Viable But Nonculturable State Research Articles

Understanding the transition to viable but non-culturable state in Escherichia coli W3110: a comprehensive analysis of potential spectrochemical biomarkers.

The viable but non-culturable (VBNC) state is considered a survival strategy employed by bacteria to endure stressful conditions, allowing them to stay alive. Bacteria in this state remain unnoticed in live cell counts as they cannot proliferate in standard culture media. VBNC cells pose a significant health risk because they retain their virulence and can revive when conditions normalize. Hence, it is crucial to develop fast, reliable, and cost-effective methods to detect bacteria in the VBNC state, particularly in the context of public health, food safety, and microbial control assessments. This research examined the biomolecular changes in Escherichia coli W3110 induced into the VBNC state in artificial seawater under three different stress conditions (temperature, metal, and antibiotic). Initially, confirmation of VBNC cells under various stresses was done using fluorescence microscopy and plate counts. Subsequently, lipid peroxidation was assessed through the TBARS assay, revealing a notable increase in peroxidation end-products in VBNC cells compared to controls. ATR-FTIR spectroscopy and chemomometrics were employed to analyze biomolecular changes, uncovering significant spectral differences in RNA, protein, and nucleic acid concentrations in VBNC cells compared to controls. Notably, RNA levels increased, while protein and nucleic acid amounts decreased. ROC analyses identified the 995cm- 1 RNA band as a consistent marker across all studied stress conditions, suggesting its potential as a robust biomarker for detecting cells induced into the VBNC state under various stressors.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.

The risk of viable but non-culturable (VBNC) enterococci and antibiotic resistance transmission during simulated municipal sludge composting

Sludge composting is a sludge resource utilization method that can reduce pollutants, such as pathogens. Enterococci are regarded as more reliable and conservative indicators of pathogen inactivation than fecal coliforms, which are typically used as indicators of fecal pollution. Non-spore pathogenic bacteria may enter a viable but non-culturable (VBNC) state during composting, leading to residual risk. The VBNC status of bacteria is related to their survival during composting. However, the survival mechanisms of enterococci during sludge composting remain unclear. Therefore, this study aimed to investigate the VBNC state of enterococci in different phases of simulated sludge composting and the fate of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during the composting process. This study is expected to provide a basis for subsequent exploration of possible methods to completely inactivate enterococci and reduce ARGs during sludge composting. Culturable enterococci were reduced in the thermophilic phase of sludge composting, but the proportion of VBNC subpopulation increased. It was reported for the first time that most VBNC enterococci were killed by extending the cooling phase of sludge compost, and by prolonging the cooling phase the types of ARG were reduced. However, there was a certain quantity (approximately 104/g dry weight) of culturable and VBNC enterococci in the compost products. In addition, MGEs and ARGs exist in both bacteria and compost products, leading to the risk of spreading antibiotic-resistant bacteria and antibiotic resistance when sludge compost products are used.

A Combination of UV and Disinfectant for Inactivating Viable but Nonculturable State Pseudomonas aeruginosa: Efficiency and Mechanisms

Conventional disinfection techniques, relying on a single disinfection step, often fail to directly eliminate microorganisms, instead causing them to enter a viable but nonculturable (VBNC) state. However, microorganisms in the VBNC state retain metabolic activity and can reactivate under suitable conditions, representing a “hidden source of contamination” that threatens drinking water safety. This study fundamentally assessed the feasibility of combined disinfection methods by integrating UV254 with disinfectant (NaClO, PAA, and PDS) for inactivating Pseudomonas aeruginosa (P. aeruginosa), an opportunistic pathogen that has been widely detected in water supply systems. The number of culturable cells was determined using the heterotrophic plate counting (HPC) method, and the number of VBNC cells was quantified using our recently developed qPCR approach. Quantitative analyses showed that combined disinfection methods can effectively reduce both culturable and VBNC cells by several orders of magnitude compared to a single disinfection step. Notably, VBNC P. aeruginosa, after 30 min of UV/NaCIO treatment, was below the detection limit (3.191 log CFU/mL) of PMA-qPCR. The reactivation experiment also confirmed that VBNC P. aeruginosa did not reactivate for 16 h after 30 min of UV/NaClO treatment under controlled laboratory conditions. The higher disinfection capacity of combined methods can be attributed to the generation of reactive radicals. This study highlighted combined disinfection as a promising approach for the inactivation of bacteria in the VBNC state, yet further studies are needed before an application can be considered for minimizing VBNC reactivation in city utility water processing or high-risk building water distribution systems.

