杭州市副溶血弧菌血清型、毒力基因、耐药性及分子分型调查

发布时间：2018-05-13 21:26

本文选题：副溶血弧菌 + 血清型　；参考：《浙江大学》2016年硕士论文

【摘要】：目的:副溶血弧菌(Vibrio parahaemolyticus,VP)是一种革兰阴性细菌,广泛分布于海洋、河口和海产品中。人类食用了被副溶血弧菌污染的海产品如花蛤等贝类食品,可能会引起腹泻或食物中毒。据报道副溶血弧菌已经在多个国家和地区暴发流行,成为世界范围内引起旅行者腹泻的主因之一。我国食源性疾病监测网站的统计数据显示副溶血弧菌已经成为导致我国食源性疾病的主要病原菌。杭州地处我国东南沿海,气候湿热多雨,居民喜欢食用花蛤等各类海产品,是副溶血弧菌导致腹泻的高发地区。本研究对杭州地区急性腹泻患者的粪便标本进行收集采样并开展对副溶血弧菌的调查,了解副溶血弧菌在杭州地区的流行现状;通过对血清型和药敏的检测,明确杭州地区副溶血弧菌血清型构成谱和耐药谱;通过毒力基因检测和分子分型研究,从分子水平来研究探讨杭州地区副溶血弧菌的毒力特征以及各菌株间的同源性和遗传关系,为副溶血弧菌防控、治疗提供依据,并为进一步的研究奠定基础。方法:收集2013年到2015年间浙江大学医学院附属第一医院门诊急性腹泻患者来源的120株副溶血弧菌,用血清凝集法对其进行血清型鉴定、用K-B法对其进行药敏试验、用PCR和基因测序来检测其毒力基因并进行多位点序列分析,对副溶血弧菌进行流行病学分析。结果:1、2013年6月至2015年9月,共收集到120株副溶血弧菌菌株,其中男性占42.5%(51株),女性占57.5%(69株);不同年龄组的分布差异较大,以18-44岁的青壮年最高,为64.2%(77株);夏季检出的菌株最多,占78.3%(94株)。2、120株副溶血弧菌中有112株(93.3%)发生O血清型凝集,分属于01、02、03、04、05五种血清群,以03为主(71.7%,80株);110株(91.7%)发生K血清型凝集,分属于17种K抗原血清,以K6为主(66.7%,80株),另外K8有8株(6.7%),K60有4株(3.3%)。共有21种O、K血清型组合(不包括OUT:KUT型别),以03:K6(80株,66.7%)为主,其次为04:K8(8株,6.7%)。另有8株(6.7%)未鉴定出O、K型别,归类于OUT:KUT型。3、副溶血弧菌对本实验的13种抗生素大都保持着较高的敏感性,尤其对亚胺培南和哌拉西林/他唑巴坦未检测到耐药菌株,敏感率达到100%;对阿米卡星的敏感率为70.8%。但是对头孢唑啉、头孢呋辛和氨苄西林的敏感率分别只有45.0%、55.0%和13.4%。本实验发现14株菌株对两种及以上的抗生素耐药,其中7株对3种以上的抗生素耐药,而且有两株分别对7种和8种抗生素耐药。4、120株副溶血弧菌均携带tlh基因,112株(93.3%)有tdh基因,仅6株(5.0%)有trh基因。本实验中,共检出致病性菌株114株(占95.0%),包括108株tdh+trh-菌株,4株tdh+trh+菌株和2株tdh-trh+菌株;同时检出非致病性菌株6株(5.0%)。大流行菌株(tdh+,trh-和toxRS/new+)共77株,占64.1%;非大流行菌株共43株,占35.6%。64株大流行菌株携带orf8基因,占所有大流行菌株的83.1%。77株大流行菌株共包括9种血清型,主要为03:K6(59株,76.6%)。5、T3SS1基因存在于所有的菌株中,其中116株(96.7%)副溶血弧菌包括了其所有的4个基因。4株(3.3%)tdh+trh-菌株缺少T3SS1的4个基因中的1个或者2个基因。T3SS2a基因大都分布在tdh+的菌株(tdh+trh-和tdh+trh+菌株)中,不存在于tdh-trh+的菌株中,但在tdh-trh-菌株中也有检出。108株tdh+trh-菌株中的107株(99.1%)包含所有T3SS2a的4个基因,只有1株缺少VP1362[vopB2]基因。T3SS2β则全部在trh+的菌株(tdh+trh+和tdh-trh+菌株)中检出,无任何一株tdh+trh-和tdh-trh-菌株携带T3SS2β基因。6、115株副溶血弧菌共检出21种序列型别(STs),其中ST3最多为80株(69.6%),其次 ST332 为 10 株(8.7%),ST88 为 5 株(4.3%),ST120 为 3 株(2.6%),其他17种STs各一株。eBURsT将本研究中得到的21种STs归类为8个克隆群(CC3、CC345、CC8、CC332等)、5个组和5个单体,其中ST3和ST572同属于克隆群CC3,且ST3为CC3的始祖(Founder);ST345、ST88和ST189同属于克隆群 CC345。另外 ST8、ST332、ST199、ST120、ST1569、ST69 分别属于克隆群CC8、CC332、CC199、CC120、CC654、CC527。ST216、ST479、ST486、ST635、ST1570 在 eBURST 中分别属于不同的组。ST545、ST712、ST1031、ST1551、ST1571各为单体。7、杭州地区流行最广泛的ST3以03:K6血清型为主(61.7%,71株),ST88以04:K8 为主(80.0%,4 株),ST120 以 03:K29 为主(66.7%,2 株)。结论:1.03:K6及04:K8是本地区腹泻患者中副溶血弧菌最主要的流行性血清型,同时杭州地区副溶血弧菌血清型具有多样性并可能存在一定的变异性。2.本地区副溶血弧菌对多数抗菌药物保持着较高的敏感性,但由于抗生素滥用,检出了对多种抗生素同时耐药的菌株。3.杭州地区副溶血弧菌最主要的毒力因子为TDH,占优势的流行株是血清型为03:K6的大流行菌株。4.杭州地区腹泻患者副溶血弧菌最流行的STs型别为ST3和ST332型,且大流行菌株的STs分布更为集中。
