Viable But Nonculturable Cells Research Articles

Reduction, Prevention, and Control of Salmonella enterica Viable but Non-culturable Cells in Flour Food.

The processing and storage conditions of flour food inevitably pose environmental stress, which promote bacteria to enter a viable but non-culturable (VBNC) state. The existence of VBNC cells causes false-negative detection in traditional culture-based detection methods, resulting in food quality and safety issues. This study aimed at investigating the influence factors including nutrition, acid, salt, and temperature for the entry into a VBNC state of Salmonella enterica and an efficient detection method. During induction with multi-stress conditions, nutrition starvation antagonizes with low-level acidity. Besides, high-level acidity was considered as an inhibitor for VBNC induction. Four inducers including nutrition starvation, salt stress, low-level acidity, and low temperature were concluded for a VBNC state. In addition, the keynote conditions for S. enterica entering a VBNC state included (i) nutrient-rich acidic environment, (ii) oligotrophic low-acidity environment, and (iii) oligotrophic refrigerated environment. Based on the keynote conditions, the environmental conditions of high acidity (1.0% v/v acetate) with low temperature (−20°C) could successfully eliminate the formation of S. enterica VBNC cells in flour food. In addition, combining with propidium monoazide pretreatment, PCR technology was applied to detect S. enterica VBNC cells. The sensitivity of the PMA–PCR technology was 105 CFU/ml in an artificially simulated food system. The results derived from this study might aid in the detection and control of VBNC state S. enterica in flour food products.

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.

Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model

The recurrence of Pseudomonas aeruginosa (PA) biofilm infections is a major issue in cystic fibrosis (CF) patients. A pivotal role is played by the presence of antibiotic-unresponsive persisters and/or viable but non-culturable (VBNC) forms, whose development might be favored by subinhibitory antibiotic concentrations. The involvement of tobramycin and ciprofloxacin, widely used to treat CF PA lung infections, in the abundance of VBNC cells was investigated in PA biofilms models. In vitro biofilms of the laboratory strain PAO1-N and the clinical strain C24 were developed and starved by subculture for 170 days in a non-nutrient (NN) broth, unsupplemented or supplemented with one-quarter minimal inhibitory concentration (MIC) of tobramycin or ciprofloxacin. VBNC cells abundance, estimated as the difference between total live (detected by qPCR and flow cytometry) and colony forming unit (CFU) counts, showed a strain- and drug-specific pattern. A greater and earlier abundance of VBNC PAO1-N cells was detected in all conditions. Exposure of the C24 strain to NN and NN + ciprofloxacin induced only a transient VBNC subpopulation, which was more abundant and stable until the end of the experiment in tobramycin-exposed biofilms. The same response to tobramycin was observed in the PAO1-N strain. These findings suggest that low tobramycin concentrations might contribute to PA infection recurrence by favoring the development of VBNC forms.

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

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

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.

Survival of staphylococci and transmissibility of their antimicrobial resistance genes in milk after heat treatments

There is growing concern that the milk heat treatments, which are the main antibacterial safeguard in the dairy industry, are not sufficient in inactivating the antimicrobial resistance (AMR) genes of staphylococci and may induce a viable but non-culturable (VBNC) state of these microorganisms. This study investigated the persistence and quantification of blaZ, mecC and tetK plasmid-mediated AMR genes copy numbers of two staphylococcal strains in both milk and Tris-EDTA (TE) buffer. During subsequent storage after pasteurization, all tested genes showed increased copy numbers. By electroporation of these genes to the Staphylococcus aureus RN42200 electro-competent strain, both mecC and tetK genes were still expressive and transferable. The formation of VBNC cells was estimated with viability staining and quantitative PCR of 16S rDNA copy numbers of both staphylococcal strains. The copy numbers increased over the storage period, which suggests that the pasteurization process was a stress factor and likely induces VBNC state in tested staphylococci. This explains the continual increase in the copy numbers of AMR genes. In contrast, after the sterilization treatment all the genes showed decreases in copy numbers. Further studies are required to evaluate the efficiency of currently practiced pasteurization and to assess the hazard level of VBNC staphylococci.

