【作者】 坎布；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 预防兽医学， 2007， 博士

【摘要】 副溶血弧菌广泛分布于世界各地,是一种常见的海产品污染菌之一。该病原菌主要导致胃肠炎和败血症。副溶血弧菌的致病性与热稳定直接溶血素(Thermostable direct hemolysin, TDH,编码基因tdh)和TDH相关溶血素(Thermostable-related hemolysin, TRH,编码基因trh)有关。但并不是所有副溶血弧菌分离株都有致病性,目前为止环境和海产品分离株及临床分离株主要毒力基因的分布及其致病能力的相关性尚不清楚。大部分临床分离株都具有由TDH引发的神奈川现象(Kanagawa phenomenon, KP),只有少数环境和海产品分离株出现此现象。另外,携带trh基因的分离株均具有尿素酶表型。很多血清型的副溶血弧菌均可携带tdh和/或trh基因。自1996年在Culcata,印度发现新03：K6流行株以来,亚洲和美国等多次爆发由该血清型引起的胃肠炎中毒事件。其它血清型如O4:K48, O4:K68, O1:K25也有潜在的致病能力,而且与03：K6亲缘关系相近。因此,分离和收集不同来源的副溶血弧菌环境和海产品分离株以及临床分离株,比较其毒力基因的分布及结构特征并对其进行分型,有助于我们更好地了解副溶血弧菌的致病机理及其分子生态特征,从而可提出控制策略,有效预防副溶血弧菌病的发生。从5个不同海产品加工厂和杭州海产品零售市场共采样766份,包括新鲜、冷冻和成品海产品以及海产品加工环节中的环境样品。经过碱性蛋白胨水的预增菌和选择性培养基(TCBS)的初步鉴定后,以具有副溶血弧菌种特异性的不耐热溶血素基因tlh和旋转酶B基因gyrB为靶目标进行双重PCR鉴定。检测结果显示163株(163/766,21.3%)为副溶血弧菌。上述结果表明,新鲜、冷冻海产品及其加工环境中均存在具有潜在致病性的副溶血弧菌。比较三种不同限制性内切酶(ApaⅠ、SmaⅠ和NotⅠ)对54株副溶血弧菌环境、海产品和临床分离株进行脉冲场凝胶电泳(PFGE)分型。结果表明NotⅠ的分型效果优于ApaⅠ或SmaⅠ,在54株分离株中,有49株具有可分型性,而另外5株不能被分型。经PFGE指纹图谱分析,可将所有菌株划分为11个脉冲型(分别为A至K)。参考株BJ1997和5株海产品分离株属于Ⅰ脉冲型,其它环境和海产品分离株则分别属于A至H、J和K PFGE型。临床分离株主要集中于A、B、D、E、G和K脉冲型。B型为主要的脉冲型,包含4株临床分离株和4株海产品分离株,同时它又可分为3个亚型,其中2株临床分离株(ZJ1和ZJ5)和2株海产品分离株((ZJ6和ZJ7)分别属于B1和B3亚型。上述结果暗示杭州地区的食物中毒事件可能因食用未煮熟的已污染副溶血弧菌的海产品所致。同时也表明PFGE分型体系可用于不同来源副溶血弧菌的遗传多样性研究。为深入了解不同来源副溶血弧菌的表型特征及基因分布情况,本研究对263株副溶血弧菌(包括4株参考菌株,32株临床株和227株环境和海产品分离株)进行表型分析和毒力相关基因的检测。结果表明,所有菌株都携带toxR和toxRS基因。28株临床菌株(28/32,87.5%)和3株海产品分离株(3/226 1.32%)为tdh阳性,3株临床株(9.4%)和7株海产品分离株(3.09%)为trh基因阳性。31 tdh-阳性的临床和海产品分离株中,30株(30/31,96.77%)为神奈川现象阳性,而10株trh-阳性中,7株(70%)为尿素酶阳性。1株海产品分离株和22临床分离株属于新流行株03：K6。比较新的03：K6流行株、01：KUT和04：K68与老的03：K6菌株toxRS序列,发现在第576至1244位碱基之间存在着6个碱基的突变。在32株临床分离株中,有26株为GS-PCR阳性,17株为orf-PCR阳性。而在海产品和环境分离株中,只有1株为GS-PCR阳性,5株为orf-PCR阳性。上述结果显示副溶血弧菌新流行株03：K6和其它具有致病性的类似血清型均存在于中国沿海地区。选取16株具有不同表型和基因型的副溶血弧菌代表性菌株进行体内外致病性研究。结果表明不同来源的菌株其致病性存在着一定的差异。临床菌株的产肠毒素能力显著高于海产品分离株(P<0.01,73.82%vs 62.07),经腹腔注射后临床菌株的小鼠毒力显著强于海产品分离株(P<0.05),LD50分别为106.62和107.13。另外,临床菌株具有更高的体外培养细胞(HeLa和Caco-2)粘附能力和更强的细胞毒性(HeLa),但无显著性差异。Tdh阳性分离株与tdh阴性菌株相比能产生更高的肠毒素(P<0.05,74.24%vs 60.55%’),对小鼠毒力更强,LD50分别为106.55和107.21,具有显著性差异(P<0.05)。另外,tdh阳性分离株具有更高的细胞粘附能力和更强的细胞毒性。体内外毒力试验表明trh阳性分离株的致病性位于tdh阳性与tdh和trh阴性菌株之间。上述结果表明副溶血弧菌临床分离株或tdh阳性菌株的体内外致病性高于海产品分离株或tdh阴性菌株。为进一步探讨tdh基因的染色体定位和结构特征,选取15株tdh阳性的副溶血弧菌代表菌株(12株临床株和3株海产品分离株)进行tdh基因的克隆和序列分析。结果表明10株临床株的tdh属于tdh1(相似性>98%),2株临床株和1株海产品分离株的tdh为tdh2(相似性>99.2%),另外2株海产品分离株的tdh则属于tdh3(相似性>98%)。此外,采用长距离PCR (LA-PCR)对海产品分离株HZ34 12 kb长的tdh临近区域(位于全基因组的VPA1312至VPA1327；)进行克隆和序列分析。结果表明该区域位于染色体2中,含有两个主要毒力相关基因,VPA1321和VPA1327,分别编码细胞杀伤因子(cytotoxic necrotising factor)和胞外酶T (exoenzyme T)。同时采用基因步移试剂盒扩增菌株F22 tdh启动子区域及其3’区域(长为5.6 kb,tdh包括基因),该菌株同时含有tdh和trh基因,且无法用LA-PCR进行目的片段的扩增。结果表明,在tdh的5’端含有2个开放阅读框架,ORF1和ORF2为类似插入序列,与副溶血弧菌RIMD2210633高度相似性(>97%),而ORF2与副溶血弧菌的TH3996的转座酶相似性为96%。另外在tdh基因的3’端发现两个未知基因,两者都编码假设蛋白(hypothetical protein)VP1767(相似性分别80.9%和55.5%),对应于副溶血弧菌RIMD2210633的染色体1中。本试验结果为深入探索副溶血弧菌的致病性、分子生态特征及基因水平转移的可能途径奠定了良好基础。更多还原

【Abstract】 Vibrio parahaemolyticus is a bacterium that common contaminant of seafoods worldwide and is ubiquitously distributed in coastal waters. It is the leading cause of seafood-associated bacterial gastroenteritis, wound infections and septicemia. Pathogenicity of V. parahaemolyticus is related to the production of a thermostable direct hemolysin (TDH) and/or a TDH related hemolytic (TRH) encoded by tdh and trh genes respectively. However, not all V. parahaemolyticus isolates are pathogenic, the relationship between their pathogenicity and distribution of major putative virulence genes among isolates from environmental, seafood and clinical sources is not well understood from the molecular epidemiologic perspectives. Most clinical isolates of V. parahaemolyticus exhibit Kanagawa phenomenon (KP) induced by TDH on Wagatsuma agar medium, while only small portion of the environmental origins