Journal articles: 'Viable but non-culturable bacteria (VBNC)'

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Hamabata, Takashi, Mitsutoshi Senoh, Masaaki Iwaki, Ayae Nishiyama, Akihiko Yamamoto, and Keigo Shibayama. "Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae." Microorganisms 9, no. 5 (April 26, 2021): 927. http://dx.doi.org/10.3390/microorganisms9050927.

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Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

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Fleischmann, Susanne, Christian Robben, Thomas Alter, Peter Rossmanith, and Patrick Mester. "How to Evaluate Non-Growing Cells—Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria." Antibiotics 10, no. 2 (January 26, 2021): 115. http://dx.doi.org/10.3390/antibiotics10020115.

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Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches.

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Wideman, Nathan E., James D. Oliver, Philip Glen Crandall, and Nathan A. Jarvis. "Detection and Potential Virulence of Viable but Non-Culturable (VBNC) Listeria monocytogenes: A Review." Microorganisms 9, no. 1 (January 19, 2021): 194. http://dx.doi.org/10.3390/microorganisms9010194.

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The detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLightTM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLightTM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is compared

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Wagley, Sariqa, Helen Morcrette, Andrea Kovacs-Simon, Zheng R. Yang, Ann Power, Richard K. Tennant, John Love, Neil Murray, Richard W. Titball, and Clive S. Butler. "Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival." PLOS Pathogens 17, no. 1 (January 13, 2021): e1009194. http://dx.doi.org/10.1371/journal.ppat.1009194.

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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.

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Grey, Brian E., and Todd R. Steck. "The Viable But Nonculturable State ofRalstonia solanacearum May Be Involved in Long-Term Survival and Plant Infection." Applied and Environmental Microbiology 67, no. 9 (September 1, 2001): 3866–72. http://dx.doi.org/10.1128/aem.67.9.3866-3872.2001.

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ABSTRACT The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearumcells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.

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Zhong, Junliang, and Xihong Zhao. "Transcriptomic Analysis of Viable but Non-Culturable Escherichia coli O157:H7 Formation Induced by Low Temperature." Microorganisms 7, no. 12 (November 30, 2019): 634. http://dx.doi.org/10.3390/microorganisms7120634.

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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.

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Hati, Revita Permata, Ratih Dewanti-Hariyadi, and L. Nuraida. "Cronobacter sakazakii local isolates response to acid stress and their resuscitability." Food Research 4, no. 1 (September 16, 2019): 244–53. http://dx.doi.org/10.26656/fr.2017.4(1).257.

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Cronobacter spp. has been reported to cause meningitis, necrotizing enterocolitis, and septicemia in a group of infants through the consumption of powder infant formula. These bacteria are reported to withstand various stress conditions such as heating, drying, low water activity, low pH, etc. A local isolate of Cronobacter sakazakii YRt2a was reportedly survived and entered Viable But Non-Culturable (VBNC) conditions during desiccation stress. This study aims to study the behavior of local isolates of Cronobacter spp. in response to acid stress and its resuscitability. C. sakazakii E2 and YRt2a were grown in TSB at pH 3.0±0.2 or 3.5±0.2. The number of culturable cells and viable cells were enumerated by the Total Plate Count and Direct Viable Count methods, respectively. Resuscitation was done by growing the stress or VBNC cells in TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer. The results showed that C. sakazakii E2 and YRt2a entered VBNC state after 60 mins of exposure to pH 3.0±0.2, while remained culturable after 120 minutes exposure to pH 3.5±0.2. TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer could resuscitate the stress or VBNC cells of C. sakazakii. Stress or VBNC state experienced by C. sakazakii in response to acid tends to be transient and can be resuscitated. C. sakazakii experiencing stress or VBNC may pose a risk for food safety.

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Highmore, Callum J., Jennifer C. Warner, Steve D. Rothwell, Sandra A. Wilks, and C. William Keevil. "Viable-but-NonculturableListeria monocytogenesandSalmonella entericaSerovar Thompson Induced by Chlorine Stress Remain Infectious." mBio 9, no. 2 (April 17, 2018): e00540-18. http://dx.doi.org/10.1128/mbio.00540-18.

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ABSTRACTThe microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-taggedListeria monocytogenesandSalmonella entericaserovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viableL. monocytogenesandSalmonellaThompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNCL. monocytogenesandSalmonellaThompson was assessed by usingCaenorhabditis elegans. Ingestion of VBNC pathogens byC. elegansresulted in a significant life span reduction (P= 0.0064 andP< 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturableL. monocytogenestreatments was observed.L. monocytogeneswas visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.IMPORTANCEMany bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogensListeria monocytogenesandSalmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed inCaenorhabditis elegansthat ingested these VBNC pathogens, with VBNCL. monocytogenesas infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.

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Dusserre, Eric, Christophe Ginevra, Sylvie Hallier-Soulier, François Vandenesch, Gabriel Festoc, Jerome Etienne, Sophie Jarraud, and Maëlle Molmeret. "A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability." Applied and Environmental Microbiology 74, no. 15 (May 30, 2008): 4817–24. http://dx.doi.org/10.1128/aem.02899-07.

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ABSTRACT Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 106 genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 105 and 102 metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.

