Viable But Non-culturable Cell Research Articles

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.

Resuscitation of viable but nonculturable bacteria promoted by ATP-mediated NAD+ synthesis

IntroductionEntry into the viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria to survive harsh environment. Although VBNC cells still have metabolic activity, they lose the ability to form colonies on nonselective culture media. Thus, conventional bacterial detection methods, such as plate counting, are unable to detect the presence of VBNC cells. When the environmental conditions are appropriate, VBNC cells can initiate resuscitation, posing a great risk to the safety of public health. The study of the VBNC resuscitation mechanism could provide new insights into the prevention and control of VBNC resuscitation. ObjectivesUncovering the molecular mechanism of VBNC cell resuscitation by investigating the role of O-antigen ligase (RfaL) in inhibiting the resuscitation of Escherichia coli O157:H7 in the VBNC state. MethodsRfaL was screened and verified as a resuscitation inhibitor of VBNC Escherichia coli O157:H7 by detecting resuscitation curve and time-lapse microscopy. The mechanism of RfaL impacts VBNC E. coli resuscitation was investigated by detecting the single cell ATP content, metabolomic changes, NAD(H) content and new protein biosynthesis of WT and ΔrfaL at different stage of resuscitation. ResultsMutation of rfaL, which encoded an O-antigen ligase, markedly shortened the resuscitating lag phase. Further studies indicated that ΔrfaL VBNC cells contained higher ATP levels, and ATP consumption during the resuscitating lag phase was highly correlated with resuscitation efficiency. Metabolomic analysis revealed that ATP was utilized to activate the Handler and salvage pathways to synthesize NAD+, balancing redox reactions to recover cell activity and promote cell resuscitation. ConclusionOur findings revealed a strategy employed by VBNC cells for revival, that is, using residual ATP to primarily recover metabolic activity, driving cells to exit dormancy. The synthesis pathway of lipopolysaccharide (LPS) in rfaL null mutant was inhibited and could supply more ATP to synthesis NAD+ and promote resuscitation.

Combatting persister cells: The daunting task in post-antibiotics era.

Over the years, much attention has been drawn to antibiotic resistance bacteria, but drug inefficacy caused by a subgroup of special phenotypic variants - persisters - has been largely neglected in both scientific and clinical field. Interestingly, this subgroup of phenotypic variants displayed their power of withstanding sufficient antibiotics exposure in a mechanism different from antibiotic resistance. In this review, we summarized the clinical importance of bacterial persisters, the evolutionary link between resistance, tolerance, and persistence, redundant mechanisms of persister formation as well as methods of studying persister cells. In the light of our recent findings of membrane-less organelle aggresome and its important roles in regulating bacterial dormancy depth, we propose an alternative approach for anti-persister therapy. That is, to force a persister into a deeper dormancy state to become a VBNC (viable but non-culturable) cell that is incapable of regrowth. We hope to provide the latest insights on persister studies and call upon more research interest into this field.

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.

Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

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

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

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Development and Application of a Simple "Easy To Operate" Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli.

O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.

Effects of quorum sensing on the biofilm formation and viable but non-culturable state

Quorum sensing exists widely in all kinds of microorganisms and is a communication channel for microorganisms. Many bacterial processes, including virulence factor expression, biofilm formation, and viable but non-culturable (VBNC) cell resuscitation, are mediated by quorum sensing, and biofilm formation complicates the treatment of various infections. Foodborne pathogens can enter VBNC state in extreme environments, and pathogens in VBNC state can evade traditional detection and resuscitate under appropriate conditions, causing potential harm to human health. The disruption of quorum sensing may decisively help control biofilm formation and VBNC cell resuscitation. This review describes the quorum sensing systems of various bacteria and major fungi, and summarizes the role of bacterial quorum sensing system in biofilm formation and VBNC resuscitation. In addition, the relationship between quorum sensing inhibitors (QSI) with biofilms and VBNC is also discussed.

Survival strategy of Cronobacter sakazakii against ampicillin pressure: Induction of the viable but nonculturable state

In a viable but nonculturable (VBNC) state, bacteria are no longer culturable on standard laboratory media, but still, remain a pathogenic potential and present possible health risks. In this study, we investigated ampicillin's ability, which is commonly used in dairy cattle disease treatment, to induce Cronobacter sakazakii into the VBNC state. After treatment with ampicillin, the counts of culturable cells decreased from 108 CFU/mL to an undetected level 7–30 days post-treatment. Meanwhile, viable cells were still approximately 104–105 cells/mL, and could be resuscitated under appropriate conditions. Fluorescence microscopy showed that VBNC cell maintained apparent cellular integrity, but that the morphology of VBNC cells differed visibly from that of normal cells. Moreover, the respiratory chain activity of VBNC cells were confirmed by flow cytometry (FCM) analysis, suggesting that cells in a VBNC state were physiologically active. Finally, transcriptomics analysis and real-time PCR (qPCR) validation were used to explore the underlying mechanisms of VBNC cell formation. Over-expression of relA, lon, ppx, and ppk in the toxin-antitoxin (TA) trigger system contributed to VBNC cell formation. In the TA trigger system, RelA and exopolyphosphatases/guanosine pentaphosphate phosphohydrolases (PPX/GPPA) synthesize ppGpp, which activates polyphosphate kinase (PPK), the cellular enzyme that accumulates plyphosphate (PolyP). PolyP combines with and stimulates Lon to degrade the antitoxins, thereby activating the toxins that induce a VBNC state. The results of our research will facilitate a better understanding of the survival strategies that bacteria develop to deal with ampicillin pressure and the health risks associated with VBNC Cronobacter sakazakii induced by antibiotics.