Induction of Salmonella Enteritidis into a Viable but Nonculturable State by Cinnamaldehyde and Its Resuscitation.

Trans-cinnamaldehyde (TC), a typical plant-derived compound, has been widely used in the control of foodborne pathogen contamination. Nevertheless, the risk associated with the occurrence of viable but nonculturable (VBNC) bacteria induced by TC remains unclear. The results of this study showed that Salmonella Enteritidis (S. Enteritidis) entered the VBNC state after being induced by TC at a minimum inhibitory concentration of 312.5 μg/mL and survived for at least 22 days under TC treatment. Enhanced resistance was found against heat treatment (75°C, 30 s), antibiotics (i.e., ampicillin, ceftriaxone sodium, chloramphenicol), and hydrogen peroxide (3%) in VBNC S. Enteritidis. A synergistic effect against VBNC S. Enteritidis occurred when TC was combined with acid treatment, including lactic acid and acetic acid (pH = 3.5). VBNC and resuscitated S. Enteritidis by sodium pyruvate treatment (100 mM) were found to retain the infectious ability to Caco-2 cells. Relative expression levels of the stress-related genes relA, spoT, ppx, lon, katG, sodA, dnaK, and grpE were upregulated in VBNC S. Enteritidis. Accumulation of reactive oxygen species (ROS) and protein aggregates was observed in VBNC cells. Besides, the resuscitation of VBNC cells was accompanied with clearance of ROS and protein aggregates. In summary, this study presents a comprehensive characterization of stress tolerance and resuscitation of VBNC S. Enteritidis induced by cinnamaldehyde, and the results provide useful information for the development of effective control strategy against VBNC pathogenic bacteria in food production.

The effects and cell phenotype changes in Escherichia coli O157:H7 induced into VBNC state by l-malic acid at low temperature

Under environmental stress conditions, microorganisms can form a viable but non-culturable (VBNC) state, which poses a potential risk to food safety and human health. l-Malic acid (L-MA) is widely found in low-temperature fruit and vegetable juices as a common natural organic acid as well as a food additive. However, there is no report on whether L-MA in food processing can promote the formation of VBNC states. In this study, we investigated the pattern of the L-MA-induced VBNC state of Escherichia coli O157:H7 as a model organism, as well as the characteristics at the cellular and molecular levels. We discovered that L-MA can induce the VBNC state in a significant number of E. coli cells, a process accelerated by low temperatures (4 °C). Our analysis revealed that VBNC cells induced by both L-MA and 4 °C resuscitated earlier than those subjected to either condition alone, indicating increased food safety risks. For survival, L-MA-induced VBNC E. coli cells became substantially smaller and exhibited a significant decrease in respiratory activity while maintaining the basic function of the cell membrane. These cellular changes were supported by molecular-level evidence, reinforcing the implications for food safety under these conditions. This study underscores the importance of recognizing the potential risks posed by L-MA and low temperatures in food preservation, particularly in terms of underestimating the presence of harmful bacteria in food products. The insights gained from this research could contribute to the development of more effective food safety measures, particularly in the handling and processing of fruit and vegetable juices.

Stress response of Salmonella Newport with various sequence types toward plasma-activated water: Viable but nonculturable state formation and outer membrane vesicle production

This study aims to investigate the response of Salmonella Newport to plasma-activated water (PAW), a novel disinfectant that attracts attention due to its broad-spectrum antimicrobial efficacy and eco-friendliness. In this work, we demonstrated that S. Newport of different sequence types (STs) could be induced into the viable but nonculturable (VBNC) state by PAW treatment. Notably, a remarkable 99.96% of S. Newport ST45 strain entered the VBNC state after a 12-min PAW treatment, which was the fastest observed among the five S. Newport STs (ST31, ST45, ST46, ST166, ST2364). Secretion of outer membrane vesicles was observed in ST45, suggesting a potential strategy against PAW treatment. Genes related to oxidative stress (sodA, katE, trxA), outer membrane proteins (ompA, ompC, ompD, ompF) and virulence (pagC, sipC, sopE2) were upregulated in the PAW-treated S. Newport, especially in ST45. A reduction of 38–65% in intracellular ATP level after PAW treatment was observed, indicating a contributor to the formation of the VBNC state. In addition, a rapid method for detecting the proportion of VBNC cells in food products based on pagC was established. This study contributes to understanding the formation mechanism of the VBNC state in S. Newport under PAW stress and offers insights for controlling microbial risks in the food industry.

