四川地区鸡源产气荚膜梭菌的分离鉴定与基因分型

目的检测四川地区健康鸡群产气荚膜梭菌的分布状况。方法从四川地区规模化鸡场采集新鲜粪便样品,按产气荚膜梭菌α、β、ε、ι毒素基因cpa、cpb、etx及iA序列,设计针对4种毒素基因的4对特异引物,应用多重PCR方法对产气荚膜梭菌进行基因分型。最后利用重复序列PCR进一步做亚型分析。结果从150份样品中分离到8株(5.3%)产气荚膜梭菌,多重PCR都只扩增出α条带,毒素基因分型结果均为A型。ERIC-PCR、REP-PCR图谱显示两者均适用于产气荚膜梭菌亚型分析,ERIC-PCR是一种更简便、快速和有效的分子流行病学调查方法。结论四川地区健康鸡群中存在的产气荚膜梭菌主要是A型。     

基于TaqMan探针的双重荧光定量PCR方法检测海产品中的副溶血弧菌

目的建立海产品中副溶血弧菌检测的双重荧光定量PCR体系。方法针对副溶血弧菌种特异性基因tlh和tdh设计引物和TaqMan探针,建立双重荧光定量PCR体系,进行特异性与敏感性研究;利用该体系检测海产品中的副溶血弧菌。结果副溶血弧菌可得到特异性扩增,而其它与副溶血弧菌共存于海产品中的细菌均未见扩增曲线。副溶血弧菌与其它细菌的混合DNA检测表明,其它细菌基因组的存在时并不干扰副溶血弧菌检测。副溶血弧菌典型菌株FJ14和BJ97的敏感性试验显示,该体系的最低检测DNA浓度分别为49.8pg与77.8pg,最低检测细菌浓度为56CFU/mL和371CFU/mL。对舟山菜市场采集的50份样本检测表明,32份为tlh基因阳性,3份为tdh基因阳性,与传统方法的检测结果相同。结论与传统检测方法相比,副溶血弧菌的双重荧光定量PCR检测方法快速准确,结果直观。     

脉冲场凝胶电泳用于副溶血性弧菌的分子流行病学研究

目的运用脉冲场凝胶电泳技术和荧光PCR技术对成都市2008-2009年分离的副溶血性弧菌进行分子分型和毒素基因检测,分析感染来源。方法利用PCR检测副溶血性弧菌分离株耐热直接溶血素(tdh)基因和耐热相关溶血素(trh)基因。用脉冲场凝胶电泳(PFGE)对菌株进行分型,所得结果用BioNumerics V 4.0软件UPGMA方法进行聚类分析。结果所试38株分离菌tdh基因阳性有31株,2株为trh阳性。以SfiⅠ酶切后PFGE分型,38株菌被分成了24个带型,大多数暴发有各自优势型别,其中有两起暴发优势型别一致;环境分离株菌型分散。结论多起暴发事件分离株之间PFGE型别差异大,成都市副溶血性弧菌暴发的感染来源复杂。     

副溶血弧菌tlh基因的克隆及序列分析

目的　构建副溶血弧菌不耐热性溶血毒素 (thermolabilehemolysin ,TLH)tlh基因重组质粒。方法　根据已知GenBank中的tlh基因序列 ,设计合成一对引物 ,用PCR方法从副溶血弧菌基因组DNA中扩增编码TLH的基因片段 ,克隆至pET32a+ 质粒 ,转化大肠杆菌DH5感受态细胞 ,经酶切及PCR鉴定 ,而后进行测序。结果　tlh基因体外扩增产物大小约1 30 0bp ,重组质粒经酶切及PCR鉴定表明获得正确重组子 ,测序结果与已知序列基本吻合。 结论　在国内首次克隆了副溶血弧菌tlh基因 ,为研究TLH的功能和探讨TLH作为作为特异性诊断靶抗原的研究奠定了基础     

澳门地区鸡源产气荚膜梭菌的污染情况及基因分型

目的 调查澳门地区鸡源性产气荚膜梭菌的污染情况及基因分型。方法 利用双管法分离产气荚膜梭菌,再作生化测试以鉴定;利用多重PCR测定分离菌株的基因分型。结果 从120个样本中检出率为37.5%,所分离到的产气荚膜梭菌均为 A 型产气荚膜梭菌。结论 初步研究指出本澳供应的鸡肠、新鲜鸡和冰鲜鸡中存在A 型产气荚膜梭菌污染,为产气荚膜梭菌所致的公共卫生问题提供了参考数据。     

