Viable But Nonculturable Research Articles

Modified in-vitro AATCC-100 procedure to measure viable bacteria from wound dressings.

Chronic wounds are reoccurring healthcare problems in the United States and cost up to $50 billion annually. Improper wound care results in complications such as wound debridement, surgical amputation, and increased morbidity/ mortality due to opportunistic infections. To eliminate wound infections, many antimicrobial dressings are developed and submitted to FDA for evaluation. AATCC-100 is a standard method widely used to evaluate cloth wound dressings. This method, requires enrichment, followed by culturing to measure the concentration of culturable organisms; a caveat to this method could result in neglected viable but nonculturable (VBNC) bacteria and overestimate the antimicrobial properties of wound dressings. Therefore, the objectives of this study were to assess this accepted protocol with quantitative real-time polymerase chain reaction (qRT-PCR), to measure time dependent antimicrobial efficacy of wound dressing, and to examine for potential viable bacteria but non-culturable as compared with traditional plating methods. The test organisms included opportunistic pathogens: Pseudomonas aeruginosa (ATCC 15692) and Staphylococcus aureus (ATCC 43300). To mimic a wound dressing environment, samples of commercially available wound dressings (McKesson Inc.) with silver ion (positive control) and dressings without silver ion (positive control) were assessed under sterile conditions. All samples were examined by the original protocol (the extended AATCC-100 method) and qRT-PCR. The expression of specific housekeeping genes was measured (proC for P. aeruginosa and 16s rRNA for S. aureus). Based on these tests, log reduction of experimental conditions was compared to identify time dependent and precise antimicrobial properties from wound dressing samples. These results showed antimicrobial properties of wound dressings diminished as incubation days are increased for both methods from day 1 PCR result of 4.31 ± 0.54 and day 1 plating result of 6.31 ± 3.04 to day 3 PCR result of 1.22 ± 0.97 and day 3 plating result of 5.89 ± 2.41. These results show that data from qRT-PCR generally produced lower standard deviation than that of culture methods, hence shown to be more precise. Complementary parallel analysis of samples using both methods better characterized antimicrobial properties of the tested samples.

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.

Development, evaluation and application of a polymerase spiral reaction (PSR) based assay on accurate detection of Vibrio parahaemolyticus viable cells and virulence factors from rice product

Vibrio parahaemolyticus is a predominate cause of microbiological food safety. This study aimed to develop a polymerase spiral reaction (PSR) based assay for rapid and visual detection on V. parahaemolyticus. Firstly, the specificity and conservativity of species-specific target tlh gene were determined. Secondly, targeting tlh and virulence factors tdh and trh, PSR assay was developed and optimized using constructed positive plasmid controls and standard strain. Visualized result determination using fluorescent dye was applied to avoid false positive issues. Thirdly, 44 strains were used for specificity evaluation. Fourthly, pure and mixed species artificial contamination systems were included to test its applicability. In addition, a propidium monoazide (PMA)-PSR assay was set up to determine viable but non-culturable (VBNC) cells. Target tlh gene acquired high specificity and PSR assay was optimized to carry out at 65ᵒC for 1 h. Detection limit of PSR assay in purified genomic DNA, plasmid and artificially contaminated food samples were 5.31–53.1 pg/μL, 100 copies, and 103 CFU/mL, respectively, demonstrating the sensitivity as 100-fold higher than regular PCR. With PMA-PSR assay, VBNC cells were accurately determined without the disruption of dead cells and food compositions. Thus, PSR is applicable in detection of V. parahaemolyticus viable cells and virulence factors.

Revealing the mechanism of citral induced entry of Vibrio vulnificus into viable but not culturable (VBNC) state based on transcriptomics

Citral has attracted much attention as a safe and effective plant-derived bacteriostatic agent. However, the ability of citral to induce the formation of VBNC state in Vibrio vulnificus has not been evaluated. In the present study, V. vulnificus was shown to be induced to form the VBNC state at 4.5 h and 3 h of citral treatment at 4MIC and 6MIC. Moreover, the citral-induced VBNC state of V. vulnificus maintained some respiratory chain activity and was able to recover well in both APW media, APW media supplemented with 5 % (v/v) Tween 80 and 2 mg/mL sodium pyruvate. Field emission and transmission electron microscopy showed that the external structure of the citral-induced VBNC V. vulnificus cells was shortened to short rods, with folded cell membrane, rough cell surface, and dense cytoplasm and loose nuclear material in the internal cell structure. In addition, the possible molecular mechanisms of citral-induced formation and recovery of V. vulnificus in the VBNC state were explored by transcriptomics. Transcriptome analyses revealed that 1118 genes were significantly altered upon entry into the VBNC state, and 1052 genes were changed after resuscitation. Most of the physiological activities related to energy production were inhibited in the citral-induced VBNC state of V. vulnificus; however, the bacteria retained its pathogenicity. The citral-induced resuscitation of V. vulnificus in the VBNC state selectively restored the activity of some genes related to bacterial growth and reproduction. Meanwhile, the expression levels of other genes may have been influenced by citral-induced resuscitation after the formation of the VBNC state. In conclusion, this study evaluated and analyzed the ability and possible mechanism of citral on the formation of VBNC state and the recovery of VBNC state of V. vulnificus, and made a comprehensive assessment for the safety of citral application in food production.

