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1
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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Ayrapetyan, Mesrop, Tiffany C. Williams, and James D. Oliver. "Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios." Applied and Environmental Microbiology 80, no. 8 (February 7, 2014): 2478–83. http://dx.doi.org/10.1128/aem.00080-14.
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ABSTRACTEntry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogenVibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNCV. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.
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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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Fakruddin, Md, Khanjada Shahnewaj Bin Mannan, and Stewart Andrews. "Viable but Nonculturable Bacteria: Food Safety and Public Health Perspective." ISRN Microbiology 2013 (September 26, 2013): 1–6. http://dx.doi.org/10.1155/2013/703813.
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The viable but nonculturable (VBNC) state is a unique survival strategy of many bacteria in the environment in response to adverse environmental conditions. VBNC bacteria cannot be cultured on routine microbiological media, but they remain viable and retain virulence. The VBNC bacteria can be resuscitated when provided with appropriate conditions. A good number of bacteria including many human pathogens have been reported to enter the VBNC state. Though there have been disputes on the existence of VBNC in the past, extensive molecular studies have resolved most of them, and VBNC has been accepted as a distinct survival state. VBNC pathogenic bacteria are considered a threat to public health and food safety due to their nondetectability through conventional food and water testing methods. A number of disease outbreaks have been reported where VBNC bacteria have been implicated as the causative agent. Further molecular and combinatorial research is needed to tackle the threat posed by VBNC bacteria with regard to public health and food safety.
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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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Shamloei, Sharareh, Ali Nabavi-Rad, Habibollah Nazem, and Abbas Yadegar. "Current Perspectives on Viable but Non-culturable Bacteria in Food Safety and Public Health." Avicenna Journal of Clinical Microbiology and Infection 9, no. 1 (March 29, 2022): 8–17. http://dx.doi.org/10.34172/ajcmi.2022.02.
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The viable but non-culturable (VBNC) state is defined as an adaptive mechanism for microorganisms adjusting to stressful conditions. Although VBNC bacteria are alive and metabolically active, they are unable to grow on routine culture media. Nevertheless, the potential capacity of VBNC pathogens to retain virulence activity and further resuscitate into the culturable state in favorable conditions constitutes a major hazard to food safety and public health. Food processing, transformation, and storage, as well as non-thermal techniques, can provoke pathogens toward VBNC induction. The distinct characteristic of VBNC bacteria led to the emergence of novel culture-independent techniques to prevent the misinterpretation of food safety. To deepen our knowledge of the molecular aspect of the VBNC state, several mechanism-oriented studies investigated the metabolic activity of VBNC bacteria and their correlation with different stressful conditions. This review aims to discuss the molecular mechanisms and genomic factors underlying the induction and resuscitation of the VBNC state. The study will further highlight innovative detection methods to provide a comprehensive perspective for future studies in the emerging fields of research concerning VBNC state, food safety, and public health.
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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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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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Šuster, Katja, and Andrej Cör. "Induction of Viable But Non-Culturable State in Clinically Relevant Staphylococci and Their Detection with Bacteriophage K." Antibiotics 12, no. 2 (February 2, 2023): 311. http://dx.doi.org/10.3390/antibiotics12020311.
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Prosthetic joint infections are frequently associated with biofilm formation and the presence of viable but non-culturable (VBNC) bacteria. Conventional sample culturing remains the gold standard for microbiological diagnosis. However, VBNC bacteria lack the ability to grow on routine culture medium, leading to culture-negative results. Bacteriophages are viruses that specifically recognize and infect bacteria. In this study, we wanted to determine if bacteriophages could be used to detect VBNC bacteria. Four staphylococcal strains were cultured for biofilm formation and transferred to low-nutrient media with different gentamycin concentrations for VBNC state induction. VBNC bacteria were confirmed with the BacLightTM viability kit staining. Suspensions of live, dead, and VBNC bacteria were incubated with bacteriophage K and assessed in a qPCR for their detection. The VBNC state was successfully induced 8 to 19 days after incubation under stressful conditions. In total, 6.1 to 23.9% of bacteria were confirmed alive while not growing on conventional culturing media. During the qPCR assay, live bacterial suspensions showed a substantial increase in phage DNA. No detection was observed in dead bacteria or phage non-susceptible E. coli suspensions. However, a reduction in phage DNA in VBNC bacterial suspensions was observed, which confirmed the detection was successful based on the adsorption of phages.
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Ncube, Pamela, Bahareh Bagheri, Wynand Johan Goosen, Michele Ann Miller, and Samantha Leigh Sampson. "Evidence, Challenges, and Knowledge Gaps Regarding Latent Tuberculosis in Animals." Microorganisms 10, no. 9 (September 15, 2022): 1845. http://dx.doi.org/10.3390/microorganisms10091845.
