空肠弯曲菌在环境压力条件下的生存机制和可培养性研究进展

空肠弯曲菌是一种重要的革兰氏阴性致病菌,可以产生几种肠毒素能侵袭小肠和大肠黏膜引起急性肠炎,据统计其导致肠炎比例是沙门氏菌的3倍。该菌污染肉类食品后,经冷冻等环境的变化会进入活的非可培养状态(viable but non-culturable state,VBNC),不能用常规方法检测到。空肠弯曲菌在pH值、湿度、温度、营养成分等条件发生极端改变的情况下可以进入VBNC状态。本研究更深入地了解空肠弯曲菌是如何抵御外力影响的,防范食品安全的潜在危害。     

细菌活的非可培养状态研究

综述了细菌活的非可培养状态的导致原因、生物学特性、检测及复苏方法,通过对活的非可培养状态的研究,使相关研究人员更加深刻的了解和认识活的非可培养状态下的细菌特性,从而对非可培养状态病原菌的防控起到积极的作用。通过细菌VBNC状态的综述进而对益生乳酸菌VBNC状态的研究提出新的思路,以解决乳酸菌发酵剂工业化生产中所面临的问题。     

金黄色葡萄球菌活的非可培养状态复苏及PMA-qPCR检测

为研究金黄色葡萄球菌活的非可培养（VBNC）状态的复苏问题,采取温度、盐度和酸度3个因素复合诱导制备VBNC菌,尝试四种复苏方法,并以荧光定量PCR和PMA-qPCR技术结合的方法进行检测。结果证明：8%无菌Tween80可以使VBNC状态金黄色葡萄球菌48 h后复苏,同时PMA-qPCR能够有效检出VBNC状态金黄色葡萄球菌,克服传统平板计数法对于VBNC菌的漏检。     

细菌的活的非可培养状态及其研究进展

细菌的活的非可培养状态是指细菌具有生物活性和毒性,但用常规培养法检测不出来的一种特殊生理状态。由于这种状态在特定条件下可以复苏,将培养出来和致病,所以研究细菌的活的非可培养状态对正确地评价食品的卫生状况,改进检验方法具有重要的意义。本文介绍了细菌进入活的非可培养状态的各种诱导因素;活的非可培养状态细菌的生理特征;已研究的能进入活的非可培养状态的细菌种类;细菌的活的非可培养状态的检测方法;最后阐述了进行细菌的活的非可培养状态研究的重要意义。     

PCR技术检测空肠弯曲菌的研究进展

空肠弯曲菌(Campylobacter jejuni)是一种人畜共患病病原菌,可以使人和动物引发多种疾病。目前,检测C.jejuni采用的国标方法是传统的培养法,但C.jejuni培养条件苛刻,且培养法存在操作繁琐、特异性不强、费时等缺点。聚合酶链式反应(polymerase chain reaction,PCR)以其快速、准确、灵敏度高、特异性强的特点,现已广泛应用于C.jejuni的检测,并成为目前快速检测临床与食品中C.jejuni最常用的的方法。本文综述了近年来利用RCR技术,包括常规PCR、多重PCR、巢式PCR、实时荧光定量PCR、PCR-酶联免疫吸附法、最大几率数-PCR、PCR-限制性片段长度多态性、PCR-变性高效液相色谱、PCR-变性梯度凝胶电泳和磁捕获-荧光PCR方法检测C.jejuni的研究进展,并针对这些PCR技术的原理、检测效果、优点和缺点等方面进行了分析比较,为有效控制和预防该菌引起的疾病提供重要信息。     

细菌活的非可培养状态的分子生物学检测技术研究进展

许多细菌在不良环境下能进入活的非可培养状态(viable but nonculturable state,VBNC)。细菌培养技术常常造成VBNC状态的细菌漏检,应用分子生物学检测技术可以提高VBNC细胞的阳性检出率。针对VBNC细胞的分子生物学检测技术,基于细菌特异性DNA分子、mRNA分子是目前常用的检测方法,而利用报告基因(如绿色荧光蛋白基因)检测VBNC细胞是一种有效的方法。最近利用叠氮溴乙锭 (ethidium monoazide bromide,EMA)或者叠氮溴化丙锭 (propidium monoazide,PMA)联合PCR技术选择性抑制细菌死细胞扩增,该方法结合了EMA(PMA)选择渗透性和PCR特异性,作为一种区分细胞死活的方法,可以有效检测细菌VBNC细胞。     