[Abstract]:Objective: Vibrio parahaemolyticus (Vibrio parahaemolyticus, VP) is a gram-negative bacterium widely distributed in marine, estuarine and marine products. Human consumption of marine products such as clams, such as clams, contaminated by Vibrio parahaemolyticus, may cause diarrhea or food poisoning. It is reported that Vibrio parahaemolyticus has been outbreaks in many countries and regions. It has become one of the main causes of traveller diarrhea worldwide. Statistics of the food borne disease surveillance website in China show that Vibrio parahaemolyticus has become the main pathogen causing food borne diseases in China. Hangzhou is located in the southeast coast of China, the climate is wet and hot and rainy, the residents like to eat all kinds of seafood, such as clams and other kinds of seafood, which are parahaemolyticus arc Bacteria caused high incidence of diarrhea. This study collected and sampled the stool specimens of acute diarrhoea in Hangzhou area and carried out a survey of Vibrio parahaemolyticus to understand the prevalence of Vibrio parahaemolyticus in Hangzhou area. Through the detection of serotype and drug sensitivity, the serotype constitution and drug resistance spectrum of Vibrio parahaemolyticus in Hangzhou area were clearly defined. To study the virulence characteristics of Vibrio parahaemolyticus in Hangzhou area and the homology and genetic relationship among the strains of Vibrio parahaemolyticus from the molecular level, and provide the basis for the prevention and control of Vibrio parahaemolyticus, and lay the foundation for further research. Fang method collected the Medical College of Zhejiang University from 2013 to 2015. 120 strains of Vibrio parahaemolyticus from patients with acute diarrhea in the first hospital were identified by serum agglutination. The drug sensitivity test was carried out by K-B method. The virulence genes were detected by PCR and gene sequencing, and the multipoint sequence analysis was carried out. The epidemiological analysis of Vibrio parahaemolyticus was carried out. The results were from June 12013 to 2015. In September, 120 strains of Vibrio parahaemolyticus were collected, of which 42.5% (51) and 57.5% (69 strains) were male, and the distribution of different age groups was different, the highest in 18-44 years old, 64.2% (77), and 78.3% (94).2120 strains of Vibrio parahaemolyticus (93.3%) of 78.3% (94) in summer (94) O serotype agglutination, belonging to 01, 02,03,04,05 five serogroups, 03 (71.7%, 80); 110 (91.7%) serotype agglutination, belonging to 17 K antigen sera, K6 (66.7%, 80), 8 (6.7%) and 4 (3.3%) of K8. There are many O, K serotype combination (excluding OUT:KUT type), 03:K6, followed by 04:K8, and another (04:K8). 