Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.     

Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios

Entry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogen Vibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNC V. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.     

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO₂-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state.     

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.     

Cold-adaptation of Antarctic microorganisms – possible involvement of viable but nonculturable state

Occurrence of the viable but nonculturable (VBNC) form of microorganisms in nature is well known. Evidence available in the literature indicates that low temperature may induce microorganisms to form VBNC cells. The VBNC state of some bacteria, collected from Antarctic lakes, has been recently reported. Hence it is postulated that formation of VBNC cells may be one of the strategies adopted by Antarctic microorganisms to survive in the extreme cold climate. Accepted: 16 October 1999     

A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7

A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e-/GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation of E. coli O157:H7 cells with PP01e-/GFP did not lead to cell lysis, while the propagation of PP01e-/GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e-/GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count.     

Induction of Viable but Nonculturable Escherichia coli O157:H7 by High Pressure CO2 and Its Characteristics

The viable but nonculturable (VBNC) state is a survival strategy adopted by many pathogens when exposed to harsh environmental stresses. In this study, we investigated for the first time that whether high pressure CO₂ (HPCD), one of the nonthermal pasteurization techniques, can induce Escherichia coli O157:H7 into the VBNC state. By measuring plate counts, viable cell counts and total cell counts, E. coli O157:H7 in 0.85% NaCl solution (pH 7.0) was able to enter the VBNC state by HPCD treatment at 5 MPa and four temperatures (25°C, 31°C, 34°C and 37°C). Meanwhile, with the improvement of treatment temperature, the time required for E. coli O157:H7 to enter VBNC state would shorten. Enzymatic activities in these VBNC cells were lower than those in the exponential-phase cells by using API ZYM kit, which were also reduced with increasing the treatment temperature, but the mechanical resistance of the VBNC cells to sonication was enhanced. These results further confirmed VBNC state was a self-protection mechanism for some bacteria, which minimized cellular energetic requirements and increased the cell resistance. When incubated in tryptic soy broth at 37°C, the VBNC cells induced by HPCD treatment at 25°C, 31°C and 34°C achieved resuscitation, but their resuscitation capabilities decreased with increasing the treatment temperature. Furthermore, electron microscopy revealed changes in the morphology and interior structure of the VBNC cells and the resuscitated cells. These results demonstrated that HPCD could induce E. coli O157:H7 into the VBNC state. Therefore, it is necessary to detect if there exist VBNC microorganisms in HPCD-treated products by molecular-based methods for food safety.     

Viability of Selected Microorganisms in Hydrocarbon Fuels

A laboratory study of the viability of selected microorganisms in a hydrocarbon fuel medium was carried out on 19 species of microorganisms, representative of the types found as natural contaminants in aircraft fuels. More species remained viable when inoculated in pure cultures than when inoculated in mixed (composite) cultures. Of the 19 species selected, 10 were still viable after 3 months and 5 were viable after 4 months in the pure culture inoculants. In the complete composite culture inoculant, the bacterial species which were viable at the end of 4 months were the same as those found in the pure culture inoculant. No fungi remained viable in the complete composite cultures after a 3-week period. The microorganisms which remain viable in a hydrocarbon fuel medium are considered indicative of a satisfactory inoculum to be used as a test culture in laboratory analysis of mechanical control techniques.     

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。     

Survival of enterococcal species in aquatic environments

Analysis of the survival ability of faecal streptococci/enterococci in the environment has almost invariably been conducted using the standard culture method (CFU counts) despite the demonstration that these microorganisms are capable of entering a viable but nonculturable (VBNC) state. In this study we evaluated the fate, in terms of culturability and viability, of different enterococcal species under laboratory stress conditions mimicking those of the aquatic environment. The results indicate that enterococcal species may activate two different survival strategies, namely starvation and the VBNC state, depending on the specific environmental condition. Moreover, the different enterococcal species can be divided into three groups on the basis of the time needed to activate the VBNC state and the resuscitation capability. The differences in activation of the two survival strategies and the different kinetics observed among the enterococcal species reaching the VBNC state should be taken into consideration when the microbiological quality of waters has to be evaluated and because of their role as faecal contamination indicators.     