空肠弯曲菌快速检测试剂盒研制及AOAC验证

本文自主研制出含内标的空肠弯曲菌实时荧光PCR快速检测试剂盒,并根据美国分析化学家协会(AOAC)微生物方法对试剂盒进行验证。结果表明,该试剂盒对115株目标菌和118株非目标菌的检测特异性为100%,灵敏度达6.4 CFU/mL。将试剂盒反复冻融30次,目的基因的Ct值无显著变化。稳健性测试结果显示,除了一株空肠弯曲菌对增菌液体积较为敏感外,增菌液体积、菌体裂解时间和DNA添加量均不影响其余10株空肠弯曲菌和5株同属的其它弯曲菌的检测结果。采用生产3 d、生产4个月和1年的3个批次试剂盒进行批间重复性测试和保质期测试,结果显示3批试剂盒对16株弯曲菌的检测结果一致,且11株空肠弯曲菌的Ct值无明显差异。对人工添加鸡肉、蔬菜和牛奶样品以及鸡肉实际样品检测结果显示,试剂盒方法与传统检测方法基本一致,未出现"假阴性"。试剂盒开放性好,可在不同品牌仪器上使用,含有的内部扩增对照可防止"假阴性",可应用于食品中空肠弯曲菌的快速检测。     

细菌活的非可培养状态的研究现状及发展趋势

微生物是危害食品质量安全的重要因素。细菌活的非可培养状态的概念提出,对于微生物学、食品质量安全、水质管理、流行病学等方面,都有重大意义。对细菌活的非可培养的研究现状及发展趋势进行了综述。     

Fla-DGGE法对食品中空肠弯曲菌和结肠弯曲菌的检测和分型

本文应用鞭毛蛋白基因flaA和flaB的变性梯度凝胶电脉(denaturing gradient gel electrophoresis,DGGE)方法对食品中空肠弯曲菌和结肠弯曲菌进行检测和分型。采用RBB(rpeated bead beating)、CTAB和丙酮-氯仿抽提三种方法提取样品基因组后进行fla-DGGE检测,结果完全一致。10份生鲜鸡、鸭肉样品中有8份含有空肠弯曲菌或结肠弯曲菌,其中3份含有两种或两种以上的空肠弯曲菌或结肠弯曲菌或它们的不同型别。克隆测序后结果表明,有7份样品被同一种空肠弯曲菌所污染,其中3份样品还被同一种结肠弯曲菌所污染,1份样品被污染情况严重,检测到含有一种结肠弯曲菌和三种空肠弯曲菌。Fla-DGGE方法快速、准确、灵敏度高,可用于食品中空肠弯曲菌和结肠弯曲菌的快速检测和分型。     

金黄色葡萄球菌活的非可培养状态的诱导和复苏

研究金黄色葡萄球菌活的非可培养状态(viable but non-culturable state,VBNC)的诱导和复苏方法。采用不同pH、温度、盐度、不同浓度的食品防腐剂山梨酸钾处理进行诱导;复苏则采用了直接升温处理、添加液体培养基升温处理、添加酵母液升温处理、添加吐温升温处理的方法。结果表明,经过不同温度和山梨酸钾诱导后的金黄色葡萄球菌进入了VBNC状态;其中诱导时间最短(76d)的条件是0.5mmol/L山梨酸钾、-20℃寡营养条件;采用BHI培养基升温的方法,以及添加0.5%和1%的吐温20和吐温80的培养液升温方法使进入VBNC状态的金黄色葡萄球菌得到复苏。VBNC状态的金黄色葡萄球菌在形态上发生了变化;复苏后的菌株除尿素酶阴性外,其他代谢特征均与标准菌株相同。     

Induction of Viable but Nonculturable State in Beer Spoilage Lactic Acid Bacteria

Strong beer spoilage strains Lactobacillus lindneri DSM 20692 and Lactobacillus paracollinoides JCM 11969^T were repeatedly subcultured in degassed beer and their culturability on MRS agar was examined. As a result, the two strains were found to show decreased culturability, suggesting that the prolonged contact with beer reduces the culturability of beer spoilage lactic acid bacteria (LAB). After 30 subcultures in degassed beer, both strains were subjected to sublethal heat treatment. As a consequence, L. lindneri DSM 20692 and L. paracollinoides JCM 11969^T were no longer detectable on MRS agar despite the presence of 460 viable cells, indicating that the viable but nonculturable (VNC) states were induced for both strains. Problematically, the heat treated VNC strains were shown to exhibit beer spoilage ability, suggesting that spoilage incidents can occur without detection by culture media. It was also shown that, once acquired, the VNC states are stably maintained in beer without further heat treatment. These results suggest the possibility that beer spoilage LAB strains remain hidden in pitching yeast and work‐in‐process products without detection. Furthermore L. lindneri DSM 20692 and L. paracollinoides JCM 11969^T in VNC states were successfully stored at ?80°C with 10% dimethyl‐sulfoxide as a cryoprotectant and reconstituted in degassed beer without losing VNC characteristics. Taken together, these findings show that valuable bioresources can be acquired from culturable beer spoilage LAB strains and maintained for long‐term storage as frozen culture stocks.     

