Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state

Changes in the outer membrane subproteome of Escherichia coli along the transition to the viable but nonculturable state (VBNC) were studied. The VBNC state was triggered by exposure of E. coli cells to adverse conditions such as aquatic systems, starvation, suboptimal temperature, visible light irradiation and seawater. The subproteome, obtained according to Molloy et al ., was analysed at the beginning of exposure (inoculum, phase 1), after a variable exposure time (95% of population culturable, phase 2) and when populations were mainly in the VBNC state (95% of cells VBNC, phase 3). Proteome changes were dependent on adverse conditions inducing the transition and were detected mainly in phase 2. The permanence of E. coli cells in seawater under illumination conditions entailed a dramatic rearrangement of the outer membrane subproteome involving 106 new spots, some of which could be identified by peptide fingerprinting. However, proteins exclusive to the VBNC state were not detected.     

Changes in intrinsic fluorescence during the production of viable but nonculturable Escherichia coli

The potential of intrinsic fluorescence spectroscopy to detect and differentiate viable but nonculturable bacteria in the presence of culturable bacteria was explored. Escherichia coli cells, starved for 210 days in nutrient-free normal saline, show new fluorescence emissions near 400 and 440 nm, and reduced emission near 340 nm. Received 7 July 1997/ Accepted in revised form 26 November 1997     

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.     

Aeromonas-Acanthamoeba interaction and early shift to a viable but nonculturable state of Aeromonas by Acanthamoeba

Aims: To investigate the hypothesis that amoeba may comprise a significant environmental reservoir for Aeromonas, Acanthamoeba–Aeromonas interaction experiments were performed. Methods and Results: Acanthamoeba were grown in monoculture and co-cultures with three different species of Aeromonas. Survival, invasion and viable but nonculturable state experiments were performed. We showed that at a low initial bacterial cell density, growth of Aeromonas spp. was inhibited by Acanthamoeba castellanii, while A. castellanii growth was unaffected. In contrast, a high initial bacterial cell density, Aeromonas hydrophila AEW44 and Aeromonas veronii biovar sobria AEW104 suppressed the growth of A. castellanii. Fluorescent and phase-contrast microscopic observations of GFP tagged Aer. hydrophila AEW44 demonstrated that the bacterial cells aggregated on A. castellanii cells after 15 min of incubation and internalized. Aeromonas hydrophila AEW44 cells were found to be actively moving. Interestingly, Aer. hydrophila AEW44 cells shifted more rapidly to a viable but nonculturable form when co-cultured with A. castellanii than in monoculture. Conclusions: We demonstrated that Aeromonas spp. are able to interact with and to infect the protozoan A. castellanii under laboratory conditions. Significance and Impact of the Study: Free-living amoeba might play a role as reservoir for Aeromonas, and thus may increase the transmission of Aeromonas by acting as a vehicle.     

Maintenance of pathogenicity during entry into and resuscitation from viable but nonculturable state in Aeromonas hydrophila exposed to natural seawater at low temperature

AIMS: To investigate the fate of Aeromonas hydrophila pathogenicity when cells switch, in nutrient-poor filtered sterilized seawater, between the culturable and nonculturable state. METHODS AND RESULTS: Aeromonas hydrophila ATCC 7966, rendered non culturable within 50-55 days of exposure to marine stress conditions, was tested for its ability to maintain haemolysin and to adhere to McCoy cells. Results showed that pathogenicity was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by the Kogure cell elongation test. However, this loss is only temporary because, following temperature shift from 5 to 23 degrees C, multiple biological activities of recovered Aer. hydrophila cells, which include their ability to lyse human erythrocytes and to attach and destroy McCoy cells were regained. During the temperature-induced resuscitation, constant total cell counts were observed. Moreover, no significant improvement in recovery yield was obtained on brain-heart infusion (BHI) agar plates amended with catalase. We suggest that in addition to the growth of the few undetected culturable cells, there is repair and growth of some mildly injured viable but nonculturable cells. CONCLUSIONS: The possibility that nonculturable cells of normally culturable Aer. hydrophila in natural marine environment may constitute a source of infectious diseases posing a public health problem was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when Aer. hydrophila cells are released in natural seawater with careful attention to the conditions in which surrounding waters gradually become warmer in late summer/early autumn.     

Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses

AIMS: This work analysed factors that influence the induction of viable but nonculturable (VBNC) state in the common enteric pathogen, Vibrio parahaemolyticus. The susceptibility of the VBNC cells to environmental stresses was investigated. METHODS AND RESULTS: Bacterium was cultured in tryptic soy broth-3% NaCl medium, shifted to a nutrient-free Morita mineral salt-0.5% NaCl medium (pH 7.8) and further incubated at 4 degrees C in a static state to induce the VBNC state in 28-35 days. The culturability and viability of the cells were monitored by the plate count method and the Bac Light viable count method, respectively. Cells grown at the optimum growth temperature and in the exponential phase better induced the VBNC state than those grown at low temperature and in the stationary phase. Low salinity of the medium crucially and markedly shortened the induction period. The VBNC cells were highly resistant to thermal (42, 47 degrees C), low salinity (0% NaCl), or acid (pH 4.0) inactivation. CONCLUSIONS: Optimal conditions for inducing VBNC V. parahaemolyticus were reported. The increase in resistance of VBNC V. parahaemolyticus to thermal, low salinity and acidic inactivation verified that this state is entered as part of a survival strategy in an adverse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods for inducing VBNC V. parahaemolyticus in a markedly short time will facilitate further physiological and pathological study. The enhanced stress resistance of the VBNC cells should attract attention to the increased risk presented by this pathogen in food.     

Formation of nonculturable Escherichia coli in drinking water

AIMS: To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. METHODS AND RESULTS: Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp-tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20-60 days in nondisinfected drinking water resulted in detection of only 0.7-5% of the initial E. coli population using standard cultivation methods, whereas 1-20% could be resuscitated to a culturable state, and 17-49% could be clearly detected using cultivation-independent methods. CONCLUSIONS: Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10-20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. SIGNIFICANCE AND IMPACT OF THE STUDY: The existence of substrate-responsive but nonculturable cells should be considered when evaluating the survival potential of E. coli in nondisinfected drinking water.     

Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells

Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells.     

Evidence for viable but nonculturable yeasts in botrytis-affected wine

AIMS: In Botrytis-affected wine, high concentrations of SO2 are added to stop the alcoholic fermentation and to stabilize the wine. During maturing in barrels or bottle-ageing, an unwanted refermentation can sometimes occur. However, results of the usual plate count of wine samples at the beginning of maturing suggest wine microbial stability. The aim of this study was to investigate the mode of yeasts survival after the addition of SO2 and to identify surviving yeasts. METHODS AND RESULTS: Using direct epifluorescence technique, we observed the behaviour of cells after SO2 addition and compared the cell number determined by this method with the result of plate counts. The persistent yeast species were identified using two methods: polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-denaturing gradient gel electrophoresis. They were identified as Saccharomyces cerevisiae and Candida stellata, and after few months of maturing, other spoiling yeasts appeared, like Rhodotorula mucilaginosa or Zygosaccharomyces bailii. CONCLUSIONS: All characteristics of the cells lead to the conclusion that yeast persisted in wine in a viable but nonculturable-like state (VBNC). Suppression of the effect of free-SO2 did not lead to the resuscitation of the cells; however, another method proved the capacity of the cells to exit from the VBNC-like state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of the presence of VBNC-like yeasts in wine. The 'refermentation' phenomenon is probably due to the exit of the VBNC state.     

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.     

Viability kinetics, induction, resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio cholerae O1 in freshwater microcosm

Aim: To study the induction of a viable but nonculturable (VBNC) state in Vibrio cholerae O1 in freshwater, in response to cold temperatures (4°C) and starvation. Methods and Results: Vibrio cholerae O1 cells were inoculated in freshwater microcosm and incubated at 4°C. The cells became coccoid, rugose and subsequently nonculturable by day 16 on tryptic soy agar (TSA) and by day 23 on TSA‐SP, while 87 and 65% of the cells retained their membrane integrity, respectively. Viable cells were observed until day 30 using direct fluorescent antibody–direct viable count method. In vitro resuscitation was demonstrated by temperature upshift. Utilizing 16S rRNA as an endogenous control, the DNA pol II (27·43‐fold), fliG (12·44‐fold), ABC transporter (27·11‐fold), relA (60·76‐fold) and flaC (15·29‐fold) were significantly up‐regulated in VBNC cells, while the expression of fadL‐3 was comparable. The expression of DNA pol II, fliG, ABC transporter, relA and flaC was 3·3, 1·1, 5·9, 5·8 and 1·2‐fold, respectively, for resuscitated cells. VBNC cells were found to be virulent, as ctxA and tcpA were expressed. Conclusions: Vibrio cholerae undergoes both phenotypic alteration and genotypic modulation to protect itself from stress in freshwater. Significance and Impact of the Study:: Induction and resuscitation of the VBNC state in freshwater is important for an understanding of the epidemiology of cholera in the freshwater environment.     

