vbnc state
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Author(s):  
Vânia Gaio ◽  
Tânia Lima ◽  
Manuel Vilanova ◽  
Nuno Cerca ◽  
Angela França

Staphylococcus epidermidis biofilm cells are characterized by increased antimicrobial tolerance and improved ability to evade host immune system defenses. These features are, in part, due to the presence of viable but non-culturable (VBNC) cells. A previous study identified genes potentially involved in VBNC cells formation in S. epidermidis biofilms, among which SERP1682/1681 raised special interest due to their putative role as a toxin–antitoxin system of the mazEF family. Herein, we constructed an S. epidermidis mutant lacking the mazEF genes homologues and determined their role in (i) VBNC state induction during biofilm formation, (ii) antimicrobial susceptibility, (iii) survival in human blood and plasma, and (iv) activation of immune cells. Our results revealed that mazEF homologue did not affect the proportion of VBNC cells in S. epidermidis 1457, refuting the previous hypothesis that mazEF homologue could be linked with the emergence of VBNC cells in S. epidermidis biofilms. Additionally, mazEF homologue did not seem to influence key virulence factors on this strain, since its deletion did not significantly affect the mutant biofilm formation capacity, antimicrobial tolerance or the response by immune cells. Surprisingly, our data suggest that mazEF does not behave as a toxin–antitoxin system in S. epidermidis strain 1457, since no decrease in the viability and culturability of bacteria was found when only the mazF toxin homologue was being expressed.


2021 ◽  
Vol 9 (12) ◽  
pp. 2618
Author(s):  
Anusuya Debnath ◽  
Shin-ichi Miyoshi

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.


2021 ◽  
Author(s):  
Yuta Morishige ◽  
Yoshiro Murase ◽  
Kinuyo Chikamatsu ◽  
Akio Aono ◽  
Yuriko Igarashi ◽  
...  

Objective: Mycobacterium tuberculosis (Mtb) H37Ra strain has been reported to rapidly enter the viable but non-culturable (VBNC) state following treatment with an NADH oxidase inhibitor (diphenyleneiodonium [DPI]) and to be resuscitated by fetal bovine serum (FBS). However, the mechanism underlying FBS-induced resuscitation is currently unclear. We tried to reveal the underlying mechanism of FBS-induced resuscitation using M. tuberculosis H37Rv. Methods: First, we evaluated the effect of DPI on culturability, viability and changes of cellular phenotypes toward H37Rv. Secondly, we measured the resuscitation-promoting effects of human serum albumin, egg-white albumin, N-acetyl-L-cysteine, and D-mannitol in DPI-induced VBNC cells, as antioxidative agents have been reported to be key molecules for resuscitation of other microbes. We also evaluated the effect of inhibition of cAMP production and protein kinase A on BSA-induced resuscitation. Results: DPI treatment successfully induced a VBNC state in H37Rv, resulting in a low proportion of culturable cells, loss of acid-fastness and lipid-accumulation but a high proportion of viable cells. Not only FBS but also bovine serum albumin (BSA) alone could resuscitate H37Rv. Contrary to our expectation, only human serum albumin had a similar resuscitative effect to BSA. The inhibition of adenylyl cyclase by SQ22536 did not have a significant effect on resuscitation; however, the inhibition of protein kinase A by H89 strongly suppressed the BSA-induced resuscitation. Conclusion: DPI-induced VBNC Mtb cells may be resuscitated via the activation of protein kinase A-dependent processes through interaction with BSA.