乳及乳制品中常见“活的非可培养态”食源致病菌研究进展

细菌在环境胁迫下能够进入一种“活的非可培养态”（viable but nonculturable state,VBNC）,常规的平板培养方法无法检测出该种状态细菌,但其仍具有可测量的代谢活性,能够在适合的条件下复苏并产生毒力。乳及乳制品因其含有丰富的营养物质而成为理想的细菌培养基,其中几种常见的食源致病菌如副溶血性弧菌、空肠弯曲杆菌、金黄色葡萄球菌、单核增生李斯特氏菌、阪崎肠杆菌等也被证实能够进入VBNC,对乳及乳制品品质安全造成严重威胁。因此,本文综述了VBNC细菌的研究进程和细胞形态、毒力等生理学特性,并介绍了乳及乳制品中几种常见VBNC致病菌的诱导与复苏因素及其检测方法。通过对VBNC细菌的综述进而为乳及乳制品中致病菌研究提供新思路,以期减少乳及乳制品中该状态致病菌对公共安全的威胁,提升乳及乳制品中生物性安全水平。     

基于iTRAQ方法分析副溶血弧菌活的非可培养状态的差异表达上调蛋白

采用低温、寡营养和食品防腐剂处理诱导副溶血弧菌进入活的非可培养状态(viable but nonculturable state,VBNC),并利用同位素标记相对与绝对定量(isobaric rags for relative and absolute quantitation,i TRAQ)蛋白组学技术分析处于VBNC状态和对数生长期的副溶血弧菌的差异表达蛋白,结合液质联用技术对蛋白进行鉴定,对差异蛋白进行GO富集分析和Pathway通路分析。结果表明,副溶血弧菌于山梨酸钾浓度为10 mmol/L的3%Na Cl寡营养培养基中,4℃条件下40~45 d即可进入到VBNC状态。通过i TRAQ蛋白组学技术共鉴定到135880个蛋白,其中定量的蛋白有1088个;在VBNC状态显著下调蛋白36个,上调蛋白15个。差异表达的上调蛋白主要是外膜蛋白、转运蛋白、铁蛋白和其他参与代谢的关键蛋白,表明VBNC状态的副溶血弧菌根据环境应激提高这些蛋白的表达以保持活性。该研究结果为更深入探讨VBNC的分子形成机制提供了理论基础。     

Bacterial dormancy in Campylobacter: abstract theory or cause for concern?

For the past 100 years, since the birth of modern microbiology, this discipline has predominantly relied on the ability to culture micro-organisms in vitro on artificial synthetic culture media under controlled conditions in the laboratory. However, sometimes it is not possible to detect foodborne pathogens using such conventional techniques. Employment of these techniques can also lead to a delay in detection of pathogens. The 'viable but non-culturable' (VNC) cellular form has been demonstrated in Campylobacter jejuni , representing a resting or dormant stage, which is induced through cell stress including starvation. This form is extremely difficult to detect and generally requires complex and sophisticated technology which is usually not available in most routine food microbiology laboratories. This review aims at examining the role of this cell form in Campylobacter , including their historical evolution, formation, physiology, detection and to discuss the challenges that this form presents to food safety.     

Validation of an Improved Method for Detection of Campylobacter jejuni in Foods

ABSTRACT:  Campylobacter jejuni ATCC 29428 and 33560 were inoculated separately into beef muscle, ground beef, and chicken skin to yield approximately 10 to 100 CFU/g of food sample. The samples were stored at 4 °C for 10 d. On days 0, 3, 7, and 10, enrichment cultures in Bolton broth supplemented with antibiotics, with and without blood supplementation were made for each sample, for 24 and 48 h following the Food and Agricultural Products Center (FAPC) and the Food and Drug Administration (FDA) protocols. Enumeration of the organisms in the enrichment cultures was done on Campylobacter Karmali selective agar after 24 and 48 h of enrichment to compare the extent of growth in both protocols. There were no significant differences between counts recovered using the FDA and the FAPC methods for detection of Campylobacter jejuni for either strain in any of the food products tested ( P > 0.05). No significant differences were observed in performance of enrichment broth supplemented with and without blood ( P > 0.05). After 48 h of enrichment, the counts recovered were similar for all products. The organisms were detectable on all days of storage in raw chicken skin, beef, and ground beef samples after both 24 and 48 h of enrichment. The results from the FAPC method for detection of C. jejuni from food were not different from the FDA method. While in the proposed method incubation at 37 °C was adequate for the strains tested it is recommended that both enrichment temperatures be used for naturally contaminated samples to ensure detection of all strains that might be present.     

双重PCR 方法检测沙门氏菌和空肠弯曲菌

为建立能够同时检测食品中沙门氏菌和空肠弯曲菌的双重PCR 方法。采用沙门氏菌鞭毛基因fimY 和空肠弯曲菌马尿酸酶基因hipO 设计特异性引物,并对影响PCR 扩增的主要因素——引物浓度、退火温度、Mg2+ 浓度因素进行优化,比较单一PCR 和双重PCR 的检测效果。结果表明：采用单一PCR 法检测沙门氏菌和空肠弯曲菌时,灵敏度分别可达到3.98pg 和4.05pg;而采用双重PCR 检测时,灵敏度较单一PCR 法有所下降,沙门氏菌和空肠弯曲菌检出限量分别为398pg 和40.5pg。本研究建立的特异性强和灵敏度高的双重PCR 检测方法,可为实现食品中沙门氏菌和空肠弯曲菌的同时检测提供新方法。