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Mycobacterium bovis and other Mycobacterium tuberculosis complex (MTBC) pathogens that cause domestic animal and wildlife tuberculosis have received considerably less attention than M. tuberculosis, the primary cause of human tuberculosis (TB). Human TB studies have shown that different stages of infection can exist, driven by host–pathogen interactions. This results in the emergence of heterogeneous subpopulations of mycobacteria in different phenotypic states, which range from actively replicating (AR) cells to viable but slowly or non-replicating (VBNR), viable but non-culturable (VBNC), and dormant mycobacteria. The VBNR, VBNC, and dormant subpopulations are believed to underlie latent tuberculosis (LTB) in humans; however, it is unclear if a similar phenomenon could be happening in animals. This review discusses the evidence, challenges, and knowledge gaps regarding LTB in animals, and possible host–pathogen differences in the MTBC strains M. tuberculosis and M. bovis during infection. We further consider models that might be adapted from human TB research to investigate how the different phenotypic states of bacteria could influence TB stages in animals. In addition, we explore potential host biomarkers and mycobacterial changes in the DosR regulon, transcriptional sigma factors, and resuscitation-promoting factors that may influence the development of LTB.
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Абдуллаева, Асият Мухтаровна, Лариса Петровна Блинкова, Борис Вениаминович Уша, Румия Камилевна Валитова, Юрий Дмитриевич Пахомов, and Дарья Борисовна Митрофанова. "Detection of Viable but Nonculturable Microbial Cells in Chicken Mince." Health, Food & Biotechnology 1, no. 4 (December 30, 2019): 26–38. http://dx.doi.org/10.36107/hfb.2019.i4.s281.
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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.
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Heim, Sabina, Maria Del Mar Lleo, Barbara Bonato, Carlos A. Guzman, and Pietro Canepari. "The Viable but Nonculturable State and Starvation Are Different Stress Responses of Enterococcus faecalis, as Determined by Proteome Analysis." Journal of Bacteriology 184, no. 23 (December 1, 2002): 6739–45. http://dx.doi.org/10.1128/jb.184.23.6739-6745.2002.
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ABSTRACT The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E. faecalis, which is activated in response to multiple environmental stresses.
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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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Pan, Hanxu, and Qing Ren. "Wake Up! Resuscitation of Viable but Nonculturable Bacteria: Mechanism and Potential Application." Foods 12, no. 1 (December 23, 2022): 82. http://dx.doi.org/10.3390/foods12010082.
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The viable but nonculturable (VBNC) state is a survival strategy for bacteria when encountered with unfavorable conditions. Under favorable environments such as nutrient supplementation, external stress elimination, or supplementation with resuscitation-promoting substances, bacteria will recover from the VBNC state, which is termed “resuscitation”. The resuscitation phenomenon is necessary for proof of VBNC existence, which has been confirmed in different ways to exclude the possibility of culturable-cell regrowth. The resuscitation of VBNC cells has been widely studied for the purpose of risk control of recovered pathogenic or spoilage bacteria. From another aspect, the resuscitation of functional bacteria can also be considered a promising field to explore. To support this point, the resuscitation mechanisms were comprehensively reviewed, which could provide the theoretical foundations for the application of resuscitated VBNC cells. In addition, the proposed applications, as well as the prospects for further applications of resuscitated VBNC bacteria in the food industry are discussed in this review.
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Grey, Brian, and Todd R. Steck. "Concentrations of Copper Thought To Be Toxic toEscherichia coli Can Induce the Viable but Nonculturable Condition." Applied and Environmental Microbiology 67, no. 11 (November 1, 2001): 5325–27. http://dx.doi.org/10.1128/aem.67.11.5325-5327.2001.
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ABSTRACT We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of Escherichia coli to enter the viable but nonculturable (VBNC) condition. Copper-induced VBNC cells could be resuscitated for up to 2 weeks after entering the VBNC state.
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WONG, HIN-CHUNG, CHI-TSUNG SHEN, CHIA-NI CHANG, YEONG-SHENG LEE, and JAMES D. OLIVER. "Biochemical and Virulence Characterization of Viable but Nonculturable Cells of Vibrio parahaemolyticus." Journal of Food Protection 67, no. 11 (November 1, 2004): 2430–35. http://dx.doi.org/10.4315/0362-028x-67.11.2430.
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Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt–0.5% NaCl medium incubated at 4°C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.
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Passerat, Julien, Patrice Got, Sam Dukan, and Patrick Monfort. "Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness." Applied and Environmental Microbiology 75, no. 16 (June 12, 2009): 5179–85. http://dx.doi.org/10.1128/aem.00334-09.