Use of flow cytometry to follow the physiological states of microorganisms in cider fermentation processes

The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes.     

Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes.     

Survival strategy of Erwinia amylovora against copper: induction of the viable-but-nonculturable state

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu(2+) was inoculated with 10(7) CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.     

Influence of pipe materials and VBNC cells on culturable bacteria in a chlorinated drinking water model system

To elucidate the influence of pipe materials on the VBNC (viable but nonculturable) state and bacterial numbers in drinking water, biofilm and effluent from stainless steel, galvanized iron, and polyvinyl chloride pipe wafers were analyzed. Although no HPC (heterotrophic plate count) was detected in the chlorinated influent of the model system, a DVC (direct viable count) still existed in the range between 3- and 4-log cells/ml. Significantly high numbers of HPC and DVC were found both in biofilm and in the effluent of the model system. The pipe material, exposure time, and the season were all relevant to the concentrations of VBNC and HPC bacteria detected. These findings indicate the importance of determining the number of VBNC cells and the type of pipe materials to estimate the HPC concentration in water distribution systems and thus the need of determining a DVC in evaluating disinfection efficiency.     

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.     

Altered cell cycle responses to insulin-like growth factor I, but not platelet-derived growth factor and epidermal growth factor, in senescing human fibroblasts

Human diploid fibroblasts (HDF) were used to study aging-related changes in the proliferative response to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor I (IGF-I, somatomedin-C) in serum-free, chemically defined culture medium. Cell cycle kinetic parameters were determined by using 5-bromodeoxyuridine incorporation and flow cytometric analysis with the DNA stain Hoechst 33258. This allowed analysis of the growth factor response to be focussed exclusively upon of the cycling faction of cells within the culture, even in senescent cell cultures which contained predominantly nondividing cells. PDGF and EGF exert their primary effect upon regulation of the proportion of cycling cells in the culture. The doses of PDGF and EGF that produced a half-maximal cycling fraction, analogous to Km, showed no large or consistent difference between young- and old-passage cells. In contrast, IGF-I primarily affects the rate of transition of cells from G1 into S phase, and the dose of IGF-I which produced a half-maximal rate of G1 exit increased up to 130-fold in older-passage cells. Unexpectedly, supraphysiologic concentrations of IGF-I were found to increase the G1 exit rate of the dividing subpopulation of cells in older-passage cultures to rates higher than those seen in young cultures. In summary, among cells capable of cycling in aging cultures, there were few changes in the regulation of the growth fraction by PDGF and EGF, but there was a greatly increased dependence on IGF-I for regulation of the rate of entry into S phase. The slower growth of the dividing population of cells in aging cultures may be related to a requirement for IGF-I at levels which are greatly above those usually supplied.     

In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus.

Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States. The bacterium occurs naturally in molluscan shellfish, and ingestion of raw oysters is typically the source of human infection. V. vulnificus is also known to enter a viable but nonculturable (VBNC) state, wherein the cells are no longer culturable on routine plating media but can be shown to remain viable. Whether or not this human pathogen remains virulent when entering the VBNC state has not been definitively demonstrated. In this study, the VBNC state was induced through a temperature downshift to 5 degrees C, with cells becoming nonculturable (< 0.1 CFU/ml) within 7 days. As they became nonculturable, virulence was determined by employing an iron overload mouse model. At the point of nonculturability (7 days), injections of the diluted microcosm population resulted in death when < 0.04 CFU was inoculated, although > 10(5) cells in the VBNC state were present in the inoculum. Culturable cells of V. vulnificus, with identification confirmed through PCR, were recovered from the blood and peritoneal cavities of mice which had died from injections of cells present in the VBNC state for at least 3 days. Thus, our data suggest that cells of V. vulnificus remain virulent, at least for some time, when present in the VBNC state and are capable of causing fatal infections following in vivo resuscitation. Our studies also indicate, however, that virulence decreases significantly as cells enter the VBNC state, which may account, at least to some extent, for the decrease in infections caused by this bacterium during winter months.     

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.     

Characterization and resuscitation of viable but nonculturable <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> VIB283

The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4°C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10¹⁰ to 10⁹ CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.