霍乱弧菌的环境生存及其监测

作为霍乱病原体的霍乱弧菌,是自然水体中的正常菌群。本文介绍了霍乱弧菌的环境生存、水体监测方法及霍乱流行的影响因素。     

Transferable Quinolone Resistance in Vibrio cholerae

Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of resistance to other agents, but its utility has been compromised by the decreasing ciprofloxacin susceptibility of Vibrio cholerae over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in gyrA, which encodes a subunit of DNA gyrase, followed by mutation in parC, which encodes a subunit of DNA topoisomerase IV. We found that ciprofloxacin activity was more recently further compromised in strains containing qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that qnrVC3 confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae and on the chromosomes of Escherichia coli transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in V. cholerae are linked to cumulative mutations in quinolone targets and most recently to a qnr gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.Cholera remains a major public health problem in many areas of the developing world. In addition to maintenance oral rehydration therapy, adjunctive antimicrobial therapy reduces the extent and duration of diarrhea, resulting in reduced fluid requirements and hospitalizations, reductions that are particularly important in resource-limited areas. Antimicrobial therapies have included tetracycline, azithromycin, and fluoroquinolones, such as ciprofloxacin, but the activity of fluoroquinolones has decreased in some areas, and this decreased activity has been associated with substantial reductions in the efficacy of ciprofloxacin relative to that of azithromycin (15, 20). To evaluate the evolution of ciprofloxacin resistance in Vibrio cholerae, we studied isolates from Bangladesh available over a 6-year period in which poor clinical responses of cholera patients to ciprofloxacin were recognized. We determined the presence of resistance mutations in genes encoding the subunits of the quinolone target enzymes DNA gyrase (gyrA and gyrB) and DNA topoisomerase IV (parC and parE) and the presence of qnr and other acquired genes that confer additional resistance to quinolones (25). Some qnr gene products have been shown to protect gyrase and topoisomerase IV from quinolone action in enteric bacteria (26, 27). qnr genes are usually located on mobile genetic elements, such as plasmids, that can transfer between strains but have been found on the chromosomes of some Vibrio spp. (5, 18). In V. cholerae, a qnr homolog, qnrVC1, has been described for isolates from Brazil (9) but has not been shown to confer transferable quinolone resistance or to be linked to incremental quinolone resistance and poor response to ciprofloxacin therapy of cholera. We show here that progressively higher levels of resistance in V. cholerae in Bangladesh were driven by accumulating mutations in topoisomerase target enzymes and by the acquisition of a quinolone resistance determinant, qnrVC3, which we identified as part of an SXT integrating conjugative element (4, 10) that also carried genes conferring resistance to tetracycline, trimethoprim-sulfamethoxazole, and streptomycin and accounted for transferable multidrug resistance that included ciprofloxacin in isolates positive for qnrVC3.     

Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

A multiplex PCR assay was developed based on atpA-sequence diversification for molecular identification of 3 major pathogenic Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. It specifically identified them from among 133 strains of various Vibrio species and other genera, and was applicable for testing seawater, suggesting its usefulness.     

一起输入性霍乱弧菌的实验室检测

目的分离培养患者粪便标本中的霍乱弧菌并检测其毒力基因及药物敏感性。 方法常规方法分离菌株，根据其生化及血清学反应结果鉴定菌株，聚合酶链反应 (PCR) 检测毒力基因IctxA、ace、tcpA、zot，/IK-B法测定药物敏感性。 结果 从患者粪便中分离到埃尔托生物型6f霍乱弧菌1株，带有4种毒力基因，对多种药物敏感，不产-内酰胺酶。 结论结果提示该株霍乱弧菌为流行株，对多种常用药物敏感，PCR可较快完成对霍乱弧菌毒力强弱的检测。     

霍乱弧菌的分子分型方法

在霍乱流行和暴发调查中,追溯传染源和调查传播途径往往需要对霍乱弧菌进行分型分析.对霍乱弧菌进行血清分型和噬菌体一生物分型,是得到菌株基本信息不可缺少的手段.如果想要揭示菌株在分子水平上的变异和进化规律,则需要进行分子分型分析.本研究对霍乱弧菌的各种分子分型方法进行逐一介绍并加以综合比较.     