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ABSTRACT The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.
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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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Debnath, Anusuya, and Shin-ichi Miyoshi. "The Impact of Protease during Recovery from Viable but Non-Culturable (VBNC) State in Vibrio cholerae." Microorganisms 9, no. 12 (December 18, 2021): 2618. http://dx.doi.org/10.3390/microorganisms9122618.
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Vibrio cholerae can survive cold stress by entering into a viable but non-culturable (VBNC) state, and resuscitation can be induced either by temperature upshift only or the addition of an anti-dormancy stimulant such as resuscitation-promoting factors (Rpfs) at suitable temperature. In this study, the role of proteinase K was analyzed as an Rpf in V. cholerae. A VBNC state was induced in V. cholerae AN59 in artificial seawater (ASW) media at 4 °C, and recovery could be achieved in filtered VBNC microcosm, called spent ASW media, merely by a temperature upshift to 37 °C. The resuscitation ability of spent ASW was further enhanced by the addition of proteinase K. The mode of action of proteinase K was investigated by comparing its effect on the growth of the VBNC and culturable state of V. cholerae in ASW and spent ASW media. The presence of proteinase K allowed culturable cells to grow faster in ASW by reducing the generation time. However, this effect of proteinase K was more pronounced in stressed VBNC cells. Moreover, proteinase K-supplemented spent ASW could also accelerate the transition of VBNC into recovered cells followed by rapid growth. Additionally, we found that dead bacterial cells were the substrate on which proteinase K acts to support high growth in spent ASW. So, the conclusion is that the proteinase K could efficiently promote the recovery and growth of dormant VBNC cells at higher temperatures by decreasing the duration of the initial lag phase required for transitioning from the VBNC to recovery state and increasing the growth rate of these recovered cells.
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Ayrapetyan, M., T. C. Williams, R. Baxter, and J. D. Oliver. "Viable but Nonculturable and Persister Cells Coexist Stochastically and Are Induced by Human Serum." Infection and Immunity 83, no. 11 (August 17, 2015): 4194–203. http://dx.doi.org/10.1128/iai.00404-15.
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ABSTRACTDormancy holds a vital role in the ecological dynamics of microorganisms. Specifically, entry into dormancy allows cells to withstand times of stress while maintaining the potential for reentry into an active existence. The viable but nonculturable (VBNC) state and antibiotic persistence are two well-recognized conditions of dormancy demonstrated to contribute to bacterial stress tolerance and, as a consequence, yield populations that are tolerant to high-dose antibiotics. Aside from this commonality, more evidence is being presented that indicates the relatedness of these two states. Here, we demonstrate that VBNC cells are present during persister isolation experiments, further indicating that these cells coexist and are induced by the same conditions. Interestingly, we reveal that VBNC cells can exist stochastically in unstressed growing cultures, a finding that is characteristic of persisters. Furthermore, human serum induces the formation of both VBNC cells and persisters, a finding not previously described for either dormancy state. Lastly, we describe the role of toxin-antitoxin systems (TAS) in the induction of the VBNC state and report that these TAS, which are classically implicated in persister cell formation, are also induced during incubation in human serum. This study provides evidence for the recently proposed “dormancy continuum hypothesis” and substantiates the physical and molecular relatedness of VBNC and persister cells in a standardized model organism. Notably, these results provide new evidence for the clinical significance of VBNC and persister cells.
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Chamanrokh, Parastoo, Rita R. Colwell, and Anwar Huq. "Loop-mediated isothermal amplification (LAMP) assay for rapid detection of viable but non-culturable Vibrio cholerae O1." Canadian Journal of Microbiology 68, no. 2 (February 2022): 103–10. http://dx.doi.org/10.1139/cjm-2021-0142.
Full textAbstract:
Vibrio cholerae, an important waterborne pathogen, is a rod-shaped bacterium that naturally exists in aquatic environments. When conditions are unfavorable for growth, the bacterium can undergo morphological and physiological changes to assume a coccoid morphology. This stage in its life cycle is referred to as viable but non-culturable (VBNC) because VBNC cells do not grow on conventional bacteriological culture media. The current study compared polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to detect and identify VBNC V. cholerae. Because it is difficult to detect and identify VBNC V. cholerae, the results of the current study are useful in showing that LAMP is more sensitive and rapid than PCR in detecting and identifying non-culturable, coccoid forms of V. cholerae. Furthermore, the LAMP method is effective in detecting and identifying very low numbers of coccoid VBNC V. cholerae in environmental water samples, with the added benefit of being inexpensive to perform.
