副溶血弧菌海产品和临床分离株的表型及溶血素相关基因分析

副溶血弧菌是(Vibrio parahaemolyticus)常见的食源性病原菌,可污染多种水产品,并引起人的食物中毒,其致病性与溶血素密切相关,如直接耐热溶血素(TDH)、TDH-相关溶血素(TRH)、不耐热溶血素(TLH)。用PCR方法对分离自浙江省部分地区的副溶血弧菌临床和海产品分离株的3种溶血素基因进行检测。结果表明,所有副溶血弧菌菌株均可检测到tlh基因;11株临床分离株均检测到tdh基因,而42株水产品分离株中只有1株检出tdh基因,携带tdh的分离株神奈川试验(KP)均为阳性。所有分离菌株中均未检测到trh基因以及其尿素酶试验呈阴性,由此可知trh基因可能与尿素酶基因连锁。副溶血弧菌分离株中致病性相关毒力因子TDH的阳性率极低,然而副溶血弧菌性食物中毒发生率较高,它们之间的关系及其发病机制还有待深入研究。     

辽宁省2014‒2016年副溶血性弧菌毒力基因及 血清型、耐药性

摘要：目的 研究辽宁省近三年副溶血性弧菌毒力基因携带、血清型分布及抗生素的耐药情况,为副溶血性弧菌疾病防治提供科学依据。方法 运用多重荧光定量PCR对2014?2016年共计317株食品中和临床分离出的副溶血性弧菌进行毒力基因tdh、trh和tlh检测,同时进行血清分型,并用肉汤稀释法测定对15种抗生素的耐药性。结果 317株分离菌中均携带tlh基因,其中有50.5％的菌株携带tdh基因。167株临床分离株中158株携带tdh基因,检出率为94.6％。317株副溶血性弧菌中85株不能进行K分型,其他菌株共分为29个血清型,167株临床分离株中71.3%为血清型为O3:K6。150株食品分离株中8%为血清型O2:K28,6%为血清型O2:K3。317株副溶血性弧菌对头孢唑啉耐药率达36.9%。结论 辽宁省副溶血性弧菌临床分离株多携带tdh毒力基因,食品分离株毒力基因tdh、trh携带率低。临床分离株中O3:K6血清型菌株均携带tdh基因,且更易产生耐药。辽宁省副溶血性弧菌食源性疾病菌株以携带tdh基因的O3:K6型菌株为主。食品分离株血清型分布比较分散,O2血清群为主要流行血清型。辽宁省地区副溶血性弧菌主要对β-内酰胺类和大环内酯类抗生素产生耐药性。     

宁波地区环境中副溶血弧菌血清学及毒力相关基因研究

目的了解宁波地区环境来源海产品中副溶血弧菌血清学特点及毒力相关基因分布。方法采集并分离2013年6-10月宁波地区海产品中副溶血弧菌,对其进行O、K抗原血清学分型;并采用PCR或多重PCR的方法来检测溶血素基因(tlh、tdh、trh)、大流行群遗传标志基因(toxRS/new、orf8)和Ⅲ型分泌系统(T3SS1、T3SS2α、T3SS2β)基因。结果从海产品样本中分离鉴定到44株副溶血弧菌的菌株,分属于20种血清型,型别多样,未见优势血清型;溶血素基因检测发现3株tdh+trh-致病性菌株,遗传标志基因检测发现1株tdh+trh-toxRS/new+大流行株,其血清型为O3:K6型;Ⅲ型分泌系统基因检测发现T3SS1基因存在于所有的副溶血弧菌菌株中,而T3SS2α基因则主要分布在tdh+的菌株中。结论宁波地区环境中副溶血弧菌致病性菌株和大流行株的检出,说明该地区具有潜在的食源性疾病爆发的风险。     

多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌

设计引物和探针,优化多重实时PCR条件,以同时检测霍乱弧菌霍乱毒素基因ctxA、副溶血弧菌种特异性基因gyrB和耐热肠毒素基因tdh。该多重实时PCR方法检测产毒素性的O1群(3株)和O139群(44株)霍乱弧菌菌株、不产毒素的O1群(12株)和O139群(6株)及非O1非O139群(7株)霍乱弧菌菌株的ctxA,阳性和阴性结果与普通PCR检测结果100%符合;检测副溶血弧菌种特异性gyrB,116株副溶血弧菌均阳性,而9株其它细菌和72株霍乱弧菌均阴性;检测tdh的阳性和阴性结果也与普通PCR结果完全一致。另外还建立了检测副溶血弧菌菌株trh1和trh2的单重实时PCR方法。     

