Viable But Non-culturable Formation Research Articles

Resistance to preservatives and the viable but non-culturable state formation of Asaia lannensis in flavored syrups.

Food security is a crucial issue that has caused extensive concern, and the use of food flavors has become prevalent over time. we used the molecular biological techniques, preservative susceptibility testing, viable but non-culturable (VBNC) state induction testing, and a transcriptome analysis to examine the bacterial contamination of favored syrup and identify the causes and develop effective control measures. The results showed that Asaia lannensis WLS1-1 is a microorganism that can spoil food and is a member of the acetic acid bacteria families. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests showed that WLS1-1 was susceptible to potassium sorbate (PS), sodium benzoate (SB), and sodium sulffte (SS) at pH 4.0. It revealed a progressive increase in resistance to these preservatives at increasing pH values. WLS1-1 was resistant to PS, SB and SS with an MIC of 4.0, 2.0 and 0.5 g/L at pH 5.0, respectively. The MIC values exceed the maximum permissible concentrations that can be added. The induction test of the VBNC state demonstrated that WLS1-1 lost its ability to grow after 321 days of PS induction, 229 days of SB induction and 52 days of SS induction combined with low temperature at 4°C. Additionally, laser confocal microscopy and a propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay showed that WLS1-1 was still alive after VBNC formation. There were 7.192 ± 0.081 (PS), 5.416 ± 0.149 (SB) and 2.837 ± 0.134 (SS) log10(CFU/mL) of viable bacteria. An analysis of the transcriptome data suggests that Asaia lannensis can enter the VBNC state by regulating oxidative stress and decreasing protein synthesis and metabolic activity in response to low temperature and preservatives. The relative resistance of Asaia lannensis to preservatives and the induction of the VBNC state by preservatives are the primary factors that contribute to the contamination of favored syrup by this bacterium. To our knowledge, this study represents the first evidence of the ability of Asaia lannensis to enter the VBNC state and provides a theoretical foundation for the control of organisms with similar types of activity.

Plasma-generated nitric oxide water: A promising strategy to combat bacterial dormancy (VBNC state) in environmental contaminant Micrococcus luteus

The viable but non-culturable (VBNC) is an inactive state, and certain bacteria can enter under adverse conditions. The VBNC state challenges the environment, food safety, and public health since VBNCs may resuscitate and pose a risk to human health. The aim of this study was to investigate the effect of plasma-generated nitric oxide water (PG-NOW) on airborne contaminant Micrococcus luteus (M. luteus) and examine its potential to induce the VBNC state. The essential conditions for bacteria to enter VBNC state are low metabolic activity and rare or no culturable counts. The results indicated that PG-NOW effectively eliminates M. luteus, and the remaining bacteria are in culturable condition. Moreover, the conventional cultured-based method combined with a propidium iodide monoazide quantitative PCR (PMAxxTM-qPCR) showed no significant VBNC induction and moderate culturable counts. Results from the qPCR revealed that gene levels in PG-NOW treated bacteria related to resuscitation-promoting factors, amino acid biosynthesis, and fatty acid metabolism were notably upregulated. PG-NOW inactivated M. luteus showed negligible VBNC formation and alleviated infection ability in lung cells. This study provides new insights into the potential use of PG-NOW reactive species for the prevention and control of the VBNC state of M. luteus.

Effect of preliminary stresses on the induction of viable but non-culturable Escherichia coli O157:H7 NCTC 12900 and Staphylococcus aureus ATCC 6538

As a novel non-thermal pasteurization technology, high pressure carbon dioxide (HPCD) has been used in food processing. However, it could induce microorganisms into a viable but nonculturable (VBNC) state, posing a potential risk to food safety and public health. In this study, we attempted to investigate the effect of various preliminary stresses including cold, heat, osmosis, acidity and oxidation on HPCD-induced VBNC formation. The results indicated that there was no effect of preliminary stresses on VBNC Staphylococcus aureus induction. However, heat, acidity and long-term (24 h) cultivation preadaptation could significantly increase the VBNC E. coli production induced by HPCD. Transcriptome analysis revealed that genes involved in ATP production were significantly decreased in these three stress-treated cells, and further ATP levels determination revealed that the ATP levels of the cell were significantly decreased after heat, acidity and long-term cultivation preadaptation, implying the decrease of ATP level caused by these stresses might be the reason for increasing VBNC production. To further study the relationship between ATP level and VBNC cell ratios after preadaptation. We artificially decreased the ATP levels, and detect their VBNC ratios after HPCD treatment. We found that with the ATP concentration decreasing after exposure to carbonyl cyanide m-chlorophenyl hydrazine (CCCP), the VBNC ratios were increased after HPCD treatment, indicating that the ATP contents were highly negatively correlated with VBNC ratios. This study proved that the preadaptation of heat, acidity and long-term cultivation could promote VBNC induction by decreasing the intracellular ATP level. In general, the obtained result gave the instruction about the storage environment for food materials, helped to further develop and optimize the HPCD processing to prevent VBNC formation and accelerate the development of HPCD technology in food industry.

