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.     

Effect of gamma radiation on native endolithic microorganisms from a radioactive waste deposit site.

A time-course experiment was conducted to evaluate the effects of gamma radiation on the indigenous microbiota present in rock obtained from Yucca Mountain, Nevada Test Site. Microcosms were constructed by placing pulverized Yucca Mountain rock in polystyrene cylinders. Continuous exposure (96 h) at a dose rate of 1.63 Gy/min was used to mimic the near-field environment surrounding waste canisters. The expected maximum surface dose rate from one unbreached canister designed to contain spent nuclear fuels is 0.06 Gy/min. Considering the current repository packing design, multiple canisters within one vault, the cumulative dose rate may well approach that used in this experiment. The microbial communities were characterized after receiving cumulative doses of 0, 0.098, 0. 58, 2.33, 4.67, 7.01 and 9.34 kGy. Radiation-resistant microorganisms in the pulverized rock became viable but nonculturable (VBNC) after a cumulative dose of 2.33 kGy. VBNC microorganisms lose the ability to grow on media on which they have routinely been cultured in response to the environmental stress imposed (i.e. radiation) but can be detected throughout the time course using direct fluorescence microscopy techniques. Two representative exopolysaccharide-producing isolates from Yucca Mountain were exposed to the same radiation regimen in sand microcosms. One isolate was much more radiation-resistant than the other, but both had greater resistance than the general microbial community based on culturable counts. However, when respiring cell counts (VBNC) were compared after irradiation, the results would indicate much more radiation resistance of the individual isolates and the microbial community in general. These results have significant implications for underground storage of nuclear waste as they indicate that indigenous microorganisms are capable of surviving gamma irradiation in a VBNC state.     

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.     

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.     

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.     

Effects of simulated solar disinfection of water on infectivity of Salmonella typhimurium

To determine whether cells of Salmonella typhimurium rendered nonculturable by simulated solar disinfection retain infectivity for mice. Bacteria suspended in water were exposed to UVA irradiation for up to 8 h. Culturability, determined by colony forming unit and Most Probable Number counts, fell by six log10 units, while cellular activity determined by the Kogure cell elongation test was retained by approximately 5% of the cells present after 8 h. Intraperitoneal doses of nonculturable cells and active but nonculturable (ABNC) cells exceeding the LD50 of the test organism and BALB/c mouse host, respectively, by 4 and 3 orders of magnitude failed to produce detectable infections. Culturable cells that had been irradiated for 1.5 h were less infective (virulent) than their nonirradiated counterparts. Nonculturable and ABNC cells of Salm. typhimurium produced by UVA irradiation do not retain infectivity for mice. Although ABNC cells could be produced by low cost solar disinfection systems, they do not appear to pose a potential infection hazard.     

Characterization and identification of actinomycetes isolated from ‘fired plots’ under shifting cultivation in northeast Himalaya, India

A total of 35 actinomycetes was isolated from soil samples collected after fire operations at agricultural sites under shifting cultivation in northeast India. More than one-half of these isolates were observed in viable but nonculturable (VBNC) state. Five isolates were always seen embedded with slimy bacteria during subculture; 11 morphologically distinct and cultivable isolates were subjected to characterization and identification. The isolates developed circular to irregular colonies of between 3 and 6 mm on tryptone yeast extract agar plates at 28 °C following 7 days of incubation. The isolates could survive at temperatures between 4 and 50 °C (optimum 28 °C), and pH 5–11 (optimum 8). The isolates varied in cell morphology, utilization of carbon sources, sensitivity to antibiotics, and salt tolerance. Based on 16S rRNA sequencing, the isolates revealed maximum similarity to the genus Streptomyces (9), and to Kitasatospora and Nocardia (1 each). Several isolates were found to be positive for production of lytic (chitinase and glucanase) and industrially important (amylase, lipase, and protease) enzymes. The occurrence of actinomycetes in VBNC state and embedded with bacteria was attributed to coping mechanisms associated with these organisms under stress (high temperature) conditions. The cultivable cultures extend the opportunity for further investigations on ecological resilience during fire operations.     

The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.     

The Viable But Nonculturable State of Ralstonia solanacearum May Be Involved in Long-Term Survival and Plant Infection

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.     

Revealing potential functions of VBNC bacteria in polycyclic aromatic hydrocarbons biodegradation

The bioremediation of polycyclic aromatic hydrocarbon (PAH)‐contaminated sites is not running smoothly, because of the lower activity of PAH‐degrading bacteria in actual bioremediation applications. The phenomenon of “viable but nonculturable” (VBNC) state may be a main limiting factor for their poor biodegradation capabilities of PAHs. Due to their abilities of entering into the VBNC state, most of bacterial populations with PAH‐degradation potential remain unculturable. Resuscitation of VBNC bacteria will enhance the degradation capability of indigenous bacteria which will eventually obtain their better capabilities in environmental bioremediation. Although evidences have been presented indicating that resuscitation of VBNC bacteria in polychlorinated biphenyl (PCB)‐contaminated environments not only significantly enhanced PCB degradation, but also obtained novel highly efficient PCB‐degrading bacteria, scanty information is available on the VBNC bacteria in PAH‐contaminated sites. VBNC bacteria, as a vast majority of potential microbial resource could be the repository of novel highly efficient PAH‐biodegraders. Therefore, studies need to be done on resuscitation of VBNC bacteria to overcome key bottlenecks in bioremediation of PAH‐contaminated sites. This mini‐review provides a new insight into the potential functions of VBNC bacteria in PAHs biodegradation.