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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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Lucena, Lucas Pontes, Géssyka Rodrigues Albuquerque, Elineide Barbosa Souza, Ana Maria Benko-Iseppon, Rodrigo Dias Oliveira Carvalho, Flávia Figueira Aburjaile, and Marco Aurélio Siqueira Gama. "Infectivity of Viable but Non-Cultivable Cells of Pigmented and Nonpigmented Xanthomonas citri pv. anacardii Strains Demonstrate the Need to Establish Indexing Protocols for Cashew Propagules." Bacteria 1, no. 4 (October 6, 2022): 207–17. http://dx.doi.org/10.3390/bacteria1040016.
Full textAbstract:
Angular leaf spot of cashew tree in Brazil has been attributed to pigmented and nonpigmented strains of Xanthomonas citri pv. anacardii. Due to the possibility of dissemination of the disease by propagating material, it is necessary to understand the survival mechanisms of the causal agent. Thus, the present study aimed to characterize the behavior of viable but non-cultivable cells (VBNC) in two pigmented strains (CCRMTAQ13 and CCRMTAQ18) and one nonpigmented strain (IBSBF2579) of X. citri pv. anacardii, integrating in silico, in vitro, and in vivo studies. Thirteen genes associated with the VBNC phase were identified in the genomes of these strains. The log phase was observed at 24, 48, and 120 h for CCRMTAQ13, CCRMTAQ18, and IBSBF2579 strains, respectively. The death phase was observed at 96 h for both pigmented strains and 168 h for the nonpigmented strain. Using qPCR analyses, it was possible to characterize the occurrence of VBNC for the three strains. When inoculated, the strains showed 100% incidence during the VBNC phase, with the IBSBF2579 strain having the longest incubation period (IP). The strains did not differ concerning final severity (FS) in the VBNC phase. To our knowledge, this is the first report of the occurrence of the VBNC mechanism in X. citri pv. anacardii strains. Furthermore, it has been demonstrated that X. citri pv. anacardii in the VBNC state is potentially infective when they meet their host’s apoplast, which points to the need to use integrated practices to detect this bacterium in cashew seedlings.
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Smith, Ben, and James D. Oliver. "In Situ and In Vitro Gene Expression by Vibrio vulnificus during Entry into, Persistence within, and Resuscitation from the Viable but Nonculturable State." Applied and Environmental Microbiology 72, no. 2 (February 2006): 1445–51. http://dx.doi.org/10.1128/aem.72.2.1445-1451.2006.
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ABSTRACT Isolation of Vibrio vulnificus during winter months is difficult due to the entrance of these cells into the viable but nonculturable (VBNC) state. While several studies have investigated in vitro gene expression upon entrance into and persistence within the VBNC state, to our knowledge, no in situ studies have been reported. We incubated clinical and environmental isolates of V. vulnificus in estuarine waters during winter months to monitor the expression of several genes during the VBNC state and compared these to results from in vitro studies. katG (periplasmic catalase) was down-regulated during the VBNC state in vitro and in situ compared to the constitutively expressed gene tufA. Our results indicate that the loss of catalase activity we previously reported is a direct result of katG repression, which likely accounts for the VBNC response of this pathogen. While expression of vvhA (hemolysin) was detectable in environmental strains during in situ incubation, it ceased in all cases by ca. 1 h. These results suggest that the natural role of hemolysin in V. vulnificus may be in osmoprotection and/or the cold shock response. Differences in expression of the capsular genes wza and wzb were observed in the two recently reported genotypes of this species. Expression of rpoS, encoding the stress sigma factor RpoS, was continuous upon entry into the VBNC state during both in situ and in vitro studies. We found the half-life of mRNA to be less than 60 minutes, confirming that mRNA detection in these VBNC cells is a result of de novo RNA synthesis.
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Mascher, Fabio, Carsten Hase, Yvan Moënne-Loccoz, and Geneviève Défago. "The Viable-but-Nonculturable State Induced by Abiotic Stress in the Biocontrol Agent Pseudomonas fluorescens CHA0 Does Not Promote Strain Persistence in Soil." Applied and Environmental Microbiology 66, no. 4 (April 1, 2000): 1662–67. http://dx.doi.org/10.1128/aem.66.4.1662-1667.2000.
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ABSTRACT The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the waterlogged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.
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Vattakaven, Thomas, Peter Bond, Graham Bradley, and Colin B. Munn. "Differential Effects of Temperature and Starvation on Induction of the Viable-but-Nonculturable State in the Coral Pathogens Vibrio shiloi and Vibrio tasmaniensis." Applied and Environmental Microbiology 72, no. 10 (October 2006): 6508–13. http://dx.doi.org/10.1128/aem.00798-06.