宁波副溶血性弧菌临床株毒力基因与多位点序列分型研究

目的 了解宁波地区副溶血性弧菌临床分离株毒力基因分布以及分子分型特征。方法 收集来源于食物中毒和散发腹泻患者副溶血弧菌菌株,利用聚合酶链式反应(polymerase chain reaction,PCR)检测耐热直接溶血素基因(tdh)和耐热直接溶血素相关溶血素基因(trh),利用多位点序列分型(multi-locus sequence typing, MLST)进行分子分型。结果 2006—2012年共分离临床株248株,选择48株进行毒力基因和MLST分型研究。42株tdh+,为93.75%;11株trh+,为22.92%。48株菌株可分为9个ST型和一个未分型,ST3有32株,占66.67%; ST265有5株,占10.42%;ST120有3株,占6.25%。ST3克隆群中tdh+/trh-菌株有25株,占78.16%。与全国其他地区比较,在宁波临床株中发现ST262。结论 tdh+型是宁波地区副溶血性弧菌优势菌株。有9种ST型,以ST3克隆为主,其次为ST265和ST120。ST3克隆中以tdh+/trh-型为主。另发现1个独特的ST262菌株。     

电泳和免疫印迹分析副溶血弧菌和溶藻弧菌主要外膜蛋白和多糖抗原

目的　考察副溶血弧菌和溶藻弧菌的主要外膜蛋白 (MOMP)及其多糖抗原 (PSA)在不同菌株间的结构特征及其免疫学特性 ,从而进一步揭示其共同的保护性抗原。方法　一步超速离心法和蛋白酶K消化法分别制备 10株副溶血弧菌和7株溶藻弧菌的MOMP和PSA ,SDS -PAGE和免疫印迹分析其结构特点。结果　两种细菌的MOMP和PSA的结构和形态菌株间差别明显 ,但也有MOMP一致的菌株 ,免疫印迹显示 ,全部的 10株副溶血弧菌和 5株溶藻弧菌都具有分子量约为36kD的主要外膜蛋白 ,他们能够被副溶血弧菌全菌抗血清所识别 ,是两种细菌共同的具有强免疫原性的主要抗原。PSA分析未见经典LPS的O侧链结构。多糖成份在菌株间的交叉反应有限 ,可能受弧菌自身表面抗原复杂性的影响 ,凝集反应结果与免疫印迹试验结果不完全一致。此外 ,副溶血弧菌与溶藻弧菌的PSA也会产生免疫学上的交叉反应。结论　 36kD的MOMP是广泛存在于副溶血弧菌和溶藻弧菌的高丰度蛋白 ;与主要外膜蛋白相比 ,多糖抗原在菌株间具有更大的异质性 ;副溶血弧菌和溶藻弧菌的MOMP可能比PSA更具研制疫苗潜力。     

2009～2011年无锡市副溶血性弧菌分离菌株的毒力检测及PFGE分析

目的了解无锡市2009～2011年副溶血性弧菌分离株携带的主要毒力因子的流行状况,并对同血清型菌株进行脉冲场凝胶电泳(PFGE)分析。方法应用PCR方法检测分离的35株副溶血性弧菌耐热性溶血毒素基因(tdh)、耐热性溶血毒素相关的溶血毒素基因(trh)和不耐热溶血毒素基因(tlh)。根据美国CDC PulseNet实验方法,用限制性内切酶SfiⅠ对O3﹕K6血清型菌株的染色体进行酶切,通过PFGE获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析。结果 35株副溶血性弧菌tdh、trh及tlh基因的携带率分别为85.7%、8.6%和100%,77.1%的副溶血性弧菌携带的毒力基因为tdh+、trh-、tlh+。PFGE图谱显示,19株O3﹕K6血清型的副溶血性弧菌共有9种PFGE带型,带型100%相同的菌株几乎都出现在同一年代相近的时间点,但也出现了跨年代菌株。结论无锡市副溶血性弧菌致病性较强,具有潜在的O3﹕K6型副溶血性弧菌暴发流行可能,需进一步加强监测管理。     