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Li, Yanlin, Jixiang Chen, Yonggang Wang, Dan Ma, and Wenhong Rui. "The effects of the recombinant YeaZ of Vibrio harveyi on the resuscitation and growth of soil bacteria in extreme soil environment." PeerJ 8 (December 21, 2020): e10342. http://dx.doi.org/10.7717/peerj.10342.

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Numerous bacteria entered the viable but non-culturable state due to the stresses of dry and salt in soils. YeaZ of Gram-negative bacteria is a resuscitation promoting factor (Rpf) homologous protein could resuscitate bacteria of natural environment in VBNC state. To investigate the promoting effect of YeaZ on the isolation of viable but non-culturable (VBNC) bacteria from soil samples in extreme environments, the recombinant YeaZ of Vibrio harveyi was prepared and added to the soil samples from volcanic soil and saline soil in Northwest China. The study has shown that YeaZ can promote the recovery and growth of soil microorganisms, and the number of cultivable bacteria in volcanic and saline soil has increased from 0.17 × 103 and 2.03 × 103 cfu⋅ml−1 to 1.00 × 103 and 5.55 × 103 cfu⋅ml−1, respectively. The 16S rDNA gene sequencing and phylogenetic analysis showed that YeaZ played an essential role in the increase of composition and diversity of bacteria. A total of 13 bacterial strains were isolated from the volcanic soil samples, which belong to phyla Actinobacteria, Firmicutes and Gamma-proteobacteria. Four species, including Ornithinimicrobium kibberense, Agrococcus citreus, Stenotrophomonas rhizophila and Pseudomonas zhaodongensis were found in the control group, while Micrococcus antarcticus, Kocuria rose, Salinibacterium xinjiangense, Planococcus antarcticus, Ornithinimicrobium kibberense and Pseudomonas zhaodongensis were isolated from the treatment groups (addition of YeaZ). Twenty-one strains were isolated from the saline soil samples, including eight species from the control group and thirteen species from the treatment groups, among which nine species were only found, including Bacillus oceanisediminis, Brevibacillus brevis, Paenibacillus xylanilyticus, Microbacterium maritypicum, B. subtilis, B. alcalophilus, B. niabensis, Oceanimonas doudoroffii and Zobellella taiwanensis. The results suggest that addition of YeaZ to soil samples can promote the recovery of VBNC. This method has the implications for the discovery of VBNC bacteria that have potential environmental functions.

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Trevors, J. T. "Viable but non-culturable (VBNC) bacteria: Gene expression in planktonic and biofilm cells." Journal of Microbiological Methods 86, no. 2 (August 2011): 266–73. http://dx.doi.org/10.1016/j.mimet.2011.04.018.

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Khan, Mohiuddin M. Taimur, Barry H. Pyle, and Anne K. Camper. "Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry." Applied and Environmental Microbiology 76, no. 15 (June 11, 2010): 5088–96. http://dx.doi.org/10.1128/aem.02932-09.

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ABSTRACT An issue of critical concern in microbiology is the ability to detect viable but nonculturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods) are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium cells after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40, and propidium iodide (PI). The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different Gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

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Blinkova, Larisa, Danik M. Martirosyan, Yury Pakhomov, Olga Dmitrieva, Rachel Vaughan, and Michael Altshuler. "Nonculturable forms of bacteria in lyophilized probiotic preparations." Functional Foods in Health and Disease 4, no. 2 (February 9, 2014): 66. http://dx.doi.org/10.31989/ffhd.v4i2.29.

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Background: Nonculturable cells are formed under stress. These viable but nonculturable (VBNC) cells retain the ability to revert to active growth and division when conditions become favorable, or after treatment with resuscitating factors. Information about the possible presence of VBNC in bacterial lyophilized probiotic preparations, foodstuffs, live vaccines, etc., indicates that human as well as animal intestines are a significant area for research.Methods: Samples were stored for different periods of time (up to 30 years) according to the manufacturers’ manuals. Total counts were conducted using the Goryaev-Thoma counting chamber and actual viability was assessed by luminescence microscopy after staining with Live/Dead® (Baclight™). CFU/ml counts were made using solid or semisolid media. Viable cells that lacked the ability to form colonies were considered VBNC.Results: We studied 11 batches of commercial probiotics (Russia) from different sources, containing lyophilized E. coli, lactobacilli, or bifidobacteria, in ampoules or vials. In E. coli preparations, depending on storage periods, the amounts of VBNC varied from 4.1% (3 years) to 99.7% (30 years) and showed different total viability (52.2 – 91.3%), as well as the percentage of VBNC cells. A different sample that had been expired for 11 years was 79.5% NC. It is also noteworthy that the 5-dose vials, 4 years past expiration, from yet another source, showed a higher amount of VBNC cells (85.5%). Two different batches that had been expired for three years contained 4.1 and 21.3% VBNC cells. 4 of the 5-dose vials of lyophilized lactobacilli were not expired and contained 58.8 – 80.4% VBNC cells. Total viability varied from 92.9 to 100%, and there was an unmistakable positive correlation between total viability and culturability. The last batch, which had expired 6 years earlier, has 23.7% viable cells and about 98% VBNC. Non-expired bifidobacterial samples contained 70.7 and 95.5% of viable cells and were 50 and 100% culturable. Conclusion: We demonstrated the presence of VBNC cells in lyophilized probiotic preparations that contained live bacteria. Probiotics stored past their expiration date may retain a high potential medical effect because they contained high numbers of viable cells. VBNC cells in studied preparations may have the potential to return to an actively growing state.Keywords: nonculturable forms of bacteria, probiotics

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Cappelier, J. M., J. Minet, C. Magras, R. R. Colwell, and M. Federighi. "Recovery in Embryonated Eggs of Viable but Nonculturable Campylobacter jejuni Cells and Maintenance of Ability To Adhere to HeLa Cells after Resuscitation." Applied and Environmental Microbiology 65, no. 11 (November 1, 1999): 5154–57. http://dx.doi.org/10.1128/aem.65.11.5154-5157.1999.