副溶血性弧菌耐热性直接溶血素(TDH)的研究进展

副溶血性弧菌(Vibrio parahaemolyticus)是海产品中一种常见的食源性致病菌,常导致水产养殖动物患病或者引起食物中毒。耐热性直接溶血素(thermotolerant direct hemolysin,TDH)是副溶血性弧菌最为重要的致病因子之一。本文围绕tdh基因在弧菌属中的广泛分布与传播、tdh基因的多样性及其表达调控、TDH的蛋白结构及其生物活性进行了综述,并对未来TDH的研究方向进行了展望。旨在进一步了解由副溶血性弧菌感染所引起的病症,为预防副溶血性弧菌的感染和临床治疗提供理论支撑。     

浙江沿海地区海产品及环境中副溶血弧菌的分离与主要毒力基因分析

2007~2008年间, 我们调查了浙江沿海地区海产品和养殖环境中副溶血弧菌的污染状况, 并分析了不同来源副溶血弧菌中主要毒力相关基因tdh、trh、ureC和T3SS2(vscC2、vcrD2)的分布特征及溶血表型与尿素酶表型。结果显示, 566份样品中共分离到395株副溶血弧菌, 检出率高达70%, 毒力相关基因分析结果发现, tdh基因阳性率为10.1%, trh与ureC基因阳性率分别为 20.0%与 11.1%, 40株tdh+菌中组成T3SS2的vscC2基因阳性率为32.5%, 其中38株tdh+菌的神奈川试验亦呈阳性; 但在44株trh+-ureC+菌株中, 尿素酶表型阳性只有6株。试验表明, 浙江沿海地区海产品及其养殖环境中副溶血弧菌污染状况比较严重, 且有相当比例的菌株携带毒力或疑似毒力基因。研究结果为深入探索副溶血弧菌的致病性、基因结构与功能(或表型)及其分子演化提供基础。     

近岸及河口环境中副溶血弧菌的生态适应性及其主要毒力基因分布特征

副溶血弧菌作为一种革兰氏阴性海洋菌,在海洋及河口环境中普遍存在,并占据了多种生态位。近年来,近岸及河口环境中副溶血弧菌引发疾病事件已成为国内外研究热点。该文综述了沿海地区副溶血弧菌的分布规律,着重阐述了影响副溶血弧菌在近岸沿海分布的主要环境生态因子,以及副溶血弧菌的主要毒力基因tdh、trh和tlh的分布特征,为开展近岸及河口环境中副溶血弧菌分布研究及沿海生态系统中副溶血弧菌相关疾病的暴发预测打下基础。     

VHH/TLH溶血素基因在海洋弧菌中分布的研究

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6 %。根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot ,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况。结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus) ,2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae) ,坎贝氏弧菌(V.campbellii) ,辛辛那提弧菌(V.cincinatiensis) ,费氏弧菌(V.fischeri) ,拟态弧菌(V.mimicus) ,飘浮弧菌(V.natriegens) ,副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei)。与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum) ,河口弧菌(V.aestuarianus) ,美人鱼发光杆菌(Photobacterium damselae subsp.damselae) ,河弧菌(V.fluvialis) ,弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus) ,而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB645 , VIB 648和SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72 ,坎贝氏弧菌VIB 285 ,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67 %~99 %和69 %~91 %。对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。     

不同温度条件下副溶血性弧菌tdh基因表达及其代谢组响应

【目的】采用RT-PCR的方法分析致病性副溶血性弧菌毒力基因表达,并应用代谢组学的方法研究毒力基因不同表达水平下致病性副溶血性弧菌代谢组的响应。【方法】本文以致病性副溶血性弧菌(Vibrio parahaemolyticus)ATCC33846为材料,分别提取不同温度(4、25和37℃)下菌体总RNA和代谢组。采用相对定量的方法检测副溶血性弧菌tdh基因在不同温度条件下的表达差异,同时应用超高压液相色谱-四级杆飞行时间质谱联用仪(UPLC/Q-TOF-MS)系统为工作平台检测其代谢组。采用主成分分析法(principal component analysis,PCA)比较副溶血性弧菌代谢组轮廓差异,并通过皮尔森和斯皮尔曼相关性分析法分析代谢组与tdh基因表达之间相关性。【结果】结果表明,不同温度条件下tdh基因表达强弱的排列顺序25℃4℃37℃;在tdh基因不同表达水平下发生显著性(P0.05)变化的主要代谢物是有机酸、氨基酸、醇、酮、酯;共得到11种代谢物与tdh基因表达高度相关(相关性系数︱r︱=1,P0.05),其中3种为负相关,8种为正相关,且醇类代谢物与tdh基因表达的正相关性最显著。【结论】本研究发现副溶血性弧菌代谢组与毒力基因表达存在一定的相关性,有望为副溶血性弧菌致病机理的深入探究提供一定的理论支持。     

