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。     

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。     

细菌“活的非可培养状态”研究进展

细菌“活的非可培养状态”（Ｖｉａｂｌｅｂｕｔｎｏｎｃｕｌ－ｔｕｒａｂｌｅｓｔａｔｅ，ＶＢＮＣ）是细菌处于不良环境条件下，细胞缩成球形，用常规方法培养不能生长繁殖，但仍然是活的一种特殊存在形式。霍乱弧菌（Ｖ．ｃ．）广泛存在于河、湖及沿岸海水中，是水环境...     

1例白血病患者血液中检出非O1非O139群霍乱弧菌

2012年8月,浦东新区儿童医学中心从1名6岁急性淋巴细胞性白血病复发患儿血液中分离到霍乱弧菌,随后浦东新区疾病预防控制中心对该分离株进行了系统生化鉴定、毒力基因及药物敏感性检测,结果为不产霍乱毒素的非O1非O139霍乱弧菌。     

一起引起食物中毒的非O1群霍乱弧菌实验室鉴定

目的 查明引发浙江省宁波市某服饰公司食物中毒事件的病原菌.方法 对采集到可疑食品和肛拭等标本参照GB/T4789-2003标准进行细菌的分离、鉴定及血清学分型;参照霍乱防治手册检测菌株的CT毒力基因.结果 6份患者大便标本与14份轻微腹泻患者或腹部不适者大便标本中检出非O1群霍乱弧菌O29血清型9株,其中腹泻患者标本检出6株,轻微腹泻患者中检出3株,检出率为45.0%.未检出志贺菌、副溶血性弧菌、致病性大肠杆菌、O1群和O139霍乱弧菌、金黄色葡萄球菌等致病菌,剩余食物未检出志贺菌、副溶血性弧菌、致病性大肠杆菌、O1群和O139霍乱弧菌、金黄色葡萄球菌等致病菌.结论 此次食物中毒为非O1群霍乱弧菌O29血清型所致.     

非O1群霍乱弧菌感染腹泻的分布及耐药性分析

为了解本地区非Ｏ１群Ｖ．ｃｈｏｌｅｒａｅ（霍乱弧菌）感染腹泻的流行状况，对我院肠道门诊腹泻病患者的粪便标本，进行了连续两年致病性弧菌的监测，并对非Ｏ１群霍乱弧菌感染的流行病学因素，进行了综合分析，结果报告如下。１材料和方法１．１标本来源１９９８年至１９９９年急性腹泻病患者的粪便标本３１６５份；患者全部为１４岁以上的成年人，年龄从１４岁至８９岁；男性患者１６８５例，女性患者 １４８０例。１．２培养基增菌及分离培养基购自北京市东城区卫生防疫站，ＭＨ琼脂购自生物梅里埃公司。新鲜脱纤维羊血和兔血本实验室…     

非O1群霍乱弧菌的鉴定及耐药性分析

对本院１９９９年６～９月在肠道门诊检出的２０株非Ｏ１群霍乱弧菌进行生化鉴定,血清学分群和药敏试验,根据１９９８年美国ＮＣＣＬＳＡ药敏法规中新增的霍乱弧菌抗生素造反和判别标准＾「２」,所选用抗生素已能满足临床治疗的需要,药敏结果揭示：抗Ｏ／１２９的非Ｏ１群霍乱弧菌对抗生素的敏感性比Ｏ／１２９敏感的非Ｏ１群霍乱弧菌有明显下降。     

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。     

河弧菌研究概况

河弧菌(Vibrio fluvialis)被认为是新发的食源性致病菌,能够引起腹泻暴发流行,对全球公共卫生安全造成潜在危险,尤其在发展中国家的沿海地区和卫生条件差的区域。经过30多年的研究和探索,人们对河弧菌及其致病性和感染的流行病学特征有了一定的了解和认识,本研究就河弧菌的发现、病原学特征,致病性、流行病学特征,临床表现等相关研究进展做一综述。     

Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays

Viable bacteria were sought in 44 Maki-negative biofilms from central venous catheters (CVCs) using epifluorescence microscopy after live/dead staining. Thirty (77%) samples contained viable but non-culturable (VBNC) cells; the majority were positive on real-time PCR specific for Staphylococcus epidermidis (one also for Staphylococcus aureus). Viable cells were significantly (p<0.01) associated with CVCs from febrile patients, three of whom showed S. epidermidis-positive blood cultures, suggesting that CVC-associated biofilms can be reservoirs for staphylococci in the VBNC state. The possible role of VBNC staphylococci in persistent infections related to medical devices requires further investigation.     

Isolation ssDNA aptamers specific for both live and viable but nonculturable state Vibrio vulnificus using whole bacteria-SEILEX technology

Vibrio vulnificus is a ubiquitous marine bacterium that may cause rapid and deadly infection, threatening lives of people living around natural bodies of water, especially in coastal regions. However, traditional culture-based methods are time-consuming and unable to detect Viable But Non-Culturable (VBNC) V. vulnificus cells. In this work, we isolated a batch of detection aptamers specifically binding to V. vulnificus in all culture status. With traditional whole bacteria-SELEX (Systematic Evolution of Ligands by EXponential enrichment), flow cytometer analysis and imaging, we identify 18 candidates and validated two of them (V8 and V13) as applicable aptamers. Their truncated sequences also showed comparable performance. The dissociation constant (KD) value of V8 is shown to be as low as 11.22 ± 1.32 nM. Optimal aptamers V8 and V13 are also validated to be effective to detect different Vibrio vulnificus strains under different binding environments using flow cytometry. As for detection parameters, the LOD of the V8 from cytometry is 29.96 CFU mL⁻¹, and the linear range is 10²–5 × 10⁵ CFU mL⁻¹. This is the first case demonstrating that aptamers can detect the existence of VBNC bacteria as well as live bacteria.

With whole-bacteria SELEX, we got aptamers that can bind to V. vulnificus in VBNC Status for the first time.     

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.     

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.