Molecular epidemiological studies of Vibrio cholerae in Bengal region

Vibrio cholerae isolates from environmental and clinical origins in the Bengal region in which epidemics of cholera break out periodically were analyzed with particular emphasis on the molecular epidemiological features. The presence of the virulence genes (ctxA, tcpA and toxR) in the isolates was analyzed by the PCR (polymerase chain reaction) method. PFGE (pulsed-field gel electrophoresis) was performed to determine the clonal relationships between the clinical and environmental strains. Antibiograms and O serovars of the isolates were also examined. O1 and O139 strains from both clinical and environmental sources were all positive for the three virulence genes while non-O1/non-O139 strains from both sources were all negative for ctxA and tcpA but positive for toxR. PFGE patterns of recent isolates of O1 and O139 were similar in each serovar regardless of origin, suggesting a clonal relationship between the clinical and environmental strains, although comparison with past isolates or isolates from different geographical area showed some differences.     

Incidence and Elimination of R Plasmids in Vibrio cholerae

Of 124 strains of Vibrio cholerae, 32 were multiply resistant to antibiotics. This resistance appeared to be determined by R plasmids on the basis of their effective elimination by sodium dodecyl sulfate, acridine orange, ethidium bromide, and ultraviolet radiation.     

R Plasmids from Asian Strains of Vibrio cholerae

Five R plasmids transferred from Asian strains of Vibrio cholerae all proved to be members of compatibility group C. A non-self-transmissible plasmid, stable in V. cholerae, was mobilized for transfer to Escherichia coli K-12 and found to be unstably inherited in that host. Plasmids of group C and P transferred to a wild V. cholerae strain were stably inherited.     

霍乱弧菌密度感应系统研究进展

密度感应(Quorum sensing,QS)系统在细菌中普遍存在,细菌细胞通过识别和密度依赖性应答其自身或周围环境中其他菌细胞分泌的一种或多种信号分子来影响一系列基因的转录表达,从而参与细菌多种生理功能的调节。革兰阴性菌的密度感应现象最早发现于海洋弧菌中,本文主要综述了烈性肠道传染病病原体-霍乱弧菌中密度感应系统的研究进展,包括系统组成、信号传递过程、调节的生理功能及与其他全局性调控因子的相互作用。     

Vibrio cholerae O1 serotype Ogawa in a neonate

In India, cholera is endemic and affects usually the 3 to 5-year-old age group. There have been occasional reports in the neonatal period with Vibrio cholerae O139 Bengal. We report here a case of Vibrio cholerae O1 diarrhea in a 2-day-old, breastfed male, who had been delivered in the hospital and developed severe dehydration.     

活的非可培养状态霍乱弧菌研究进展

霍乱弧菌是引起霍乱的病原菌,并广泛存在于水生环境中。生存环境的恶劣,可以导致霍乱弧菌进入一种特殊的状态——活的非可培养(viable but nonculturable,VBNC)状态,使用常规的实验室检测技术不能检出这种特殊状态的霍乱弧菌,但其仍具有致病性,能够通过动物肠道复苏从而致病。本文从生物学特性、检测方法、致病性、转录组表达等方面详细介绍了霍乱弧菌VBNC状态,并阐述了霍乱弧菌VBNC状态对公共卫生安全的重要意义。     

Determination of the cytotoxic activity of Vibrio cholerae]

Cytotoxic activities of broth culture supernatants of 39 V. cholerae strains 01 and non 01 were studied in L-929 cell cultures. The examined strains differed by the cytotoxic factor production, which factors were identified (with the use of anti-cytolysin serum) as cytolysin. The strains capable of choleric enterotoxin secretion did not produce cytolysin.     

