宁波北仑地区急性腹泻患者副溶血弧菌表型及分子特征研究

发布时间：2018-07-02 11:49

本文选题：急性腹泻 + 副溶血弧菌　；参考：《浙江大学》2014年硕士论文

【摘要】：目的：副溶血弧菌(Vibrio parahaemolyticus, VP)是一种革兰阴性嗜盐性弧菌,广泛存在于海水和海产品中。1950年在日本副溶血弧菌首次发现并被认为是引起食物中毒的重要致病菌,在我国,副溶血弧菌感染引发的暴发事件居微生物食源性疾病爆发的首位。浙江宁波位于我国东南沿海,海产品消费比较盛行,人们又喜欢食用生的或者未煮熟的海产品,因此副溶血弧菌是引起食物中毒最主要的病原菌。本课题通过收集宁波北仑区急性腹泻患者粪便标本,通过检测血清学凝集试验,可以知道宁波北仑区溶血弧菌血清型构成谱；通过毒力基因检测及分子分型分析,可以为副溶血弧菌感染的防控提供分子流行病学依据,这为开展副溶血弧菌的持续监测研究提供依据,并为进一步的研究工作奠定基础。方法： 2012年4月至2012年10月,从北仑区人民医院采集急性腹泻患者的粪便标本,用常规微生物检验程序对常见肠道致病菌进行分离培养及鉴定。对分离的副溶血弧菌菌株开展血清学凝集试验、利用PCR技术检测毒力基因检测及多位点序列分型(MLST)分析。结果： 1.2012年4-10月共收集腹泻患者的粪便共962份,共分离出副溶血弧菌71株,分离率为7.4%(71/962),列细菌性病原第二位。感染病例男女性别比为1.73:1(45例：26例),年龄范围为16-61岁,其中主要集中在20-45岁(69.0%,49/71)。 2.本次研究共有16种O、K血清型组合,其中03:K6型是最主要的血清型,共44株,占62.0%；04:K8(5株,7.0%)、O1:K56(3株,4.2%),另有3(4.2%)株未鉴定出O、K型别,归类于OUT:KUT型。 3.利用PCR对71株副溶血弧菌进行tdh.trh.tlh毒力基因检测发现：所有副溶血弧菌tlh基因均阳性,98.6%的菌株(70株)tdh基因阳性,仅3株为trh阳性。基因型表现为：tlh+tdh+trh-菌株67株,tlh+tdh+trh+菌株3株和tlh+tdh-trh-菌株1株。通过对大流行群菌株遗传标志性基因的检测发现,宁波北仑区急性腹泻患者感染的副溶血弧菌主要是大流行菌株(tdh+,trh-和toxRS/new+),共47株(66.2%)；非大流行菌株共24株(33.8%)。36株大流行菌株携带orf8基因,占所有大流行菌株的76.6%。 4.在本次研究中,71株急性腹泻患者分离的副溶血弧菌进行副溶血弧菌MLST分析,全部成功分型,共分为13个STs,其中ST3共45株(63.4%),ST885株,ST479、ST8和ST120各4株,另外还包括ST176、ST199、ST216、ST332、ST417、 ST610、ST712和ST80等8种STs。结论： 1.本地区急性腹泻患者中副溶血弧菌检出率高达7.4%,因此在本地区腹泻病例中有必要开展副溶血弧菌的检测,降低副溶血性弧菌引起的食源性疾病的发生和传播的有效途径。 2.TDH是宁波北仑地区副溶血弧菌最主要的致病因子,大流行株是本地区最主要的流行株,血清型03:K6的副溶血弧菌是其优势菌株。 3.通过MLST分型发现宁波北仑区急性腹泻患者中副溶血弧菌ST3最多,是优势型别。主要优势型别与世界上许多国家流行的型别一致,但同时又存在多种亚型。副溶血弧菌流行亚型的多样性是其在宁波北仑区呈高发流行的一个主要原因。
[Abstract]:Objective:
Vibrio parahaemolyticus (Vibrio parahaemolyticus, VP), a gram-negative halophilic Vibrio, is widely found in sea water and marine products for the first time in Japanese Vibrio parahaemolyticus (Vibrio parahaemolyticus) and is considered to be an important pathogen for food poisoning. In China, the outbreak of Vibrio parahaemolyticus caused by the outbreak of Vibrio parahaemolyticus is in the outbreak of food borne diseases. Zhejiang Ningbo is located in the southeast coast of China, and the consumption of seafood is more prevalent. People like to eat raw or uncooked seafood. Therefore, Vibrio parahaemolyticus is the main cause of food poisoning. By collecting the stool specimens from patients with acute diarrhea in Beilun District of Ningbo, the serological agglutination test can be carried out. To know the composition spectrum of serotype of Vibrio haemolyticus in Beilun District of Ningbo, and to provide molecular epidemiological basis for the prevention and control of Vibrio parahaemolyticus through the detection of virulence gene and molecular typing, which provides the basis for the continuous monitoring and research of Vibrio parahaemolyticus and for further research work.
Method:
From April 2012 to October 2012, the fecal specimens of patients with acute diarrhea were collected from the people's Hospital of Beilun District, and the common intestinal pathogenic bacteria were isolated, cultured and identified by routine microbiological test procedure. The serological agglutination test was carried out on the isolated Vibrio parahaemolyticus strain, and the PCR technique was used to detect the virulence gene and the multipoint sequence classification (M LST) analysis.