Deletion of codY impairs Staphylococcus epidermidis biofilm formation, generation of viable but non-culturable cells and stimulates cytokine production in human macrophages.

Introduction. Staphylococcus epidermidis biofilms are one of the major causes of bloodstream infections related to the use of medical devices. The diagnosis of these infections is challenging, delaying their treatment and resulting in increased morbidity and mortality rates. As such, it is urgent to characterize the mechanisms employed by this bacterium to endure antibiotic treatments and the response of the host immune system, to develop more effective therapeutic strategies. In several bacterial species, the gene codY was shown to encode a protein that regulates the expression of genes involved in biofilm formation and immune evasion. Additionally, in a previous study, our group generated evidence indicating that codY is involved in the emergence of viable but non-culturable (VBNC) cells in S. epidermidis.Gap statement/Hypothesis. As such, we hypothesized that the gene codY has have an important role in this bacterium virulence.Aim. This study aimed to assess, for the first time, the impact of the deletion of the gene codY in S. epidermidis virulence, namely, in antibiotic susceptibility, biofilm formation, VBNC state emergence and in vitro host immune system response.Methodology. Using an allelic replacement strategy, we constructed and then characterized an S. epidermidis strain lacking codY, in regards to biofilm and VBNC cell formation, susceptibility to antibiotics as well as their role in the interaction with human blood and plasma. Additionally, we investigate whether the codY gene can impact the activation of innate immune cells by evaluating the production of both pro- and anti-inflammatory cytokines by THP-1 macrophages.Results. We demonstrated that the deletion of the gene codY resulted in biofilms with less c.f.u. counts and fewer VBNC cells. Furthermore, we show that although WT and mutant cells were similarly internalized in vitro by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner.Conclusion. Our results indicate that codY contributes to S. epidermidis virulence, which in turn may have an impact on our ability to manage the biofilm-associated infections caused by this bacterium.

Effect of physicochemical and microbiological factors on the development of viable but non-culturable and resuscitation states of Vibrio cholerae.

Vibrio cholerae can endure harsh environmental conditions by transitioning into viable but non-culturable (VBNC) form and resuscitate upon return of appropriate conditions. In this study, we assessed the impact of physicochemical and microbiological factors, on the development of low temperature-induced VBNC state and subsequent recovery by temperature upshift. In estuarine water, Vibrio cholerae exhibits a slower decline in culturability over a period of 77 days as compared to 10 days in fresh water. When variable cell numbers from different growth phases were used for VBNC induction, it was observed that the higher inoculum size (106-107 cfu ml-1) from the late log phase culture appears to be crucial for entering the VBNC state. Conversely, starved cells could enter the VBNC state with an initial inoculum of 104-105 cfu ml-1, followed by resuscitation as well. The addition of glucose, GlcNAc and mannitol differentially affects progression into VBNC, while the addition of tryptone, yeast extract and casamino acid facilitated early entry into the VBNC state and shortened the length of the recovery period. Altogether these findings demonstrated that the ionic strength of water, inoculum size and the availability of nutrients played distinct roles during VBNC induction and resuscitation.