2004-2013年北京市外环境及食品中霍乱弧菌监测结果分析

目的 了解北京市外环境及食品中霍乱弧菌污染状况,为人间霍乱疫情防控提供科学依据。方法 2004-2013年,每月均在全市的16个区(县)采集地表水、游泳池水、养殖池水、水产品、熟食、其他等6类样品,分别进行霍乱弧菌培养,对培养结果应用描述流行病学的方法进行分析。结果 2004-2013年养殖池水和其他类别样品的霍乱阳性率最高,分别为1.16%(44/3808)和1.48%(61/4135);游泳池水中未检出霍乱弧菌;外环境疫情在北京市集中在城近郊区,呈环形分布;牛蛙和甲鱼检测阳性率较高,霍乱毒素(cholera toxin, CT)阳性疫情较少,占检测总起数的9.09%(5/55),且均为水产品涂抹疫情。2007年以来,霍乱外环境疫情较少,O139群霍乱弧菌污染呈下降趋势,O1群稻叶型则有所增多。结论 北京市霍乱弧菌污染较轻,且检出菌株也以CT阴性菌株为主,对人体威胁较小。     

An immunoenzyme method of detecting Vibrio cholerae cytolysin]

Enzyme immunoassay was used in detection of V. cholerae cytolysin. Conjugate of immunoglobulins to purified cytolysin with horseradish peroxidase was used, obtained by the periodate technique. The method sensitivity is 2 ng/ml of purified cytolysin. The results of hemolytic activity measurements in supernatants of 40 V. cholerae strains and enzyme immunoassay findings were in high correlation.     

Experimental non-O group 1 Vibrio cholerae gastroenteritis in humans.

In this study, 27 volunteers received one of three non-O group 1 Vibrio cholerae strains in doses as high as 10(9) CFU. Only one strain (strain C) caused diarrhea: this strain was able to colonize the gastrointestinal tract, and produced a heat-stable enterotoxin (NAG-ST). Diarrhea was not seen with a strain (strain A) that colonized the intestine but did not produce NAG-ST, nor with a strain (strain B) that produced NAG-ST but did not colonize. Persons receiving strain C had diarrhea and abdominal cramps. Diarrheal stool volumes ranged from 154 to 5,397 ml; stool samples from the patient having 5,397 ml of diarrhea were tested and found to contain NAG-ST. The median incubation period for illness was 10 h. There was a suggestion that occurrence of diarrhea was dependent on inoculum size. Immune responses to homologous outer membrane proteins, lipopolysaccharide, and whole-cell lysates were demonstrable with all three strains. Our data demonstrate that V. cholerae of O groups other than 1 are able to cause severe diarrheal disease. However, not all strains are pathogenic for humans: virulence of strain C may be dependent on its ability both to colonize the intestine and to produce a toxin such as NAG-ST.     

霍乱弧菌活的非可培养状态研究进展

霍乱弧菌是霍乱的病原体,广泛存在于海洋及江河入海口环境中。在不良环境中霍乱弧菌会进入活的非可培养状态(viable but non-culturable state, VBNC),常规分离方法检测不到,在条件合适时VBNC状态的霍乱弧菌会复苏为可培养状态。本文对近年霍乱弧菌VBNC相关研究做了回顾,以期能够为霍乱弧菌越冬机制以及霍乱的监测提供参考。     

Chemiosmotic mechanism of antimicrobial activity of Ag(+) in Vibrio cholerae

Although the antimicrobial effects of silver salts were noticed long ago, the molecular mechanism of the bactericidal action of Ag(+) in low concentrations has not been elucidated. Here, we show that low concentrations of Ag(+) induce a massive proton leakage through the Vibrio cholerae membrane, which results in complete deenergization and, with a high degree of probability, cell death.