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ABSTRACT The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.

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Ordax, Mï¿½nica, Ester Marco-Noales, Marï¿½a M. Lï¿½pez, and Elena G. Biosca. "Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State." Applied and Environmental Microbiology 72, no. 5 (May 2006): 3482–88. http://dx.doi.org/10.1128/aem.72.5.3482-3488.2006.

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ABSTRACT Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.

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Arvizu, Ignacio Servando, and Sean Richard Murray. "A simple, quantitative assay for the detection of viable but non-culturable (VBNC) bacteria." STAR Protocols 2, no. 3 (September 2021): 100738. http://dx.doi.org/10.1016/j.xpro.2021.100738.

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Rahman, Farjana, and Rashed Noor. "Prevalence of pathogenic bacteria in common salad vegetables of Dhaka Metropolis." Bangladesh Journal of Botany 41, no. 2 (January 21, 2013): 159–62. http://dx.doi.org/10.3329/bjb.v41i2.13442.

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Microbial quality of common salad vegetables (viz. carrot, cucumber, tomato and lettuce) collected from Dhaka metropolis was analysed to detect the presence of bacterial pathogens. The occurrence of huge numbers of fecal coliforms (1.0×104 - 4.09×106 cfu/g), Escherichia coli (1.0×104 - 5.0×108 cfu/g), Staphylococcus aureus (2.0×105 - 5.95×107 cfu/g), and Listeria spp. (1.5×106 - 6.5×107 cfu/g) were detected in all the tested samples. Interestingly, occurrence of viable but non-culturable (VBNC) bacteria was also noticed. DOI: http://dx.doi.org/10.3329/bjb.v41i2.13442 Bangladesh J. Bot. 41(2): 159-162, 2012 (December)

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Fernández-Delgado, Milagro, María Alexandra García-Amado, Monica Contreras, Renzo Nino Incani, Humberto Chirinos, Héctor Rojas, and Paula Suárez. "SURVIVAL, INDUCTION AND RESUSCITATION OF Vibrio cholerae FROM THE VIABLE BUT NON-CULTURABLE STATE IN THE SOUTHERN CARIBBEAN SEA." Revista do Instituto de Medicina Tropical de São Paulo 57, no. 1 (February 2015): 21–26. http://dx.doi.org/10.1590/s0036-46652015000100003.

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The causative agent of cholera, Vibrio cholerae, can enter into a viable but non-culturable (VBNC) state in response to unfavorable conditions. The aim of this study was to evaluate the in situ survival of V. cholerae in an aquatic environment of the Southern Caribbean Sea, and its induction and resuscitation from the VBNC state. V. cholerae non-O1, non-O139 was inoculated into diffusion chambers placed at the Cuare Wildlife Refuge, Venezuela, and monitored for plate, total and viable cells counts. At 119 days of exposure to the environment, the colony count was < 10 CFU/mL and a portion of the bacterial population entered the VBNC state. Additionally, the viability decreased two orders of magnitude and morphological changes occurred from rod to coccoid cells. Among the aquatic environmental variables, the salinity had negative correlation with the colony counts in the dry season. Resuscitation studies showed significant recovery of cell cultivability with spent media addition (p < 0.05). These results suggest that V. cholerae can persist in the VBNC state in this Caribbean environment and revert to a cultivable form under favorable conditions. The VBNC state might represent a critical step in cholera transmission in susceptible areas.

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Noll, Matthias, Katharina Trunzer, Antje Vondran, Szilvia Vincze, Ralf Dieckmann, Sascha Al Dahouk, and Carolin Gold. "Benzalkonium Chloride Induces a VBNC State in Listeria monocytogenes." Microorganisms 8, no. 2 (January 28, 2020): 184. http://dx.doi.org/10.3390/microorganisms8020184.

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The objective of our study was to investigate the effects of benzalkonium chloride (BC) adaptation of L. monocytogenes on the susceptibility to antimicrobial agents and on the viable but non culturable (VBNC) state of the bacterial cells. We adapted L. monocytogenes SLCC2540 to BC by applying BC below minimum inhibitory concentration (MIC) to above minimum bactericidal concentration (MBC). The culturable fractions and the susceptibility of adapted and parental cells to BC were assessed. In addition, cell membrane permeability and glucose uptake were analyzed by multi parametric flow cytometry using the fluorescent agents SYTO9, propidium iodide, and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG). Adapted cells displayed a two-fold MIC increase of BC and reduced antibiotic susceptibility. At high BC concentrations, the decrease in the number of colony forming units was significantly lower in the population of adapted cells compared to parental cells. At the same time, the number of metabolically active cells with intact membranes was significantly higher than the number of culturable cells. Growth-independent viability assays revealed an adapted subpopulation after BC application that was not culturable, indicating increased abundance of viable but nonculturable (VBNC) cells. Moreover, adapted cells can outcompete non-adapted cells under sublethal concentrations of disinfectants, which may lead to novel public health risks.

