“活的非可培养态”食源致病细菌的研究进展

食源致病细菌是影响食品质量与安全的重要生物污染源。食品加工过程中的低温、寡营养及食品防腐等条件可诱使食源致病细菌进入活的非可培养状态(viable but non-culturable state,VBNC),处于此状态的细菌最大的特点就是丧失了平板上的生长繁殖能力,但生命体征是活的,并且保留原菌的毒力和致病性。研究表明,活的非可培养态细菌在细胞形态、基因表达、蛋白表达等方面发生了复杂变化,并且一定条件下可复苏为可培养状态,成为逃避检测的"隐性传染源",对食品安全构成潜在威胁。文中对食源性致病菌VBNC态的诱导条件及复苏情况、生物学特性变化、现代检测技术的发展等方面的研究进行了综述。期望为解决当下由活的非可培养态细菌引起的食品安全问题提供理论参考。     

弧菌VBNC状态的研究进展

细菌的活的非可培养(VBNC,VNC)状态是在不良环境下细菌具有生物活性和毒性,用常规培养法无法检出,却在一定条件下可以复苏并保存致病性的一种特殊生理状态。本文综述了三种常见致病性弧菌的活的非可培养状态下的生物学特性、形成机制及其复苏的研究。运用现代生物技术手段,深入地研究细菌的VBNC状态,对食品卫生与安全具有重要的意义。     

活的非可培养状态副溶血性弧菌实时荧光逆转录PCR检测方法的建立及其毒力研究

目的 建立检测活的非可培养(VBNC)状态副溶血性弧菌的荧光逆转录PCR方法并研究其毒力基因表达.方法 将副溶血性弧菌AS079菌株添加到陈化海水中,4℃冰箱内培养,使其进入VBNC状态,针对其管家基因、鉴定基因和毒力基因分别设计实时荧光逆转录PCR引物,通过不同的PCR反应程序和引物浓度组合试验,摸索最佳反应体系条件,用于VBNC状态副溶血性弧菌的检测和毒力研究.结果 用所建立的检测VBNC状态副溶血性弧菌的实时荧光逆转录PCR方法,对接种于陈化海水培养的不同时期的AS079副溶血性弧菌进行荧光定量逆转录PCR扩增,结果显示,随着培养时间的延长,毒力基因tdh2和鉴定基因toxR表达水平持续下降,但即使细菌进入VBNC状态,这两个基因也能够得到很好的扩增,说明以toxR基因和tdh2基因对进入VBNC状态的副溶血性弧菌进行检测和毒力研究方法可行.灵敏度试验表明,鉴定基因toxR的最低检测限可达到48 cfu/ml,研究毒力基因tdh2的表达需要细菌浓度至少为4.8×102cfu/ml,同时试验证明此方法与其他相近食源性致病菌无交叉反应.结论 该实时荧光逆转录PCR方法检测快速、特异性强、敏感度高,适用于VBNC状态副溶血性弧菌的检测和毒力分析.     

食源性致病菌活的不可培养状态研究进展

在食品加工、产品运输及贮藏过程中,极端的环境条件可能会导致食品中的致病菌进入活的不可培养(viable but non-culturable,VBNC)状态,如大肠杆菌O157:H7、副溶血性弧菌、沙门氏菌、金黄色葡萄球菌等常见的食源性致病菌。食源性致病菌一旦进入了VBNC状态,常规的培养检测方法不能将其检出,易造成VBNC状态致病菌漏检,威胁食品安全和人体健康。本文归纳了食品加工过程中可能致使食源性致病菌进入VBNC状态的相关因素,概括了食源性致病菌VBNC状态的生物学特性,并着重介绍VBNC食源性致病菌的检测方法,以期为食源性致病菌的安全防控提供科学依据。     

