本文選題：副溶血弧菌 + 血清型��； 參考：《浙江大學(xué)》2016年碩士論文

【摘要】：目的:副溶血弧菌(Vibrio parahaemolyticus,VP)是一種革蘭陰性細(xì)菌,廣泛分布于海洋、河口和海產(chǎn)品中。人類食用了被副溶血弧菌污染的海產(chǎn)品如花蛤等貝類食品,可能會(huì)引起腹瀉或食物中毒。據(jù)報(bào)道副溶血弧菌已經(jīng)在多個(gè)國(guó)家和地區(qū)暴發(fā)流行,成為世界范圍內(nèi)引起旅行者腹瀉的主因之一。我國(guó)食源性疾病監(jiān)測(cè)網(wǎng)站的統(tǒng)計(jì)數(shù)據(jù)顯示副溶血弧菌已經(jīng)成為導(dǎo)致我國(guó)食源性疾病的主要病原菌。杭州地處我國(guó)東南沿海,氣候濕熱多雨,居民喜歡食用花蛤等各類海產(chǎn)品,是副溶血弧菌導(dǎo)致腹瀉的高發(fā)地區(qū)。本研究對(duì)杭州地區(qū)急性腹瀉患者的糞便標(biāo)本進(jìn)行收集采樣并開(kāi)展對(duì)副溶血弧菌的調(diào)查,了解副溶血弧菌在杭州地區(qū)的流行現(xiàn)狀;通過(guò)對(duì)血清型和藥敏的檢測(cè),明確杭州地區(qū)副溶血弧菌血清型構(gòu)成譜和耐藥譜;通過(guò)毒力基因檢測(cè)和分子分型研究,從分子水平來(lái)研究探討杭州地區(qū)副溶血弧菌的毒力特征以及各菌株間的同源性和遺傳關(guān)系,為副溶血弧菌防控、治療提供依據(jù),并為進(jìn)一步的研究奠定基礎(chǔ)。方法:收集2013年到2015年間浙江大學(xué)醫(yī)學(xué)院附屬第一醫(yī)院門(mén)診急性腹瀉患者來(lái)源的120株副溶血弧菌,用血清凝集法對(duì)其進(jìn)行血清型鑒定、用K-B法對(duì)其進(jìn)行藥敏試驗(yàn)、用PCR和基因測(cè)序來(lái)檢測(cè)其毒力基因并進(jìn)行多位點(diǎn)序列分析,對(duì)副溶血弧菌進(jìn)行流行病學(xué)分析。結(jié)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);不同年齡組的分布差異較大,以18-44歲的青壯年最高,為64.2%(77株);夏季檢出的菌株最多,占78.3%(94株)。2、120株副溶血弧菌中有112株(93.3%)發(fā)生O血清型凝集,分屬于01、02、03、04、05五種血清群,以03為主(71.7%,80株);110株(91.7%)發(fā)生K血清型凝集,分屬于17種K抗原血清,以K6為主(66.7%,80株),另外K8有8株(6.7%),K60有4株(3.3%)。共有21種O、K血清型組合(不包括OUT:KUT型別),以03:K6(80株,66.7%)為主,其次為04:K8(8株,6.7%)。另有8株(6.7%)未鑒定出O、K型別,歸類于OUT:KUT型。3、副溶血弧菌對(duì)本實(shí)驗(yàn)的13種抗生素大都保持著較高的敏感性,尤其對(duì)亞胺培南和哌拉西林/他唑巴坦未檢測(cè)到耐藥菌株,敏感率達(dá)到100%;對(duì)阿米卡星的敏感率為70.8%。但是對(duì)頭孢唑啉、頭孢呋辛和氨芐西林的敏感率分別只有45.0%、55.0%和13.4%。本實(shí)驗(yàn)發(fā)現(xiàn)14株菌株對(duì)兩種及以上的抗生素耐藥,其中7株對(duì)3種以上的抗生素耐藥,而且有兩株分別對(duì)7種和8種抗生素耐藥。4、120株副溶血弧菌均攜帶tlh基因,112株(93.3%)有tdh基因,僅6株(5.0%)有trh基因。本實(shí)驗(yàn)中,共檢出致病性菌株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;同時(shí)檢出非致病性菌株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株大流行菌株攜帶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種血清型,主要為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個(gè)基因。4株(3.3%)tdh+trh-菌株缺少T3SS1的4個(gè)基因中的1個(gè)或者2個(gè)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-菌株中也有檢出。108株tdh+trh-菌株中的107株(99.1%)包含所有T3SS2a的4個(gè)基因,只有1株缺少VP1362[vopB2]基因。T3SS2β則全部在trh+的菌株(tdh+trh+和tdh-trh+菌株)中檢出,無(wú)任何一株tdh+trh-和tdh-trh-菌株攜帶T3SS2β基因。6、115株副溶血弧菌共檢出21種序列型別(STs),其中ST3最多為80株(69.6%),其次 ST332 為 10 株(8.7%),ST88 為 5 株(4.3%),ST120 為 3 株(2.6%),其他17種STs各一株。eBURsT將本研究中得到的21種STs歸類為8個(gè)克隆群(CC3、CC345、CC8、CC332等)、5個(gè)組和5個(gè)單體,其中ST3和ST572同屬于克隆群CC3,且ST3為CC3的始祖(Founder);ST345、ST88和ST189同屬于克隆群 CC345。另外 ST8、ST332、ST199、ST120、ST1569、ST69 分別屬于克隆群CC8、CC332、CC199、CC120、CC654、CC527。ST216、ST479、ST486、ST635、ST1570 在 eBURST 中分別屬于不同的組。ST545、ST712、ST1031、ST1551、ST1571各為單體。7、杭州地區(qū)流行最廣泛的ST3以03:K6血清型為主(61.7%,71株),ST88以04:K8 為主(80.0%,4 株),ST120 以 03:K29 為主(66.7%,2 株)。結(jié)論:1.03:K6及04:K8是本地區(qū)腹瀉患者中副溶血弧菌最主要的流行性血清型,同時(shí)杭州地區(qū)副溶血弧菌血清型具有多樣性并可能存在一定的變異性。2.本地區(qū)副溶血弧菌對(duì)多數(shù)抗菌藥物保持著較高的敏感性,但由于抗生素濫用,檢出了對(duì)多種抗生素同時(shí)耐藥的菌株。3.杭州地區(qū)副溶血弧菌最主要的毒力因子為T(mén)DH,占優(yōu)勢(shì)的流行株是血清型為03:K6的大流行菌株。4.杭州地區(qū)腹瀉患者副溶血弧菌最流行的STs型別為ST3和ST332型,且大流行菌株的STs分布更為集中。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R446.5

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