Viable But Non-culturable Bacteria Research Articles

Exploring the distribution and co-occurrence of rpf-like genes and nitrogen-cycling genes in water reservoir sediments.

Water reservoir sediments represent a distinct habitat that harbors diverse microbial resources crucial for nitrogen cycling processes. The discovery of resuscitation promoting factor (Rpf) has been recognized as a crucial development in understanding the potential of microbial populations. However, our understanding of the relationship between microorganisms containing rpf-like genes and nitrogen-cycling functional populations remains limited. The present study explored the distribution patterns of rpf-like genes and nitrogen-cycling genes in various water reservoir sediments, along with their correlation with environmental factors. Additionally, the co-occurrence of rpf-like genes with genes associated with the nitrogen cycle and viable but non-culturable (VBNC) formation was investigated. The findings indicated the ubiquitous occurrence of Rpf-like domains and their related genes in the examined reservoir sediments. Notably, rpf-like genes were predominantly associated with Bradyrhizobium, Nitrospira, and Anaeromyxobacter, with pH emerging as the primary influencing factor for their distribution. Genera such as Nitrospira, Bradyrhizobium, Anaeromyxobacter, and Dechloromonas harbor the majority of nitrogen-cycling functional genes, particularly denitrification genes. The distribution of nitrogen-cycling microbial communities in the reservoir sediments was mainly influenced by pH and NH4 +. Notably, correlation network analysis revealed close connections between microorganisms containing rpf-like genes and nitrogen-cycling functional populations, as well as VBNC bacteria. These findings offer new insights into the prevalence of rpf-like genes in the water reservoir sediments and their correlation with nitrogen-cycling microbial communities, enhancing our understanding of the significant potential of microbial nitrogen cycling.

Multiple factors trigger the formation and resuscitation of the VBNC state in alcohol-producing Klebsiella pneumoniae.

Klebsiella pneumoniae can enter a viable but nonculturable (VBNC) state to survive in unfavorable environments. Our research found that high-, medium-, and low-alcohol-producing K. pneumoniae strains are associated with nonalcoholic fatty liver disease. However, the presence of the three Kpn strains has not been reported in the VBNC state or during resuscitation. In this study, the effects of different strains, salt concentrations, oxygen concentrations, temperatures, and nutrients in K. pneumoniae VBNC state were evaluated. The results showed that high-alcohol-producing K. pneumoniae induced a slower VBNC state than medium-alcohol-producing K. pneumoniae, and low-alcohol-producing K. pneumoniae. A high-salt concentration and micro-oxygen environment accelerated the loss of culturability. Simultaneously, both real-time quantitative PCR and droplet digital PCR were developed to compare the quantitative comparison of three Kpn strain VBNC states by counting single-copy gene numbers. At 22°C or 37°C, the number of culturable cells decreased significantly from about 108 to 105-106 CFU/mL. In addition, imipenem, ciprofloxacin, polymyxin, and phiW14 inhibited cell resuscitation but could not kill VBNC-state cells. These results revealed that the different environments evaluated play different roles in the VBNC induction process, and new effective strategies for eliminating VBNC-state cells need to be further studied. These findings provide a better understanding of VBNC-state occurrence, maintenance, detection, and absolute quantification, as well as metabolic studies of resuscitation resistance and ethanol production.IMPORTANCEBacteria may enter VBNC state under different harsh environments. Pathogenic VBNC bacteria cells in clinical and environmental samples pose a potential threat to public health because cells cannot be found by routine culture. The alcohol-producing Kpn VBNC state was not reported, and the influencing factors were unknown. The formation and recovery of VBNC state is a complete bacterial escape process. We evaluated the influence of multiple induction conditions on the formation of VBNC state and recovery from antibiotic and bacteriophage inhibition, and established a sensitive molecular method to enumerate the VBNC cells single-copy gene. The method can improve the sensitivity of pathogen detection in clinical, food, and environmental contamination monitoring, and outbreak warning. The study of the formation and recovery of VBNC-state cells under different stress environments will also promote the microbiological research on the development, adaptation, and resuscitation in VBNC-state ecology.