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。     

VBNC状态啤酒易感乳杆菌的诱导及复苏

本文研究了一种典型的难培养啤酒易感乳杆菌-耐酸乳杆菌(Lactobacillus acetotolerans)形成"活的但不可培养"(VBNC)的诱导方式,以及从此状态复苏至可培养状态的方法。将培养至对数期的耐酸乳杆菌2011-11菌株采用啤酒内连续传代驯化法还原其在啤酒酿造过程中的实际生存状况,并通过荧光染料染色对诱导后的耐酸乳杆菌细胞进行活性检测;利用逐步升温和添加促进物法对VBNC菌进行复苏试验。当连续传代至第19代,MRS检测平板上可培养菌数降为零,此时菌体多数仍存在呈现绿色荧光的活细胞,表明耐酸乳杆菌已形成VBNC状态,且仍具有污染啤酒能力;将刚进入VBNC状态的耐酸乳杆菌菌悬液经过氧化氢酶处理后涂布于MRS平板,于26℃厌氧培养,可使耐酸乳杆菌复苏至可培养状态。本研究证实,可通过啤酒内连续传代诱导获得VBNC状态耐酸乳杆菌,而添加过氧化氢酶改良常规MRS平板是一种有效的复苏方法。     

物理场加工中细菌活的非可培养状态相关研究进展

物理场加工技术是一类使用加热、加压和辐射等物理学方法的技术, 其中热加工、超声波、超高压和紫外辐照等技术已被广泛应用于食品加工。近年来多个报道显示, 在物理场加工中细菌可进入活的非可培养状态(viable but non-culturable, VBNC)。VBNC状态指微生物为了适应逆境而进入的一种特殊休眠状态, 在合适条件下能复苏, 甚至可能依然保持致病能力, 这提示我们应该对物理场加工食品的微生物安全予以高度重视。本文主要综述了物理场加工中食源性致病菌和腐败菌VBNC状态的研究进展, 并从共性机制的角度出发分析其形成原因。本文旨在为更好的应用物理场加工技术并保障食品安全提供新思路和新方向。     

植物乳杆菌活的非可培养状态的初步研究

利用16S rDNA序列分析法测得实验室保藏的乳酸菌的16S rDNA序列,并与基因库中基因序列进行同源性比较,经鉴定此菌为植物乳杆菌.并研究了植物乳杆菌进入活的非可培养状态(Viable but Non-cuhurable,VBNC)的诱导条件和复苏条件.结果显示,在诱导条件为MRS液体培养基、pH5.5～6.2、温度-20℃和有氧环境时,植物乳杆菌在12d之后进入了VBNC状态;复苏条件为添加有6％吐温-80的MRS液体培养基和普通MRS液体培养基分别在48h和96h之内可以使VBNC的植物乳杆菌复苏.     

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。     

活的非可培养状态细菌的培养和复苏条件的研究

本文研究食品常用的消杀条件对大肠杆菌和金黄色葡萄球菌进入活的非可培养状态(viabile but nonculturable,VBNC)的诱导,并探讨高温诱导的VBNC菌的复苏条件。将对数生长期的大肠杆菌、金黄色葡萄球菌通过温度、盐度以及不同浓度的山梨酸钾处理进行诱导;对高温条件诱导的VBNC菌液添加维生素类物质,研究复苏VBNC菌的条件。结果表明,大肠杆菌在-20℃培养可形成VBNC菌,在温度超过65℃水浴30 min 存在VBNC菌; NaCl浓度大于15%环境下培养15ｄ可诱导VBNC菌的形成;山梨酸钾浓度高于0.15％即可诱导大肠杆菌进入VBNC;金黄色葡萄球菌在相同条件下诱导菌落数变化缓慢,未出现VBNC菌。复苏研究中,添加0.1‰-0.18‰范围的维生素C或0.252%的泛酸或0.06‰生物素可使VBNC的大肠杆菌复苏;VBNC的金黄色葡萄球菌复苏则需要添加0.2‰-0.25‰范围的维生素C或0.248%的泛酸或0.06‰生物素。本研究表明食品的消杀条件均有形成VBNC菌的可能,不同属的VBNC菌复苏在相同复苏物质条件下所需的浓度不同。为保证食品安全在食品检测环节可通过添加复苏条件以确保检测结果的准确性,或在运输储藏过程中避免复苏物质的存在,本研究有望为企业对食品的消杀处理以及检测、储藏运输等条件改善提供科学依据。     

