蛭弧菌对溶藻弧菌的消除作用研究

本实验研究了盐度(NaCl浓度,以下同),Ca2+和Mg2+)对海洋蛭弧菌5#-12和5#-sh06生长活性和裂解溶藻弧菌能力的影响,测定了两株蛭弧菌对11株致病性溶藻弧菌的裂解谱,同时研究了其对牡蛎养殖水体和肠道中溶藻弧菌的清除作用.结果表明,在1.0-3.5%的盐度范围内,蛭孤菌均能生长,最适盐度为3.0%;ca2+和Mg2+浓度为3mM时,5#-sh06具有最好的生长活性,而5#-12在Ca2+和Mg2+浓度为4mM时生长活性达到最强;Ca2+和Mg2+浓度为4 mM时,蛭弧菌5#-12和5#-sh06的裂解能力平均分别提高了11.8%和22.6%;5#-12和5#-sh06分别能裂解其中9株和10株溶藻弧菌,而两株蛭弧菌协同作用,能裂解全部11株溶藻弧菌;蛭弧菌能够有效降低牡蛎肠道和养殖水体中弧菌的数量,试验第7 d,实验组牡蛎肠道和养殖水体中弧菌浓度对数值比对照组分别低7.24和7.97.本研究结果揭示了蛭弧菌作为生物消除剂在控制和消除食源致病性溶藻弧菌等病原菌方面具有潜在的应用价值.     

应用蛭弧菌清除牡蛎中潜在致病弧菌的研究

本实验以从深圳湾海底沉积物中分离得到的两株蛭弧菌菌株506、菌株512,对31株常见的牡蛎致病弧菌进行了裂解能力实验,探索了它们的裂解谱。结果表明,蛭弧菌506可裂解31株试验宿主菌中的29株,512则可以裂解其中24株,而两株蛭弧菌的协同裂解能力高达100%。该结果展示了蛭弧菌在消除牡蛎致病菌及牡蛎活体内残留食源性致病菌的可行性。此外,我们测定了盐度对这两株蛭弧菌生长情况的影响,结果表明在10-45‰的盐度范围内,蛭弧菌均能生长,最适盐度为30‰;同时我们还研究了其对牡蛎养殖水体和肠道中副溶血弧菌的清除作用,数据表明蛭弧菌可以有效地清除牡蛎养殖环境和体内的副溶血弧菌,试验第7d,实验组水体中和牡蛎肠道副溶血弧菌浓度对数值比对照组分别低5.59和3.39,显示了其在清除牡蛎中弧菌的能力。     

蛭弧菌的分离鉴定及其对非O1霍乱弧菌的消除作用研究

从海洋环境中分离到2株蛭弧菌(A-1和A-2),研究了其对6株不同来源非O1霍乱弧菌的消除作用,以探索其对由非O1霍乱弧菌所引起的腹泻疾病的预防效果。实验结果表明,在20～35℃温度条件下,蛭弧菌均有较好的裂解效果,30℃时裂解能力最强;在pH6.5～8.1条件下,蛭弧菌均有较好的裂解能力,pH为7.2时,达到最佳裂解效果;A-1能裂解其中5株非O1霍乱弧菌,A-2能裂解其中4株非O1霍乱弧菌,而2株蛭弧菌协同作用,能全部裂解6株非O1霍乱弧菌。本研究结果揭示了蛭弧菌作为生物消除剂在预防由非O1霍乱弧菌所引起的腹泻疾病方面具有潜在应用价值。     

蛭弧菌的分离及消除沙门氏菌的初步研究

蛭弧菌具有裂解病原菌的功效,可以用作生物净化因子.本实验从海洋环境中分离到1株蛭弧菌(BDK-1).利用蛭弧菌BDK-1进行消除鸡蛋表面的沙门氏菌的人工模拟实验,结果表明,蛭弧菌BDK-1在其浓度为104～1010PFU/mL时,可有效消除鸡蛋表面的沙门氏菌,显示了蛭弧菌在食品卫生安全应用方面的潜在价值.     