Unveiling the distribution characteristics of rpf-like genes and indigenous resuscitation promoting factor production in PCB-contaminated soils

Resuscitation promoting factors (Rpfs), known for their anti-dormancy cytokine properties, have been extensively investigated in the medical field. Although the Rpf from Micrococcus luteus has been successfully utilized to resuscitate and stimulate microbial populations for the degradation of polychlorinated biphenyls (PCBs), the presence of indigenous Rpf homologs in PCB-contaminated soils has not been established. In this study, the distribution characteristics of rpf-like genes and indigenous strain capable of producing Rpf in PCB-contaminated soils were explored. The results revealed the widespread presence of Rpf-like domains and their associated genes, particularly in close association with heavy metals and PCBs. The rpf-like genes were predominantly found in Proteobacteria and displayed a positive correlation with genes involved in PCB degradation and viable but non-culturable (VBNC) formation. Notably, the recombinant Rpf-Ac protein derived from the indigenous strain Achromobacter sp. HR2 exhibited muralytic activity and demonstrated significant efficacy in resuscitating the growth of VBNC cells, while also stimulating the growth of normal cells. These findings shed light on the prevalent presence of Rpf homologs in PCB-contaminated soils and their potential to resuscitate functional populations in the VBNC state, thereby enhancing in situ bioremediation.

Resuscitation of baijiu pit mud bacteria based on Rpf protein of Umezawaea beigongshangensis.

The pit mud in the Baijiu fermentation cellar is an abundant microbial resource that is closely related to the quality of baijiu. However, many naturally existing species might be in a viable but nonculturable (VBNC) state, posing challenges to the isolation and application of functional species. Herein, a previously isolated strain from baijiu mash, Umezawaea beigongshangensis, was found to contain the rpf gene that encodes resuscitation promotion factor (Rpf). Therefore, the gene was cloned and heterologously expressed, and the recombinant protein (Ub-Rpf 2) was purified. Ub-Rpf 2 was found to significantly promote the growth of resuscitated VBNC state Corynebacterium beijingensis and Sphingomonas beigongshangensis. To determine the resuscitation effect of Ub-Rpf 2 on real ecological samples, the protein was supplemented in pit mud for enrichment culture. Results revealed that specific families and genera were enriched in abundance upon Ub-Rpf 2 incubation, including a new family of Symbiobacteraceae and culturable Symbiobacterium genus. Furthermore, 14 species belonging to 12 genera were obtained in Ub-Rpf 2 treated samples, including a suspected novel species. This study lays a foundation for applying Rpf from U. beigongshangensis to exploit microbial resources in baijiu pit mud.

Bacteriostasis of nisin against planktonic and biofilm bacteria: Its mechanism and application.

Food safety incidents caused by bacterial contamination have always been one of the public safety issues of social concern. Planktonic cells, viable but non-culturable (VBNC) cells, and biofilm cells of bacteria can coexist in food or food processing, posing more serious challenges to public health and safety by increasing bacterial survival and difficulty in detection. As a non-toxic, no side effect, and highly effective bacteriostatic substance, nisin has received wide attention from researchers. In this review, we summarized the species and biosynthesis of nisin, the effects of nisin alone or in combination with other treatments on planktonic and biofilm cells, and its applications in the fields of food, feed, and medicine by consulting numerous studies. Meanwhile, the mechanism of nisin on planktonic and biofilm cells was proposed based on existing researches. Nisin not only has antibacterial activity against most G+ bacteria but also exhibits a bacteriostatic effect on G- bacteria when combined with other antibacterial treatments. In addition to planktonic cells, nisin also has significant effects on bacterial cells in biofilms by changing the thickness, density, and composition of biofilms. Based on the three action processes of nisin on biofilms, we summarized the changes of bacteria in biofilms, including the causes of bacterial death and the formation of the VBNC state. We consider that research on the relationship between nisin and VBNC state should be strengthened.

Assessing viability of Escherichia coli in mining-impacted surface waters

Multiple threats to surface water quality, including both fecal pollution and acid mine drainage (AMD) are frequently coincident in mining regions, such as parts of the Appalachian region of the U.S. While it has been established that AMD can have toxic effects to fecal bacteria in surface waters, there is also evidence that AMD may induce a viable but non-culturable (VBNC) state, leaving fecal bacteria undetectable by common standard methods. This study aims to better understand the occurrence of VBNC Escherichia coli in mining-impacted waters (MIW) through three objectives: (1) assess the relationship between MIWs and concentrations of culturable fecal bacteria, (2) compare standard methods for quantification of E. coli in MIW, and (3) investigate whether MIW can induce a VBNC state in E. coli. An analysis of historic data from the Deckers Creek and West Run Watersheds of West Virginia revealed a moderate correlation between pH and E. coli abundance determined via culture and enzyme-based methods. In samples collected from sites within the two watersheds impacted by historic mining activities, the E. coli uidA gene was measured via quantitative polymerase chain reaction (qPCR) and found to be significantly more abundant than E. coli concentrations measured via culture or enzyme substrate coliform test. However, this discrepancy between methods was greatest in samples with a low pH. Further, experiments in which an environmental strain of E. coli was dosed into samples of MIW diluted with saline at different concentrations confirmed that the culturability of E. coli significantly decreased with increasing MIW concentration, while the total number of intact cells, determined via "live/dead” staining and microscopy, remained high. This finding suggests that culture and enzyme methods may underestimate viable E. coli levels in AMD-impacted waters.