Treatment with disinfectants may induce an increase in viable but non culturable populations of Listeria monocytogenes in biofilms formed in smoked salmon processing environments

The objectives of this study were 1) to evaluate the impact of two industrial disinfectants on the viability of Listeria monocytogenes populations in biofilm and 2) to investigate the viability state of L. monocytogenes cells present on contact surfaces in the smoked salmon processing environment. In the first step, we cultured mono species and mixed species biofilms containing L. monocytogenes on stainless steel or polyvinyl chloride (PVC) at 8 °C for 48h. The biofilms were then exposed to quaternary ammonium- and hydrogen peroxide-based disinfectants. Residual total populations of L. monocytogenes were measured by qPCR, and viable culturable (VC) cell populations were quantified using standard microbiological culture-based techniques and by a quantitative PCR (qPCR) assay coupled with a propidium monoazide treatment. Decreases in VC populations and the appearance of viable but non culturable (VBNC) populations were observed in response to treatment with the disinfectants. An 8 month sampling campaign in 4 smoked salmon processing plants was also carried out to detect L. monocytogenes in environmental samples. VBNC cells were detected mainly after the cleaning and disinfection operations. This study showed that industrial disinfectants did not inactivate all L. monocytogenes cells on inert surfaces. The presence of VBNC populations of L. monocytogenes in the smoked salmon processing environment is a public health concern.

Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae.

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.

Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation.

The significance of viable but non-culturable (VBNC) cells in the food industry is not well known, mainly because of the lack of suitable detection methodologies to distinguish them from dead cells. The study aimed at the selection of the method to differentiate dead and VBNC cells of Listeria monocytogenes in process wash water (PWW) from the fruit and vegetable industry. Different methodologies were examined including (i) flow cytometry, (ii) viability quantitative polymerase chain reaction (v-qPCR) using an improved version of the propidium monoazide (PMAxx) dye as DNA amplificatory inhibitor, and (iii) v-qPCR combining ethidium monoazide (EMA) and PMAxx. The results showed that the flow cytometry, although previously recommended, was not a suitable methodology to differentiate between dead and VBNC cells in PWW, probably because of the complex composition of the water, causing interferences and leading to an overestimation of the dead cells. Based on results obtained, the v-qPCR combined with EMA and PMAxx was the most suitable technique for the detection and quantification of VBNC cells in PWW. Concentrations of 10 μM EMA and 75 μM PMAxx incubated at 40°C for 40 min followed by a 15-min light exposure inhibited most of the qPCR amplification from dead cells. For the first time, this methodology was validated in an industrial processing line for shredded lettuce washed with chlorine (10 mg/L). The analysis of PWW samples allowed the differentiation of dead and VBNC cells. Therefore, this method can be considered as a rapid and reliable one recommended for the detection of VBNC cells in complex water matrixes such as those of the food industry. However, the complete discrimination of dead and VBNC cells was not achieved, which led to a slight overestimation of the percentage of VBNC cells in PWW, mostly, due to the complex composition of this type of water. More studies are needed to determine the significance of VBNC cells in case of potential cross-contamination of fresh produce during washing.

The potential advantages of using a poly(HPMA) brush in urinary catheters: effects on biofilm cells and architecture

Infections related to bacterial colonization of medical devices are a growing concern given the socio-economical impacts in healthcare systems. Colonization of a device surface with bacteria usually triggers the development of a biofilm, which is more difficult to eradicate than free-floating or adhered bacteria and can act as a reservoir for subsequent infections. Biofilms often harbor Viable but nonculturable (VBNC) cells that are likely to be more resistant to antibiotic treatment and that can become active in more favorable conditions causing infection. Biofilm formation is dependent on different factors, chiefly the properties of the surface and of the surrounding medium, and the hydrodynamic conditions. In this work, the antifouling performance of a poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brush was evaluated in vitro in conditions that mimic a urinary catheter using Escherichia coli as a model organism. The results obtained with the brush were compared to those obtained with two control surfaces, polydimethylsiloxane (PDMS) (the most common material for catheters) and glass. A decrease in initial adhesion and surface coverage was observed on the brush. This antifouling behavior was maintained during biofilm maturation and even in a simulated post-bladder infection period when the reduction in total cell number reached 87 %. Biofilms were shown to adapt their architecture during that period and VBNC cells adsorbed weakly on the brushes and were completely washed away. Taken together, these results suggest that the use of the poly(HPMA) brush in urinary tract devices such as catheters and stents may reduce biofilm formation and possibly render the formed biofilms more susceptible to antibiotic treatment and with reduced infectivity potential.