Assessing the effectiveness and mechanisms of Fe/PS, Fe/H2O2, and O3 treatment for water disinfection: Spotlight on VBNC bacteria

Advanced oxidation processes (AOPs) exhibit significant potential for water disinfection due to their generation of large quantities of highly oxidizing free radicals. However, the neglect of viable but nonculturable (VBNC) cells obscures their true disinfection efficacy and potential environmental health risks. Therefore, the study evaluated the disinfection effectiveness and mechanisms of typical AOPs, including Fe/H2O2, Fe/persulfate (PS), and O3, from the perspective of the production of VBNC bacteria. The results indicate that Fe/PS exhibits the strongest bacterial inactivation rate (99.94%), and the cells lose their ability to reactivate. Fe/H2O2 and O3 induce more cells to enter the VBNC state compared to Fe/PS. Moreover, different AOPs result in varying levels of free radical production and utilization efficiency, with SO4•− and O3 exhibiting greater selectivity in deactivating bacteria compared to HO•. The inhibition of VBNC bacteria production by Fe/PS treatment may be attributed to the combined action of HO• and SO4•− on microorganisms, leading to oxidative stress and metabolic disruption in bacteria through the inhibition of biofilm formation and aminoacyl-tRNA biosynthesis (p < 0.05), thereby causing direct bacterial death rather than entry into the VBNC state. In contrast, Fe/H2O2 and O3 result in the upregulation of the metabolism of alanine, aspartate, and glutamate, as well as styrene degradation capacity by the bacteria, leading to the production of more VBNC bacteria. Overall, the study offers insights into mitigating potential biological risks in water disinfection and developing environmentally friendly and efficient disinfection technologies.

Nanowire-assisted electroporation via inducing cell destruction for inhibiting formation of VBNC bacteria: Comparison with chlorination

The induction of viable but nonculturable (VBNC) bacteria with cellular integrity and low metabolic activity by chemical disinfection causes a significant underestimation of potential microbiological risks in drinking water. Herein, a physical Co3O4 nanowire-assisted electroporation (NW-EP) was developed to induce cell damage via the locally enhanced electric field over nanowire tips, potentially achieving effective inhibition of VBNC cells as compared with chemical chlorination (Cl2). NW-EP enabled over 5-log removal of culturable cell for various G+/G- bacteria under voltage of 1.0 V and hydraulic retention time of 180 s, and with ∼3–6 times lower energy consumption than Cl2. NW-EP also achieved much higher removals (∼84.6 % and 89.5 %) of viable Bacillus cereus (G+) and Acinetobacter schindleri (G-) via generating unrecoverable pores on cell wall and reversible/irreversible pores on cell membrane than Cl2 (∼28.6 % and 41.1 %) with insignificant cell damage. The residual VBNC bacteria with cell wall damage and membrane pore resealing exhibited gradual inactivation by osmotic stress, leading to ∼99.8 % cell inactivation after 24 h storage (∼59.4 % for Cl2). Characterizations of cell membrane integrity and cell morphology revealed that osmotic stress promoted cell membrane damage for the gradual inactivation of VBNC cells during storage. The excellent adaptability of NW-EP for controlling VBNC cells in DI, tap and lake waters suggested its promising application potentials for drinking water, such as design of an external device on household taps.

GP-fatty acid axis regulating the adaptive morphogenesis of VBNC bacteria in response to chlorine stress in the process of chlorination disinfection