蛭弧菌的生长特性及其培养条件的优化

为了将从海洋环境中分离得到的4株蛭弧茵发酵制备为微生态制剂,应用于水产食品安全生产,本实验研究了不同条件对蛭弧茵生长的影响.结果表明,4株蛭弧茵的最适pH为7.1～7.4;最适NaCl浓度为2.5%～3%;最适温度为30℃;二价金属离子Ca2+浓度的提高可有效增强蛭弧茵的生长及裂解能力;4株蛭弧菌对青霉素、土霉素、氯霉素和诺氟沙星均敏感;有机污染(苯酚和尿素)和重金属离子(Pb2+)污染对蛭弧菌的生长繁殖能力均有显著影响.并在单因素实验的基础上,运用响应面分析法确定了BDZ-100的最佳生长条件为:宿主菌浓度1xlO10 CFU/mL,,摇床速度250 r/min,培养液pH7,温度31℃,Nacl浓度3.1%,Ca2+浓度8.5mmol/L.从而为蛭弧菌微生态制剂的发酵制备和应用提供理论基础.     

天津主要河流中耐高温蛭弧菌的分离及生物学特性

从天津4条主要河流(独流减河,南运河,,北运河,子牙河)中分离出了耐高温的蛭弧菌并进行了纯化.通过采用宿主双层琼脂平板法对从河水中筛选出耐高温菌株并对其进行宿主范围、pH、温度等生物学特性的测定,结果表明:耐高温蛭弧菌在pH 7.0～7.5生长最好;对热致死的大肠杆菌,嗜水气单胞菌,荧光假单胞菌等3种细菌仍有裂解作用且...     

霍乱弧菌噬菌体在消除海产品中相关弧菌的潜在应用研究

噬菌体具有裂解病菌的作用,可用作生物净化因子.本实验以霍乱弧菌为宿主菌,采用双层平板法从鲍鱼养殖环境中分离到5株霍乱弧茵噬菌体.以23株弧菌为宿主菌,本文研究了霍乱弧菌噬菌体对海产品中常见弧茵的裂解消除能力,同时针对宽裂解谱噬菌体进行包括热失活、pH稳定性、紫外照射、不同镁离子浓度、不同柠檬酸钠浓度等理化因素对其裂解能力的影响,以探索其裂解消除弧茵的最优条件.实验结果表明噬菌体在消除霍乱弧菌方面有潜在的应用价值,紫外照射、柠檬酸钠对噬菌体裂解有不同程度的抑制作用,温度和pH值对噬茵体裂解也有不同程度的影响,而适度的镁离子则有促进作用.     

2006-2008年广东省水产品和食物中毒患者副溶血性弧菌分离株血清分型研究

目的研究2006-2008年广东地区副溶血性弧菌食物中毒患者分离株和水产品分离株的血清型分布。方法采用血清玻片凝集试验对2006-2008年我中心收集的279株副溶血性弧菌进行血清分型,这些菌株均由广东省食品污染物监测网和省食源性致病菌监测网分离获得,分离地区覆盖广东的粤西、粤北、粤东和珠三角地区,其中食物中毒患者分离株176株,水产品分离株103株。结果2006-2008年收集的279株副溶血性弧菌中,在176株食物中毒患者分离株中完全分型168株,分型率为95.45%,共分出15个血清型,以O3:K6为主,占73.30%(129/176);在103株水产品分离株中完全分型57株,分型率为55.34%,其血清型别分布较分散,未见优势菌型,共分出40个血清型,主要集中在O1,O2,O3,O4,O5,O10,O11等7个菌群。结论2006-2008年广东各地区上送的副溶血性弧菌食物中毒患者分离株以O3:K6为主,血清型地区差异不明显;水产品分离株血清型呈多样性,且与食物中毒患者分离株的血清型不一致。     

上海市闵行地区副溶血性弧菌分子流行病学特征分析

目的 分析上海市闵行地区副溶血性弧菌临床和食源性分离株的分子流行病学特征,了解本地区副溶血性弧菌的流行规律.方法 对2007-2010年临床来源菌株(食物中毒患者和散发腹泻病例)以及食源性样品中分离的184株副溶血性弧菌进行血清分型、耐热直接溶血素基因(tdh)和耐热相关溶血素基因(trh)检测.对102株临床来源O3:K6型菌株及41株食源性样品来源株,应用脉冲场凝胶电泳(PFGE)进行分子分型和溯源分析.结果 在调查的52起副溶血性弧菌引起的食物中毒事件中,tdh阳性、trh阴性的O3:K6血清型引起的有46起(88.5％),tdh阳性、trh阴性的O4:K8血清型引起的有5起(9.6％).在散发腹泻病例中,tdh阳性、trh阴性的O3:K6、O4:K8和O3:KUT菌株分别占60.3％(38/63)、19.0％(12/63)和15.9％(10/63).41株食源性样品株分属9种O血清群,未见O3:K6和O4:K8血清型菌株,而且tdh和trh均为阴性.PFGE聚类分析显示闵行地区食物中毒O3:K6分离株中存在着遗传关系密切相关的优势流行克隆,散发腹泻患者O3:K6分离株中存在着与食物中毒优势株相同谱型,而所有食源性样品分离株与临床患者来源株亲缘关系都较远.结论 临床和食源性样品来源的副溶血性弧菌在血清分型、毒力基因和分子特征等方面存在显著差异,一大群遗传关系密切、tdh阳性、trh阴性的O3:K6血清型菌株在闵行地区呈优势流行.     