副溶血性弧菌食物中毒及腹泻菌株III型分泌系统的检测及分析

目的了解副溶血性弧菌食物中毒和临床腹泻株Ⅲ型分泌系统的分布以及耐药特征。方法对食物中毒和临床腹泻分离到的共21株副溶血性弧菌进行毒力基因tdh、trh、T3SS1、T3SS2α、T3SS2β和toxR检测,并用VITEK 2 compact全自动微生物鉴定系统进行了耐药性分析。结果 21株菌株中tdh+/trh-占90.48%(19/21),tdh-/trh+和tdh-/trh-分别占4.76%、4.76%,未检测到tdh+/trh+菌株。T3SS1广泛存在于所有菌株中。T3SS2α存在于tdh+/trh-菌株,T3SS2β存在于tdh-/trh+菌株。1株食物中毒菌株毒力基因携带情况为tdh-/trh-/T3SS2α-/T3SS2β-。21株副溶血性弧菌对阿莫西林、头孢吡肟、抗菌素B、庆大霉素、环丙沙星和复方新诺明敏感,对氨苄西林完全耐药。结论食物中毒和临床腹泻分离到的菌株大多携带tdh基因,T3SS2α与tdh相关,而T3SS2β则存在于trh+菌株。未携带tdh和trh基因的食物中毒分离株表明副溶血性弧菌不仅仅依赖TDH和TRH发挥毒力作用,其致病机制具有多样性和复杂性。     

Tn5介导的致病性副溶血弧菌溶血能力差异突变株表型分析

【目的】构建致病性副溶血弧菌(Vibrio parahaemolyticus,Vp)突变体库,分析溶血能力差异突变株表型特征,为深入挖掘和认识tdh的调控机制提供研究基础。【方法】利用双亲本接合法,将Mini-Tn5-Km2片段随机插入致病性Vp (ATCC 33846)基因组,并以含有卡那霉素(Km)和氨苄青霉素(Amp)的TCBS选择性培养基进行突变株(KmRAmpS)筛选;结合PCR方法对突变菌株进行Km基因筛查,构建在不同基因位点随机插入突变的Vp突变体库。以我妻氏血平板筛选溶血表型变化菌株,并对其生长曲线、菌膜形成能力和运动能力进行测定。【结果】采用双亲本接合法,成功建立包含490株Vp突变株的突变体库,并获得5株溶血表型变化稳定的菌株(2株为溶血能力上调,3株为下调)。5株突变株在生长速率、菌膜形成能力及运动能力方面与亲本株有显著性差异。其中,2株溶血能力上调菌株及1株溶血能力下调菌株在运动能力、生长速率和菌膜形成能力方面较亲本株显著降低(P<0.05);另2株溶血能力下调菌株菌膜形成能力较亲本株显著提高(P<0.05)。【结论】Tn5转座子可用于建立Vp突变体库;Vp溶血能力与其表型特征具有相关性;研究所获得的5株溶血表型突变株为进一步探讨Vp tdh的调控机制奠定基础。     

Manifestation of the Kanagawa phenomenon, the virulence-associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene

Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh 2 gene, one of the two tdh genes ( tdh 1 and tdh 2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain. The tdh 1– and tdh 2– lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh 2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh 2 and tdh 1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP-negative strains are close to the tdh 2 promoter; they differ at position −34 but have the same base at position −24 as the tdh 2 promoter. We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position −34 is sufficient to increase the expression of these genes to the KP-positive level. Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.     

Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.     

基于分子马达生物传感器技术的副溶血性弧菌分子分型方法的初步研究

[目的]建立基于分子马达技术的简便快速的分子分型方法,对携带和非携带毒力基因的副溶血性弧菌进行快速分类.[方法]以F0F1-ATPase为核心构建分子马达,以副溶血性弧菌毒力基因tdh、trh和种特异性基因tlh、toxR为靶基因设计4个探针.通过生物素-亲和素系统将探针与分子马达连接构建F0F1-ATPase分子马达生物传感器,对10株副溶血性弧菌分离株进行分类,并与PCR-电泳-凝胶成像结果进行比较;同时对生物传感器的检测灵敏度和特异性进行研究.[结果]10株试验菌株中10株tdh阳性,0株trh阳性,而10株菌都携带tlh和toxR,与PCR-电泳-凝胶成像结果一致;分子马达生物传感器的最低检测限为1 pg/反应体系,且能够对副溶血性弧菌特异性识别,PCR-电泳-凝胶成像方法的最低检测限为10 pg/PCR反应体系.[结论]建立了基于分子马达的分子分型方法,能够对副溶血性弧菌的致病性进行快速诊断,检测灵敏度比PCR-电泳-凝胶成像方法高了10倍,而且特异性非常高.该方法简便、快速、省时、省力,适用于地方疾控部门和口岸检疫部门的基层实验室开展副溶血性弧菌监测和流行病学溯源工作.     