Result:
A total of 962 diarrhoea patients were collected in 1.2012 years and 4-10 months, and 71 strains of Vibrio parahaemolyticus were isolated. The separation rate was 7.4% (71/962) and second were bacterial pathogens. The sex ratio of male and female in the infected cases was 1.73:1 (45 cases, 26 cases) and the age range was 16-61 years old, mainly in 20-45 years (69%, 49/71).
2. a total of 16 kinds of O, K serotype combination, of which type 03:K6 is the most important serotype, a total of 44, 62%, 04:K8 (5, 7%), O1:K56 (3, 4.2%), and 3 (4.2%) strains did not identify O, K type, classified in OUT:KUT type.
3. tdh.trh.tlh virulence gene detection of Vibrio parahaemolyticus by PCR showed that all Vibrio parahaemolyticus TLH gene was positive, 98.6% strains (70 strains) were TDH positive and only 3 were TRH positive. The genotypes were 67 strains of tlh+tdh+trh- strain, 3 strains of tlh+tdh+trh+ strain and 1 strains of tlh+tdh-trh- strain. The detection of genetic markers found that the main strains of Vibrio parahaemolyticus infected by acute diarrhea in Beilun District, Ningbo, were mainly pandemic strains (tdh+, trh- and toxRS/new+), 47 (66.2%), 24 strains of non - pandemic strains (33.8%).36 strains carried the ORF8 gene, accounting for the 76.6%. of the pandemic strain.
4. in this study, 71 strains of Vibrio parahaemolyticus isolated from Vibrio parahaemolyticus isolated from acute diarrhea patients were analyzed with Vibrio parahaemolyticus (Vibrio parahaemolyticus MLST), all of which were divided into 13 STs, including 45 ST3 (63.4%), ST885, ST479, ST8 and ST120, including ST176, ST199, ST216, ST332, ST417, ST417, 8, etc.
Conclusion:
1. the detection rate of Vibrio parahaemolyticus in patients with acute diarrhea in the region is up to 7.4%. Therefore, it is necessary to detect Vibrio parahaemolyticus in local diarrhea cases and reduce the effective way of the occurrence and transmission of foodborne diseases caused by Vibrio parahaemolyticus.
2.TDH is the most important pathogenic factor of Vibrio parahaemolyticus in Beilun, Ningbo. The pandemic strain is the main epidemic strain in the region, and Vibrio parahaemolyticus of serotype 03:K6 is the dominant strain.
3. it was found that Vibrio parahaemolyticus ST3 was the most dominant type of parahaemolyticus in acute diarrhea patients in Beilun District of Ningbo Province, which was the dominant type. The main dominant type was in accordance with the popular types in many countries in the world, but there were many subtypes at the same time. The diversity of Vibrio parahaemolyticus epidemic subtype is one of the main reasons for its high prevalence in Beilun District of Ningbo.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R446.5;R572

【共引文献】