两种弧菌显色培养基在疑似副溶血性弧菌鉴定中的效果比较

为了比较环凯和科玛嘉弧菌显色培养基对食品中可疑菌株的初步鉴定作用,本文按国标(GB/T4789.7-2008)程序,以环凯和科玛嘉弧菌显色培养基初步鉴定硫代硫酸盐-柠檬酸盐-胆盐-蔗糖琼脂(TCBS)的副溶血性弧菌可疑菌株,最后用API和MID鉴定证实。在97份食品样品的定性与定量检测中,TCBS疑似菌株有109株,对应样品有31份。环凯和科玛嘉弧菌显色培养基对109株TCBS可疑菌株初步鉴定的结果相同,均检出64株显紫色的可疑菌株,对应样品22份。经API和MID确认,阳性菌株60株,阳性样品为22份,阳性率为22.68%。两种培养基初步鉴定TCBS可疑菌株的敏感性、特异性和符合性均分别为100.00%、91.84%和96.33%。环凯和科玛嘉弧菌显色培养基对TCBS的可疑菌株具有较好的初步鉴定效果。     

Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction-Universal Array approach

Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumption were successfully screened, thus demonstrating that the developed microarray-based platform could be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood.     

海产品中溶藻弧菌的分离及多种方法鉴定效果的比较

目的了解北京市市售海产品中溶藻弧菌的污染情况,并对使用不同方法鉴定溶藻弧菌的效果进行比较。方法样品经碱性蛋白胨水增菌后,分别于硫代硫酸盐柠檬酸盐胆盐蔗糖(TCBS)琼脂培养基和科玛嘉弧菌显色培养基上划线培养,实时荧光聚合酶链式反应(PCR)法鉴定可疑菌落。以rpoB基因测序方法为参考,比较了实时荧光PCR和VITEK两种方法的鉴定效果。结果对北京市水产品市场随机采集的116份海产品进行了检测,实时荧光PCR法鉴定出溶藻弧菌阳性样品95份,检出率高达82%。使用科玛嘉弧菌显色培养基的检出率高于TCBS培养基,分别为82%(95/116)和72%(83/116)。经rpoB基因核苷酸序列测定确定95株疑似菌株为溶藻弧菌。采用VITEK 2 COMPACT GN鉴定卡对这95株菌株进行鉴定,31株鉴定为溶藻弧菌,其余未能得到准确的鉴定结果。结论北京市市售海产品中溶藻弧菌检出率高。科玛嘉弧菌显色培养基比TCBS琼脂培养基更适于溶藻弧菌的分离,实时荧光PCR法的鉴定效果优于VITEK法。     

Occurrence and expression of virulence-related properties of Vibrio species isolated from widely consumed seafood products

In this study, widely consumed fresh seafood products were examined for the presence of Vibrio spp. Thirteen percent of the samples examined were found to be contaminated with halophilic vibrios belonging to the species V. alginolyticus (81.48%), V. parahaemolyticus (14.8%) and V. cholerae non 0:1 (3.7%). A greater isolation frequency (18.9%) was found for mussels. Significant adhesiveness and strong cytotoxicity factors were revealed in a significant number of the Vibrio spp. isolated. These results confirm that the presence of Vibrio spp. in seafood products is common, and suggest that routine examination of such products for these pathogenic agents would be advisable.     

中国部分水产品副溶血性弧菌毒力基因的分布特征

目的了解中国部分水产品中副溶血性弧菌毒力基因的分布情况。方法通过聚合酶链反应测定从中国部分水产品中分离的192株副溶血性弧菌是否携带毒力基因tdh、trh,及种特异性基因tlh、toxR。结果192株实验菌株tdh全阴性,4株trh阳性,毒力基因携带率2.08%;而tlh、toxR的携带率均为100%。结论中国副溶血性弧菌水产品分离株毒力基因携带率非常低。     

Prevalence of potentially pathogenic Vibrio species in the seafood marketed in Malaysia