Investigating the specific metabolic regulatory strategy of viable but non-culturable (VBNC) bacteria is crucial for understanding adaptive morphogenesis, processes of shrinking body size and maintaining cell membrane integrity. Metabolomic studies have revealed an upregulation in the metabolic competence of glycerophospholipids (GP) and fatty acid catabolism in VBNC bacteria. Disruption of the GP-fatty acid axis, as evidenced by scanning electron microscopy, hinders the maintenance of adaptive morphology. VBNC bacteria minimize cell size by consuming fatty acids to sustain intracellular energy metabolism. Concurrently, GP metabolism replenishes fatty acid consumption and upholds cell membrane integrity, preventing HClO intrusion. Regulation of the GP-fatty acid axis also sustains intracellular antioxidants (e.g. glutathione and ubiquinone), preventing lipid peroxidation. Moreover, disruption of the GP-fatty acid axis impedes biofilm formation (reduced by 94.77 %) and reactivation (reduced by 95.73 %) of VBNC bacteria, with negligible effects on culturable bacteria. The study underscores the GP-fatty acid axis regulation to form adaptive morphology is a fundamental survival strategy for VBNC bacteria. Developing an efficient disinfection method targeting the GP-fatty acid axis emerges as a promising strategy to mitigate the health risk of VBNC bacteria.

Benzalkonium chloride disinfectant residues stimulate biofilm formation and increase survival of Vibrio bacterial pathogens.

Vibrio spp. are opportunistic human and animal pathogens found ubiquitously in marine environments. Globally, there is a predicted rise in the prevalence of Vibrio spp. due to increasing ocean temperatures, which carries significant implications for public health and the seafood industry. Consequently, there is an urgent need for enhanced strategies to control Vibrio spp. and prevent contamination, particularly in aquaculture and seafood processing facilities. Presently, these industries employ various disinfectants, including benzalkonium chloride (BAC), as part of their management strategies. While higher concentrations of BAC may be effective against these pathogens, inadequate rinsing post-disinfection could result in residual concentrations of BAC in the surrounding environment. This study aimed to investigate the adaptation and survival of Vibrio spp. exposed to varying concentrations of BAC residues. Results revealed that Vibrio bacteria, when exposed, exhibited a phenotypic adaptation characterized by an increase in biofilm biomass. Importantly, this effect was found to be strain-specific rather than species-specific. Exposure to BAC residues induced physiological changes in Vibrio biofilms, leading to an increase in the number of injured and alive cells within the biofilm. The exact nature of the "injured" bacteria remains unclear, but it is postulated that BAC might heighten the risk of viable but non-culturable (VBNC) bacteria development. These VBNC bacteria pose a significant threat, especially since they cannot be detected using the standard culture-based methods commonly employed for microbiological risk assessment in aquaculture and seafood industries. The undetected presence of VBNC bacteria could result in recurrent contamination events and subsequent disease outbreaks. This study provides evidence regarding the role of c-di-GMP signaling pathways in Vibrio adaptation mechanisms and suggests that c-di-GMP mediated repression is a potential avenue for further research. The findings underscore that the misuse and overuse of BAC may increase the risk of biofilm development and bacterial survival within the seafood processing chain.

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 (&gt;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.

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.

Analyzing the pressure resistant, sublethal injury and resuscitable viable but non-culturable state population of Escherichia coli, Staphylococcus aureus, Bacillus amyloliquefaciens and Lactiplantibacillus plantarum under high pressure processing

This study aimed to analyze and reduce the pressure resistance (PR), sublethal injury (SLI), and viable but non-culturable (VBNC) populations during HPP. Escherichia coli, Staphylococcus aureus, Bacillus amyloliquefaciens and Lactiplantibacillus plantarum were selected for evaluation of PR, SLI and VBNC cell counts and proportions during HPP. The results revealed that the bactericidal efficiency against these strains gradually improved as the processing pressure increased. However, viable bacteria could still be detected, suggesting that there may involve the presence of resistant population that difficult to be killed or revived from SLI. Further detecting the quantity and proportion of PR, SLI and VBNC bacteria found that these state of cells were present during whole HPP treatment. Additionally, the more resistant a bacterial species was to high pressure, the fewer SLI and more resuscitable VBNC (RVBNC) populations it generated, and vice versa. Therefore, correlation analysis was also employed to make the relationship between log reduction, SLI and RVBNC population ratios clearer. The results demonstrated that the log reduction was highly positive correlation with SLI population ratios, and negative correlation with RVBNC population within our detected species at 500 MPa. Furthermore, CO2 and Nisin were employed to combined with HPP to reduce these survivors. Comparing with 233, 218, 241 and 259 MPa for HPP treatment, it took 37, 89, 135 and 229 MPa for HPP + CO2, and 189, 161, 199 and 292 MPa for HPP + Nisin to the first decimal reduction for E. coli, S.aureus, B. amyloliquefaciens and L. plantarum, respectively. The results indicated that HPP combined with CO2 or Nisin could significantly reduce the quantity of PR, SLI, and RVBNC cells during HPP, and provide better bactericidal effects. In conclusion, we quantified the presence of PR, SLI, and VBNC bacteria after high pressure treatment and investigate the effectiveness of HPP combined with CO2 or Nisin to enhance the inactivation of bacteria and reduce the occurrence of PR, SLI, and RVBNC bacteria.