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Dinu, Laura-Dorina, and Susan Bach. "Induction of Viable but Nonculturable Escherichia coli O157:H7 in the Phyllosphere of Lettuce: a Food Safety Risk Factor." Applied and Environmental Microbiology 77, no. 23 (September 30, 2011): 8295–302. http://dx.doi.org/10.1128/aem.05020-11.

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ABSTRACTEscherichia coliO157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate ofE. coliO157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction ofE. coliO157:H7 Tn7gfptransformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), bothE. coliO157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (109and 106E. coliO157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log10cells but did not detect culturable cells. These findings indicate thatE. coliO157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.

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Kaur, Jasjeet, R. Karthikeyan, and R. Smith. "Assessment of Escherichia coli reactivation after photocatalytic water disinfection using flow cytometry: comparison with a culture-based method." Water Supply 13, no. 3 (May 1, 2013): 816–25. http://dx.doi.org/10.2166/ws.2013.071.

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The photocatalytic process generates highly reactive oxidative species, such as hydroxyl radicals, which enable mineralization of cellular compounds. Microorganisms often tend to lose their culturability after disinfection, but could remain viable to proliferate under optimum conditions. Estimation of bacterial counts using culture-based methods pose limitations in differentiating viable, non-viable, and viable but non-culturable (VBNC) cells. Presence of viable and VBNC state cells in disinfected water could pose a potential health risk and accurate estimation of these cells through a molecular method is critical. Assessment of live/dead states of an indicator waterborne pathogen, Escherichia coli (ATCC®10798) after disinfection was conducted using flow cytometry. Photocatalysis was carried out under low pressure ultraviolet (LP UV) radiation alone and at four titanium dioxide (TiO2) concentrations (1, 0.75, 0.5 and 0.1 g/L). During the repair period, flow cytometry showed 4–5 log10 higher cell counts than the culture-based method. Photocatalysis using 0.1 g/L TiO2 resulted in 50% cells with intact cell membrane during the repair period and lowered the repair rate of the ATCC®10798, E. coli after disinfection.

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Li, Yanmei, Ling Yang, Jie Fu, Muxia Yan, Dingqiang Chen, and Li Zhang. "The novel loop-mediated isothermal amplification based confirmation methodology on the bacteria in Viable but Non-Culturable (VBNC) state." Microbial Pathogenesis 111 (October 2017): 280–84. http://dx.doi.org/10.1016/j.micpath.2017.09.007.

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Oliver, James D., Maya Dagher, and Karl Linden. "Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater." Journal of Water and Health 3, no. 3 (September 1, 2005): 249–57. http://dx.doi.org/10.2166/wh.2005.040.

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We examined the effects of chlorine disinfection on Escherichia coli and Salmonella typhimurium in secondary-treated wastewater to determine whether such treatment might induce these bacteria into the viable but nonculturable (VBNC) state. In this state, cells lose culturability but retain viability and the potential to revert to the metabolically active and infectious state. To examine the effects of chlorination on cells in different physiological states, cells from the logarithmic and stationary phases, or nutrient starved, or grown in natural wastewater, were studied. Isogenic cells with and without plasmids were also examined. Whereas a mixture of free and combined chlorine, as occurs under typical wastewater disinfection, was found to be rapidly lethal to most cells, regardless of their physiological state or plasmid content, c. 104 of the original 106 cells ml−1 did survive in the VBNC state. While we were not successful in resuscitating these cells to the culturable state, the presence of such nonculturable cells in treated wastewater offers a potential public health hazard.

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Mangiaterra, Gianmarco, Elisa Carotti, Salvatore Vaiasicca, Nicholas Cedraro, Barbara Citterio, Anna La Teana, and Francesca Biavasco. "Contribution of Drugs Interfering with Protein and Cell Wall Synthesis to the Persistence of Pseudomonas aeruginosa Biofilms: An In Vitro Model." International Journal of Molecular Sciences 22, no. 4 (February 5, 2021): 1628. http://dx.doi.org/10.3390/ijms22041628.

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The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung.

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Giagnoni, Laura, Mariarita Arenella, Erica Galardi, Paolo Nannipieri, and Giancarlo Renella. "Bacterial culturability and the viable but non-culturable (VBNC) state studied by a proteomic approach using an artificial soil." Soil Biology and Biochemistry 118 (March 2018): 51–58. http://dx.doi.org/10.1016/j.soilbio.2017.12.004.

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Orruño, Maite, Claudia Parada, Vladimir R. Kaberdin, and Inés Arana. "The Effect of Visible Light on Cell Envelope Subproteome during Vibrio harveyi Survival at 20 °C in Seawater." Microorganisms 9, no. 3 (March 13, 2021): 594. http://dx.doi.org/10.3390/microorganisms9030594.