Persistence and viable but non-culturable state induced by streptomycin in Erwinia amylovora.

Persister cell and viable but non-culturable (VBNC) state of bacteria are survival strategies against antibiotics and various environmental stresses, respectively, but they tend to be ignored in agriculture fields, even though bacteria can regain their abilities to survive and produce disease once those stresses disappear. This study was carried out to determine whether persister cell and VBNC state in Erwinia amylovora are present after exposures to streptomycin, the length of their persistence, and the steps needed to decrease the inoculum. Persister cells were observed using biphasic killed growth curve for 4-8 h when the late stationary phase cells of E. amylovora were cultured in liquid medium containing streptomycin. This state was maintained for up to 12 h based on the colony forming units (CFUs) of the colonies that grew on the mannitol glutamate yeast extract (MGY) medium after streptomycin was removed. The CFUs on the MGY medium were lower than the total count determined using the LIVE/DEAD Kit, suggesting that persister cells and VBNC state might co-exist for up to 12 h after exposure to streptomycin. However, after 12 h, E. amylovora cells did not continue to grow on the medium for 9 days, suggesting that they entered a VBNC state at that time and remained in a persistent state. In addition, based on the Redox Sensor Green staining method, the presence of both states was confirmed for up to 12 h, and only then did the VBNC state became apparent. Furthermore, persister cells were observed for up to 24 h, and damaged cells reduced when E. amylovora cells were culture in distilled water with streptomycin, indicating that the uptake of lower nutrients in E. amylovora led to prolonged persister cells and VBNC state, which are more likely to survive after streptomycin treatments. The addition of sucrose and oxytetracycline to distilled water containing streptomycin reduced persister cells than other sources did. Thus, to inhibit the spread of fire blight, management techniques must consider the hazards of using streptomycin treatments that induce dormancy, such as persister cells and VBNC state, beyond the development of resistant strain.

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.

Quantitative Detection of VBNC State Pseudomonas aeruginosa Contributing to Accurate Assessment of Microbial Inactivation in Drinking Water Disinfection

In harsh environments, bacteria often enter a viable but nonculturable (VBNC) state, which cannot be detected using heterotrophic plate counting (HPC). Importantly, VBNC bacteria can potentially resuscitate under favorable conditions, posing a risk to drinking water safety. This study introduces an innovative approach, combining improved quantitative polymerase chain reaction (qPCR) with propidium monoazide (PMA) dye and HPC to accurately quantify VBNC Pseudomonas aeruginosa (P. aeruginosa). The method was applied to assess the ability of various disinfection techniques to induce P. aeruginosa into the VBNC state. Different disinfection methods, including ultraviolet radiation (UV), sodium hypochlorite (NaClO), and peracetic acid (PAA), significantly reduced bacterial culturability (>99.9%), with the majority entering the VBNC state. Notably, under favorable conditions, UV-induced VBNC cells were resuscitated faster than those induced by NaClO. VBNC P. aeruginosa exhibited relatively high intracellular adenosine triphosphate (ATP) levels, indicating ongoing metabolic activity. Scanning electron microscopy (SEM) reveals that some bacteria maintained cellular integrity for UV and PAA treatment, while evident membrane disruption was observed after NaClO disinfection. This study represents a significant advancement in quantitatively detecting VBNC state P. aeruginosa, contributing to an accurate assessment of microbial inactivation during drinking water disinfection.