Detection of a functional insertion sequence responsible for deletion of the thermostable direct hemolysin gene (tdh) in Vibrio parahaemolyticus

The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh(+) strains and a tdh(-) strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh(-) strain was derived from the tdh(+) strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh(+) sequences in the tdh(-) strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh(+) strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh(-) strain.     

Occurrence, characterisation and detection of potential virulence determinants of emerging aquatic bacterial pathogens from the Philippines and Thailand

Strains of Aeromonas spp., 'non-cholera vibrios' (NCVs) and Plesiomonas shigelloides isolated from aquatic environments, fish and human diarrhoeal cases in the Philippines and Thailand were characterised for potential virulence markers. Thus, the production of cytotoxin, cell-associated and cell-free haemolysin and their capacity to adhere to human intestinal (Henle 407) cells in vitro was investigated. In addition, the occurrence of tlh and tdh haemolysin genes and urease activity among V. parahaemolyticus strains was investigated. The results showed that strains recovered from clinical sources (human and fish) produced these virulence factors, whereas these are absent in environmental strains.     

Molecular characterization of thermostable direct haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters in Mangalore, India

Pathogenic Vibrio parahaemolyticus strains producing either or both of a thermostable direct haemolysin (TDH) and a TDH-related haemolysin (TRH) encoded by tdh and trh genes, respectively, are isolated at a low rate from the environment. However, recently we observed that a considerable percentage of APW (alkaline peptone water) enrichment broths of oysters collected off Mangalore India, were trh(+), rather than tdh(+) by PCR. In order to further investigate the prevalence and genetic diversity of trh bearing V. parahaemolyticus in our coast, we attempted to isolate and characterize trh(+)V. parahaemolyticus from oysters. A total of 27 trh(+) strains were isolated during the period between March 2002 and February 2004, of which nine were also tdh(+). All the trh(+) isolates were positive for urease phenotype. The isolates belonged to diverse phenotypes. In order to explore the possible presence of heterogeneity in the trh gene region among trh(+)V. parahaemolyticus, a 1.5 kb region around trh gene was PCR amplified and restriction digested using selected restriction enzymes. The whole genome comparison of strains was performed by randomly amplified polymorphic DNA PCR (RAPD PCR). The PCR-RFLP results revealed fairly well conserved nature of the trh gene region studied in different serotypes. Though 11 strains were positive by PCR for a genomic fragment that has been reported to be amplified in pandemic strains, all strains were negative by group-specific PCR (GS-PCR), orf8 PCR and showed a different RAPD pattern compared with pandemic strains. The results suggest that genetically diverse V. parahaemolyticus carrying virulence genes are associated with the aquatic environment in this region.     

Comparison of the pathogenic potentials of environmental and clinical vibrio parahaemolyticus strains indicates a role for temperature regulation in virulence

Although the presence of pathogenic Vibrio spp. in estuarine environments of northern New England has been known for some time (C. H. Bartley and L. W. Slanetz, Appl. Microbiol. 21: 965-966, 1971, and K. R. O'Neil, S. H. Jones, and D. J. Grimes, FEMS Microbiol. Lett. 60:163-167, 1990), their virulence and the relative threat they may pose to human health has yet to be evaluated. In this study, the virulence potential of 33 Vibrio parahaemolyticus isolates collected from the Great Bay Estuary of New Hampshire was assessed in comparison to that of clinical strains. The environmental isolates lack thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are encoded by tdh and trh, respectively. Though not hemolytic, they do possess putative virulence factors, such type III secretion system 1, and are highly cytotoxic to human gastrointestinal cells. The expression of known and putative virulence-associated traits, including hemolysin, protease, motility, biofilm formation, and cytotoxicity, by clinical reference isolates correlated with increased temperature from 28°C to 37°C. In contrast, the environmental isolates did not induce their putative virulence-associated traits in response to a temperature of 37°C. We further identified a significant correlation between hemolytic activity and growth phase among clinical strains, whereby hemolysin production decreases with increasing cell density. The introduction of a tdh::gfp promoter fusion into the environmental strains revealed that they regulate this virulence-associated gene appropriately in response to temperature, indicating that their existing regulatory mechanisms are primed to manage newly acquired virulence genes.