Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 °C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10⁴ cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 °C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection.The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

Cell Wall Chemical Composition of Enterococcus faecalis in the Viable but Nonculturable State

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.     

Induction and Resuscitation of the Viable but Nonculturable State in a Cyanobacteria-Lysing Bacterium Isolated from Cyanobacterial Bloom

The viable but nonculturable (VBNC) state has been found to be a growth strategy used by many aquatic pathogens; however, few studies have focused on VBNC state on other aquatic bacterial groups. The purpose of this study was to explore the VBNC state of cyanobacteria-lysing bacteria and the conditions that regulate their VBNC state transformation. Three cyanobacteria-lysing heterotrophic bacterial strains (F1, F2 and F3) were isolated with liquid infection method from a lake that has experienced a cyanobacterial bloom. According to their morphological, physiological and biochemical characteristics and results of 16SrDNA sequence analysis, F1, F2 and F3 were identified as strains of Staphylococcus sp., Stappia sp. and Microbacterium sp., respectively. After being co-cultured with the axenic cyanobacterium, Microcystis aeruginosa 905, for 7 days, strains F1, F2 and F3 exhibited an inhibition effect on cyanobacterial growth, which was expressed as a reduction in chlorophyll concentration of 96.0%, 94.9% and 84.8%, respectively. Both autoclaved and filtered bacterial cultures still showed lytic effects on cyanobacterial cells while centrifuged pellets were less efficient than other fractions. This indicated that lytic factors were extracelluar and heat-resistant. The environmental conditions that could induce the VBNC state of strain F1 were also studied. Under low temperature (4°C), distilled deionized water (DDW) induced almost 100% of F1 cells to the VBNC state after 6 days while different salinities (1%, 3% and 5% of NaCl solution) and lake water required 18 days. A solution of the cyanobacterial toxin microcystin-LR (MC-LR) crude extract also induced F1 to the VBNC state, and the effect was stronger than DDW. Even the lowest MC-LR concentration (10 μg L⁻¹) could induce 69.7% of F1 cells into VBNC state after 24 h. On the other hand, addition of Microcystis aeruginosa cells caused resuscitation of VBNC state F1 cells within 1 day, expressed as an increase of viable cell number and a decrease of VBNC ratio. Both VBNC state and culturable state F1 cells showed lytic effects on cyanobacteria, with their VBNC ratio varying during co-culturing with cyanobacteria. The findings indicated that VBNC state transformation of cyanobacteria-lysing bacteria could be regulated by cyanobacterial cells or their toxin, and the transformation may play an important role in cyanobacterial termination.     

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...     

Modification of the Peptidoglycan of Escherichia coli in the Viable But Nonculturable State

The aim of this study was to analyse the chemical composition of peptidoglycan and the state of some of the enzymes involved in its metabolism in Escherichia coli KN126 in the viable but nonculturable (VBNC) state which is a survival strategy adopted by bacteria (including those of medical interest) when exposed to environmental stresses. When entering the VBNC state, E. coli cells miniaturised and became coccus-shaped. Analysis of peptidoglycan chemical composition, by separation in HPLC of muropeptides released by muramidase digestion of purified peptidoglycan, indicated a high degree of cross-linking, a threefold increase in unusual DAP–DAP cross-linking, an increase in muropeptides bearing covalently bound lipoprotein, and a shortening of the average length of glycan strands in comparison with dividing cells. Analysis of penicillin-binding proteins (PBPs), enzymes involved in the terminal stage of peptidoglycan assembly showed the disappearance of high-molecular-weight PBPs 1A, 1B, 2, and 3 in VBNC cells. Finally, VBNC cells displayed an autolytic capability which was far higher than that of exponentially growing cells. It is suggested that part of these alterations of peptidoglycan may be connected with the VBNC state. Received: 20 March 2001 / Accepted: 7 June 2001     

氧化胁迫诱导苍白杆菌JP1形成VBNC状态及其特征

石油降解菌在各种有害环境因素作用下会进入活的非可培养(viable but non-culturable, VBNC)状态，从而影响其生长及石油降解率。为了研究有害环境因素对石油降解菌生长及石油降解率的影响，采用分光光度法、荧光染色-激光共聚焦显微镜观测H₂O₂胁迫下苍白杆菌(Ochrobactrum sp.)JP1细胞的生长及VBNC状态形成情况。结果表明，不同浓度H₂O₂对其生长有一定抑制作用，当培养液中H₂O₂浓度为75.0 mmol/L时，可有效抑制苍白杆菌JP1生长，处理12 h后苍白杆菌JP1进入VBNC状态。VBNC状态的苍白杆菌JP1细胞缩小变成球体，周质间隙增大；在适宜条件下，VBNC状态苍白杆菌JP1能够复苏为可培养状态，添加丙酮酸钠能够促进VBNC状态细菌细胞的复苏。复苏后的苍白杆菌RJP1具有良好的环境适应性和石油降解能力，为石油污染生物修复的菌种筛选及应用提供了新的策略。     

Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 degrees C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10(4) cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 degrees C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection. The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

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.     