Degradation of 16S rRNA and attributes of viability of viable but nonculturable probiotic bacteria

Aims:  To assess the stability of 16S rRNA of viable but nonculturable (VBNC) probiotics during storage when compared with different attributes of viability.
Methods and Results:  Levels of RNA of the probiotic strains Bifidobacterium longum 46, B. longum 2C and B. animalis subsp. lactis Bb-12 were monitored during storage in fermented and nonfermented foods. Cells which gradually lost their culturability in fermented products retained high level of rRNA, whereas rRNA of acid-killed control cells decreased at faster rate. Furthermore, the viability of B. longum 2C was monitored during storage by measuring changes in reductase activity, cytoplasmic membrane integrity and esterase activity using a flow cytometer. All of the culture-independent viability assays suggested that the cells remained viable during storage. In nonfermented media, the observed losses in culturability were smaller, and the changes in cell counts were comparable with the changes in rRNA levels.
Conclusions:  Viable but nonculturable probiotics maintain high levels of rRNA and retain properties of viable bacteria including reductase activity. Quantification of 16S rRNA complements culture-independent viability assays.
Significance and Impact of the Study:  Culture-independent viability assays allow the detection of VBNC probiotics, and can be used parallel to conventional culture-dependent methods to obtain accurate information on probiotic viability.     

Beta-D-glucuronidase activity assay to assess viable Escherichia coli abundance in freshwaters

AIMS: The relationships between the beta-D-glucuronidase (GLUase) activity, the abundance of culturable Escherichia coli and the number of viable E. coli were investigated in river and wastewater samples. METHODS AND RESULTS: GLUase activity was measured as the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide. Culturable E. coli were enumerated by the most probale number (MPN) microplate method. Viable E. coli were estimated by fluorescent in situ hybridization (FISH) coupled with a procedure of viability testing (DVC-FISH procedure). Significant correlations were found between the log of GLUase activity and both, the log culturable E. coli and the log of viable E. coli. CONCLUSIONS: GLUase activity per viable E. coli gave a broadly constant value from low to highly contaminated waters while GLUase activity per culturable E. coli strongly increased at low contaminated waters because of an underestimation of the number of active E. coli by the culture-based method. SIGNIFICANCE AND IMPACT OF THE STUDY: GLUase activity is a reliable parameter for the rapid quantification of viable E. coli in waters.     

Isolation of a Vibrio cholerae transposon-mutant with an altered viable but nonculturable response

Abstract We have isolated more than 2500 mutants of Vibrio cholerae by using transposon mutagenesis. Mutants were screened under low nutrient conditions in artificial seawater for an altered viable but nonculturable response, compared to the wild-type. Mutant JR09H1 entered the viable but nonculturable state more rapidly than the wild-type at both 25°C and 4°C.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Demonstration of viable but nonculturable Vibrio cholerae O1 in fresh water environment of India using ciprofloxacin DFA-DVC method

Aim: To demonstrate the presence of culturable and nonculturable viable pathogenic Vibrio cholerae O1 in fresh water environments of a cholera‐endemic region in India. Methods and Results: Conventional culture and ciprofloxacin DFA–DVC were utilized to investigate the existence of V. cholerae O1. We isolated pathogenic culturable V. cholerae O1 from water samples collected from cholera‐affected areas. No culturable V. cholerae O1 was isolated from water and plankton samples from natural fresh water bodies. Ciprofloxacin was used for DFA–DVC as V. cholerae O1 are 100% resistant to nalidixic acid in our region. The viable but nonculturable O1 cells were demonstrated in 2·21 and 40·69% samples from natural water bodies and cholera‐affected areas, respectively. Conclusion: Vibrio cholerae O1 VBNC could be demonstrated using modified DFA–DVC technique. Ciprofloxacin is preferable to nalidixic acid for DVC in view of existing high‐level resistance to nalidixic acid in cholera‐endemic areas. Significance and Impact of the study: We endorse that for public health surveillance, cholera outbreak investigation and disease control water samples in addition to culture should be tested for V. cholerae using DFA–DVC.     

Morphology of the viable but nonculturable Vibrio cholerae as determined bt the freeze fixation technique

Abstract The morphology of the nonculturable Vibrio cholerae strain TSI-4 was examined by the freeze fixation technique of electron microscopy and subsequently four unique structures were found in the fine structure s of this bacterium. The size of the cell was about 2 3 of the growing cell. Although the cell was observed to have an outer membrane as well as the cell membrane and cytoplasm, the outer membrane was undulated and had a surface layer of fine fibers. The peptidoglycan layer was thick and more electron dense than that of normal cells.