Current Perspectives on Viable but Non-Culturable Foodborne Pathogenic Bacteria: A Review.

Foodborne diseases caused by foodborne pathogens pose risks to food safety. Effective detection and efficient inactivation of pathogenic bacteria has always been a research hotspot in the field of food safety. Complicating these goals, bacteria can be induced to adopt a viable but non-culturable (VBNC) state under adverse external environmental stresses. When in the VBNC state, pathogens cannot form visible colonies during traditional culture but remain metabolically active and toxic. The resulting false negative results in growth-related assays can jeopardize food safety. This review summarizes the latest research on VBNC foodborne pathogens, including induction conditions, detection methods, mechanism of VBNC formation, and possible control strategies. It is hoped that this review can provide ideas and methods for future research on VBNC foodborne pathogenic bacteria.

Identification of determinants for entering into a viable but nonculturable state in Vibrio alginolyticus by Tn-seq.

The viable but nonculturable (VBNC) state is a dormant state of nonsporulating bacteria that enhances survival in adverse environments. Systematic genome-wide research on the genetic basis of VBNC formation is warranted. In this study, we demonstrated that the marine bacterium Vibrio alginolyticus lost culturability but remained viable and entered into the VBNC state when exposed to low nutrient concentrations for prolonged periods of time. Using transposon-insertion sequencing (Tn-seq), we identified 635 determinants governing the formation of the VBNC state, including 322 genes with defective effects on VBNC formation and 313 genes contributing to entry into the VBNC state. Tn-seq analysis revealed that genes involved in various metabolic pathways were shown to have an inhibitory effect on VBNC formation, while genes related to chemotaxis or folate biosynthesis promoted entry into the VBNC state. Moreover, the effects of these genes on the formation of VBNC were validated with the growth of deletion mutants of eight selected genes under nutrient-limited conditions. Interestingly, fleQ and pyrI were identified as essential for entry into the VBNC state, and they affected the formation of the VBNC state independent of RpoE or ToxR regulation. Collectively, these results provide new insights into the mechanism of VBNC formation. KEY POINTS: • Vibrio alginolyticushas the ability to enter into the VBNC state under low nutrient conditions at lowtemperature. • The 635 determinants for entry into the VBNC state were systematically identified bytransposon-insertion sequencing. • PyrI and FleQ were validated to play significant roles in the formation of the VBNC state.

Acid shock protein Asr induces protein aggregation to promote E. coli O157:H7 entering viable but non-culturable state under high pressure carbon dioxide stress

Under stressful conditions, bacteria can enter viable but non-culturable (VBNC) state to survive. VBNC cells lost ability to grow on routine culture medium but are still alive and may revive in suitable conditions. The revived cells can consume nutrients or produce toxins, leading to food spoilage or human illness, posing great risk to food safety and public health. Previously, we have reported that high pressure carbon dioxide (HPCD), an environment-friendly sterilization technology, can induce VBNC formation. However, the underlying mechanism is unclear. By performing a comprehensive transcriptomic analysis, we revealed that HPCD initiated high expression of asr, encoding an acid shock protein, could promote VBNC formation of E. coli O157:H7. Quantitative reverse transcription PCR analysis suggested that high expression of asr (i) inhibited acid resistance (AR) systems, resulting in endogenous proton accumulation; (ii) inhibited hchA expression, a protein stabilizing factor. The two effects resulted in endogenous protein aggregation, which was highly correlated to VBNC formation. Accordingly, HPCD-stressed cells exhibited decreased efficiency of electron transfer chain and ATP production, which was also contributory to cytoplasmic protein aggregation. Taken together, HPCD-initiated high expression of Asr coupled with decreased ATP production led to protein aggregation, finally promoted the cells to enter VBNC state.