Flow-cytometry analysis reveals persister resuscitation characteristics

BackgroundPersisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics. Although both cell types are stained as live and often appear as nongrowing during antibiotic treatment, the only distinguishing feature is the ability of persisters to recolonize in standard culture media in the absence of antibiotics. Despite considerable progress in the characterization of persister formation mechanisms, their resuscitation mechanisms remain unclear due to technical limitations in detecting and isolating these cell types in culture environments that are highly heterogeneous.ResultsIn this study, we used a methodology integrating flow cytometry, fluorescent protein expression systems and ampicillin-mediated cell lysing technique to monitor persister resuscitation at the single-cell level. With this method, we were able to investigate the effects of various culture conditions (e.g., antibiotic treatment time, the length of the stationary phase in overnight pre-cultures, or pretreatment of cells with a metabolic inhibitor) on persister resuscitation. Although we observed long-term pre-cultures have many more VBNC cells compared to short-term pre-cultures, only a small fraction of non-lysed cells was able to resuscitate in all conditions tested. Regardless of pre-culturing and ampicillin treatment times, these persister cells started to resuscitate within 1 hour, after they were transferred to fresh liquid media, with the same doubling time that normal cells have. Our analysis further showed that ampicillin was not able to lyse the cells in the presence of arsenate, a metabolic inhibitor commonly used to increase bacterial persistence. However, the removal of arsenate during antibiotic treatment resulted in cell lysis and a reduction in persister levels despite the significant decrease in ATP levels in the cells.ConclusionsThe strategy presented in this study helps us monitor persister resuscitation at the single-cell level, and simultaneously quantify persister, VBNC and dead cell subpopulations in ampicillin-treated cultures. Our results indicate that the characterization of persister resuscitation with flow cytometry will enhance the current molecular-level understanding of persistence and its evolution.

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

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

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

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

Detection of viable but nonculturable Vibrio parahaemolyticus in shrimp samples using improved real-time PCR and real-time LAMP methods

Accurate and highly sensitive detection of viable but nonculturable (VBNC) Vibrio parahaemolyticus is crucial for preventing seafood-related outbreaks. The VBNC state may occur due to bacterial susceptibility to cold shock during food storage. Propidium monoazide (PMA) has been widely applied to detect VBNC foodborne pathogens. In this study, we developed and compared real-time PCR (qPCR) and real-time LAMP (qLAMP) methods combined with an improved propidium monoazide (PMAxx) to detect pathogenic VBNC V. parahaemolyticus. The designed primers and probes were determined to be highly selective for V. parahaemolyticus strains. The combined PMAxx-qPCR method, requiring 100 min, demonstrated a quantification limit of 10.5 colony-forming units (CFU)/mL in pure culture and 28 CFU/g in raw shrimp respectively, which were 10-fold lower than the PMAxx-qLAMP method (45min). When testing mixtures containing different ratios of VBNC to dead V. parahaemolyticus, PMAxx-based methods were notably superior at distinguishing between VBNC and dead bacteria when VBNC cell concentrations were low. Therefore, PMAxx is an effective means for improving the detection and quantification of VBNC V. parahaemolyticus by qPCR and qLAMP. Pretreatment with PMAxx was shown to be suitable for detection of VBNC V. parahaemolyticus in complex food samples, including raw shrimp.

Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy

BackgroundClonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population’s resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment.MethodsHere, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment.ResultsWe show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism.ConclusionOur data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.

Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent

The detection and quantification of viable Escherichia coli cells in wastewater treatment plant effluent is very important as it is the main disinfection efficacy parameter for assessing its public health risk and environmental impact. The aim of this study was to develop a sensitive and false-positive free propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay to quantify the viable but non-culturable (VBNC) E. coli present in secondary wastewater effluent after chlorine disinfection. The qPCR target was the E. coli uidA gene, and native Taq was used to eliminate false positives caused by the presence of contaminant E. coli DNA in recombinant Taq polymerase reagents. Due to issues with qPCR inhibitors in wastewater, this study explored several pre-DNA extraction treatment methods for qPCR inhibitor removal. PMA-qPCR validation was done using salmon testes DNA (Sketa DNA) as an exogenous control added directly to the wastewater samples and amplified using a separate qPCR assay. After disinfection of secondary effluent with 2ppm chlorine at the plant, the mean Log10 CFU reduction in E. coli was 2.85 from a mean CFU of 3.48/10mL compared to 0.21 Log10 CCE mean reduction of the uidA gene from a mean CCE of 3.16/10mL. The VBNC cell concentrations were calculated as 2.32 Log10/10mL by subtracting the colony forming units (CFU) obtained from membrane filtration from the calculated CFU equivalent (CCE) values obtained from PMA-qPCR. These results demonstrate the effective use of a PMA-qPCR method for the quantification of the E. coli uidA gene and indicate there are high numbers (2.01×103CCE/100mL) of VBNC E. coli cells leaving the wastewater treatment plant in the final effluent after chlorine treatment. VBNC bacterial cells are of concern as they have the potential to resuscitate and grow, regain virulence, affect natural microbiome in the discharge sites, and pass on antimicrobial resistant genes to other microorganisms.

Functional magnetic nanoparticles for facile viable but nonculturable bacteria separation and purification

Viable But Nonculturable (VBNC) Bacteria, which represent a unique population of microorganisms in drinking water systems, have become a potential threat to human health. Current studies on VBNC cells usually fail to obtain pure VBNC state bacteria, which may lead to inaccurate results. We therefore introduce a novel method of VBNC cell separation and purification in this paper. PAH-coated magnetic nanoparticles (MNPs) were synthesized and found to be capable of capturing and releasing bacterial cells with high efficiency. With the aid of an additional incubation step, VBNC cells were easily isolated and purified from normal bacteria using functional MNPs. Our method represents a new technique that can be utilized in studies of VBNCs. Open image in new window

Induction of the viable but nonculturable state in Clavibacter michiganensis subsp. michiganensis and in planta resuscitation of the cells on tomato seedlings

Bacterial canker of tomato is an economically important seedborne disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm). Copper‐based bactericides and seed treatment with hydrochloric acid are commonly used for bacterial canker management. Recent studies have shown that some bacteria can enter a viable but nonculturable (VBNC) state, and fail to form colonies on microbiological agar media. Bacteria in the VBNC state can recover their culturability when returned to favourable conditions. This study reports the induction of the VBNC state in Cmm by CuSO4 and low pH, and resuscitation of VBNC cells on tomato seedlings. Flow cytometry using the nucleic acid dyes SYTO 9 and propidium iodide, combined with agar plating, was used to assess VBNC cell counts. It was demonstrated that CuSO4 and low pH induced the VBNC state in Cmm and the rate of induction increased with copper ion concentration and acidity. Pathogenicity tests showed that some of the VBNC cells induced by CuSO4 retained their ability to colonize tomato seedlings but failed to produce typical bacterial canker symptoms by 2 months post‐inoculation. This was probably due to low levels of resuscitation of VBNC Cmm cells resulting in low levels of initial inoculum. This study has improved understanding of the VBNC state of Gram‐positive phytopathogenic bacteria. Most importantly, because copper‐based chemicals and low pH conditions are used for disease management, induction of the VBNC state and subsequent resuscitation of Cmm cells on tomato seedlings may limit pathogen detection by culture‐based assays yet present a risk for disease development in the field.

Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR

The well-known human pathogenic bacterium, Vibrio cholerae, can enter a physiologically viable but non-culturable (VBNC) state under stress conditions. The differentiation of VBNC cells and nonviable cells is essential for both disease prevention and basic research. Among all the methods for detecting viability, propidium monoazide (PMA) combined with real-time PCR is popular because of its specificity, sensitivity, and speed. However, the effect of PMA treatment is not consistent and varies among different species and conditions. In this study, with an initial cell concentration of 1×108CFU/ml, time and dose–effect relationships of different PMA treatments were evaluated via quantitative real-time PCR using live cell suspensions, dead cell suspensions and VBNC cell suspensions of V. cholerae O1 El Tor strain C6706. The results suggested that a PMA treatment of 20μM PMA for 20min was optimal under our conditions. This treatment maximized the suppression of the PCR signal from membrane-compromised dead cells but had little effect on the signal from membrane-intact live cells. In addition to the characteristics of PMA treatment itself, the initial concentration of the targeted bacteria showed a significant negative influence on the stability of PMA-PCR assay in this study. We developed a strategy that mimicked a 1×108CFU/ml cell concentration with dead bacteria of a different bacterial species, the DNA of which cannot be amplified using the real time PCR primers. With this strategy, our optimal approach successfully overcame the impact of low cell density and generated stable and reliable results for counting viable cells of V. cholerae in the VBNC state.

Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios

Entry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogen Vibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNC V. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.