人艰难梭菌毒素B抗原表位抗体的制备及其ELISA检测方法的建立

目的制备人艰难梭菌TcdB抗原表位抗体,建立针对人艰难梭菌TcdB双抗体夹心ELISA检测方法。方法通过生物信息学等方法预测人艰难梭菌TcdB蛋白的抗原表位,固相多肽合成法合成抗原并制备抗体Anti-TcdB1和Anti-TcdB2,ELISA检测效价并验证其特异性。用辣根过氧化物酶(Horseradish peroxidase,HRP)标记TcdB2抗体,建立ELISA双抗体夹心法。利用棋盘滴定法筛选一抗最佳包被浓度、最佳样品稀释倍数及最适二抗工作浓度等条件,优化ELISA检测方法并确定临界值,用已知阳性、阴性标本及重组B蛋白验证该方法的特异性、灵敏度和重复性。结果间接ELISA确定Anti-TcdB1的效价为1∶512 000,抗TcdB2抗体为1∶2 048 000。棋盘滴定等方法确定ELISA一抗最佳包被浓度0.5μg/ml,样品最佳稀释倍数1∶2,酶标抗体最佳稀释度为1∶20 000。该诊断方法特异,不与其它肠道细菌感染出现交叉反应;重组B蛋白最低检测限为32.25ng/ml;试验的重复性良好,批内和批间变异系数均小于10%。结论本研究建立的检测人艰难梭菌TcdB的ELISA双抗体夹心法敏感、特异,重复性良好,可用于鉴别产毒素B艰难梭菌。     

复合聚合酶链反应同时检测二个腹泻毒力因子

为检测腹泻病原菌产毒性大肠杆菌（ＥＴＥＣ）和副溶血弧菌的两个毒力因子基因热稳肠毒素和热稳直接溶血素，用标准模板建立复合聚合酶链反应（ＰＣＲ），并检测了模拟标本和８８份临床腹泻大便标本。结果热稳肠毒素和热稳直接溶血素基因引物扩增各自的标准模板分别产生１８６ｂｐ和４２５ｂｐ的片段，一元的复合ＰＣＲ都只扩增热稳肠毒素和热稳直接溶血素基因ＤＮＡ并产生特异片段，对其它细菌无扩增。热稳肠毒素和热稳直接溶血素的     

Human gastroenteritis associated with Vibrio parahaemolyticus in Recife, Brazil]

A study was carried out on the occurrence of Vibrio parahaemolyticus in 1,100 diarrheal feces, routinely sent to a private clinical laboratory for microbiologic diagnosis, in Recife. V. parahaemolyticus was isolated from 14 (1.3%) fecal samples. However, if we considered only the specimens from adult patients, the isolation rate of V. parahaemolyticus rose to 7.1%. In most cases (92.86%), V. parahaemolyticus was the only enteropathogen recognized. Among the isolates, seven K antigen serovars were demonstrated, and three were untypable. Only two human isolates, both ureolytic, did not produce the thermostable direct hemolysin. We concluded that V. parahaemolyticus is an important cause of sea food linked diarrhea among adults in Recife.     

Characterization of Vibrio parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.     

Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

AIM： To develop a new, rapid and accurate reverse dot blot （RDB） method for the detection of intestinal pathogens in fecal samples. METHODS： The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157：H7, Clostridium botulinum, Bacillus cereus, Clostridium perfringens , Vibrio parahaemolyticus , Shigella spp., Yersinia enterocolitica , Vibrio cholerae , Listeria monocytogenes and Staphylococcus aureus. The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested. Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay. RESULTS： The methods could identify the 12 intestinal pathogens specifically, and the detection limit was as low as 103 CFUs. The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%. CONCLUSION： The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.     

Adaptive and inflammatory immune responses in patients infected with strains of Vibrio parahaemolyticus

In patients with diarrhea caused by Vibrio parahaemolyticus, antibody-secreting cell responses to thermostable direct hemolysin (TDH), lipopolysaccharide (LPS), and whole-cell bacteria were seen. TDH- and LPS-specific responses were seen in serum samples, and immunoglobulin A antibody responses were observed in stool. Levels of C-reactive protein and nitric oxide metabolites increased in the systemic circulation at the onset of illness. Tumor necrosis factor-alpha and lactoferrin levels were high during the acute stage in mucosal secretions and in plasma, whereas interleukin-1beta levels were high only in mucosal secretions. Duodenal and rectal biopsy specimens obtained at the onset of illness showed an acute inflammatory response. The lamina propria showed edema, congestion of blood vessels, and hemorrhage, with an increase in levels of polymorphonuclear neutrophils and macrophages. Strains belonging to different serotypes exhibited varying resistance to killing by serum; the O8:K21 strain was most sensitive. Infection with V. parahaemolyticus results in B cell responses and an acute inflammatory response that is self-limiting.     