are KP positive and the presence of trh gene in the isolates is strongly correlated with urease phenotypes. The strains bearing tdh and/or trh genes belong to diverse serotypes. However, a pandemic clone of O3:K6 is a major serotype which was first detected in Culcata, India, and has been responsible for many outbreaks in Asia and the United State since 1996. The other serotypes O4:K48, O4:K68, O1:K25 and 01.KUT (K untypable) have similar potential to cause outbreaks of gastroenteritis, and are genetically closed to the new O3:K6 clone. Therefore, it is essential to characterize a substantial amount of V. parahaemolyticus isolates from clinical samples, seafoods and environments, compare the distribution of major putative virulence genes among those isolates and type the V. parahaemolyticus isolates from different origins for better understanding of the pathogenesis and molecular ecology of this bacterium. Thus, crucial control strategies could be established for reducing risk of V. parahaemolyticus infections associated with seafood consumptionA total of 766 fresh, frozen seafood and seafood processing environmental samples were collected from five different seafood factories and Hangzhau’s retail markets. The samples were then examined for the presence of V. parahaemolyticus using alkaline peptone water enrichment and selective agar medium (thiosulfate-citrate-bile salts-sucrose, TCBS) followed by duplex PCR targeting the thermolabile hemolysin (tlh) and Gyrase B (gyrB) genes specific for this bacterium. Finally,163 (21.3%) out of 766 samples were identified as V. parahaemolyticus. The results indicate that V. parahaemolyticus with potential pathogenicity are present in fresh and frozen seafood as well as in seafood processing environments.Fifty-four selected V. parahaemolyticus isolates from clinical samples, seafoods and environments (including reference strain) were characterized by pulsed-field gel electrophoresis (PFGE) using Apal, Smal and Notl for DNA digestion. The enzyme Notl was found more discriminatory power than Smal or ApaⅠfor V. parahaemolyticus typing. Out of 54 strains, only 49 strains were typable, while other 5 strains were untypable. PFGE patterns after Notl digestion could be grouped into 11 distinct pulsetypes (types A to K). Five seafood isolates and the reference strain BJ1997 were grouped as cluster I, while the other seafood and environmental isolates were grouped as A to H, J and K. The clinical strains were grouped into clusters A, B, D, E, G and K respectively. Cluster B was the main cluster and comprised of 4 clinical isolates and 4 seafood isolates. It could be further divided into three subtypes with 2 clinical isolates (strains ZJ1 and ZJ5) belonging to subtype B1 and 2 seafood isolates (strains ZJ6 and ZJ7) grouping into B3. These results implied that the food poisoning in Hangzhou might be due to the consumption of improperly cooked seafoods contaminated with the potentially pathogenic V. parahaemolyticus. Our results also reveal that PFGE typing scheme could be used for the characterization of the high genetic diversity among the V. parahaemolyticus isolates from different origins.Two hundred and sixty-three V. parahaemolyticus (including 4 reference,32 clinical isolates and 227 seafood and environmental isolates) were examined for their hemolytic and urea-producing phenotypes together with the presence of major putative virulence genes by PCRs. All V. parahaemolyticus isolates were positive for both toxR and toxRS genes. Twenty-eight of clinical isolates (28/32,87.5%) and three seafood isolates (3/226,1.32%) were tdh-positive while only three of the clinical isolates (9.4%) and seven of the seafood isolates (3.09%) were positive for trh gene. Among the isolates,30 (96.77%) out of 31 tdh-positive