The fate of cold‐stressed or tetracycline‐resistantVibrio spp. in precooked shrimp during frozen storage

AbstractWe compared the fate of cold‐stressed (CS) or tetracycline‐resistant (TR) Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae in precooked shrimp during frozen storage. The recovery ability of viable but nonculturable (VBNC) Vibrio cells was compared at 25°C. Each suspension of nonstressed (NS), CS, or TR Vibrio cells inoculated into cooked shrimps were subjected to frozen storage at −20°C or three freeze–thaw cycles. CS and TR cells were more rapidly converted to VBNC state than NS cells. Most of VBNC Vibrio cells were observed as viable cells after frozen storage. Although there were differences in the recovery extent of cells depending on the types of stress and strain, VBNC cells were resuscitated at 25°C. The most resistant to tetracycline and the lowest injury rate were observed in V. cholerae cells during frozen storage.

Characterization and potential mechanisms of highly antibiotic tolerant VBNC Escherichia coli induced by low level chlorination

Escherichia coli is an important pathogenic indicator in drinking water. Viable but non-culturable (VBNC) E. coli induced by low level chlorination was found to have higher antibiotic tolerance. The emerging of VBNC bacteria in drinking water systems is posing challenges to the control of bio-safety. It is necessary to study the underlying mechanisms of VBNC state E. coli under actual residual chlorine condition of drinking water pipe network. In this study, we investigated the changes of morphology and gene expressions that might present such state. The results indicated that the size of VBNC E. coli was not remarkably changed or recovered culturability under favorable environmental conditions. Results from transcriptomic analysis revealed that the regulated genes related to fimbrial-like adhesin protein, putative periplasmic pilin chaperone, regulators of the transcriptional regulation, antibiotic resistance genes and stress-induced genes, rendering VBNC cells more tolerant to adverse environmental conditions. In total of 16 genes were significantly up-regulated under the VBNC state, including three genes encoding toxic protein (ygeG, ibsD, shoB), indicating that VBNC E. coil was still a threat to human. The work is of great relevance in the context of better understanding this poorly understood physiological state.

Quantitative detection of viable but nonculturable state Escherichia coli O157:H7 by ddPCR combined with propidium monoazide

Escherichia coli O157:H7, the causative agent of haemorrhagic colitis and haemolytic uremic syndrome in humans, has been implicated in large food-borne outbreaks all over the world. When confronted with harsh environmental stresses, it can enter into a viable but nonculturable (VBNC) state, which poses a great risk to food safety and public health since conventional methods are invalid to detect VBNC cells. Herein, a system for detecting VBNC state Escherichia coli O157:H7 was established by combining droplet digital PCR (ddPCR) with propidium monoazide (PMA). This system took advantage of an absolute quantification without external calibration of ddPCR and DNA binding, modification capacity of PMA after penetrating into compromised cells, providing a sensitive detection limit of 5 copies/μL, good accuracy and specificity. The PMA-ddPCR platform could be applied not only on detection of VBNC cells induced by non-thermal pasteurization technique, high pressure CO2 (HPCD), but also quantification of viable E. coli O157:H7 in food samples including prawn, squid, lettuce and pear juice. Taken together, our research provides an effective method for detecting VBNC pathogenic bacteria during food pasteurization process or in food samples.

Susceptibility of foodborne pathogens to sanitizers in produce rinse water and potential induction of viable but non-culturable state

Chemical sanitizers are commonly used for fresh produce washing to reduce potential presence of foodborne pathogens and spoilage bacteria, and to prevent cross-contamination. In this study, we evaluated the susceptibility of foodborne pathogens, including Shiga-toxigenic Escherichia coli, Salmonella enterica, and Listeria monocytogenes, to free chlorine (FC) and peracetic acid (PAA) in spinach and romaine lettuce rinsates as simulated leafy green wash water. All inoculated pathogens exhibited notably enhanced tolerance (over 2 log of increased recovery) to FC, but not to PAA, when treated in fresh produce rinsates in comparison to that in sterile water. Both FC and PAA effectively inactivated the inoculated foodborne pathogens, rendering them undetectable after 30 s exposure to 10 mg/L FC or 30 mg/L PAA, by selective plating or non-selective enrichment. However, potentially viable cells for all the inoculated foodborne pathogens were detected by PMA-qPCR, especially in wash water samples treated with PAA at concentrations up to 50 mg/L. While laser confocal microscopy after differential staining suggested significant presence of viable but non-culturable (VBNC) populations in PAA treated wash water samples, resuscitation in vitro of the potential VBNC cells was not achieved. This study provides scientific data to better understand the risks of potential VBNC foodborne pathogens during fresh produce washing.