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ABSTRACT We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20°C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17°C), the VBNC state was not induced by incubation at 4°C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30°C), was induced into the VBNC state by incubation at 4°C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20°C, and V. shiloi became VBNC after incubation for 126 days at 20°C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.
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Fleischmann, Susanne, Christian Robben, and Patrick Mester. "Resistenzbestimmung nicht-kultivierbarer Bakterien." BIOspektrum 28, no. 2 (March 2022): 144–46. http://dx.doi.org/10.1007/s12268-022-1727-4.
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AbstractTo cope with environmental stress, bacteria can enter resistant dormancy states such as the viable but non-culturable (VBNC) state, in which cells do not divide but remain alive with the potential to resuscitate. Present in both food and clinical environments, VBNC cells are a serious health risk and a challenge for conventional analytical methods. By detecting growth-independent viability parameters, it is possible to investigate resistance of VBNC cells and identify effective antimicrobials.
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del Mar Lleò, Maria, Sabrina Pierobon, Maria Carla Tafi, Caterina Signoretto, and Pietro Canepari. "mRNA Detection by Reverse Transcription-PCR for Monitoring Viability over Time in an Enterococcus faecalis Viable but Nonculturable Population Maintained in a Laboratory Microcosm." Applied and Environmental Microbiology 66, no. 10 (October 1, 2000): 4564–67. http://dx.doi.org/10.1128/aem.66.10.4564-4567.2000.
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ABSTRACT The viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria when they are exposed to hostile environmental conditions. It has been shown that VBNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. It is thus necessary to develop methods capable of detecting nonculturable bacteria and of establishing their viability when the microbiological quality of environments is monitored. In this study we demonstrated that a gene was expressed during the VBNC state in a low-nutrient-concentration microcosm through detection of Enterococcus faecalis pbp5 mRNA by reverse transcription-PCR over a 3-month period. The presence of mRNA correlated with metabolic activity and resuscitation capability, indicating the viability of the VBNC cells.
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Morishige, Yuta, Atsushi Koike, Ai Tamura-Ueyama, and Fumio Amano. "Induction of Viable but Nonculturable Salmonella in Exponentially Grown Cells by Exposure to a Low-Humidity Environment and Their Resuscitation by Catalase." Journal of Food Protection 80, no. 2 (January 24, 2017): 288–94. http://dx.doi.org/10.4315/0362-028x.jfp-16-183.
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ABSTRACTSalmonella is a major cause of foodborne disease that sometimes occurs in massive outbreaks around the world. This pathogen is tolerant of low-humidity conditions. We previously described a method for induction of viable but nonculturable (VBNC) Salmonella enterica serovar Enteritidis by treatment with hydrogen peroxide (H2O2) and subsequent resuscitation with 0.3 mM sodium pyruvate. Here, we report a new method for the induction of the VBNC state in Salmonella Enteritidis cells, one involving dehydration. Exposure of Salmonella Enteritidis cells to dehydration stress under poor nutritional conditions (0.9% [wt/vol] NaCl) and 10 to 20% relative humidity at room temperature decreased the presence of culturable population to 0.0067%, but respiratory and glucose uptake active populations were maintained at 0.46 and 1.12%, respectively, meaning that approximately 1% may have entered the VBNC state. Furthermore, these VBNC cells could be resuscitated to acquire culturability by incubation with catalase in M9 minimal medium without glucose in a manner dependent on the dose of catalase but not sodium pyruvate. These results suggest that a low-humidity environment could cause Salmonella Enteritidis cells to enter the VBNC state and the cells could then be resuscitated for growth by treatment with catalase, suggesting a potential risk of Salmonella Enteritidis to survive in low water activity foods in the VBNC state and to start regrowth for foodborne illness.
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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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Ordax, M�nica, Ester Marco-Noales, Mar�a M. L�pez, 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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Signoretto, Caterina, Maria del Mar Lleò, Maria Carla Tafi, and Pietro Canepari. "Cell Wall Chemical Composition ofEnterococcus faecalis in the Viable but Nonculturable State." Applied and Environmental Microbiology 66, no. 5 (May 1, 2000): 1953–59. http://dx.doi.org/10.1128/aem.66.5.1953-1959.2000.
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ABSTRACT The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall ofEnterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.
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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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Du, Meng, Jixiang Chen, Xiaohua Zhang, Aijuan Li, Yun Li, and Yingeng Wang. "Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate." Applied and Environmental Microbiology 73, no. 4 (December 22, 2006): 1349–54. http://dx.doi.org/10.1128/aem.02243-06.
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ABSTRACT Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4�C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 109 CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.