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A number of Vibrio spp. belong to the well-studied model organisms used to understand the strategies developed by marine bacteria to cope with adverse conditions (starvation, suboptimal temperature, solar radiation, etc.) in their natural environments. Temperature and nutrient availability are considered to be the key factors that influence Vibrio harveyi physiology, morphology, and persistence in aquatic systems. In contrast to the well-studied effects of temperature and starvation on Vibrio survival, little is known about the impact of visible light able to cause photooxidative stress. Here we employ V. harveyi ATCC 14126T as a model organism to analyze and compare the survival patterns and changes in the protein composition of its cell envelope during the long-term permanence of this bacterium in seawater microcosm at 20 °C in the presence and absence of illumination with visible light. We found that V. harveyi exposure to visible light reduces cell culturability likely inducing the entry into the Viable but Non Culturable state (VBNC), whereas populations maintained in darkness remained culturable for at least 21 days. Despite these differences, the starved cells in both populations underwent morphological changes by reducing their size. Moreover, further proteomic analysis revealed a number of changes in the composition of cell envelope potentially accountable for the different adaptation pattern manifested in the absence and presence of visible light.

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Shammi, Tahmina. "Detection of Vibrio spp., Salmonella spp., and Shigella spp. among the frozen food samples employing enrichment culture technique." Stamford Journal of Microbiology 5, no. 1 (March 1, 2016): 26–29. http://dx.doi.org/10.3329/sjm.v5i1.26917.

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Freezing has long been an established method for food preservation. Freezing temperature may act as a stress factor for microbial cells, transforming the cells into injured or dormant state. Upon inoculation, these debilitated cells cannot grow on solid media and hence produce false negative results. Foods contaminated with injured cells of pathogenic bacterial strains are of potential health risk. Employing enrichment cultivation technique, present study attempted to detect such injured, dormant or viable but non culturable (VBNC) cells in different frozen food samples, collected from local markets and super-shops of Dhaka metropolis. Compared to the conventional cultivation means, the enrichment procedure revealed a significant increase in bacterial burden as well as increase in the pathogenic load. A maximum of 3 log increase in case of total bacterial load while 4 log, 5 log and 2 log increase in case of Vibrio spp., Salmonella spp. and Shigella spp., consecutively were observed. These findings clearly demonstrated the presence of injured cells in frozen foods which could be lethal under normal condition thereby posing public health risk.Stamford Journal of Microbiology, Vol.5(1) 2015: 26-29

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Sciuto, Emanuele Luigi, Pasqualina Laganà, Simona Filice, Silvia Scalese, Sebania Libertino, Domenico Corso, Giuseppina Faro, and Maria Anna Coniglio. "Environmental Management of Legionella in Domestic Water Systems: Consolidated and Innovative Approaches for Disinfection Methods and Risk Assessment." Microorganisms 9, no. 3 (March 11, 2021): 577. http://dx.doi.org/10.3390/microorganisms9030577.

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Legionella is able to remain in water as free-living planktonic bacteria or to grow within biofilms that adhere to the pipes. It is also able to enter amoebas or to switch into a viable but not culturable (VBNC) state, which contributes to its resistance to harsh conditions and hinders its detection in water. Factors regulating Legionella growth, such as environmental conditions, type and concentration of available organic and inorganic nutrients, presence of protozoa, spatial location of microorganisms, metal plumbing components, and associated corrosion products are important for Legionella survival and growth. Finally, water treatment and distribution conditions may affect each of these factors. A deeper comprehension of Legionella interactions in water distribution systems with the environmental conditions is needed for better control of the colonization. To this purpose, the implementation of water management plans is the main prevention measure against Legionella. A water management program requires coordination among building managers, health care providers, and Public Health professionals. The review reports a comprehensive view of the state of the art and the promising perspectives of both monitoring and disinfection methods against Legionella in water, focusing on the main current challenges concerning the Public Health sector.

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Teixeira, Pilar, Bruna Fernandes, Ana Margarida Silva, Nicolina Dias, and Joana Azeredo. "Evaluation by Flow Cytometry of Escherichia coli Viability in Lettuce after Disinfection." Antibiotics 9, no. 1 (December 31, 2019): 14. http://dx.doi.org/10.3390/antibiotics9010014.

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Foodborne outbreaks due to the consumption of ready-to-eat vegetables have increased worldwide, with Escherichia coli (E. coli) being one of the main sources responsible. Viable but nonculturable bacteria (VBNC) retain virulence even after some disinfection procedures and constitute a huge problem to public health due to their non-detectability through conventional microbiological techniques. Flow cytometry (FCM) is a promising tool in food microbiology as it enables the distinction of the different physiological states of bacteria after disinfection procedures within a short time. In this study, samples of lettuce inoculated with E. coli were subject to disinfection with sodium hypochlorite at free chlorine concentrations of 5, 10, 25, 50, and 100 mg·L−1 or with 35% peracetic acid at concentrations of 5, 10, 25, and 50 mg·L−1. The efficiency of these disinfectants on the viability of E. coli in lettuce was evaluated by flow cytometry with LIVE/DEAD stains. Results from this study suggest that FCM can effectively monitor cell viability. However, peracetic acid is more effective than sodium hypochlorite as, at half the concentration, it is enough to kill 100% of bacteria and always induces a lower percentage of VBNC. Finally, we can conclude that the recommended levels of chemical disinfectants for fresh fruit and vegetables are adequate when applied in lettuce. More importantly, it is possible to ensure that all cells of E. coli are dead and that there are no VBNC cells even with lower concentrations of those chemicals. These results can serve as guidance for lettuce disinfection, improving quality and the safety of consumption.