Modeling the survival of Campylobacter jejuni in raw milk considering the viable but non‐culturable cells (VBNC)

AbstractCampylobacter spp. cannot grow in raw milk, but it is able to transform into a viable but non‐culturable (VBNC) state enabling the survival in such harsh conditions. In this study, Campylobacter jejuni survival in raw milk was investigated taken into consideration colony‐forming units (CFUs) and VBNC cells. CFU from two different strains of C. jejuni (DSM 4688 and BfR‐CA‐18043) were enumerated at three temperatures (5°C, 8°C, and 12°C). In parallel, a viability real‐time PCR was conducted to quantify intact and putatively infectious units (IPIUs) (comprising CFU and VBNC bacteria). The data generated were used to model the viability of C. jejuni during raw milk storage. Here, a one‐step fitting approach was performed using parameter estimates from an intermediate two‐step fit as starting values to generate tertiary models. Different primary model equations (Trilinear and Weibull) were required to fit the CFU and the IPIU data. Strain‐specific linear secondary models were generated to analyze the effect of storage temperature on the maximum specific inactivation rate of the CFU data. The time of the first decimal reduction parameter of the IPIU models could be modeled by a strain‐independent linear secondary model. The developed tertiary models for CFU and IPIU differ significantly in their predictions, for example, for the time required for a one log10 reduction. Taken into consideration that VBNC could revert to a culturable state during the raw milk storage, our results underline the importance of considering IPIU and not only CFU to avoid underestimation of the survival of C. jejuni in raw milk.

Phenol degradation at high salinity by a resuscitated strain Pseudomonas sp. SAS26: kinetics and pathway

Exploration of novel salt-tolerant phenol-degrading bacterial strains are often limited by unculturability of functional bacteria, as they are prone to entering a viable but non-culturable (VBNC) state in response to complex stressful environmental conditions. Thus, resuscitating the VBNC bacteria provides a huge microbial source to screen effective degraders for bioaugmentation. In this study, a resuscitated strain Pseudomonas sp. SAS26, which exhibited the highest phenol-degrading ability, was selected to investigate its phenol degradation performance at high salinity. The results indicated that SAS26 was capable of efficiently degrading phenol at NaCl concentrations of 0–30 g/L, with decreased efficiency observed at 40–60 g/L. At 10 g/L NaCl, complete degradation of 1800 mg/L phenol was achieved within 48 h. At a higher NaCl concentration of 30 g/L, SAS26 could fully degrade 1500 mg/L phenol within 60 h, with the highest degradation rate of 0.360 g/(g CDW·h) observed at approximately 24 h. The modeling of phenol degradation at high salinity demonstrated that the Webb model was the most appropriate for describing the degradation kinetics, followed by the Yano and Aiba models. Furthermore, the analysis of the enzyme activities and catA gene expression suggested that SAS26 initially converted phenol to catechol via phenol hydroxylase, and then oxidized catechol by the catalysis of catechol 1,2-dioxygenase via the ortho-cleavage pathway. These results revealed the high-efficient phenol-degrading capability of the resuscitated strain SAS26 at high salinity, which shed light on cultivating effective bio-inoculants for treating high-saline phenolic wastewater.