strains were exhibited Kanagawa phenomenon,7 (70%) out of 10 trh-positive strains were urease positive. One tdh-positive of seafood isolate as well as 22 clinical isolates belonged to the new pandemic clone O3:K6 according to serotyping and sequencing of the toxRS locus. The new O3:K6 clone,01:KUT and O4:K68 shared the same characteristic variations from old O3:K6 clone at six base positions from 576 to 1244 of the toxRS locus. Twenty-six and 17 out of 32 clinical isolates (81.3%) were positive for GS-PCR and orf8-PCR respectively, while only one (0.44%) of the seafood and environmental isolates were positive for GS-PCR and five (2.21%) positive for orf8. These results demonstrate the new pandemic O3:K6 clone and other pathogenic serotypes of V. parahaemolyticus isolated from clinical and seafood sources are also present in the Chinese coastal areas.Sixteen representative V. parahaemolyticus having different phenotypes and genotypes were examined for their pathogenicity in in vitro and in vivo systems. The pathogenicity varied considerably among isolates. The clinical isolates were apparently more enteropathogenic (P<0.01 with I/B ratio,73.82% vs 62.07%) and more virulent than their seafoods counterparts to mice via intraperitoneal route with P<0.05, log LD50 6.62 vs 7.13). They were also more adherent to in vitro cultured cells and of higher cytotoxicity as measured by LDH release of the HeLa cells although there were no statistical differences. The tdh-positive V. parahaemolyticus isolates were of higher enteropathogenicity (P<0.05,74.24% vs 60.55%) and more virulent via intraperitoneal route (P＜0.05, log LD50 6.55 vs 7.21) than tdh-negative isolates. The tdh-positive isolates were generally more cytotoxic and adhesive to the cultured cell lines as well. From the in vitro and in vivo pathogenicity profiles, trh-positive isolates seemed to line between tdh-positive isolates and those without tdh or trh. These results suggest that clinical isolates or isolates having the tdh genotype were more pathogenic in in vitro or in vivo models than seafood or tdh-negative isolates.Tdh gene from 15 selected tdh-positive strains (12 clinical isolates and 3 seafood isolates) was sequenced in order to verify its genetic characteristics. The tdh of ten clinical isolates belong to tdhl (>98%), while tdh of another 3 isolates (1 seafood and 2 clinical) and 2 seafood isolates (F22 and F29) tended to be tdh2 (>99.2%) and tdh3 (>98%) respectively after sequence analysis. Furthermore, a DNA region with full length of 12 kb (corresponding to whole genome VPA1312 to VPA1327; ca.12 kb) was cloned and sequenced via the long and accurate (LA-PCR) from one seafood isolate HZ34. Two major putative virulence associated genes, encoding the cytotoxic necrotising factor and exoenzyme T, respectively, were found to be located on chromosome 2, which were corresponding to VPA1321 and VPA1327. In addition, genome walking kit was used to amplify the tdh-promoter and 3’end regions of tdh gene (ca.5.6 kb, including) from the strain F22 which possessed both tdh and trh genes. The results show that both 5’and 3’end regions contain both two open reading frames (ORFs). Two ORFs in the 5’end region are insertion sequence like element (IS), which were 97% and 99% homologous to V. parahaemolyticus TH3996 and V. parahaemolyticus RIMD2210633, respectively. We found two novel genes in the 3’end region of tdh locus which encode hypothetical proteins VP1767 (80.9% and 55.5%, respectively) on chromosome 1 of V. parahaemolyticus RIMD2210633.In summary, results obtained in these studies have laid good foundation for further exploration of the pathogenicity of V. parahaemolyticus from seafood isolates, regulation of virulence gene expression and the possibility of horizontal gene transfers.更多还原