Extracellular pH decline introduced by high pressure carbon dioxide is a main factor inducing bacteria to enter viable but non-culturable state

High pressure carbon dioxide (HPCD) has been used in food processing as a non-thermal pasteurization technology. However, the potential of HPCD to induce viable but non-culturable (VBNC) cells limits its application. The objective of this study was to explore the roles of extracellular pH of 3 (pHex3) and high pressure (HP) of HPCD during VBNC induction and the underlying molecular mechanism. By using the model organism Escherichia coli O157:H7, we found that the combined effects of pHex3 and HP could mimic the effect of HPCD for VBNC induction. Further investigation of the individual effect of pHex3 and HP on VBNC induction revealed that pHex3 could induce a higher proportion of VBNC cells with a slower induction rate compared with HPCD, whereas HP was unable to induce VBNC formation. Notably, the cells treated by pHex3 and HPCD had similar morphological changes, and VBNC cells induced by pHex3 and HPCD had similar stress resistance characteristics. These results strongly indicated that pHex3 introduced by HPCD was a main factor for VBNC induction. Additionally, we found that HP played the role in accelerating VBNC formation in the process of HPCD treatment. Transcriptomic analysis revealed 85, 263 and 529 differentially expressed genes (DEGs) for HP-, pHex3- and HPCD-treated cells compared with untreated ones. 59 DEGs shared by pHex3 and HPCD treatment might be responsible for VBNC induction, and they were mainly involved in cellular transport and localization.

Roles of virulence regulator ToxR in viable but non-culturable formation by controlling reactive oxygen species resistance in pathogen Vibrio alginolyticus

Under adverse circumstances, bacteria enter the viable but non-culturable (VBNC) state, a dormancy-like state for survival. The altered gene regulation underlying the entry of the VBNC state has not yet been well elucidated. Here, we reported that a subpopulation of cells (23.8 %) in Vibrio alginolyticus cultures enters the VBNC state in response to nutrient limitation at alkaline pH. The proteolysis of pivotal virulence regulator ToxR at these conditions is associated with VBNC formation. Meantime, ToxR abrogation impaired the mobility and the expression of virulence-associated genes, resulting in attenuated virulence in V. alginolyticus. RNA-seq and ChIP-seq analyses of the cells grown in VBNC-inducing conditions revealed that ToxR directly controls the expression of ∼8 genes including ahpC and dps involved in reactive oxygen species (ROS) resistance. ToxR binds to the promoter regions of kdgR, ppiC, ahpC, and dps and further controls their respective expression under oxidative stress conditions. The cells with impaired ToxR accumulated detrimental intracellular ROS. Moreover, these genes contribute to bacterial culturability as their in-frame deletion strains exhibiting severely decreased plate counts and the complementary strain showed rescued viability. Collectively, this study revealed the role of ToxR in switching on the VBNC state by sensing unfavorable environmental signals such as endogenous ROS (hydrogen peroxide, H2O2) in V. alginolyticus and provided mechanistic insights into Vibrio lifestyle adaptation in the marine environment.

In silico analysis revealing CsrA roles in motility-sessility switching and tuning VBNC cells in Vibrio parahaemolyticus

The formation of biofilms is a survival strategy employed by bacteria to help protect them from changing or unfavourable environments. In this research, 319 genes which govern biofilm formation in V. parahaemolyticus, as reported in 1,625 publications, were analysed using protein-protein-interaction (PPI) network analysis. CsrA was identified as a motility-sessility switch and biofilm formation regulator. Through robust rank aggregation (RRA) analysis of GSE65340, the generation of viable but non-culturable (VBNC) cells that may enhance cell tolerance to stress, was found to be associated with the TCA cycle and carbon metabolism biological pathways. The finding that CsrA is likely to play a role in the development of VBNC cells improves understanding of the molecular mechanisms of VBNC formation in V. parahaemolyticus and contributes to on-going efforts to reduce the hazard posed by this foodborne pathogen.

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.

First Report on the Rapid Detection and Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) in Viable but Non-culturable (VBNC) Under Food Storage Conditions.

Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.

Synergistic role of abiotic factors driving viable but non-culturable Vibrio cholerae.