毒素-抗毒素系统对微生物活的非可培养状态形成的影响研究进展

毒素-抗毒素（toxin-antitoxin，TA）系统广泛存在于微生物中，由稳定的毒素和不稳定的同源抗毒素组成，参与应激反应并可调控微生物生长。活的非可培养（viable but non-culturable，VBNC）状态是某些微生物在不良环境中形成的一种休眠状态，处于VBNC状态的微生物在常规培养基上不生长，但VBNC细胞仍具有代谢活性，且在适宜条件下能够恢复可培养性，因此VBNC状态的食源性致病菌对食品安全和人类健康构成潜在威胁。本文综述了TA系统和微生物VBNC状态形成关系的研究，进一步提出了TA系统对微生物VBNC状态形成的可能影响机制，以期为VBNC状态形成机制的研究提供一定参考。     

Induction,detection, formation,and resuscitation of viable but non‐culturable state microorganisms

The viable but nonculturable (VBNC) state has been recognized as a strategy for bacteria to cope with stressful environments; in this state, bacteria fail to grow on routine culture medium but are actually alive and can resuscitate into a culturable state under favorable conditions. The VBNC state may pose a great threat to food safety and public health. To date, more than 100 VBNC microorganism species have been proven to exist in fields of food safety, environmental application, and agricultural diseases. Most harsh conditions can induce these microorganisms into the VBNC state, including food processing and preservation methods, adverse environmental conditions, and plant‐disease controlling means. The characteristics of VBNC state cells differ from those of normally growing cells and dead cells, based on which of the various detection methods are developed, and they are of great significance for potential risk assessment. To provide molecular level insights into this state, many studies on induction and resuscitation mechanisms have emerged over the past three decades, including research on omics, specific genes, or proteins involved in VBNC state formation and the roles of promoters in resuscitation from the VBNC state. In this review, microorganism species, induction and resuscitation factors, detection methods, and formation and resuscitation mechanisms of the VBNC state are comprehensively and systematically summarized.     

Practical Food Safety Interventions for Dairy Production

Consumers are increasingly concerned about the safety of their food and uncertain about food production practices. Potential threats to human health related to dairy products and dairy farming include errors in pasteurization, consumption of raw milk products, contamination of milk products by emerging heat-resistant pathogens, emergence of antimicrobial resistance in zoonotic pathogens, chemical adulteration of milk, transmission of zoonotic pathogens to humans through animal contact, and foodborne disease related to cull dairy cows. Most dairy farmers feel responsible for the safety of milk and beef that originate on their farms, but linkage between farm production practices and the quality of processed products have been weak. The safety of dairy products can be enhanced by adoption of a number of management practices. Sources of microbial contamination of milk must be minimized by adoption of hygienic standards that can be easily evaluated. Uniform adoption of milking practices that reduce microbial contamination of milk should be emphasized. The diagnosis of salmonellosis or listeriosis on a dairy farm should be regarded as an indication that other potentially infected animals may be present in the herd. Coliform counts on bulk tank milk should be routinely performed as an indicator of fecal contamination. A reduction in the national regulatory limit for somatic cells in bulk tank milk should be considered based on potential enhancements in milk safety. Dairy farmers must take responsibility for the market cattle leaving their farms. The inappropriate or prophylactic use of antimicrobial agents must be minimized to ensure that antimicrobial resistance does not develop in animal pathogens. Consumers can have confidence in food safety programs on dairy farms that promote awareness and accountability for the products that are produced.     

Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt-0.5% NaCl medium incubated at 4 degrees C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.     