6.7%) O, K type, and OUT:KUT type.3 were not identified, and Vibrio parahaemolyticus was most sensitive to the 13 antibiotics in this experiment, especially for imipenem and piperacillin / tazobactam, the susceptibility rate reached 100%, and the sensitivity to Amikacin was 70.8%. but cefazolin, cefuroxime and ammonia. The susceptibility rate of benzicillin was only 45%, 55% and 13.4%. found that 14 strains were resistant to two or more antibiotics, 7 of which were resistant to more than 3 antibiotics, and two strains of 7 and 8 antibiotic resistant Vibrio parahaemolyticus all carried TLH genes, 112 (93.3%) had TDH gene, only 6 (5%) had TRH base. In this experiment, 114 pathogenic strains (95%) were detected, including 108 tdh+trh- strains, 4 tdh+trh+ strains and 2 tdh-trh+ strains, 6 strains of non pathogenic strains (5%), 77 strains of pandemic strains (tdh+, trh- and toxRS/new+), and 43 strains of non pandemic strains, accounting for ORF8 gene carrying 35.6%.64 strains. The 83.1%.77 strain of all the pandemic strains consisted of 9 serotypes, mainly 03:K6 (59, 76.6%).5, and the T3SS1 gene existed in all strains, of which 116 (96.7%) Vibrio parahaemolyticus included 1 or 2 gene.T3SS2a genes of all 4 gene.4 strains (3.3%) tdh+trh- strains lacking T3SS1. All of the strains of tdh+ (tdh+trh- and tdh+trh+ strains) were not found in the strains of tdh-trh+, but 107 of the.108 strain tdh+trh- strains (99.1%) contained 4 genes of all T3SS2a, and only 1 lacking VP1362[vopB2] gene.T3SS2 beta were all detected in trh+ strains (tdh+trh+ and isolates). 21 strains of Vibrio parahaemolyticus (STs) were detected by tdh+trh- and tdh-trh- strains with T3SS2 beta gene.6115, of which ST3 was 80 (69.6%), ST332 was 10 (8.7%), ST88 was 5 (4.3%), ST120 was 3 (2.6%), and 17 STs each was classified as 8 of 21 STs. The clone group (CC3, CC345, CC8, CC332, etc.), 5 groups and 5 monomers, of which ST3 and ST572 belong to the clone group CC3, and ST3 is the ancestor of CC3. EBURST belongs to different groups of.ST545, ST712, ST1031, ST1551, ST1571 each.7. The most popular ST3 in Hangzhou region is dominated by 03:K6 serotype (61.7%, 71 strains), ST88 is 04:K8 (80%, 4), ST120 is mainly (66.7%, 2 strains). The serotypes of Vibrio parahaemolyticus in Hangzhou area have diversity and may exist a certain variety of.2.. Vibrio parahaemolyticus is highly sensitive to most antibiotics. However, due to the abuse of antibiotics, the bacterial Vibrio parahaemolyticus in Hangzhou area, Hangzhou, is the most important strain that is resistant to a variety of antibiotics simultaneously. The virulence factor is TDH, and the dominant epidemic strain is the serotype 03:K6. The most popular STs type of Vibrio parahaemolyticus in Hangzhou Hangzhou area is ST3 and ST332, and the distribution of STs in the pandemic strains is more concentrated.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.5

【参考文献】