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Naser, Iftekhar Bin, Tushar Ahmed Shishir, Shah Nayeem Faruque, M. Mozammel Hoque, Anamul Hasan, and Shah M. Faruque. "Environmental prevalence of toxigenic Vibrio cholerae O1 in Bangladesh coincides with V. cholerae non-O1 non-O139 genetic variants which overproduce autoinducer-2." PLOS ONE 16, no. 7 (July 2, 2021): e0254068. http://dx.doi.org/10.1371/journal.pone.0254068.

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Prevalence of toxigenic Vibrio cholerae O1 in aquatic reservoirs in Bangladesh apparently increases coinciding with the occurrence of seasonal cholera epidemics. In between epidemics, these bacteria persist in water mostly as dormant cells, known as viable but non-culturable cells (VBNC), or conditionally viable environmental cells (CVEC), that fail to grow in routine culture. CVEC resuscitate to active cells when enriched in culture medium supplemented with quorum sensing autoinducers CAI-1 or AI-2 which are signal molecules that regulate gene expression dependent on cell density. V. cholerae O1 mutant strains with inactivated cqsS gene encoding the CAI-1 receptor has been shown to overproduce AI-2 that enhance CVEC resuscitation in water samples. Since V. cholerae non-O1 non-O139 (non-cholera-vibrios) are abundant in aquatic ecosystems, we identified and characterized naturally occurring variant strains of V. cholerae non-O1 non-O139 which overproduce AI-2, and monitored their co-occurrence with V. cholerae O1 in water samples. The nucleotide sequence and predicted protein products of the cqsS gene carried by AI-2 overproducing variant strains showed divergence from that of typical V. cholerae O1 or non-O1 strains, and their culture supernatants enhanced resuscitation of CVEC in water samples. Furthermore, prevalence of V. cholerae O1 in the aquatic environment was found to coincide with an increase in AI-2 overproducing non-O1 non-O139 strains. These results suggest a possible role of non-cholera vibrios in the environmental biology of the cholera pathogen, in which non-O1 non-O139 variant strains overproducing AI-2 presumably contribute in resuscitation of the latent pathogen, leading to seasonal cholera epidemics. Importance. Toxigenic Vibrio cholerae which causes seasonal epidemics of cholera persists in aquatic reservoirs in endemic areas. The bacteria mostly exist in a dormant state during inter-epidemic periods, but periodically resuscitate to the active form. The resuscitation is enhanced by signal molecules called autoinducers (AIs). Toxigenic V. cholerae can be recovered from water samples that normally test negative for the organism in conventional culture, by supplementing the culture medium with exogenous AIs. V. cholerae belonging to the non-O1 non-O139 serogroups which do not cause cholera are also abundant in natural waters, and they are capable of producing AIs. In this study we characterized V. cholerae non-O1 non-O139 variant strains which overproduce an autoinducer called AI-2, and found that the abundance of the cholera pathogen in aquatic reservoirs correlates with an increase in the AI-2 overproducing strains. Our results suggest a probable role of these variant strains in the environmental biology and epidemiology of toxigenic V. cholerae, and may lead to novel means for surveillance, prevention and control of cholera.

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Kumar, Amit, Daphne Ng, and Bin Cao. "Fate ofEnterococcus faecalisin stormwater matrices under ultraviolet-A (365 nm) irradiation." Environmental Science: Water Research & Technology 4, no. 5 (2018): 639–43. http://dx.doi.org/10.1039/c8ew00010g.

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Moumita, Anika Bushra, Nafisa Tabassum, and Ifra Tun Nur. "In Vitro demonstration of Pseudomonas Growth and Phenotypic Examinations of the Cells Under Cold Shock." Bangladesh Journal of Microbiology 34, no. 2 (January 1, 2019): 119–24. http://dx.doi.org/10.3329/bjm.v34i2.39623.

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The present study attempted to detect the effect of cold shock on Pseudomonas aeruginosa, P. fluorescence, and P. putida; to deduce the culturable cells, the possible dead cells and viable but non-culturable (VBNC) cells; i. e., living but not capable of forming the colony forming units (CFUs) at 0 oC and at 4 oC. Estimation of VBNC at low temperature was performed by deducting the number of culturable cells (formed as CFUs) at 0 °C and at 4 oC from the culturable cells at 37 °C. Maximum culturable cells (around 106 CFU/ ml) appeared at 24-48 hours, and after that, a gradual decline in the numbers of the culturable cells was observed. A minor but significant growth reduction was noticed in Minimal Broth compared to that in Luria Broth. Significant reduction in the culturable cells of all three strains was noticed under cold shock. However, a fraction of the population of each strain was observed to survive under cold stress. Interestingly while the number of culturable cells decreased in course of time under cold shock, the number of VBNC apparently seemed to be constant or to be increased. A comparatively lower number of VBNC was noticed under cold shock in case of P. putida which in turn affected culturability at ambient temperature. Bangladesh J Microbiol, Volume 34 Number 2 December 2017, pp 119-124

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Meier, Thomas, and Bernd Bendinger. "Survival of pathogens in drinking water plumbing systems: impact factors and sanitation options." Water Supply 16, no. 4 (March 24, 2016): 931–41. http://dx.doi.org/10.2166/ws.2016.040.