Characterization of two predicted lipid metabolism genes (FTL_1569 and FTL_1570) in the intracellular pathogen, Francisella tularensis LVS.

Many species of bacteria enter a viable but non-culturable (VBNC) state when exposed to stressful conditions. The VBNC state acts as an adaptive strategy that allows for long-term survival of bacteria in unfavorable environments. VBNC bacteria are difficult to detect in the environment due to an inability to grow on nutrient agar. Although the ability to enter the VBNC state may be advantageous for bacteria, it poses a risk to human health. Francisella tularensis, transitions rapidly and spontaneously to the VBNC state, giving it the potential to be an excellent model organism for the study of this phenomenon. Microscopic observations reveal that changes in cell morphology are coincident with the VBNC state. To characterize these morphological changes, fluorescence microscopy imaging using membrane lipid staining has been used in the live vaccine strain of F. tularensis. Microscopy imaging suggested that lipid metabolism may be important for the transition into the VBNC state, and the change in morphology. To investigate further, genetic deletion mutants have been constructed from two predicted lipid metabolism genes, FTL_1569 (phosphoglycolate phosphatase) and FTL_1570 (phospholipase D family protein). Gentamicin protection assays with immune cells and red blood cells have been conducted to investigate the role of these genes in virulence of F. tularensis. Our preliminary results suggest that lipid metabolism may play roles in both persistence and in invasion in F. tularensis. (This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Applying RNA-Seq to investigate the transition into a Viable But Non Culturable State (VBNC) for the intracellular pathogen, Francisella tularensis LVS.

Many species of bacteria, under conditions of stress or nutrient limitation, enter a state of dormancy referred to as viable but non-culturable (VBNC). VBNC bacteria persist in the environment, are difficult to detect and identify by many standard laboratory methods and can be altered in their susceptibility to antibiotics. Entry into the VBNC state is often accompanied by morphological changes; however, the mechanisms underlying this are poorly understood. Francisella transitions rapidly and spontaneously to a VBNC state, and therefore has the potential to be an excellent model organism for the study of this phenomenon. To investigate the transcriptome of VBNC F. tularensis LVS we extracted RNA from culturable and VBNC cells and carried out an RNA-Seq analysis using both Long-read Nanopore and Illumina Sequencing. Differentially expressed genes (DEGs) were identified using a DE-Seq pipeline. Over 300 genes were significantly upregulated and ~100 genes were down regulated in VBNC cells. Amongst the upregulated genes were some involved in the transport of metals or small molecules, and we have also identified putative transcriptional regulators that may be master controllers of the VBNC process. Our data represents the first transcriptomic analysis of F. tularensis LVS as it transitions into the VBNC state. Identifying genes that are involved in this transition is a critical first step in understanding the mechanisms that drive F. tularensis LVS into the VBNC state and will help us identify factors that allow the resuscitation of these bacteria. This will have important implications for understanding both environmental persistence and pathogenicity of Francisella species. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Glyoxylate cycle maintains the metabolic homeostasis of Pseudomonas aeruginosa in viable but nonculturable state induced by chlorine stress