Author(s):  
Vânia Gaio ◽  
Nathalie Lopes ◽  
Nuno Cerca ◽  
Angela França

Staphylococcus epidermidis biofilm cells can enter a physiological state known as viable but non-culturable (VBNC), where, despite being alive, they do not grow in conventional laboratory media. As such, the presence of VBNC cells impacts the diagnosis of S. epidermidis biofilm-associated infections. Previous transcriptomics analysis of S. epidermidis strain 9142 biofilms with higher proportions of VBNC cells suggested that the genes pdhA, codY and mazEF could be involved in the induction of the VBNC state. However, it was previously demonstrated that VBNC induction is strain-dependent. To properly assess the role of these genes in VBNC induction, the construction of mutant strains is necessary. Thus, herein, we assessed if VBNC cells could be induced in strain 1457, a strain amenable to genetic manipulation, and if the previously identified genes were involved in the modulation of the VBNC state in this strain. Furthermore, we evaluated the formation of VBNC cells on planktonic cultures. Our results showed that despite being commonly associated with biofilms, the proportion of VBNC cells can be modulated in both biofilm and planktonic cultures and that the expression of codY and pdhA was upregulated under VBNC inducing conditions in both phenotypes. Overall, our study revealed that the formation of VBNC cells in S. epidermidis is independent of the mode of growth and that the genes codY and pdhA seem to be relevant for the regulation of this physiological condition.


Author(s):  
Victor Jayeola ◽  
J. M. Farber ◽  
S. Kathariou

Salmonella can become viable but non-culturable (VBNC) in response to environmental stressors but the induction of the VBNC state in Salmonella contaminating ready-to-eat dried fruit is poorly characterized. Dried apples, strawberries and raisins were mixed with a five-strain cocktail of Salmonella at 4% volume per weight of dried fruit at 10 9 CFU/g. The inoculated dried fruit were then dried in desiccators at 25°C until the water activity (a w ) approximated that of the uninoculated dried fruit. However, Salmonella could not be recovered after drying, not even after enrichment, suggesting a population reduction of approx. 8 log CFU/g. To assess the potential impact of storage temperature on survival, dried apples were spot-inoculated with the Salmonella cocktail, dried under ambient atmosphere at 25°C and stored at 4 and 25°C. Spot-inoculation permitted recovery of Salmonella on dried apple after drying, with the population of Salmonella decreasing progressively on dried apples stored at 25°C until it was undetectable after about 46 days, even following enrichment. The population decline was noticeably slower at 4°C, with Salmonella being detected until 82 days. However, fluorescence microscopy and laser scanning confocal microscopy with the LIVE-DEAD Bac Light Bacterial Viability system at timepoints at which no Salmonella could be recovered on growth media even following enrichment, showed that a large proportion (56-85%) of the Salmonella cells on the dried fruit were viable. The data suggest that the unique combination of stressors in dried fruit can induce large numbers of VBNC cells of Salmonella . IMPORTANCE Salmonella is a leading foodborne pathogen globally causing numerous outbreaks of foodborne illnesses and remains the leading contributor to deaths attributed to foodborne disease in the United States and other industrialized nations. Therefore, efficient detection methods for Salmonella contaminating food are critical for public health and food safety. Culture-based microbiological methods are considered the gold standard for the detection and enumeration of Salmonella in food. Findings from this study suggest that unique stressors on dried fruit can induce the VBNC state in Salmonella , thus rendering it undetectable with culture-based methods even though the bacteria remain viable. Therefore, strong consideration should be given to using, in addition to culture-based methods, microscopic and molecular methods for the accurate detection of all viable and/or culturable cells of Salmonella contaminating dried fruit, as all these cells have the potential to cause human illness.


Author(s):  
Shuo Zhao ◽  
Jingyun Zhang ◽  
Zhe Li ◽  
Yu Han ◽  
Biao Kan

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.


mBio ◽  
2021 ◽  
Author(s):  
Liselot Dewachter ◽  
Celien Bollen ◽  
Dorien Wilmaerts ◽  
Elen Louwagie ◽  
Pauline Herpels ◽  
...  

While persistence and the viable but nonculturable (VBNC) state are currently investigated in isolation, our results strongly indicate that these phenotypes represent different stages of the same dormancy program and that they should therefore be studied within the same conceptual framework. Moreover, we show here for the first time that the dynamics of protein aggregation perfectly match the onset and further development of bacterial dormancy and that different dormant phenotypes are linked to different stages of protein aggregation.