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Coutard, François, Solen Lozach, Monique Pommepuy, and Dominique Hervio-Heath. "Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but Nonculturable Vibrio parahaemolyticus after Recovery of Culturability." Applied and Environmental Microbiology 73, no. 16 (June 8, 2007): 5183–89. http://dx.doi.org/10.1128/aem.02776-06.
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ABSTRACT A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4°C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37°C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state.
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Xiao, Xing-long, Cong Tian, Yi-gang Yu, and Hui Wu. "Detection of viable but nonculturable Escherichia coli O157:H7 using propidium monoazide treatments and qPCR." Canadian Journal of Microbiology 59, no. 3 (March 2013): 157–63. http://dx.doi.org/10.1139/cjm-2012-0577.
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Escherichia coli O157:H7 can enter into a viable but nonculturable (VBNC) state under stress conditions. The aims of the present study were to examine the influences of environmental factors on the survivability and culturability of E. coli O157:H7 and to develop an approach for accurate detection of VBNC E. coli O157:H7. The E. coli O157:H7 strain ATCC 6589 was inoculated into 3 induction microcosm models: (i) Luria–Bertani broth, (ii) sterilized tap water, and (iii) sterilized physiological saline solution. Our results showed that low temperature and nutritional starvation significantly impacted on the survivability of E. coli O157:H7 cells and that the in-vitro-induced VBNC cells were capable of resuscitating under normal temperature and appropriate nutrients. We tested the effectiveness of an approach combining propidium monoazide (PMA) treatment with real-time polymerase chain reaction (PMA–qPCR) for accurate quantification of total, viable, dead, and VBNC cells under different induction microcosm models. Our results indicated different threshold cycle (Ct) values for PMA-treated cells and untreated cells (ΔCt = 4.97, 4.29, and 3.30 for Luria–Bertani broth, sterilized tap water, and sterilized physiological saline solution, respectively). We determined the quantification limit of this PMA–qPCR approach to be 1 × 102 cells·mL–1, providing sufficient sensitivity for detection of VBNC E. coli O157:H7 cells to no less than 100 cells·mL–1. This study clearly demonstrated the feasibility and effectiveness of using PMA–qPCR to accurately quantify E. coli O157:H7 in a VBNC state.
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Warner, Jennifer M., and James D. Oliver. "Randomly Amplified Polymorphic DNA Analysis of Starved and Viable but Nonculturable Vibrio vulnificusCells." Applied and Environmental Microbiology 64, no. 8 (August 1, 1998): 3025–28. http://dx.doi.org/10.1128/aem.64.8.3025-3028.1998.
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ABSTRACT Vibrio vulnificus is an estuarine bacterium capable of causing a rapidly fatal infection in humans. Because of the low nutrient levels and temperature fluctuations found in the organism’s natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of V. vulnificus strains grown in rich media and has been applied to starved and VBNC cells of V. vulnificusin the present study. As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period. Most noticeable was a uniform loss of RAPD amplification products. By 4 h of starvation, the cells were undetectable by the RAPD method. Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm. The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state. VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method. The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states. This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify V. vulnificus DNA in these states.
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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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Signoretto, Caterina, Gloria Burlacchini, Carla Pruzzo, and Pietro Canepari. "Persistence of Enterococcus faecalis in Aquatic Environments via Surface Interactions with Copepods." Applied and Environmental Microbiology 71, no. 5 (May 2005): 2756–61. http://dx.doi.org/10.1128/aem.71.5.2756-2761.2005.
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ABSTRACT Several human pathogens and fecal-pollution indicators may persist as viable organisms in natural environments, owing to their ability to activate different types of survival strategies. These strategies include adhesion on both abiotic and biotic surfaces and the entrance to the so-called viable but nonculturable (VBNC) state. In an 18-month survey for the detection of enterococci in both lake water and seawater, C. Signoretto et al. (Appl. Environ. Microbiol. 70:6892-6896, 2004) have shown that Enterococcus faecalis was detected mostly bound to plankton and in the VBNC state. In the present study, we show that in vitro adhesion of E. faecalis to copepods accelerated the entry of cells into the VBNC state relative to that of planktonic bacteria. VBNC E. faecalis cells maintained their adhesive properties to copepods and chitin (the main component of the copepod carapace), though to a reduced extent in comparison with growing cells. Sugar competition experiments showed interference with adhesion to both copepods and chitin by GlcNAc and only to copepods by d-mannose. Four enterococcal cell wall proteins present in both growing and VBNC cells and lipoteichoic acid were shown to be capable of binding chitin. The results indicate that copepods may represent an additional environmental reservoir of enterococci, thus suggesting the advisability of redesigning the protocols currently used for microbial detection during the evaluation of the microbiological quality of environmental samples.
