自然界中处于VBNC状态微生物的研究进展*

自然界中大约有 90 %以上的微生物尚未被分离鉴定。 80年代初提出VBNC状态微生物的存在 ,所谓的VBNC是指微生物在自然界中生存的一种状态 ,本文综述了在实验室条件下VBNC状态的形成条件、培养性的恢复及处于VBNC状态微生物的检测方法等方面的研究进展 ,并介绍了第一个微生物生长因子Rpf。     

自然界中处于VBNC状态微生物的研究进展

自然界中大约有90％以上的微生物尚未被分离鉴定。80年代初提出VBNC状态微生物的存在,所谓的VBNC是指微生物在自然界中生存的一种状态,本综述了在实验室条件下VBNC状态的形成条件、培养性的恢复及处于VBNC状态微生物的检测方法等方面的研究进展,并介绍了第一个微生物生长因子Rpf.     

细菌VBNC态检测技术的研究进展

细菌在一些不良压力条件下会进入"活的非可培养状态"(viable but non-culturable,VBNC),其仍具有活力但不能采用常规的平板培养基进行培养,VBNC态细菌一旦培养条件适宜仍能继续生长繁殖,有些致病菌依旧具有毒力。如果检测方法不当,会造成假阳性或假阴性的结果,需要采取适合的检测方法进行检测。文章概述了几种检测VBNC态细菌的方法,如活菌直接计数法、核酸染料检测法、呼吸检测法、分子生物学法、免疫学法、流式细胞仪检测法等,并对检测方法的应用现状进行了评述。     

微生物VBNC状态形成及复苏机制

99%以上的微生物因处于活的但非可培养(viable but non-culturable,VBNC)状态而无法分离培养。复苏促进因子(resuscitation-promoting factors,Rpfs)是培养获取VBNC菌的最重要突破。结合课题组近十余年从环境功能视角利用Rpf复苏培养VBNC菌的研究,本文在阐述微生物VBNC状态的形成及复苏进展的基础上,从VBNC菌形成及复苏过程出发,探究"探索因子"与群体感应的内在关系。并总结了课题组利用Rpf所复苏培养的具有潜在环境功能的VBNC菌种。本论文将为揭示微生物VBNC状态的形成及复苏机制提供新的思路,并为认识和重新评价Rpf法复苏培养VBNC菌在污染环境微生物修复中的作用提供理论依据。     

致病微生物应对环境胁迫形成的VBNC状态及其对风险评估的潜在影响

防止食源性致病菌对食品的污染是保障食品安全的重要策略之一。研究表明,环境胁迫下微生物可能形成"活的不可培养状态"(VBNC状态),成为食品安全的潜在风险。本文对致病微生物VBNC状态的最近发现进行了归纳和总结,重点分析了不同环境胁迫(包括自然胁迫以及食品加工和保藏过程中的各种胁迫等)因素下菌体进入VBNC状态的普遍性、诱导条件和生理特点等,并对现行的基于微生物可培养性而建立起来的常规检测方法的局限性及其对食品安全风险评估潜在影响进行了讨论。     

环境中"活的非可培养(VBNC)"细菌的研究进展

1982年徐怀恕等通过对V.cholerae和E.coli存活规律的研究,首次发现并提出了细菌“活的非可培养”状态(Viable but non—culturable state,VBNC),即细菌处于不良环境条件下,其细胞通常缩成球形(最近许多研究中发现还有些细菌表现为体积增大,细胞伸长),用常规方法培养不能使其生长繁殖,但仍然具有代谢活性,在DNA合成抑制剂的作用下,添加一定量的营养物培养时,这些细胞虽然不能分裂,但仍可以伸长生长,证明其仍然存活,并非死亡。处于VBNC状态的细菌,在一定的条件下可以复苏,并具有潜在的致病性,如V.cholerae即通过这种形式实现越冬。至今已有多种细菌进入VBNC状态的报道,许多G^-菌及某些不形成芽胞的G^ 菌在不良环境条件下都可能进入该状态。这些细菌通过进入VBNC状态,得以渡过难关并存活下来,成为细菌的一种特殊存活形式。现在,细菌VBNC状态已作为微生物学的一个全新概念,受到极大的关注,它对传统的微生态学、食品安全、水质监测、菌种保藏及流行病学研究等均提出了新的挑战,也为人们应用基因工程菌的危险性评估提供了新的认识,本文对环境中VBNC细菌的研究进展进行了综述。     