德氏乳杆菌保加利亚亚种ND02活的非可培养态诱导和复苏

为有效提高乳酸菌发酵剂菌种在生产和应用过程中的活性,对一株具有优良发酵特性的德氏乳杆菌保加利亚亚种ND02(保加利亚乳杆菌ND02)进行了活的非可培养(viable but non-culturable,VBNC)态诱导和复苏的初步研究。将活化的保加利亚乳杆菌ND02在不同复合条件下进行诱导,同时采用平板计数法和荧光显微镜两种计数方法对其进行检测,将诱导成功的样本通过改变温度及改变培养基成分进行复苏实验。结果表明:保加利亚乳杆菌ND02在液体MRS中4℃诱导190 d便可以进入VBNC态,并发现10%脱脂乳+0.1%酵母粉是有效的复苏方法。为保加利亚乳杆菌ND02在应用过程中避免VBNC态的产生及活性的提高提供一定的参考依据。     

Viable but non-culturable forms of food and waterborne bacteria: Quo Vadis?

There is increasing evidence for a viable but non-culturable (VBNC) state in microbes, particularly in the stressing environment presented by modern foods with their varied pre-treatment and packaging strategies. This is a cause for concern because of evidence that microbial pathogens in such a state may retain their capacity to cause infections after ingestion by the consumer despite their inability to grow under the conditions employed in procedures for determining their presence in foods. Heavily stressed pathogenic species of bacteria in a VBNC or not immediately culturable state are potentially dangerous public health problems, particularly as stressed cells may be more virulent than well-fed bacteria. In this viewpoint article, I wish to focus on possible procedures for detecting such organisms and assessing their physiological state.     

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。     

牛奶发酵过程中阪崎克罗诺杆菌可形成活的非可培养状态

该研究以保加利亚乳杆菌为牛奶发酵剂,研究了牛奶发酵过程中阪崎克罗诺杆菌与保加利亚乳杆菌的相互作用以及阪崎克罗诺杆菌存活量的变化情况,并结合16S rDNA高通量测序技术和PMA-qPCR方法分析阪崎克罗诺杆菌在牛奶发酵过程中是否形成“活的非可培养”状态。结果显示,保加利亚乳杆菌在牛奶中发酵48 h后pH值可达到3.40,酸度值可达212.00。在发酵前期接种阪崎克罗诺杆菌,48 h后pH值和酸度值分别为3.50和193.30;在发酵中期接种阪崎克罗诺杆菌,48 h后pH值和酸度值分别为3.47和214.30。发酵前期阪崎克罗诺杆菌的污染对保加利亚乳杆菌发酵结果的影响更大。阪崎克罗诺杆菌在牛奶中生长48 h后菌浓度可稳定在9.08 lg(CFU/mL);不论在牛奶经发酵前期或中期接种阪崎克罗诺杆菌,发酵后期平板计数法均检测不到阪崎克罗诺杆菌,但16s rDNA和PMA-qPCR技术均可检测到阪崎克罗诺杆菌活菌的存在。该研究结果说明,阪崎克罗诺杆菌在牛奶发酵后进入了“活的非可培养”状态,该状态的存在会导致该菌检测时活菌数量的低估,从而引发乳及乳制品的安全隐患。     

一种适用于乳制品基因组DNA快速提取方法的研究

目的对比NaOH裂解法、PBS裂解法以及直接煮沸法3种方法提取乳制品中核酸的提取效果,优化提取条件,确定一种更适用于现场检测、简便快速的的乳制品DNA快速提取技术。方法以牛奶、水牛奶、牦牛奶、羊奶、骆驼奶、以及驴奶6种常见的乳制品为材料,分别用NaOH裂解法、PBS裂解法以及直接煮沸法3种提取方法提取乳制品中的DNA,并根据裂解液用量和裂解时间进行优化,通过PCR扩增和琼脂糖凝胶电泳分析,检测DNA提取的质量和灵敏度。结果 NaOH裂解法能够提取所有物种的乳制品DNA,而且可以在最佳裂解条件下提取模拟掺假混合乳的DNA进行检测,发现其检测限能达到1%的牛奶含量。结论该方法取样量小,成本低,在15 min内即可完成快速提取,为实验室乳制品DNA定性或定量鉴别,以及乳制品的现场掺假鉴别提供了一种快速灵敏低成本的样品前处理技术。