First-time characterization of viable but non-culturable Proteus mirabilis: Induction and resuscitation

Pathogenic bacteria can enter into a viable but non-culturable (VBNC) state under unfavourable conditions. Proteus mirabilis is responsible for dire clinical consequences including septicaemia, urinary tract infections and pneumonia, but is not a species previously known to enter VBNC state. We suggested that stress-induced P. mirabilis can enter a VBNC state in which it retains virulence. P. mirabilis isolates were incubated in extreme osmotic pressure, starvation, low temperature and low pH to induce a VBNC state. Resuscitation was induced by temperature upshift and inoculation in tryptone soy broth with Tween 20 and brain heart infusion broth. Cellular ultrastructure and gene expression were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR), respectively. High osmotic pressure and low acidity caused rapid entry into VBNC state. Temperature upshift caused the highest percentage of resuscitation (93%) under different induction conditions. In the VBNC state, cells showed aberrant and dwarf morphology, virulence genes and stress response genes (envZ and rpoS) were expressed (levels varied depending on strain and inducing factors). This is the first-time characterization of VBNC P. mirabilis. The ability of P. mirabilis pathogenic strains to enter a stress-induced VBNC state can be a serious public health threat.     

Effect of temperature and plasmid carriage on nonculturability in organisms targeted for release

Abstract: The ability to track genetically modified bacteria released into the environment is essential for assessing their persistence and dispersal. Some bacteria can enter a 'viable but nonculturable' (VBNC) state in which the cells remain viable while losing the ability to grow on routine culture media. Thus, VBNC cells are not detectable by standard plating methods. In order to determine what conditions, if any, induce this state in Pseudomonas fluorescens, Pseudomonas syringae , and Escherichia coli , cells were 'marked' with lux genes, either chromosomally or on one of two different plasmids. Variations in temperature, but not nutrient or NaCl concentrations, affected culturability of these strains and induced the VBNC state. The temperature which induced the VBNC state in the two pseudomonads depended on whether or not the cell carried one of the two lux -marked plasmids. This effect was shown not to be due to the presence of the lux genes, as their removal from the plasmid had no effect on entry into the VBNC state. Instead, the effect appeared to depend on the location of the plasmid DNA, as a strain of P. fluorescens with the same plasmid integrated into the chromosome behaved identically to the parent strain. The fact that plasmids may have such a dramatic effect on culturability has significant implications for the monitoring of genetically modified bacteria intended for environmental release.     

Recovery in Embryonated Eggs of Viable but Nonculturable Campylobacter jejuni Cells and Maintenance of Ability To Adhere to HeLa Cells after Resuscitation

The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.     

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。     

Recovery in embryonated eggs of viable but nonculturable Campylobacter jejuni cells and maintenance of ability to adhere to HeLa cells after resuscitation.

The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.     

Adherence of Yersinia enterocolitica to Mammalian Epithelial Cell Lines

Yersinia enterocolitica RIMD 2501003 grown at 25 C avidly adhered to various kinds of cultured epithelial cell lines (HeLa, FL, Y-1 adrenal, human intestine, human conjunctiva) but the bacteria grown at 37 C did not adhere. This phenomenon paralleled the temperature-dependent motility of the bacteria. To clarify the adherence mechanism, we obtained two kinds of mutants, an immobile mutant and a nonadherent mutant, by treatment with A-methyl-A-nitro-A-nitrosoguanidine. The immobile mutant did not move on soft agar but retained the capacity to adhere to cultured epithelial cells when grown at 25 C. The nonadherent mutant did not adhere to cultured epithelial cells but retained the ability to move on soft agar when grown at 25 C. When the bacteria were killed by heat, ultraviolet light irradiation or formaldehyde they lost their capacity to adhere to the cultured epithelial cells. Antiserum against Y. enterocolitica RIMD 2501003 grown at 25 C was absorbed with the bacteria grown at 37 C, with the bacteria grown at 25 C, with the nonadherent mutant grown at 25 C and with the bacteria killed by various means. Only the antiserum absorbed with bacteria grown at 37 C inhibited the adherence of bacteria. These data indicate that motility does not correlate with adherence of Y. enterocolitica. It appears that the adherence factor involves both a temperature-dependent surface factor and a factor synthesized de novo during the interaction of susceptible cells with the bacteria.     

Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry

The present study reports the disinfection effects of chemically and electrochemically dosed chlorine on two models for typical water-borne bacteria (Escherichia coli and Legionella beliardensis) by plating and flow cytometry (FCM) in combination with different fluorescence dyes. The residual effect on various cell functions, including cultivability, esterase activity, membrane polarization, and integrity, was tested at different free chlorine concentrations. In comparison, chemical disinfection yielded on average 60% more E. coli cells entering the viable but nonculturable (VBNC) state than electrochemical disinfection. Here, VBNC is defined as those cells with intact cell membrane but which cannot be cultured on solid nutrient agar plates. L. beliardensis was about five times more resistant to chlorine disinfection than E. coli. The results also suggested the two methods result in different disinfection mechanisms on L. beliardensis, i.e., chemically dosed chlorine targeted cell membrane integrity before enzyme activity, while electrochemically dosed chlorine acted the other way round. In addition, both bacteria lost the integrity of their cell membranes at three times lower chlorine concentration over a longer contact time (i.e., 40 vs. 10 min) by the chemical method. Our results showed that FCM is an appropriate tool to evaluate the effects of water disinfection and the percentage of cells in VBNC in a matter of hours. Electrochemical disinfection is suggested to be a favorable alternative for chemical disinfection.     

Exploring the potential environmental functions of viable but non-culturable bacteria

A conventional plate count is the most commonly employed method to estimate the number of living bacteria in environmental samples. In fact, judging the level of viable culture by plate count is limited, because it is often several orders of magnitude less than the number of living bacteria actually present. Most of the bacteria are in “viable but non-culturable” (VBNC) state, whose cells are intact and alive and can resuscitate when surrounding conditions are more favorable. The most exciting recent development in resuscitating VBNC bacteria is a bacterial cytokine, namely, the resuscitation-promoting factor (Rpf), secreted by Micrococcus luteus, which promotes the resuscitation and growth of high G+C Gram-positive organisms, including some species of the genus Mycobacterium. However, most of studies deal with VBNC bacteria only from the point of view of medicine and epidemiology. It is therefore of great significance to research whether these VBNC state bacteria also possess some useful environmental capabilities, such as degradation, flocculation, etc. Further studies are needed to elucidate the possible environmental role of the VBNC bacteria, rather than only considering their role as potential pathogens from the point view of epidemiology and public health. We have studied the resuscitation of these VBNC bacteria in polluted environments by adding culture supernatant containing Rpf from M. luteus, and it was found that, as a huge microbial resource, VBNC bacteria could provide important answers to dealing with existing problems of environmental pollution. This mini-review will provide new insight for considering the potentially environmental functions of VBNC bacteria.     

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.     

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.     

Entry of Vibrio cincinnatiensis into viable but nonculturable state and its resuscitation

Aims:  The aim was to characterize the viable but nonculturable (VBNC) state of Vibrio cincinnatiensis and its resuscitation.
Methods and Results:  Vibrio cincinnatiensis VIB287 was cultured in sterilized seawater microcosms at 4°C. Plate counts, direct viable counts and total counts were used. A large population of the V. cincinnatiensis became nonculturable after approx. 50 day at 4°C. Electron microscopy revealed that the VBNC cells changed from rod to coccoid and decreased in size. Resuscitation of VBNC cells was achieved by temperature upshift in nutrition of yeast extract and peptone by addition of catalase or compound vitamin B. The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the group inoculated with the VBNC cells.
Conclusions:  Vibrio cincinnatiensis VIB287 could enter VBNC state in adverse environments. Resuscitation of VBNC cells occurred by addition of compound vitamin B or catalase to VBNC cells containing nutrient. The resuscitative cells might retain their pathogenicity.
Significance and Impact of the Study:  The study confirmed that V. cincinnatiensis could enter into VBNC state in seawater at low temperature and resuscitated. The resuscitative cells retained their pathogenicity, which may be important in future studies of ecology of V. cincinnatiensis .     

The survival response of Escherichia coli K12 in a natural environment

To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4°C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4°C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28°C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28°C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28°C.