Vibrio cholerae O1, a natural inhabitant of estuarine environments, is found in a dormant, viable but non-culturable (VBNC) state during interepidemic periods. Although the individual roles of abiotic factors affecting VBNC formation have been extensively studied, their interplay in driving this phenomenon remains largely unaddressed. Here, we identified that major abiotic factors synergize with low nutrient conditions governing entry of cells into the VBNC state. Specifically, V. cholerae cells exposed to a combination of alkaline pH and high salinity under aeration at low temperatures (VBNC-inducing conditions) synergize to facilitate rapid entry into VBNC, whereas the opposite conditions prevented entry into the state. The major virulence regulator ToxR, and the stringent response protein RelA played opposing roles, repressing and facilitating VBNC entry respectively. Further, VBNC-inducing conditions negated the effects of ToxR and RelA, facilitating rapid formation of VBNC cells. In summary, this study highlights the synergy between critical abiotic factors and identified ToxR and RelA as two associated regulators, allowing for the persistence of V. cholerae in aquatic environments. Insights obtained in this study will help better understand environmental survival non-sporulating bacteria and transmission of facultative bacterial pathogens.

Formation and Control of the Viable but Non-culturable State of Foodborne Pathogen Escherichia coli O157:H7.

As a common foodborne pathogen, Escherichia coli O157:H7 produces toxins causing serious diseases. However, traditional methods failed in detecting E. coli O157:H7 cells in the viable but non-culturable (VBNC) state, which poses a threat to food safety. This study aimed at investigating the formation, control, and detection of the VBNC state of E. coli O157:H7. Three factors including medium, salt, and acid concentrations were selected as a single variation. Orthogonal experiments were designed with three factors and four levels, and 16 experimental schemes were used. The formation of the VBNC state was examined by agar plate counting and LIVE/DEAD® BacLightTM bacterial viability kit with fluorescence microscopy. According to the effects of environmental conditions on the formation of the VBNC state of E. coli O157:H7, the inhibition on VBNC state formation was investigated. In addition, E. coli in the VBNC state in food samples (crystal cake) was detected by propidium monoazide–polymerase chain reaction (PMA-PCR) assays. Acetic acid concentration showed the most impact on VBNC formation of E. coli O157:H7, followed by medium and salt concentration. The addition of 1.0% acetic acid could directly kill E. coli O157:H7 and eliminate its VBNC formation. In crystal cake, 25, 50, or 100% medium with 1.0% acetic acid could inhibit VBNC state formation and kill E. coli O157:H7 within 3 days. The VBNC cell number was reduced by adding 1.0% acetic acid. PMA-PCR assay could be used to detect E. coli VBNC cells in crystal cake with detection limit at 104 CFU/ml. The understanding on the inducing and inhibitory conditions for the VBNC state of E. coli O157:H7 in a typical food system, as well as the development of an efficient VBNC cell detection method might aid in the control of VBNC E. coli O157:H7 cells in the food industry.

The Association of Cell Division Regulated by DicC With the Formation of Viable but Non-culturable Escherichia coli O157:H7.

The viable but non-culturable (VBNC) state, in which bacteria fail to grow on routine culture media but are actually alive, has been widely recognized as a strategy adopted by bacteria to cope with stressful environments. However, little is known regarding the molecular mechanism of VBNC formation. Here, we aimed to elucidate the specific roles of cell division regulatory proteins and the cell growth rate during VBNC Escherichia coli O157:H7 formation. We have previously found that expression of dicC is reduced by 20.08-fold in VBNC E. coli O157:H7 compared to non-VBNC cells. Little is known about DicC except that it, along with DicA, appears to act as a regulator of cell division by regulating expression of the cell division inhibitor DicB. First, our results showed that the VBNC cell number increased in the ΔdicC mutant as well as the DicA-overexpressing strain but decreased in the DicC-overexpressing strain induced by high-pressure carbon dioxide, acid, and H2O2. Furthermore, the growth rates of both the DicA-overexpressing strain and the ΔdicC mutant were higher than that of the control strain, while DicC-overexpressing strain grew significantly more slowly than the vector strain. The level of the dicB gene, regulated by dicA and dicC and inhibiting cell division, was increased in the DicC-overexpressing strain and decreased in the ΔdicC mutant and DicA-overexpressing strain, which was consistent with the growth phenotypes. In addition, the dwarfing cell morphology of the ΔdicC mutant and DicA-overexpressing strain were observed by SEM and TEM. Taken together, our study demonstrates that DicC negatively regulates the formation of the VBNC state, and DicA enhances the ability of cells to enter the VBNC state. Besides, the cell growth rate and dwarfing cell morphology may be correlated with the formation of the VBNC state.