细菌“活的不可培养状态”的生态意义及研究进展

"活的不可培养(VBNC)"状态是细菌在不良条件下的一种生存方式.VBNC状态作为细菌的一种生理状态,对传统微生物学产生了深远的影响.进入VBNC状态的细胞发生了一系列变化,无法继续用常规培养方法检测,在医学健康,环境科学等领域产生了巨大的影响,改进检测方法具有重要的意义.本文介绍了进入VBNC状态细菌在DNA、蛋白质组成等方面发生的变化,复苏过程.同时还介绍了VBNC状态的最新检测方法,最后对VBNC状态未来的研究方法进行了讨论.     

细菌有活力但不可培养状态及其机制研究进展

有活力但不可培养(viable but non-culturable,VBNC)状态是细菌遭遇逆境时进入的一种特殊状态,该状态下的菌体在条件适宜时可复苏并恢复其致病性,被认为是细菌躲避不良环境的一种生存策略。VBNC状态菌体对人类医学和工农业生产具有巨大的潜在威胁,开展关于VBNC状态的检测及诱导、复苏及其机制研究可为减少或避免该状态细菌的危害提供理论基础。本文简要综述了细菌VBNC状态在诱导、复苏及致病性等方面的研究进展,并结合本实验室及国内外相关团队近年来在植物病原细菌VBNC状态研究中的结果,详细总结了VBNC状态细菌的形成和复苏机制,对植物病原细菌在环境胁迫下的存活机制、病害田间初侵染来源分析及VBNC状态菌体在病害循环中的作用等相关研究具有重要参考意义。     

氧化压力下沙门氏菌可培养性检测及其活的非可培养状态形成情况

【背景】氧化压力会导致细菌进入活的非可培养(viable but non-culturable,VBNC)状态,菌落形成能力可能受到亚致死损伤的影响。目前对于VBNC态细菌的定量检测是基于活菌数与可培养数的差值,因此可培养数的检测对于VBNC态定量研究很关键,培养基组成不合适可能会造成漏检。【目的】分析培养基组成对氧化压力下亚致死损伤细菌检测的重要影响;探究常见食源性致病菌肠炎沙门氏菌在氧化压力下形成VBNC态的情况。【方法】分别采用Luria-Bertani (LB)、beef peptone yeast (BPY)和Salmonella Shigella (SS)培养基检测并比较肠炎沙门氏菌的可培养数;采用RT-qPCR、荧光染色-激光共聚焦显微镜观测氧化压力下肠炎沙门氏菌形成VBNC态的情况。【结果】非选择性培养基LB和BPY能检出亚致死细菌,SS培养基中牛胆盐导致可培养数减少;肠炎沙门氏菌经53°C过氧化氢处理1.5 h后进入VBNC态的比例显著高于53°C过氧化氢+亚铁离子和过氧化氢+柠檬酸处理(P<0.05)。【结论】在对VBNC态的检测中应选择合适的固体培养基检测可...     

活的但非可培养(VBNC) 状态菌的研究进展

VBNC(viable but non-culturable)是指处于"活的但非可培养"状态的微生物,此微生物体的细胞仍有代谢活性,但用常规方法无法分离培养.本文阐述了VBNC状态菌的形成机理、转变与种类、复苏、研究意义及其应用展望.并报道了我们在十余年间针对生态环境中处于VBNC状态菌的复苏、可培养化、系统进化关系及潜在功能等方面的一些研究成果,拟为微生物资源的开发与应用提供新的科学依据.     

