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。     

浙南沿海地区人群霍乱弧菌感染及外环境污染调查

目的了解浙南沿海地区腹泻病例霍乱弧菌感染和外环境霍乱弧菌污染情况,为防控霍乱传播流行提供科学依据。方法通过现场采样和标本分离培养方法,对温州市所属沿海地区医院肠道门诊腹泻病例及其环境标本进行霍乱弧菌分布调查。结果 2010-2013年度从温州市104个肠道门诊45 921份病人标本中分离出5份霍乱弧菌阳性,感染率为0.01%。从210份与霍乱病人密切接触者标本中分离出2例霍乱弧菌阳性,阳性率为0.95%。从9 497份水体和食品标本中分离出4例霍乱弧菌阳性,阳性率0.04%,分离出的霍乱弧菌有O139群、O1群稻叶型、O1群小川型等。结论浙南沿海地区为霍乱老疫区,人群和外环境目前仍受霍乱弧菌感染潜在威胁,应加强肠道门诊早期诊断,采取积极防控措施。     

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。     

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。     

一起霍乱暴发疫情的采样与实验室检测分析

目的通过对聚餐引起的霍乱暴发疫情的实验室检测分析,讨论采样对实验室结果的影响。方法霍乱弧菌的培养、血清学鉴定、生化反应按照《霍乱防治手册》第5版进行。结果 7份肛拭子或粪便中1份分离出O139群霍乱弧菌,4份食品中1份分离出O139群霍乱弧菌,1份水标本未分离出O139群霍乱弧菌。结论由聚餐引起的霍乱暴发疫情,采样应由经过专业培训的人员参加,应早期、无菌、适量、安全采集,由专门人员送到实验室以确保检测结果准确,为现场提供有力的支撑。     

浙江省海盐县一起聚餐引起的霍乱疫情调查

目的　通过对海盐县霍乱疫情的调查处置， 分析疫情原因， 控制疫情蔓延。 方法　根据GB 15984-1995标准培养鉴定霍乱弧菌；对患者、带菌者、聚餐人员、密切接触者、周围人群、外环境全面开展流行病学调查，对聚餐食物危险因素调查分析，并采取有效的疫情控制措施。 结果　共发病1 例，健康带菌者9例，为小川型霍乱弧菌，噬菌体-生物分型为15f，毒力基因ctx为阳性。结论　本次是一起农村聚餐引起的霍乱疫情，聚餐人员感染的霍乱弧菌具有产生霍乱肠毒素的能力，是一种少见的非流行株，聚餐时所用的白鸡在加工、 烹调过程中交叉污染而引发。     

山西省从粪便中分离到产霍乱毒素的拟态弧菌

目的从山西省霍乱样粪便中分离到拟态弧菌,并检测其是否携带霍乱毒素基因。方法采集患者粪便、患者家用自来水、金鱼和鱼缸水标本,按照WS289-2008和《霍乱防治手册》(第5版)方法 ,对标本进行增菌和选择性培养基的分离培养,疑似菌落以霍乱弧菌血清凝集试验检测,并用API20E系统鉴定菌种。同时,疑似菌落用PCR检测O1、O139群特异性和是否携带霍乱毒素基因。结果粪便标本经增菌后接种选择性培养基,在庆大琼脂和碱性琼脂上均有霍乱弧菌疑似菌落,但在TCBS琼脂上为绿色、透明、中等大小、光滑、湿润的菌落;血平板上可见灰白色、湿润菌落,有透明溶血环;染色镜检湿片暗视野下未见流星状运动细菌。氧化酶和粘丝实验阳性,霍乱弧菌O1、O139群血清凝集试验阴性。菌株经API20E鉴定为拟态弧菌(93.5%可能性)。环境标本中未分离和检测到拟态弧菌。PCR检测该拟态弧菌中携带霍乱毒素基因,而O1、O139群特异性为阴性。结论从急性霍乱样腹泻粪便中分离到1株拟态弧菌,对于临床类似霍乱样症状的病例标本,当病原的霍乱弧菌血清凝集为阴性时,应进一步做霍乱毒素的检测。     

一起食源性霍乱暴发疫情的菌型鉴定及药敏分析

目的对霍乱暴发疫情进行霍乱弧菌菌型鉴定及药敏分析,为霍乱的防控工作提供参考。方法采集患者、密切接触者和外环境标本进行菌型鉴定,并对菌株进行临床常用的20种抗菌药物进行药敏分析。结果共采集45份标本,其中9份患者肛拭子和1份外环境拭子检出O1群ElTor霍乱弧菌稻叶型1d,密切接触者标本中未检出霍乱弧菌;10株霍乱弧菌对20种药物的敏感度完全一致,其中100%耐药的抗生素只有复方新诺明1种,敏感的抗生素有菌必治、庆大霉素等11种,中敏的抗生素有氨苄西林等8种。结论引起本次食源性霍乱暴发疫情的霍乱弧菌菌型是O1群ElTor霍乱弧菌稻叶型1d;菌必治、庆大霉素等可为抗菌治疗和预防性服药的首选药物,而复方新诺明则不应被选用。     