Detection of Viable but Nonculturable Microbial Cells in Chicken Mince

The relevance of the study and the presence of gaps in the relevant knowledge on the topic. Potential existence of hazardous viable but nonculturable (VBNC) cells of pathogenic microorganisms in foodstuffs that can be formed under the influence of various factors, their detection and determination of conditions for formation of VBNC cells of various contaminant bacteria are relevant for preventing contamination of meats.Methods. In the study, a search was conducted for VBNC cells in chicken mince in real time and during experimental infection of it by Staphylococcus aureus 209P. In order to detect VBNC cells in chicken mince, total number of microbes, number of bacterial colonies (CFU), and the portion of living (dead) cells were determined in 1g of the product using a commercial set of fluorescent dyes. A second study was carried out after 5 h of incubation of tested samples at room temperature.Results and discussion. In samples of minced meat on the 4th day after production, more than 99% of all detected living cells were VBNC. After 5-hour incubation of the sample, the number of CFU/g increased by 22.5 times, but the portion of VBNC cells remained higher than 99% of viable bacteria. During artificial infection of the same batch of mince with S. aureus in broth culture at the stage of logarithmic growth, the amount of VBNC cells for 0 hours was 97.3%. After 5 hours their number increased to 99.99%. Probably, in the introduced culture of Staphylococcus at the stage of active reproduction, formation of VBNC bacteria did not occur, which initially reduced their number in the sample. After 5-h incubation, transition of bacteria to VBNC state was accelerated, possibly due to unfavorable conditions for the cell population (changes in trophic substrate, temperature, pH, etc.).Conclusions. Experimental data confirm presence of VBNC bacteria in chicken products that don’t grow on traditional nutrient media, and showing a false negative result in traditional microbiological expertise. Because of the biohazard of such dormant cells, it is advisable to provide regulated testing of foodstuffs for presence of VBNC cells.

Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood

The present study compared different concentrations of propidium monoazide (PMA), time of exposure to light and different light intensities to determine the optimal conditions for the quantification of viable Escherichia coli in cell suspension and in food matrix. The influence of cell density and the effectiveness of PMA in viable but non-culturable (VBNC) E. coli cells were evaluated and also applied in food matrix. For that purpose, different concentrations of PMA (20 μM, 40 μM, 50 μM, 60 μM and 80 μM) under different times of exposure (5 min, 10 min, 15 min, 20 min and 30 min) to lights of different intensities (500 W and 650 W) were evaluated. After determining the optimal conditions, the PMA-qPCR methods were applied to different compositions of live and heat-killed E. coli suspensions (v:v; 0:1; 1:0; 1:1) in concentrations ranging from 3 Log to 7 Log CFU/mL. The same dilutions were prepared with E. coli in VBNC state and applied in food matrix. The results obtained from qPCR, PMA-qPCR and plate counts were compared. The results suggested that a PMA treatment of 50 μM PMA for 15 min under 650 W light intensity was optimal under our conditions. For E. coli cell suspensions, the amplification of heat-killed cells was inhibited greatly by PMA when concentrations were ≤ 5 Log CFU/mL. For the samples of oyster inoculated with heat-killed cells, E. coli was not detected by PMA-qPCR in concentrations ≤4 Log CFU/g. Regarding the results with VBNC state, we considered the PMA-qPCR method to be applicable for enumerating E. coli VBNC cells in oyster samples. Based on our findings, we further recommend the use of PMA-qPCR with the aim of reducing the amplification of dead cells for improving its performance, since false-positives could still occur depending on the level of E. coli in the sample. The application of the PMA-qPCR for quantification of bacteria, compared to the use of culture-dependent methods, is quite promising. However, further studies are recommended, especially using different food matrices.

Transcriptomic Analysis of Viable but Non-Culturable Escherichia coli O157:H7 Formation Induced by Low Temperature.

Escherichia coli O157:H7 is one of the most common pathogenic bacteria that pose a threat to food safety. The aim of this study was to investigate the mechanisms of the formation of viable but non-culturable (VBNC) E. coli O157:H7 induced by low temperature (−20 °C) using RNA sequencing (RNA-Seq) transcriptomics analysis. The results of the present investigation revealed the presence of 2298 differentially expressed genes in VBNC cells, accounting for 46.03% of the total number of genes. Additionally, GO function and KEGG pathway enrichment analysis were performed to investigate the functional and related metabolic pathways of the differentially expressed genes. We found that the ion transport, protein synthesis, and protein transmembrane transport activities were significantly improved in the VBNC cells, indicating that E. coli O157:H7 cells synthesized a considerable amount of protein to maintain the levels of their functional metabolic processes and life activities in the VBNC state. In conclusion, we suggest that the increased synthesis of proteins such as SecY, FtsY, and Ffh might indicate that they are the key proteins involved in the improvement of the transmembrane transport activities in VBNC E. coli O157:H7 cells, maintaining their functional metabolism in the VBNC state and enhancing their survival ability under low temperatures.