Uncovering the molecular basis of viable but non culturable (VBNC) cells

Viable but non-culturable (VBNC) cells are cells that are metabolically active, but are unable to form colonies on standard culture media. Following environmental stimuli, such as temperature upshift, some VBNC cells can ‘resuscitate’ restoring their ability to grow on media. Currently, over 80 bacterial species are reported to enter the VBNC state. The ability of VBNC cells to go undetected by conventional microbiological practices could lead to an underestimation of total viable cells in environmental and clinical samples. Furthermore, their capacity to retain virulence potential and their ability for renewed metabolic activity means the VBNC state in pathogens may pose a risk to human health and thus warrants further investigation. This research project has investigated the ability of the human pathogen Vibrio parahaemolyticus to form VBNC cells when exposed to stressful conditions. V. parahaemolyticus is a bacterium that is present in the marine environment and can be found in seawater, shellfish (such as oysters and mussels) and in crustacea (such as crab). This bacterium is the leading cause of seafood associated gastroenteritis worldwide and often results in watery/bloody diarrhea and vomiting. We have developed robust models to generate V. parahaemolyticus VBNC cells in the laboratory and report that different sub populations of VBNC cells can occur based upon their metabolic activity, cell shape and the ability to grow and cause disease in Galleria mellonella. Using mass spectrophotometry we have identified several proteins which may play roles in VBNC formation and resuscitation in V. parahaemolyticus.

RNA-seq-based monitoring of gene expression changes of viable but non-culturable state of Vibrio cholerae induced by cold seawater.

Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the acute diarrheal disease cholera. Entry into a viable but non-culturable (VBNC) state is a survival strategy by which V. cholerae withstands natural stresses and is important for the transition between the aquatic and host environments during the V. cholerae life cycle. In this study, the formation of VBNC V. cholerae induced by cold seawater exposure was investigated using RNA sequencing (RNA-seq). The analysis revealed that the expression of 1420 genes was changed on VBNC state formation. In the VBNC cells, genes related to biofilm formation, chitin utilization and stress responses were upregulated, whereas those related to cell division, morphology and ribosomal activity were mainly downregulated. The concurrent acquisition of a carbon source and the arrest of cell division in cells with low metabolic activity help bacteria increase their resistance to unfavourable environments. Moreover, two transcriptional regulators, SlmA and MetJ, were found to play roles in both VBNC formation and intestinal colonization, suggesting that some genes may function in both processes. This acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance and may help control future cholera infections and outbreaks.

Modeling the effect of light and salinity on viable but non-culturable (VBNC) Enterococcus

Enterococci have been recommended as suitable bacteria indicators for assessing the microbial quality of recreational waters. However, recent studies have shown that bacteria, including enterococci, are able to enter a viable but non-culturable (VBNC) state under environmentally stressed conditions, where they may remain undetected if culture-based methods are employed. To appreciate the extent of transformation of these cells in surface waters, a model Enterococcus organism, E. faecalis, was examined in laboratory controlled microcosms under different light and salinity conditions. Cells were detected by both standard culture-based and PMA-qPCR (propidium monoazide quantitative PCR) methods so that the VBNC cells could be enumerated. The decay rates from the culture based method (kc) and PMA-qPCR method (kp) were established for the different conditions. In general, the kC values (ranging from 0.0088 hr−1 to 0.9755 hr−1) were always higher than the kP values (0.0019 hr−1 to 0.2373 hr−1), implying that cells were able to retain their viability for much longer periods than what is shown by the culture-based method. In both cases, the k values generally showed an increasing trend with an increase in light irradiation, implying greater die-off with light. For freshwater microcosms, the kp values were 3–6 times lower than the kc values for different irradiation conditions, whereas for seawater the difference was up to 12 times, showing that E. faecalis adapts well to seawater. The kinetic data were used to develop models to describe the dynamics of VBNC formation in natural waters. At low light intensities (less than about 20 Wm-2), the proportion of VBNC cells was found to steadily increase to as high as 50%, even after 4 days. However, at higher light levels, this proportion was achieved more quickly (less than 5 h) but also diminished more rapidly. Hence, at high light levels, the percentage of VBNC cells is expected to be significant only for a few hours, whereas at low light levels, the VBNC cells can be expected to be present for a long period of time. These results have implications on the interpretation of microbial water quality data that are based on culture based methods.