Bacteria enter a viable but non-culturable (VBNC) state with low metabolic activity to cope with environmental stress (e.g., chlorine disinfection). Elucidating the mechanism and key pathway of VBNC bacteria maintaining low metabolic competence is of great significance to realize their effective control and reduce their environmental and health risks. This study discovered that the glyoxylate cycle is a key metabolic pathway for VBNC bacteria, but not for culturable bacteria. And blocking the glyoxylate cycle pathway inhibited the reactivation and led to the death of VBNC bacteria. The main mechanisms involved the breakdown of material and energy metabolism and the antioxidant system. Gas chromatography-tandem mass spectrometry analysis showed that blocking the glyoxylate cycle led to a disruption of carbohydrate metabolism and fatty acid catabolism in VBNC bacteria. As a result, the energy metabolism system of VBNC bacteria collapsed and the abundance of energy metabolites (ATP, NAD+ and NADP+) decreased significantly. Moreover, the decrease in the level of quorum sensing signaling molecules (quinolinone and N-Butanoyl-D-homoserine lactone) inhibited the synthesis of extracellular polymeric substances (EPSs) and biofilm formation. And the downregulation of glycerophospholipid metabolic competence increased the permeability of cell membranes, leading to the entry of large amounts of hypochlorous acid (HClO) into the bacteria. In addition, the down-regulation of nucleotide metabolism, glutathione metabolism, and the reduction of antioxidant enzyme content resulted in the inability to scavenge reactive oxygen species (ROS) generated by chlorine stress. The large production of ROS and the reduction of antioxidants together led to the breakdown of the antioxidant system of VBNC bacteria. In short, the glyoxylate cycle is the key metabolism pathway of VBNC bacteria for stress resistance and maintaining cellular metabolic balance, and targeting the glyoxylate cycle represents an attractive strategy for developing new and efficient disinfection methods for the control of VBNC bacteria.

Induction of Viable but Non-Culturable State in Clinically Relevant Staphylococci and Their Detection with Bacteriophage K

Prosthetic joint infections are frequently associated with biofilm formation and the presence of viable but non-culturable (VBNC) bacteria. Conventional sample culturing remains the gold standard for microbiological diagnosis. However, VBNC bacteria lack the ability to grow on routine culture medium, leading to culture-negative results. Bacteriophages are viruses that specifically recognize and infect bacteria. In this study, we wanted to determine if bacteriophages could be used to detect VBNC bacteria. Four staphylococcal strains were cultured for biofilm formation and transferred to low-nutrient media with different gentamycin concentrations for VBNC state induction. VBNC bacteria were confirmed with the BacLightTM viability kit staining. Suspensions of live, dead, and VBNC bacteria were incubated with bacteriophage K and assessed in a qPCR for their detection. The VBNC state was successfully induced 8 to 19 days after incubation under stressful conditions. In total, 6.1 to 23.9% of bacteria were confirmed alive while not growing on conventional culturing media. During the qPCR assay, live bacterial suspensions showed a substantial increase in phage DNA. No detection was observed in dead bacteria or phage non-susceptible E. coli suspensions. However, a reduction in phage DNA in VBNC bacterial suspensions was observed, which confirmed the detection was successful based on the adsorption of phages.

Wake Up! Resuscitation of Viable but Nonculturable Bacteria: Mechanism and Potential Application.

The viable but nonculturable (VBNC) state is a survival strategy for bacteria when encountered with unfavorable conditions. Under favorable environments such as nutrient supplementation, external stress elimination, or supplementation with resuscitation-promoting substances, bacteria will recover from the VBNC state, which is termed "resuscitation". The resuscitation phenomenon is necessary for proof of VBNC existence, which has been confirmed in different ways to exclude the possibility of culturable-cell regrowth. The resuscitation of VBNC cells has been widely studied for the purpose of risk control of recovered pathogenic or spoilage bacteria. From another aspect, the resuscitation of functional bacteria can also be considered a promising field to explore. To support this point, the resuscitation mechanisms were comprehensively reviewed, which could provide the theoretical foundations for the application of resuscitated VBNC cells. In addition, the proposed applications, as well as the prospects for further applications of resuscitated VBNC bacteria in the food industry are discussed in this review.

Recent Methods for the Viability Assessment of Bacterial Pathogens: Advances, Challenges, and Future Perspectives

Viability assessment is a critical step in evaluating bacterial pathogens to determine infectious risks to public health. Based on three accepted viable criteria (culturability, metabolic activity, and membrane integrity), current viability assessments are categorized into three main strategies. The first strategy relies on the culturability of bacteria. The major limitation of this strategy is that it cannot detect viable but nonculturable (VBNC) bacteria. As the second strategy, based on the metabolic activity of bacteria, VBNC bacteria can be detected. However, VBNC bacteria sometimes can enter a dormant state that allows them to silence reproduction and metabolism; therefore, they cannot be detected based on culturability and metabolic activity. In order to overcome this drawback, viability assessments based on membrane integrity (third strategy) have been developed. However, these techniques generally require multiple steps, bulky machines, and laboratory technicians to conduct the tests, making them less attractive and popular applications. With significant advances in microfluidic technology, these limitations of current technologies for viability assessment can be improved. This review summarized and discussed the advances, challenges, and future perspectives of current methods for the viability assessment of bacterial pathogens.