Evidence of a Viable but Nonculturable (VBNC) Phase in B. abortus S19 under Oxidative Stress (H2O2, -Fe2+, Bleach) and under Non-Oxidative Inhibitory Conditions (Isopropanol, Erythritol, Selenite).

The effect of oxidative stress on the survival of various Brucella species has not been fully investigated yet. We here conducted a study in which we investigated the effect of different types of oxidative stress (Fe2+, H2O2, bleach) versus non-oxidative inhibitory effects (selenite, erythritol, and isopropanol) on the survival of B. abortus S19, B. abortus S19 ∆mglA 3.14, and B. neotomae 5K33. The work focuses on the appearance of ATP-CFU quotient imbalances indicating the existence of a viable but nonculturable (VBNC) form of B. abortus S19, as has previously been shown.

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.

Viable Campylobacter jejuni on Eggshells and Its Potential to Cross-contaminate Egg White and Yolk When Using a Manual Separation Technique, Determined by Culture and Propidium Monoazide (PMA) qPCR

Manual separation of egg yolk from egg white using the eggshell is common practice in private households. For this, the egg is cracked and both components are separated by passing the egg yolk back and forth between the two halves of the eggshell, allowing the egg white to drip down while the egg yolk remains in the shell. During this process, the egg content naturally gets in contact with the outside of the eggshell, which might lead to a cross-contamination with its microorganisms, thus was correspondingly assessed in this study. Campylobacter jejuni is one of the most important zoonotic pathogens that can be found on eggshells. Therefore, this bacterium was used to artificially contaminate the eggshells (n = 22) with concentrations of 3.1 ± 0.6 log10 cfu/g. After separating the egg yolk from the egg white, cross-contamination was determined using culture and qPCR. Altogether, cross-contaminations with C. jejuni were found in 15 egg white (68%) and in three egg yolk (14%) samples. Afterward, 90 eggs from 30 egg packs from different producers in and around Munich (Germany) were obtained for field study purposes. To address the problem of culturing due to a possible viable but nonculturable (VBNC) status of C. jejuni, a method to differentiate viable and dead C. jejuni on eggshell using 10 µM propidium monoazide (PMA) and qPCR was developed. As a result, seven egg packs (23%) were positive for C. jejuni. Of these, only one (3%) was contaminated with viable cells, but still in a concentration of 3.3 log10 cells/g shell. According to these results and considering that eggshells might also be naturally contaminated with other pathogens, the authors recommend avoiding the manual separation technique of egg white and yolk by the eggshell. Especially if raw egg white or yolk is used for preparation of not sufficiently heated foods, where contaminating pathogens are not inactivated during processing, this technique might be a safety hazard for the consumer.

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.

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.

Improved Canker Processing and Viability Droplet Digital PCR Allow Detection of Erwinia amylovora Viable Nonculturable Cells in Apple Bark.

The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 102 to 7.6 × 107 cells mL-1, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management.

Effect of flux and shear rate on E. coli recovery in tangential flow filtration through a single hollow fiber.

Pathogenic bacteria which enter a viable but non-culturable (VBNC) state impede efforts to reach detectable concentrations required for PCR methods. This motivated a strategy for tangential flow filtration to concentrate bacteria in aqueous samples while maintaining the bacteria in a viable state, maximizing their recovery and achieving high fluxes through a single hollow fiber membrane. Filtrations were carried out for green fluorescent protein (GFP) E. coli at high shear rates (up to 27,000 sec-1) through 0.2 μm cut-off polyethersulfone (PES) microfilter membranes or 50 kDa polysulfone (PS) ultrafilter membranes. High shear minimized bacterial attachment on membrane surfaces, which would otherwise occur due to forced convection of the particles to the membrane surface at high flux conditions. Single fiber filter modules were constructed to facilitate concentration of Escherichia coli at fluxes ranging from 55 to 4500 L m-2 h-1. The effect of high shear rates on bacterial viability was found to be minimal with bacterial losses during filtration caused principally by their accumulation on the membrane surface. Recoveries of 90% were achievable at high shear rates when the average flux was ≤300 L m-2 h-1. This corresponded to a 3-h filtration time for a 225 mL sample through a single hollow fiber. Detectable bacteria concentrations of 1800 colony-forming unit (CFU)/mL were achieved for starting concentrations of 140 CFU/mL.

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 (>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.

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.

High pressure processing plus technologies: Enhancing the inactivation of vegetative microorganisms.

High pressure processing (HPP) is a non-thermal technology that can ensure microbial safety without compromising food quality. However, the presence of pressure-resistant sub-populations, the revival of sub-lethally injured (SLI) cells, and the resuscitation of viable but non-culturable (VBNC) cells pose challenges for its further development. The combination of HPP with other methods such as moderate temperatures, low pH, and natural antimicrobials (e.g., bacteriocins, lactate, reuterin, endolysin, lactoferrin, lactoperoxidase system, chitosan, essential oils) or other non-thermal processes (e.g., CO2, UV-TiO2 photocatalysis, ultrasound, pulsed electric fields, ultrafiltration) offers feasible alternatives to enhance microbial inactivation, termed as "HPP plus" technologies. These combinations can effectively eliminate pressure-resistant sub-populations, reduce SLI or VBNC cell populations, and inhibit their revival or resuscitation. This review provides an updated overview of microbial inactivation by "HPP plus" technologies and elucidates possible inactivation mechanisms.