Distribution of thermostable direct hemolysin (TDH)- and TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in coastal Shimane Prefecture and TDH and TRH V parahaemolyticus contamination of retail shellfish

We studied distribution of thermostable direct hemolysin (TDH)- and TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in coastal sea water, sediment, and shellfish and related retail shellfish contamination in Shimane Prefecture, Japan, between 2002 and 2004. V. parahaemolyticus was isolated from > 80% of sea water, sediment, and shellfish. The detection of TDH gene (tdh) and TRH gene (trh)-positive V parahaemolyticus in sea water was 11%, in sediment 16%, and in shellfish 26%. The number of genes and gene-related in seawater was 23 MPN/L, in sediment 29 MPN/100 g, and in shellfish 460 MPN/10 g. TDH- and TRH-producing V. parahaemolyticus detected in seawater was 5%, in sediment 11% and in shellfish 14%. The continuous distribution of TDH-producing O2:K28, O4:K88, O4:K37, and O4:KUT organisms on the western coast and TRH2-producing O5:k30, O5:K43, O10:K19, O10:KUT, O11:K40, O11:KUT, and OUT:KUT organisms on the Oki Island coast suggested the settlement of these organisms in these coastal environments. From 7 (12%) of 59 retail short-necked clam samples, we isolated TDH-producing O 1:KUT, O3:K6 (2 strains from 2 samples imported from Korea), O4:K12, OUT:K8, and TRH2-producing OUT:K40 and OUT:K51 organisms. These findings suggested that TDH- and TRH-producing V. parahaemolyticus are widely distributed along the coast of this prefecture and are transported by contaminated retail shellfish from other areas.     

Isolation of thermostable direct hemolysin producing Vibrio parahaemolyticus from food using screening by PCR in food-borne outbreaks

The producibility of thermostable direct hemolysin (TDH) is the most important pathogenic factor in Vibrio parahaemolyticus. TDH (+) V. parahaemolyticus is usually isolated from patients having V. parahaemolyticus food-borne disease. TDH (+) V. parahaemolyticus is, however, very difficult to isolate from food and environmental samples. In the 5 years from 2000 to 2004 in Tokyo, V. parahaemolyticus was isolated from food samples related to 67 of 227 V parahaemolyticus food-borne outbreaks. In these outbreaks, TDH (+) strains were also tried to isolate using PCR as the screening methods. TDH (+) V. parahaemolyticus strains were able to isolate from enrichment broth in which toxR and tdh genes become positive in PCR. TDH (+) strains of the same serotype with patients were able to be isolated from 23 food samples related to 11 outbreaks (16.4%); 3 outbreaks in 2000, 2 in 2001, 2 in 2002, 1 in 2003, and 3 in 2004. The serotypes of V. parahaemolyticus isolated from food were O3 : K6 (10 samples), O3 : K5 (6 samples), O1 : K25 (4 samples), O3 : K29 (2 samples), O4 : K 8 (1 sample), and O4 : K11 (1 sample). The isolation rate of the TDH (+) strain from enrichment broth differed with samples. In several samples TDH (+) strains were isolated easily only by examining 3 colonies, hence no TDH (+) strains were isolated in spite of the examination of 250 colonies. No correlation was seen between the number of V. parahaemolyticus and the isolation rate of TDH (+) strains in food samples. Screening using PCR is very effective method for isolating TDH (+) V. parahaemolyticus from food samples.     

Symptoms of food-borne diseases and gastroenteritis in Kyushu, Japan

In this study we analyzed the symptoms of gastroenteritis or food-borne disease caused by the 10 most prevalent pathogens: Norovirus, Salmonella, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, Shiga toxin-producing Escherichia coli (STEC), enterotoxigenic E. coli (ETEC), Shigella sonnei/flexneri (Shigella), Staphylococcus aureus, and emetic-type Bacillus cereus. The symptoms diarrhea, vomiting, fever, abdominal pain, and headache, and the incubation period in 646 cases in 10 districts of Kyushu between January 2000 and December 2004 were recorded. The pathogen with the shortest mean incubation period was B. cereus (0.8 h), and was followed by S. aureus (3.3 h), C. perfringens (10.7 h) and V. parahaemolyticus (16.4 h). All the patients infected with B. cereus and S. aureus developed symptoms within 6 hours, and those infected with V. parahaemolyticus and C. perfringens developed symptoms within 24 hours. Bloody diarrhea was associated with STEC and Shigella, but rare with other pathogens. Vomiting was associated with almost all cases of S. aureus and B. cereus infection, and occurred in 71.5% of the Norovirus cases and 56.1% of the V. parahaemolyticus cases. Vomiting was less common in the C. perfringens (22.0%) and the ETEC and STEC (both about 5%). Bloody diarrhea, abdominal pain, and vomiting were statistically significantly more common with STEC 0157 infection than with STEC non-0157 infection. Since the cases analyzed in this study included all degrees of illness, mild to severe, and a wide range of ages, the information obtained will serve as a good reference material for administrative and laboratory work when an outbreak takes place.