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The objective was to determine impact factors that would allow Pseudomonas aeruginosa and Legionella pneumophila to survive sanitation measures of household drinking water systems. Therefore, different disinfectant concentrations were tested under different operating conditions in close-to-practice experiments. Particular attention was paid to the viable but non-culturable (VBNC) state of the pathogens. Legionella pneumophila was able to survive disinfection with ClO2 in a culturable state in the biofilm at 37 °C while successful sanitation could be achieved at 11 °C, however non-culturable cells were still present. Culturable P. aeruginosa outlasted disinfection in biofilms of pipes at elevated nutrient concentrations and in built-in parts at 37 °C. Overall, the VBNC state was not the predominant factor for its survival. Additional experiments showed that the lack of an autochthonous biofilm may promote the growth of P. aeruginosa. This emphasizes the importance of localization and elimination of contamination sources in a plumbing system before disinfection is performed and the need for compliance with operating conditions and construction requirements defined by generally recognized standards of good practice in Germany.

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Mangiaterra, Gianmarco, Nicholas Cedraro, Salvatore Vaiasicca, Barbara Citterio, Roberta Galeazzi, Emiliano Laudadio, Giovanna Mobbili, Cristina Minnelli, Davide Bizzaro, and Francesca Biavasco. "Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model." Antibiotics 9, no. 7 (July 10, 2020): 399. http://dx.doi.org/10.3390/antibiotics9070399.

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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.

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Navarro, Yurena, María-Jesús Torija, Albert Mas, and Gemma Beltran. "Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts." Foods 9, no. 10 (September 27, 2020): 1373. http://dx.doi.org/10.3390/foods9101373.

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The use of controlled mixed inocula of Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations (S. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima), comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.

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Gin, Karina Yew-Hoong, and Shin Giek Goh. "Modeling the effect of light and salinity on viable but non-culturable (VBNC) Enterococcus." Water Research 47, no. 10 (June 2013): 3315–28. http://dx.doi.org/10.1016/j.watres.2013.03.021.

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MAGAJNA, BRENDA, and HEIDI SCHRAFT. "Evaluation of Propidium Monoazide and Quantitative PCR To Quantify Viable Campylobacter jejuni Biofilm and Planktonic Cells in Log Phase and in a Viable but Nonculturable State." Journal of Food Protection 78, no. 7 (July 1, 2015): 1303–11. http://dx.doi.org/10.4315/0362-028x.jfp-14-583.

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Despite being considered fragile and fastidious, Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in the developed world. C. jejuni survives stresses by forming biofilms or entering a viable but nonculturable (VBNC) state. To investigate the number of viable cells in samples exposed to low nutrient and temperature stress, a novel method, propidium monoazide quantitative PCR (PMAqPCR), was compared with BacLight biovolume analysis and conventional plate counting for the enumeration of C. jejuni–removed biofilm cells and separately grown planktonic cells in late log phase (24 h). There were no significant differences between viable cell counts obtained using PMAqPCR and those from plate counts or BacLight biovolume analyses for each sample, confirming that this method provides results consistent with those from accepted enumeration methods (P > 0.05). To induce a VBNC state, C. jejuni planktonic cells and dislodged and washed biofilm cells were separately incubated in phosphate-buffered saline at 4°C for up to 60 days. Even when cells exposed to stress were provided with enrichment in Bolton broth before plating, treated biofilm cells lost culturability by day 10, whereas their planktonic counterparts remained culturable to day 60. The nonculturable biofilm cells remained viable in high numbers to day 60, and viable cell counts from the PMAqPCR (6.15 log cells per ml) were not significantly different from those obtained using the BacLight assay (6.98 log cells per ml) (P > 0.05), confirming that this novel method is also reliable for cells exposed to stress for extended periods. PMAqPCR shows promise for analysis where C. jejuni exists in biofilms or in the VBNC state. Adopting PMAqPCR in routine monitoring, in conjunction with improved biofilm cell collection methods, will allow for more accurate enumeration of viable and potentially virulent cells, leading to improved sanitation and reduced incidence of infection.

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Morishige, Yuta, Ko Fujimori, and Fumio Amano. "Differential Resuscitative Effect of Pyruvate and its Analogues on VBNC (Viable But Non-Culturable) Salmonella." Microbes and Environments 28, no. 2 (2013): 180–86. http://dx.doi.org/10.1264/jsme2.me12174.

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Talebi Bezmin Abadi, Amin. "Viable but Non-culturable Bacteria: Clinical Practice and Future Perspective." Research in Molecular Medicine 5, no. 2 (May 1, 2017): 1–2. http://dx.doi.org/10.29252/rmm.5.2.1.

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Keep, N. H., J. M. Ward, G. Robertson, M. Cohen-Gonsaud, and B. Henderson. "Bacterial resuscitation factors: revival of viable but non-culturable bacteria." Cellular and Molecular Life Sciences 63, no. 22 (September 29, 2006): 2555–59. http://dx.doi.org/10.1007/s00018-006-6188-2.

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Chen, Sheng, Jie Zeng, Yahong Wang, Chengsong Ye, Shuai Zhu, Lin Feng, Shenghua Zhang, and Xin Yu. "Modelling the effect of chlorination/chloramination on induction of viable but non-culturable (VBNC) Escherichia coli." Environmental Technology 41, no. 26 (May 9, 2019): 3443–55. http://dx.doi.org/10.1080/09593330.2019.1611939.