Seafood samples obtained in seafood markets and supermarkets at 11 sites selected from four states in Malaysia were examined for the presence of nine potentially pathogenic species from the genus Vibrio between July 1998 and June 1999. We examined 768 sample sets that included shrimp, squid, crab, cockles, and mussels. We extensively examined shrimp samples from Selangor State to determine seasonal variation of Vibrio populations. Eight potentially pathogenic Vibrio species were detected, with overall incidence in the samples at 4.6% for V. cholerae, 4.7% for V. parahaemolyticus, 6.0% for V. vulnificus, 11% for V. alginolyticus, 9.9% for V. metschnikovii, 1.3% for V. mimicus, 13% for V. damsela, 7.6% for V. fluvialis, and 52% for a combined population of all of the above. As many as eight Vibrio species were detected in shrimp and only four in squid and peel mussels. The overall percent incidence of any of the eight vibrios was highest (82%) in cockles (Anadara granosa) among the seafoods examined and was highest (100%) in Kuching, Sarawak State, and lowest (25%) in Penang, Pulau Penang State, among the sampling sites. Of 97 strains of V. cholerae isolated, one strain belonged to the O1 serotype and 14 to the O139 serotype. The results indicate that the various seafood markets in Malaysia are contaminated with potentially pathogenic Vibrio species regardless of the season and suggest that there is a need for adequate consumer protection measures.     

Biological characterization of two marine Bdellovibrio-and-like organisms isolated from Daya bay of Shenzhen, China and their application in the elimination of Vibrio parahaemolyticus in oyster

Bdellovibrio-and-like organisms (BALOs) are a group of highly motile delta-proteobacteria that prey on other gram-negative bacteria. However, nothing is known of the application potential of marine BALOs in safeguarding seafood safety. Here, biological characterization of two marine BALOs strains and their application in the elimination of Vibrio parahaemolyticus in oyster (Crassostrea ariakensis) at the laboratory scale were investigated.BALOs strains BDH12 and BDHSH06 were isolated from sediment of Daya bay in Shenzhen of China, with Shewanella putrefaciens strain 12 and V. parahaemolyticus strain SH06 as preys, respectively, when using double layer agar technique. They were identified as BALOs morphologically by transmission electron microscopy, while partial 16S rDNA sequencing analysis revealed that they showed no close relationships with members of the known genera Bdellovibrio, Bacteriolyticum, Bacteriovorax, or Peredibacter.Biological characterizations revealed that both strains had the optimal pH, salinity and temperature at 7.2, 3% and 30 °C, correspondingly. They could not utilize autoclaved, dead cells as hosts. Prey range analysis revealed that individually, BDH12 and BDHSH06 lysed 82.5% (47 strains) and 84.2% (48 strains) of the total 57 preys tested respectively. In combination, they lysed 98.2% (56 of 57) strains. All strains of V. parahaemolyticus, Vibrio cholerae and Vibrio alginolyticus tested could be lysed by both strains.A 7-day laboratory-scale V. parahaemolyticus elimination experiment in oyster showed that in the control, the cell counts of total vibrios and V. parahaemolyticus strain Vp plus in water and in oyster intestines were on the rise, whereas in the BALOs treated groups, their numbers were down from 8.09 ± 0.05 log CFU/ml and 8.02 ± 0.04 log CFU/ml to 2.39 ± 0.01 log CFU/ml and 2.33 ± 0.01 log CFU/ml, respectively. The same patterns could also be observed in oyster intestines. Results of this study indicate the feasibility of using BALOs to biologically control or even eliminate V. parahaemolyticus in seafood oyster.     

2016年北京市海产品中创伤弧菌的污染调查及两种检测方法比较

目的 对北京市市售海产品的创伤弧菌污染情况进行调查,并比较实时荧光聚合酶链式反应(RT-PCR)法与VITEK鉴定方法检测结果的一致性。方法 采用传统检验方法结合分子生物学方法对在北京市市场随机采集的105份海产品进行创伤弧菌检验,并比较了RT-PCR法和VITEK鉴定方法的准确性。结果 105份海产品中,有40份样品检出创伤弧菌,检出率为38.10%;其中,虾类产品检出率高达52.38%(11/21),其次为贝类产品(37.88%,25/66)和鱼类产品(22.22%,4/18)。经rpoB基因测序验证,RT-PCR和VITEK方法的准确率分别为100.00%(40/40)和67.50%(27/40)。结论 北京市海产品中存在创伤弧菌的污染,应对海产品中创伤弧菌引起食源性污染的潜在风险进行评估,预防食物中毒的发生。     

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.