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Lleò, Maria del Mar, Barbara Bonato, Caterina Signoretto, and Pietro Canepari. "Vancomycin Resistance Is Maintained in Enterococci in the Viable but Nonculturable State and after Division Is Resumed." Antimicrobial Agents and Chemotherapy 47, no. 3 (March 2003): 1154–56. http://dx.doi.org/10.1128/aac.47.3.1154-1156.2003.
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ABSTRACT Stressed vancomycin-resistant enterococci (VRE) can activate a survival strategy known as the viable but nonculturable (VBNC) state and are able to maintain vancomycin resistance. During restoration of division they continue to express the vancomycin resistance trait. We suggest that VBNC enterococci may constitute further reservoirs of VRE and therefore represent an additional risk for human health.
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Chaveerach, P., A. A. H. M. ter Huurne, L. J. A. Lipman, and F. van Knapen. "Survival and Resuscitation of Ten Strains of Campylobacter jejuni and Campylobacter coli under Acid Conditions." Applied and Environmental Microbiology 69, no. 1 (January 2003): 711–14. http://dx.doi.org/10.1128/aem.69.1.711-714.2003.
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ABSTRACT The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4′,6′-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.
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Liu, Yanming, Ainslie Gilchrist, Jing Zhang, and Xing-Fang Li. "Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water." Applied and Environmental Microbiology 74, no. 5 (January 18, 2008): 1502–7. http://dx.doi.org/10.1128/aem.02125-07.
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ABSTRACT A sensitive method for specific detection of viable Escherichia coli O157:H7 cells, including viable but nonculturable (VBNC) cells, in water samples was developed. This method involved capture of the bacterial cells on a low-protein-binding membrane and direct extraction and purification of RNA followed by reverse transcription-PCR and electronic microarray detection of the rfbE and fliC genes of E. coli O157:H7. It detected as few as 1 CFU of E. coli O157:H7 in diluted cultures, 3 to 4 CFU/liter in tap water, 7 CFU/liter in river water, and 50 VBNC cells in 1 liter of river water, demonstrating the best limit of detection reported to date for VBNC cells in environmental water samples.
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Allegra, Séverine, Françoise Berger, Philippe Berthelot, Florence Grattard, Bruno Pozzetto, and Serge Riffard. "Use of Flow Cytometry To Monitor Legionella Viability." Applied and Environmental Microbiology 74, no. 24 (October 10, 2008): 7813–16. http://dx.doi.org/10.1128/aem.01364-08.
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ABSTRACT Legionella viability was monitored during heat shock treatment at 70°C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.
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Fischer-Le Saux, Marion, Dominique Hervio-Heath, Solen Loaec, Rita R. Colwell, and Monique Pommepuy. "Detection of Cytotoxin-Hemolysin mRNA in Nonculturable Populations of Environmental and Clinical Vibrio vulnificus Strains in Artificial Seawater." Applied and Environmental Microbiology 68, no. 11 (November 2002): 5641–46. http://dx.doi.org/10.1128/aem.68.11.5641-5646.2002.
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ABSTRACT The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of Vibrio vulnificus. For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of V. vulnificus and targeted the cytotoxin-hemolysin virulence gene vvhA. Three strains, two environmental, IF Vv10 and IF Vv18, and one clinical, C7184, were used in this study. Artificial seawater, inoculated with mid-log-phase cells, was maintained at 4°C. VBNC cells resulted after 3, 6, and 14 days for C7184, IF Vv18, and IF Vv10, respectively. Our data indicate that seminested RT-PCR is sensitive for the detection of vvhA mRNA in artificial seawater when exclusively nonculturable bacteria are present. This is the first report of the expression of a toxin gene in VBNC V. vulnificus. Moreover, vvhA transcripts were shown to persist in nonculturable populations over a 4.5-month period, with a progressive decline of the signal over time. This result indicates that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk.
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Zeng, Bin, Guozhong Zhao, Xiaohong Cao, Zhen Yang, Chunling Wang, and Lihua Hou. "Formation and Resuscitation of Viable but NonculturableSalmonella typhi." BioMed Research International 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/907170.
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Salmonella typhiis a pathogen that causes the human disease of typhoid fever. The aim of this study was to investigate the viable but nonculturable (VBNC) state ofS. typhi. Some samples were stimulated at 4°C or −20°C, while others were induced by different concentrations of CuSO4. Total cell counts remained constant throughout several days by acridine orange direct counting; however, plate counts declined to undetectable levels within 48 hours by plate counting at −20°C. The direct viable counts remained fairly constant at this level by direct viable counting. Carbon and nitrogen materials slowly decreased which indicated that a large population of cells existed in the VBNC state and entered the VBNC state in response to exposure to 0.01 or 0.015 mmol/L CuSO4for more than 14 or 12 days, respectively. Adding 3% Tween 20 or 1% catalase enabled cells to become culturable again, with resuscitation times of 48 h and 24 h, respectively. The atomic force microscope results showed that cells gradually changed in shape from short rods to coccoids, and decreased in size when they entered the VBNC state. Further animal experiments suggested that resuscitated cells might regain pathogenicity.