Unveiling the PCB biodegradation potential and stress survival strategies of resuscitated strain Pseudomonas sp. HR1

The exploration of resuscitated strains, facilitated by the resuscitation promoting factor (Rpf), has substantially expanded the pool of cultivated degraders, enhancing the screening of bio-inoculants for bioremediation applications. However, it remains unknown whether these resuscitated strains can re-enter the viable but non-culturable (VBNC) state and the specific stress conditions that trigger such a transition. In this work, the whole genome, and polychlorinated biphenyl (PCB)-degrading capabilities of a resuscitated strain HR1, were investigated. Notably, the focus of this exploration was on elucidating whether HR1 would undergo a transition into the VBNC state when exposed to low temperature and PCBs, with and without the presence of heavy metals (HMs). The results suggested that the resuscitated strain Pseudomonas sp. HR1 harbored various functional genes related to xenobiotic biodegradation, demonstrating remarkable efficiency in Aroclor 1242 degradation and strong resistance against stress induced by low temperature and PCBs. Nevertheless, when exposed to the combined stress of low temperature, PCBs, and HMs, HR1 underwent a transition into the VBNC state. This transition was characterized by significant decreases in enzyme activities and notable changes in both morphological and physiological traits when compared to normal cells. Gene expression analysis revealed molecular shifts underlying the VBNC state, with down-regulated genes showed differential expression across multiple pathways and functions, including oxidative phosphorylation, glycolysis, tricarboxylic acid cycle, amino acid metabolism, translation and cytoplasm, while up-regulated genes predominantly associated with transcription regulation, membrane function, quorum sensing, and transporter activity. These findings highlighted the great potential of resuscitated strains as bio-inoculants in bioaugmentation and shed light on the survival mechanisms of functional strains under stressful conditions, which should be carefully considered during bioremediation processes.

Staphylococcus aureus enters viable-but-nonculturable state in response to chitooligosaccharide stress by altering metabolic pattern and transmembrane transport function

Although chitooligosaccharide (COS) has attracted the attention of some researchers due to its good solubility and broad-spectrum antibacterial activity, our study found that Staphylococcus aureus treated with low concentration of COS actively entered the viable-but-nonculturable (VBNC) state to resist this environmental stress. In this study, the transcriptome of VBNC-state S. aureus after COS treatment was analyzed by RNA-sequencing. Compared with the control group, pathway enrichment analysis showed that COS-treated S. aureus adopted a series of adaptive adjustment strategies for survival, including significant up-regulation of the differential genes' expression of such as ABC transporters (metI, tagG), Sec dependent transport pathway (secDF), peptidoglycan synthesis pathway (murG) and alteration of their physiological metabolic patterns, where ATP depletion played a key role in the formation of the VBNC-state S. aureus. Further, by using oxidative phosphorylation uncoupling agent to adjust the initial level of ATP in S. aureus, it was found that the reduction of intracellular ATP level could accelerate the formation of VBNC state. Overall, our results preliminarily elucidated the molecular mechanism of COS inducing the VBNC-state S. aureus. It provided an important theoretical reference for further achieving effective bacterial inactivation by COS.

Detection Of Viable but Non Culturable (VBNC) Bacteria Using of Propidium Monoazide PCR: A Review

Microorganisms in their natural habitats are subjected to a broad range of stressors. In order to survive and retain their vitality under those demanding conditions, they enter a state known as viable but nonculturable (VBNC). Traditional laboratory techniques cannot identify microbes in the VBNC state, but they can be brought back to life in the right circumstances. As a result, VBNC pathogens seriously jeopardize public health and food safety. More than 100 microbe species have been found to have entered the VBNC state to date, owing to a variety of chemical and physical processes. Since the VBNC syndrome was first discovered forty years ago, new methods for inducing, detecting, resuscitating, and understanding its molecular pathways have been created. To determine their effect on public health, live bacterial foodborne pathogens must be found and measured. Due to the existence of viable but nonculturable cells (VBNC), which can retain metabolic and pathogenic activity, traditional growth-based approaches have the potential to significantly underestimate the true numbers. The similar issue arises when using different culture-independent molecular techniques, such PCR, which can detect DNA from dead cells in addition to VBNC bacteria. Viability dyes and PCR have been used to selectively identify and quantify viable foodborne microorganisms, overcoming this drawback.

The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria.

Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.