NBIC - Viable but Nonculturable State (VBNC) Flashcards
What are VBNCs? (3)
- Bacterial cells that enter a survival state in response to stress
- Defined by their inability to grow in the lab
- There is little consensus on other aspects of the state
What are some characteristics of VBNC cells? (7)
- Are moribund; state of decline or near death
- Can resuscitate
- Are culturable but starved.
- Are attenuated (weakened/reduced) in pathogenicity.
- Retain virulence.
- Are persister cells.
- Differ from persister cells.
Contradictory nature of these statements highlights the complexity and ongoing debate surrounding VBNC cells
What factors induce the VBNC state? (8)
- Low temperature
- Osmotic pressure
- Antibiotic pressure
- UV irradiation
- Ultrasound
- Starvation
- pH changes
- Chlorine exposure
bold - main ones
What physiological changes occur in the VBNC state? (4)
- Altered fatty acid composition
- Reduction in cell size
- Reduction in gene expression
- Reduction in metabolism (has been argued against)
How do VBNCs compare to spores? (4)
Similarity
- Both form in response to stress
Difference
- Spores are metabolically dormant and hardy
- Spores have a distinct morphology
- Spores are separate from their parent cells
How do VBNCs compare to persister cells? (4)
- Both help bacterial populations survive eradication
- Molecular links via toxin-antitoxin systems
- Some evidence of stochastic (random) formation
- Readily revert to culturability (VBNC cells require longer resuscitation time)
What is the role of the toxin-antitoxin (TA) system in VBNCs? (5)
- Regulates gene expression in bacteria
- Consists of a protein toxin and an antitoxin
- If antitoxin is RNA, it binds to toxin mRNA, preventing its production
- Free toxin inhibits translation and restricts cell growth
- Less antitoxin = less culturability
What are the possible transitions between VBNC and persister cells?
Linked to toxin-antitoxin system
- Active → Persister (e.g. by stress or high toxin levels) - some released toxin
- Persister → Active (favorable conditions, resuscitation time required)
- Persister → VBNC (under more severe stress, deeper dormancy) - a lot of released toxin
- VBNC → Persister (resuscitation possible, longer recovery time)
- Persister (from VBNC) → Active (eventual reactivation under suitable conditions)
How do biofilms relate to the persister and VBNC state? (4)
- Provide protection for bacterial populations
- Low pH, low oxygen, and nutrient scarcity stress bacteria (stressful environments)
- Stress induces persister and VBNC states
- Helps bacteria survive antibiotic treatments
What are molecular markers of the VBNC state? (4)
- RpoS
- oxyR
- envZ
- ompW
What is the role of RpoS in VBNC induction? (4)
- Transcriptional regulator, responsible for ~10% of the genome in stress response (Salmonella)
- Lower RpoS activity increases likelihood of VBNC induction (negative correlation with VBNC induction)
- VBNC cells still express rpoS; rpoS-deficient mutants die more easily than VBNC cells
- Exact role of RpoS in VBNC state is still unclear
What is the role of OxyR in VBNC cells? (3)
- Regulates production of catalase, which breaks down hydrogen peroxide (H₂O₂)
- In Vibrio vulnificus, catalase loses activity at low temperatures, making it difficult to culture
- OxyR-deficient Vibrio could not grow on agar due to H₂O₂ in the media
What is the role of EnvZ in VBNC cells? (2)
- EnvZ is a transmembrane protein in E. coli that monitors osmolarity
- EnvZ-deficient mutants could not enter the VBNC state in response to:
- Osmolarity changes
- pH shifts
- Starvation
What is the role of OmpW in VBNC cells? (3)
- OmpW (Outer Membrane Protein W) in E. coli O157:H7 is expressed in the VBNC state under oxidative stress
- Deletion of OmpW increased culturability
- Overexpression of OmpW decreased recovery
What key VBNC features influence detection methods? (7)
- Do not grow on agar plates
- Reduced but persistent metabolism
- Smaller size and coccoid morphology
- Continued gene expression with altered profiles
- Changes in fatty acid composition of their cell wall
- Increased tolerance to antibiotics and oxidative stress
- VBNC cells may be able to resuscitate back to a culturable state.
What are some direct methods to detect VBNC cells? (7)
- Microscopy
- Transcriptomic and metabolic analyses
- Molecular marker identification
- Flow cytometry (e.g., FACS)
- Infectivity assays
- Raman spectroscopy possibly
- Resuscitate and culture
What is the Direct Viable Count (DVC) method? (4)
- Compares culture tests to total living cells under a microscope
- Difference between culturable and total viable cells = VBNC population
- Time-consuming and subjective
- Poor sampling can lead to inaccurate estimates
What are omics-based methods to study VBNCs? (4)
- Transcriptomics – Confirms viability via differential gene expression
- Proteomics – Identifies protein markers for VBNCs
- Lipidomics – Potential role in detecting fatty acid changes
- Metabolomics – Detects metabolic activity to confirm viability
Can VBNC cells remain infectious? (3)
- The ability of VBNCs to cause disease is debated
- Infectivity studies use foodborne pathogens in mice and mammalian cell cultures
- Example: Liu et al. assessed E. coli O157:H7 cytotoxicity in water-induced VBNC state
What role does Flow Cytometry play in VBNC detection? (3)
- Measures and sorts cells based on characteristics
- Staining combined with fluorescence-activated cell sorting (FACS)
- Can rapidly count VBNC populations
How does PMA-qPCR help detect VBNCs? (4)
- qPCR quantifies gene copies in bacterial samples by amplifying the DNA
- PMA dye binds DNA in dead cells, preventing amplification
- Only viable cells are detected
- Used to detect Listeria in chlorinated water (Truchado et al) and Vibrio in shrimp (Zhao et al)
How might Raman spectroscopy and microfluidics help study VBNCs? (2)
- Single-cell analysis using microfluidic sorting
- Raman spectroscopy could detect VBNC-specific biochemical signatures
What are key factors for VBNC resuscitation? (4)
-
Resuscitation Promoting Factor (Rpf) – Highly conserved protein triggering revival;
- secreted into growing medium by growing cells and attaches to surface receptors of VBNCs, triggering their resuscitation.
- Sodium pyruvate – Antioxidant that neutralizes hydrogen peroxide
- Temperature increase – Restores culturability in temperature-induced VBNCs
- Host incubation – VBNC Listeria resuscitated in embryonated eggs