Inability of Escherichia coli to resuscitate from the viable but nonculturable state

After induction of the viable but nonculturable (VBNC) state in Escherichia coli populations, we analysed abiotic and biotic factors suggested to promote the resuscitation process. The response to the stressing conditions implied the formation of three subpopulations, culturable, VBNC and nonviable. In most adverse situations studied, the VBNC subpopulation did not represent the dominant fraction, decreasing with time. This suggests that, in most cases, the VBNC is not a successful phenotype. Combining methods of dilution and inhibition of remaining culturable cells, we designed a working protocol in order to distinguish unequivocally between regrowth and resuscitation. Reversion of abiotic factors inducing nonculturability as well as prevention of additional oxidative stress did not provoke resuscitation. Participation of biotic factors was studied by addition of supernatants from different origin without positive results. These results indicate that the E. coli strain used is not able to resuscitate from the VBNC state. VBNC cells release into the surrounding medium, and could thus aid in the survival of persisting culturable cells. The formation of a VBNC subpopulation could thus be considered as an adaptive process, designed for the benefit of the population as a whole.     

Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co‐culture with eukaryotic cells

VBNC Vibrio cholerae O139 VC‐280 obtained by incubation in 1% solution of artificial sea water IO at 4°C for 74 days converted to the culturable state when co‐cultured with CHO cells. Other eukaryotic cell lines, including HT‐29, Caco‐2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC‐280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC‐280 was incubated in 1% IO at 4°C for up to 91 days, the number of cells converted by co‐culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed.     

Survival strategies of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Vibrio</Emphasis> spp.: contribution of the viable but nonculturable phenotype to their stress-resistance and persistence in adverse environments

In their natural ecosystems, bacteria are continuously exposed to changing environmental factors including physicochemical parameters (e.g. temperature, pH, etc.), availability of nutrients as well as interaction(s) with other organisms. To increase their tolerance and survival under adverse conditions, bacteria trigger a number of adaptation mechanisms. One of the well-known adaptation responses of the non-spore-forming bacteria is the acquisition of the viable but non-culturable (VBNC) state. This phenotype is induced by different stress factors (e.g. low temperature) and is characterized by the temporal loss of culturability, which can potentially be restored. Moreover, this response can be combined with the bust and boom strategy, which implies the death of the main population of the stressed cells (or their entry into the VBNC state) upon stress, thus enabling the remaining cells (i.e. residual culturable population) to subsist at the expense of the dead or/and VBNC cells. In this review, we discuss the characteristics of the VBNC state, its biological significance and contribution to bacterial survival.     

Viable but non-culturable Listeria monocytogenes on parsley leaves and absence of recovery to a culturable state

AIMS: To investigate the presence of viable but non-culturable Listeria monocytogenes during survival on parsley leaves under low relative humidity (RH) and to evaluate the ability of L. monocytogenes to recover from VBNC to culturable state under satured humidity. METHODS AND RESULTS: Under low RH (47-69%) on parsley leaves, the initial number of L. monocytogenes populations counted on non selective media (10(9) L. monocytogenes per leaf on TSA) was reduced by 6 log10 scales in 15 days, whereas number of viable L. monocytogenes counted under the microscope was reduced by 3-4 log10 scales, indicating the presence of VBNC cells. This was demonstrated on three L. monocytogenes strains (EGDe, Bug 1995 and LmP60). Changing from low to 100% RH permitted an increase of the culturable counts of L. monocytogenes and this growth was observed only when residual culturable cells were present. Moreover, VBNC L. monocytogenes inoculated on parsley leaves did not become culturable after incubation under 100% RH. CONCLUSIONS: Dry conditions induced VBNC L. monocytogenes on parsley leaves but these VBNC were likely unable to recover culturability after transfer to satured humidity. SIGNIFICANCE AND IMPACT OF STUDY: Enumeration on culture media presumably under-estimates the number of viable L. monocytogenes on fresh produce after exposure to low RH.     

Stepwise changes in viable but nonculturable Vibrio cholerae cells

Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC‐state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT‐29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co‐cultivation with HT‐29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co‐cultivation with HT‐29. These characteristic changes in VBNC‐state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.     

Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.