Significance and Impact of the Study

As the vast majority microbial resource, viable but nonculturable (VBNC) bacteria, which showed their potential functions in polycyclic aromatic hydrocarbons (PAHs) biodegradation, can be of great significance in environmental bioremediation. It is therefore important to resuscitate VBNC bacteria for their better capabilities. Meanwhile, preventing the indigenous functional community from entering into the VBNC state will also maintain the high activity of PAH‐degrading bacteria in actual bioremediation applications. Undoubtedly, much more work needs to be done to reveal indigenous micro‐organisms in the VBNC state from the perspective of environmental functions.     

The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis,as determined by proteome analysis

The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E. faecalis, which is activated in response to multiple environmental stresses.     

Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination.

BACKGROUND: Vibrio alginolyticus is known to enter into a viable but nonculturable (VBNC) state in response to environmental conditions unfavorable to the growth. Cells in VBNC condition pose a public health threat because they are potentially pathogenic. METHODS: We constructed a pathway for the identification of the most significant variables and the characterization of those variables able to discriminate the groups under investigation. Different parameters measured by the image processing software were chosen as the most representative of V. alginolyticus cell morphology (length index for dimension) and metabolic activity (density profile indexes). To detect relationships between the groups of treatment performed, we carried out a principal components analysis (PCA). RESULTS: The PCA analysis indicated that increasing coccoid shape transformation was related to both metabolic and dimension variations, delineating a well defined graph profile. Indeed, we discovered that specific morphological variations occur when cells in the culturable state pass into VBNC condition, namely comma-shaped culturable bacteria are converted into coccoid-shaped VBNC cells. The results were also supported by scanning electron microscopy analysis. CONCLUSIONS: This technique allows the analysis of a large number of vibrio samples in a short period of time. The obtained multiparameter information may complement genetic/molecular analyses facilitating, in an automatic fashion, further studies to evaluate the potential risk of this pathogen in the environment. It may also be a useful tool for large-scale cell biology studies and high content screening.     

Induction of the Viable but Nonculturable State of Ralstonia solanacearum by Low Temperature in the Soil Microcosm and Its Resuscitation by Catalase

Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state.     

Proteomic Analysis of Protein Expression in the Induction of the Viable But Nonculturable State of Vibrio harveyi SF1

Vibrio harveyi has been reported to enter into a viable but nonculturable (VBNC) state. One marine V. harveyi strain, SF1 became nonculturable when incubated in seawater microcosm at 4 °C within 60 days. We investigated protein expression in the exponential phase of V. harveyi SF1 and compared it to the VBNC state. Cytosolic proteins were resolved by two-dimensional polyacrylamide gel electrophoresis using pH 4–7 linear gradients. Among these proteins, sixteen proteins which were strongly downregulated or upregulated in the VBNC cells were identified by MALDI-TOF-TOF mass spectrometry. The results indicated that the differentially expressed proteins were mainly focused on stress response proteins and key components of central and intermediary metabolism, like carbohydrate metabolism, transport, and translation. This study provided clues for understanding the mechanism of adaptation to the VBNC state.     

Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples.

The gfp-tagging method and lux-tagging method were compared to select a better method for verifying a viable but nonculturable (VBNC) state of bacteria in the environment. An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescent protein (GFP). The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi. Using the same method, S. typhi was chromosomally marked with luxAB genes encoding luciferase. The survival of gfp-tagged S. typhi introduced into groundwater microcosms was examined by GFP-based plate count, total cell count, and a direct viable count method. In microcosms containing lux-tagged S. typhi, luminescence-based plate count and the measurement of bioluminescence of each microcosm sample were performed. In microcosms containing lux-tagged S. typhi, viable but nonculturable cells could not be detected by using luminometry. As no distinguishable luminescence signals from the background signals were found in samples containing no culturable cells, a VBNC state of S. typhi could not be verified in lux-based systems. However, comparison between GFP-based direct viable counts and plate counts was a good method for verifying the VBNC state of S. typhi. Because GFP-based direct viable count method provided a direct and precise estimation of viable cells of introduced bacteria into natural environments, it can be used for verifying the VBNC state of bacteria in environmental samples.     

Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor

Escherichia coli O157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate of E. coli O157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction of E. coli O157:H7 Tn7gfp transformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), both E. coli O157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (10(9) and 10(6) E. coli O157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log(10) cells but did not detect culturable cells. These findings indicate that E. coli O157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.     

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