某地霍乱防治检测资料分析

目的本文就标本采集、增菌时间控制、分离培养、抗菌药物、培养基的选择等影响霍乱弧菌检出的因素和对今后疫区监测的实验室工作的一些认识加以介绍、分析、讨论,以期对霍乱防治检测工作有所裨益。方法用常规分离培养方法对疫区内采集的254例粪便标本和16例环境水样进行检测。结果所有标本均分离出01群小川型霍乱弧菌。结论有利于及时、准确地检出霍乱弧菌,提高检出率,对控制霍乱流行有重要意义。     

霍乱弧菌越冬模拟标本分离培养基的研制

本文报道了以霍乱弧菌越冬机理研究为目的，进行霍乱弧菌实验性越冬体分离培养基的研制。秋冬季在外环境水体中的霍乱弧菌由于受到寒冷等恶劣刺激,菌体发生改变。这时很难应用常规分离方法培养成功，给霍乱病原监测带来困难。本试验从激活酶系统，提高酶活性，改善代谢水平，补充与酶活性有关的辅酶和辅机以及加入生长刺激物质，维持高渗环境的方法分离培养取得成功。使肌吐高渗多胨琼脂培基的分离率达53-73%，而对照的庆大霉素琼脂培养基仅13%。     

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.     

Vancomycin resistance is maintained in enterococci in the viable but nonculturable state and after division is resumed

Stressed vancomycin-resistant enterococci (VRE) can activate a survival strategy known as the viable but nonculturable (VBNC) state and are able to maintain vancomycin resistance. During restoration of division they continue to express the vancomycin resistance trait. We suggest that VBNC enterococci may constitute further reservoirs of VRE and therefore represent an additional risk for human health.     

Contradictory responses in induction of delayed type hypersensitivity in orally immunized mice

Induction of delayed type hypersensitivity (DTH) in mice fed with sheep red blood cells (SRBC) and live Vibrio cholerae (V. cholerae) both forcedly and ad lib was comparatively investigated. Although suppression of DTH to SRBC or V. cholerae was induced in mice fed forcedly for 1 or 2 weeks, mice fed ad lib could produce the positive footpad reactions to antigens. Furthermore, the suppression of DTH in forcedly fed mice showed an antigenic specificity. These observations indicated that the induction of unresponsiveness to DTH in orally immunized mice was markedly influenced by the oral administration.     

GM1-dot-EIA for the detection of toxin-producing Vibrio cholerae strains

A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.     

产色素O139群霍乱弧菌环境分离株的亲缘关系分析

目的 筛检霍乱弧菌O139血清群产褐色色素的环境分离菌株并分析其遗传近缘关系。 方法 在LBA平板上检测了1992-2010年分离保存的全国1530株O139群霍乱弧菌的色素产生能力,对其中能够产生水溶性褐色色素的菌株的马尿酸氧化酶基因VC1345进行测序,并用脉冲场凝胶电泳(PFGE)、多基因序列分型分析色素产生菌株之间的亲缘关系。 结果 筛选到不同年份不同地区的16株产色素的非产毒O139群菌株。所有的色素产生菌株具有完全一致的VC1345基因、相似的PFGE带型,多基因序列分型显示仅有2种型别。 结论 分离到的O139群产色素菌株来源于近缘的克隆群,而这些菌株之间已存在一定程度的变异。在水体中的持续存在显示其较强的环境生存能力,这些特征提示在霍乱弧菌的环境监测中需提高对这类菌株的关注。     

The use of micromethods for the identification of Vibrios]

The authors recommend micromethods for laboratory studies of Vibrio; such methods may be widely used at bacteriologic laboratories for examinations of biochemical characteristics of these microorganisms, for rapid identification of V. cholerae 01, and for serologic identification (typing) of V. cholerae non 01, since they accelerate the diagnosis and are much simpler than macromethods.     

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.     

Identification of Vibrio mimicus bacteriophages

Lysogeny was studied in Vibrio mimicus; the indicator V. cholerae El Tor strain was selected to identify phages. New V. mimicus phages were obtained and identified, which had a morphological similarity and an antigen affinity for morphological group I cholerae phages. Phage differentiation revealed that morphological group I V. mimicus phages showed certain differences manifested as their lytic activity against V. cholerae strain 1322-69 of serovar 37 while this property was absent in cholerae phages.