Metabolic characteristics and markers in viable but nonculturable state of Pseudomonas aeruginosa induced by chlorine stress

Many Gram-negative pathogens enter the viable but nonculturable (VBNC) state to resist external environmental stress (such as disinfection). However, little is known about the metabolic properties, especially for the metabolic markers, of VBNC bacteria, which impedes the development of efficient disinfection technologies and causes more potential health risks. In this study, we analyzed the metabolic characteristics of chlorine stress-induced VBNC Pseudomonas aeruginosa at the population and single-cell levels. The overall metabolic activity of VBNC bacteria showed a downward trend, but the glyoxylate cycle, fatty acid and glycerophospholipid metabolism pathways were up-regulated. Based on the metabolic profiles of VBNC bacteria, nine metabolic markers (pyruvate, glyoxylate, guanine, glutamate, sn glycero-3-phos-phocholine, fatty acid, D-alanine, glutathione, N-Butanoyl-D-homoserine lactone) were determined. The results of single-cell Raman spectroscopy showed that the metabolic activity of VBNC bacteria was significantly reduced, but showed more significant metabolic heterogeneity. The redshift of the Raman peaks of 15N and 13C labeled VBNC bacteria was significantly weaker than that of the culturable bacteria, suggesting that the VBNC bacteria have a reduced ability to synthesize proteins, nucleotides, phospholipids, and carbohydrates. The result of this study can help to better understand the metabolic mechanisms and energy management strategy of VBNC bacteria, to achieve precise identification and effective control of VBNC bacteria.

Exploring potential bacterial populations for enhanced anthraquinone dyes biodegradation: a critical review.

Anthraquinone dyes, which include an anthraquinone chromophore group, are the second-largest among dye classes, which is often employed in textile manufacturing. A significant number of anthraquinone dyes get into the environment, creating severe pollution since many of these dyes have intricate and stable structures. Currently, microbiological treatment of wastewater is an economically and feasibly viable solution for treating printing and dyeing wastewater, and there are growing reports of biodegradation of anthraquinone dyes. In this review, we outline the current advances in the biodegradation of anthraquinone dyes, summarizes dyes biodegradation by bacterial, fungal, and algae strains, factors influencing dyes biodegradation, current methods in enhancing dyes biodegradation, resuscitation of viable but non-culturable (VBNC) bacteria for better microbial performance, and potentials of VBNC bacteria in degrading dyes. Finally, future directions and important areas for study are given, and such efforts are anticipated to improve the anaerobic degradation process.

Current Perspectives on Viable but Non-culturable Bacteria in Food Safety and Public Health

The viable but non-culturable (VBNC) state is defined as an adaptive mechanism for microorganisms adjusting to stressful conditions. Although VBNC bacteria are alive and metabolically active, they are unable to grow on routine culture media. Nevertheless, the potential capacity of VBNC pathogens to retain virulence activity and further resuscitate into the culturable state in favorable conditions constitutes a major hazard to food safety and public health. Food processing, transformation, and storage, as well as non-thermal techniques, can provoke pathogens toward VBNC induction. The distinct characteristic of VBNC bacteria led to the emergence of novel culture-independent techniques to prevent the misinterpretation of food safety. To deepen our knowledge of the molecular aspect of the VBNC state, several mechanism-oriented studies investigated the metabolic activity of VBNC bacteria and their correlation with different stressful conditions. This review aims to discuss the molecular mechanisms and genomic factors underlying the induction and resuscitation of the VBNC state. The study will further highlight innovative detection methods to provide a comprehensive perspective for future studies in the emerging fields of research concerning VBNC state, food safety, and public health.