Author(s):  
Ann L. Power ◽  
Daniel G. Barber ◽  
Sophie R. M. Groenhof ◽  
Sariqa Wagley ◽  
Ping Liu ◽  
...  

Bacteria modify their morphology in response to various factors including growth stage, nutrient availability, predation, motility and long-term survival strategies. Morphological changes may also be associated with specific physiological phenotypes such as the formation of dormant or persister cells in a “viable but non-culturable” (VBNC) state which frequently display different shapes and size compared to their active counterparts. Such dormancy phenotypes can display various degrees of tolerance to antibiotics and therefore a detailed understanding of these phenotypes is crucial for combatting chronic infections and associated diseases. Cell shape and size are therefore more than simple phenotypic characteristics; they are important physiological properties for understanding bacterial life-strategies and pathologies. However, quantitative studies on the changes to cell morphologies during bacterial growth, persister cell formation and the VBNC state are few and severely constrained by current limitations in the most used investigative techniques of flow cytometry (FC) and light or electron microscopy. In this study, we applied high-throughput Imaging Flow Cytometry (IFC) to characterise and quantify, at single-cell level and over time, the phenotypic heterogeneity and morphological changes in cultured populations of four bacterial species, Bacillus subtilis, Lactiplantibacillus plantarum, Pediococcus acidilactici and Escherichia coli. Morphologies in relation to growth stage and stress responses, cell integrity and metabolic activity were analysed. Additionally, we were able to identify and morphologically classify dormant cell phenotypes such as VBNC cells and monitor the resuscitation of persister cells in Escherichia coli following antibiotic treatment. We therefore demonstrate that IFC, with its high-throughput data collection and image capture capabilities, provides a platform by which a detailed understanding of changes in bacterial phenotypes and their physiological implications may be accurately monitored and quantified, leading to a better understanding of the role of phenotypic heterogeneity in the dynamic microbiome.


Author(s):  
Mengqi Xie ◽  
Luning Xu ◽  
Rong Zhang ◽  
Yan Zhou ◽  
Yeyuan Xiao ◽  
...  

Microbial degradation plays an important role in the environmental remediation. However, most microorganisms’ pollutant-degrading capability is weakened due to their entry into a viable but non-culturable (VBNC) state. Despite there is some evidence for the VBNC state of pollutant-degrading bacteria, limited studies have been conducted to investigate the VBNC state of pollutant degraders among fungi. In this work, the morphological, physiological and molecular changes of a phenol-degrading yeast strain LN1 exposed to high phenol concentrations were investigated. The results confirmed that Candida sp. strain LN1, which possessed highly efficient capability in degrading 1000 mg/L of phenol, as well as high potential for aromatic compounds degradation, entered into the VBNC state after 14 h of incubation at 6000 mg/L phenol. Resuscitation of VBNC cells can restore their phenol degradation performance. Compared to normal cells, significant dwarfing, surface damage and physiological changes of VBNC cells were observed. Molecular analysis indicated that the down-regulated genes were related to oxidative stress response, xenobiotics degradation, carbohydrate and energy metabolism, whereas the up-regulated genes were related to RNA polymerase, amino acid metabolism, DNA replication and repair. This report revealed that a pollutant-degrading yeast strain entered into the VBNC state under high concentrations of contaminants, providing new insights into its survival status and bioremediation potential under stress. Importance The viable but non-culturable (VBNC) state is known to affect the culturability and activity of microorganisms. However, limited studies have been conducted to investigate the VBNC state of other pollutant degraders such as fungi. In this study, the VBNC state of a phenol-degrading yeast strain was discovered. In addition, comprehensive analysis of the morphological, physiological and molecular changes of the VBNC cells were performed. This study provides new insight into the VBNC state of pollutant degraders, and how they restored the activities that was inhibited by stressful conditions. Enhanced bioremediation performance of indigenous microorganisms could be expected by preventing and controlling the formation of VBNC state.


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