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Ghit, Diaa. "Detection of Viable but Non-Culturable (Vbnc) Escherichia Coli from Beef Liver Using Pma-PCR Assay." Alexandria Journal of Veterinary Sciences 69, no. 2 (2021): 1. http://dx.doi.org/10.5455/ajvs.75980.

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Zolfaghari, Mehdi, Masoud Rezaei, Ashraf Mohabbati Mobarez, Mehdi Forozandeh Moghaddam, Hedayat Hosseini, and Mohammad Khezri. "Virulence genes expression in viable but non-culturable state of Listeria monocytogenes in fish meat." Food Science and Technology International 26, no. 3 (October 4, 2019): 205–12. http://dx.doi.org/10.1177/1082013219877267.

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This study aimed to evaluate the fate of Listeria monocytogenes in water microcosm and rainbow trout fillet under salinity stress of 0% and 30% NaCl at refrigerator temperature (4 ± 2 ℃). Bacterial culturability was studied by standard culture and colony count method. Reverse transcription-PCR (RT-PCR) of 16 S rRNA gene was used to detect viability of non-culturable bacteria. Also, the qualitative expression of pathogenic genes ( hly and inlA) was studied using RT-PCR. The results showed that bacteria in water microcosm lost their culturability at 13 days under 0% salinity (starvation or distilled water) and at 27 days under 30% salinity; however, bacteria in rainbow trout fillet remained culturable under 0% and 30% NaCl. RT-PCR of 16 S rRNA gene was positive for all treatments during the period of this study, indicating the entering of L. monocytogenes into the viable but non-culturable state in water microcosm under 0% and 30% NaCl. Also, viable but non-culturable L. monocytogenes retained the expression of hly and inlA genes. So, it could be concluded that L. monocytogenes in viable but non-culturable state can cause serious health problems and further investigation is necessary to elucidate the effects of other processing and storage conditions (light, dark, smoking, etc.) on behavior of L. monocytogenes in smoked and salted fish.

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Berry, C., B. J. Lloyd, and J. S. Colbourne. "Effect of Heat Shock on Recovery of Escherichia coli from Drinking Water." Water Science and Technology 24, no. 2 (July 1, 1991): 85–88. http://dx.doi.org/10.2166/wst.1991.0034.

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Recent studies on the survival of bacteria in the aquatic environment have suggested that bacteria in water may enter a ‘viable non-culturable' phase in response to stress. Such bacteria are a cause of concern if pathogenic, because they cannot be detected using traditional culture methods. It has been found that mild heat shock causes viable non-culturable Legionella pneumophila to grow on laboratory media. The survival and culturability of Escherichia coli in sterile drinking water was investigated. The isolates used were from the environment rather than clinical. As expected, the count by culture on nutrient agar declined with time while the microscopy count remained approximately constant. Under laboratory conditions the E. coli isolates survived up to three months or more. After three months a portion of each suspension was heat shocked at 35°C for 20 minutes and then assayed by culture and microscopy immediately. An average increase of three log cycles was noted in the count by culture. There was no corresponding increase in the count by microscopy. Thus E. coli appears to exhibit viable non-culturable behaviour. Heat shock causes non-culturable bacteria to regain their ability to grow on artificial media. Such a finding may have several implications for the water industry and microbiology in general.

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Jo, Hye Jin, Hye Ri Jeon, and Ki Sun Yoon. "Behavior of Campylobacter jejuni Biofilm Cells and Viable But Non-Culturable (VBNC) C. jejuni on Smoked Duck." Journal of the Korean Society of Food Science and Nutrition 45, no. 7 (July 31, 2016): 1041–48. http://dx.doi.org/10.3746/jkfn.2016.45.7.1041.

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Dietersdorfer, Elisabeth, Alexander Kirschner, Barbara Schrammel, Anna Ohradanova-Repic, Hannes Stockinger, Regina Sommer, Julia Walochnik, and Sílvia Cervero-Aragó. "Starved viable but non-culturable (VBNC) Legionella strains can infect and replicate in amoebae and human macrophages." Water Research 141 (September 2018): 428–38. http://dx.doi.org/10.1016/j.watres.2018.01.058.

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HIRAISHI, AKIRA. "Distibution of viable but non-culturable bacteria in wastewater treatment systems." Microbes and environments 14, no. 2 (1999): 91–99. http://dx.doi.org/10.1264/jsme2.14.91.

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McKay, A. M. "Viable but non-culturable forms of potentially pathogenic bacteria in water." Letters in Applied Microbiology 14, no. 4 (April 1992): 129–35. http://dx.doi.org/10.1111/j.1472-765x.1992.tb00667.x.

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Su, Xiaomei, Xi Chen, Jinxing Hu, Chaofeng Shen, and Linxian Ding. "Exploring the potential environmental functions of viable but non-culturable bacteria." World Journal of Microbiology and Biotechnology 29, no. 12 (June 4, 2013): 2213–18. http://dx.doi.org/10.1007/s11274-013-1390-5.

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Morishige, Yuta, Masaaki Tanda, Ko Fujimori, Yoshiki Mino, and Fumio Amano. "Induction of Viable but Non-culturable (VBNC) State in Salmonella Cultured in M9 Minimal Medium Containing High Glucose." Biological and Pharmaceutical Bulletin 37, no. 10 (2014): 1617–25. http://dx.doi.org/10.1248/bpb.b14-00322.

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Journal articles on the topic 'Viable but non-culturable bacteria (VBNC)'

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