ITRAQ-based proteomic analyses of the plant-pathogenic bacterium Acidovorax citrulli during entrance into and resuscitation from the viable but nonculturable state

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) disease, infects cucurbit crops including watermelon and melon. This bacterium can enter the viable but nonculturable (VBNC) state following exposure to copper sulfate. Moreover, copper-induced VBNC A. citrulli cells can be resuscitated by EDTA. In this study, isobaric tag for relative and absolute quantification (iTRAQ) was used to compare protein profiles of VBNC cells, resuscitated cells at different stages and log-phase cells of the A. citrulli model strain AAC00-1. A total of 2672 proteins were identified, with 60 being differentially abundant in VBNC cells compared with log-phase cells, and 469 being differentially abundant in resuscitated cells compared with VBNC cells. Proteins involved in the arginine and proline metabolism pathway and degradation of aromatic compounds could be important for the VBNC cells. In the early resuscitation process, proteins associated with carbon metabolism, and degradation of naphthalene and aromatic compounds were significantly enriched, while proteins involved in oxidative phosphorylation, bacterial chemotaxis, ABC transporters and quorum sensing were significantly enriched at the late resuscitation stages. This is the first study reporting thorough protein profile analyses of VBNC and resuscitating cells of a plant-pathogenic bacterium. Biological significanceThe VBNC state is a dormant-like condition that was reported to occur in many bacterial species, upon facing a variety of environmental stresses. Acidovorax citrulli is a seed borne pathogenic bacterium that threatens cucurbit production worldwide. Moreover, A. citrulli can enter into the VBNC state after treatment of copper sulfate, thus increasing its survival and dissemination probabilities. This study enriches our understanding of the mechanisms of entrance into and resuscitation from the VBNC state of this important plant-pathogenic bacterium. This knowledge could be exploited in the future to develop novel approaches to interfere with these processes, thus contributing to a more efficient management of this pathogen. In a broader perspective, the knowledge emerging from this study has implications to the general understanding of the VBNC state in bacteria.

Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC

In this study, a rapid and sensitive real-time loop-mediated isothermal amplification (Rti-LAMP) assay was developed for quantitative and evaluation of viable but non-culturable (VBNC) Salmonella. Four micrograms per milliliter of ethidium bromide monoazide (EMA) could significantly inhibit DNA amplification derived from dead cells in Rti-LAMP assays. The EMA-Rti-LAMP was used to monitor the culturable and VBNC Salmonella cells induced by 4 °C and − 20 °C, as direct fluorescence method (DEM) and plate counting method as controls. When 1.3 × 104 CFU/mL Salmonella was 5 cycles of freeze-thaw, the cells were all dead. However, Salmonella in 1.3 × 106 CFU/mL gradually transferred into VBNC state reaching 6.0 × 102 CFU/mL (0.05%) after 5 cycles of freeze-thaw. Keeping Salmonella 1.3 × 104 CFU/mL and 1.3 × 106 CFU/mL in 0.85% NaCl at 4 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells generated gradually from 0 to 4.2 × 103 CFU/mL and 1.3 × 105 CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage, respectively. While in − 20 °C, 1.3 × 104 CFU/mL Salmonella sharply inactivated during 20 days, but 1.3 × 106 CFU/mL increasingly transferred into VBNC state reaching 3.5 × 104 CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage. The results indicated that the EMA-Rti-LAMP had similar accuracy with DEM in rapidly detecting viable including VBNC cells, and the former had specificity but the latter did not. The EMA-Rti-LAMP combined with bentonite-coated activated carbon (BCAC) treatment could detect as low as 35 CFU/g VBNC Salmonella derived from contaminated chicken, and the entire assay completed in 5 h. Furthermore, four identical samples were Salmonella positive from 24 retail frozen chicken samples detected by plate culture (GB4789.4-2016), BCAC-Rti-LAMP, and BCAC-EMA-Rti-LAMP. The BCAC-EMA-Rti-LAMP had one more sample for Salmonella positive than that of plate culture, but less two samples than that of BCAC-Rti-LAMP. Noticeably, the BCAC-EMA-Rti-LAMP had much more accuracy as plate counting than that of BCAC-Rti-LAMP in detection of viable Salmonella derived from chicken. These results strongly suggested that the BCAC-EMA-Rti-LAMP assay could be a rapid and sensitive method for detection of viable Salmonella including VBNC cells in chicken without enrichment.