STEC timely monitoring and comparing the different sources and to analyze the molecular epidemiological characteristic, is helpful to the analysis of genetic relationship, grasp the variation of, And provide some guidance for clinical use. Objective: 1. through STEC isolates collected from different sources, understand the detection and prevalence of STEC was obtained, laboratory data, a comprehensive analysis of the technical route to explore different sources of STEC, lay the foundation for its long-term monitoring; 2. to understand the status of drug sensitivity of STEC and multiple drug resistance, to provide the results of STEC type molecular reference.3. analysis for clinical medication, explore the relationship between the strains of different sources, and to compare the molecular typing and phenotype of the relation between the result. The method of 1. sample sources: (1) collected in rural area of Shandong Province, Weishan in Jining county and Yantai Laizhou City backyard poultry and livestock (fresh stool samples; 2) based on the Shandong province food safety risk monitoring, collecting food source Escherichia coli; (3) based on the work of active surveillance of foodborne diseases in Shandong Province, collected from patients with foodborne E. The separation of E.coli.2.STEC strains: (1) for animal fecal samples, respectively using immunomagnetic capture method and ECC color plate culture method for screening O157:H7 and non 0157:H7 type STEC suspicious colonies, MALDI-TOF-MS suspicious colony strains, detection of virulence genes by PCR amplification, identification of Escherichia coli carrying one or two STX virulence gene STEC; (2) the source of foodborne diseases and food source Escherichia coli MALDI-TOF-MS collected strains, detection of virulence gene was amplified by PCR, carrying the identification of Escherichia coli virulence genes analysis of STEC.3. biochemical characteristics: identification and analysis of.4. serotypes by DBI-09 lapactic E. coli for biochemical identification kit of isolated STEC was a series of biochemical characteristics by specific primer PCR amplification sequencing method combined with serum agglutination method confirmed serotype.5. strain STEC Drug sensitivity test: broth dilution method of 7 kinds of 16 kinds of antibiotics, drug sensitive test, get the minimum inhibitory concentration (MIC) values, analyze the drug sensitivity of.6. molecular typing: STEC to the strains of PFGE and MLST classification. Results 1. from 1022 animal fecal samples 26 strains of STEC were isolated from 157 strains of food; Escherichia coli detected in 10 strains of pathogenic Escherichia coli, did not detect STEC positive strains; 3 strains were identified as STEC from 726 strains of Escherichia coli were the source of foodborne diseases; the other 6 strains isolated from 2007-2012 years history of animal STEC.2. 35 STEC strains, 23 strains carried STX2 gene, 2 strains carried STX1 gene; 9 strains carrying STX2 and adhesion gene EAE; 1 strains carrying STX1, STX2 and EAE three genes of 3.35 STEC strains in 8 strains were identified as serotype 0157:H7 strains,.27 strains accounted for 22.86% of non 0157:H7 S TEC strain serotype distribution, no serotype of diarrheagenic Escherichia coli isolated from the dominant serotype.10 strain food source in different.4.35 STEC strains have different degrees of resistance, resistance to sulfamethoxazole was the highest (65.71%), followed by tetracycline (60%), SMZ-TMP (57.14%) and naphthalene nalidixic acid (54.29%). The overall effect of beta lactam antibiotic resistance rate showed low levels of sulfa antibiotic resistance rate showed higher levels of.10 were the food source of diarrheogenic Escherichia coli on sulfamethoxazole resistance rate was the highest (90%), to other antibiotics were low.35 strain STEC of the 24 strains multi drug resistant strains, accounting for 68.57%; 10 strains of food source of diarrheogenic Escherichia coli 2 strains of multidrug-resistant bacteria, accounting for 20.00%.5.35 STEC strains and 10 strains of food source diarrheogenic Escherichia coli molecular typing results is more complex, all 45 strains belong to 30 gene type 36 PFGE gene Fingerprint, homology between 57.6%-100%, reflects a certain degree of genetic polymorphism of.SD025 and SD028 genotypes of the strains were 4 strains (up to 8.89%); followed by SD011, SD021 and SD023, were 3 strains (6.67%). All the 45 strains there were 24 genotypes of MLST. 35 of them were STEC 14 kinds of MLST type, ST11 type was most, contains 8 strains (22.86%), followed by ST5133 and ST540, including 4 strains (11.43%) type MLST results of diarrheagenic Escherichia coli isolated from.10 strains in food are different. Conclusion 1.STEC strains of serotype distribution, serum no advantage type phenotype results reflect the variability of certain strains of.2.STEC strain of beta lactam antibiotic resistance rate showed low levels of sulfa antibiotic resistance rate showed a high level of 3.; molecular typing results showed the polymorphism and complexity of STEC strain variability is high, STEC strains from different sources were not On the whole, the typing ability of PFGE was higher than that of MLST, and the correlation between the MLST typing and serotype was higher.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

【相似文献】