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Tholozan, J. L., J. M. Cappelier, J. P. Tissier, G. Delattre, and M. Federighi. "Physiological Characterization of Viable-but-Nonculturable Campylobacter jejuniCells." Applied and Environmental Microbiology 65, no. 3 (March 1, 1999): 1110–16. http://dx.doi.org/10.1128/aem.65.3.1110-1116.1999.
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ABSTRACT Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (107 cells ml−1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride–4′,6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 μl mg of protein−1 for the culturable form and 10.96 μl mg of protein−1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.
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Wang, Hen-Wei, Chun-Hui Chung, Tsung-Yong Ma, and Hin-chung Wong. "Roles of Alkyl Hydroperoxide Reductase Subunit C (AhpC) in Viable but Nonculturable Vibrio parahaemolyticus." Applied and Environmental Microbiology 79, no. 12 (April 5, 2013): 3734–43. http://dx.doi.org/10.1128/aem.00560-13.
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ABSTRACTAlkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for the detoxification of reactive oxygen species that form in bacterial cells or are derived from the host; thus, AhpC facilitates the survival of pathogenic bacteria under environmental stresses or during infection. This study investigates the role of AhpC in the induction and maintenance of a viable but nonculturable (VBNC) state inVibrio parahaemolyticus. In this investigation,ahpC1(VPA1683) andahpC2(VP0580) were identified in chromosomes II and I of this pathogen, respectively. Mutants with deletions of these twoahpCgenes and their complementary strains were constructed from the parent strain KX-V231. The growth of these strains was monitored on tryptic soy agar–3% NaCl in the presence of the extrinsic peroxides H2O2andtert-butyl hydroperoxide (t-BOOH) at different incubation temperatures. The results revealed that bothahpCgenes were protective againstt-BOOH, whileahpC1was protective against H2O2. The protective function ofahpC2at 4°C was higher than that ofahpC1. The times required to induce the VBNC state (4.7 weeks) at 4°C in a modified Morita mineral salt solution with 0.5% NaCl and then to maintain the VBNC state (4.7 weeks) in anahpC2mutant and anahpC1 ahpC2double mutant were significantly shorter than those for the parent strain (for induction, 6.2 weeks; for maintenance, 7.8 weeks) and theahpC1mutant (for induction, 6.0 weeks; for maintenance, 8.0 weeks) (P< 0.03). Complementation with anahpC2gene reversed the effects of theahpC2mutation in shortening the times for induction and maintenance of the VBNC state. This investigation identified the different functions of the twoahpCgenes and confirmed the particular role ofahpC2in the VBNC state ofV. parahaemolyticus.
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Juhna, Talis, Dagne Birzniece, and Janis Rubulis. "Effect of Phosphorus on Survival of Escherichia coli in Drinking Water Biofilms." Applied and Environmental Microbiology 73, no. 11 (April 6, 2007): 3755–58. http://dx.doi.org/10.1128/aem.00313-07.
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ABSTRACT The effect of phosphorus addition on survival of Escherichia coli in an experimental drinking water distribution system was investigated. Higher phosphorus concentrations prolonged the survival of culturable E. coli in water and biofilms. Although phosphorus addition did not affect viable but not culturable (VBNC) E. coli in biofilms, these structures could act as a reservoir of VBNC forms of E. coli in drinking water distribution systems.
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Yao, Meiling, Fei Wang, and Gheorghe Arpentin. "The Effect of pesticide usage on grape yeast." BIO Web of Conferences 53 (2022): 05001. http://dx.doi.org/10.1051/bioconf/20225305001.
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For the conventional vineyard, reducing the pesticide usage is the most important issue for converting to organic vineyard. In order to study the effect of pesticide usage on grape yeast, an investigation were designed in vintage 2020, ten vineyards (include two organic vineyards) with different treatment frequency index (TFI) were chosen, and the yeast population counted by two method: cultivate method and the direct epifluorescence technique (DEFT). The results show, the grape yeast from Moldova exist the viable but non-culturable (VBNC) state. In organic vineyards, less proportion yeast reach the VBNC, however the opposite conclusion draw from conventional vineyards. High TFI with high level VBNC yeast population, inversely, less culturable yeast. No matter in which yeast state, the treatment of powdery mildew is the most influential factor on yeast population.