NBIC - Viable but Nonculturable State (VBNC) II

Question 1

Why do VBNC foodborne pathogens matter? (4)

Answer 1

• If they can retain virulence while in the VBNC state.
• If they can resuscitate back to a culturable state.
• VBNCs (and persisters) can survive antibiotic treatments.
• VBNCs can evade quality control checks in food processing.

Question 2

Why is VBNC pathogen testing in food challenging? (2)

Answer 2

• The microbial safety of food is dominated by culture techniques.
• Alternative techniques (PCR, immunoassays) require enrichment steps (short period of growth in culture media to amplify bacteria).
• VBNC cells are excluded from all these tests.

Question 3

Where in the farm-to-fork chain are VBNCs produced? (4)

Answer 3

The field

• UV irradiation
• Low nutrients
• Temperature fluctuations

The factory environment

• Starvation on factory surfaces
• Sanitisers used on equipment

Decontamination process

• Chlorine stress
• Other chemical stress (e.g., ozone)
• UV irradiation

The fridge

• Low temperature

Question 4

In what foods are VBNCs a big threat? (3)

Answer 4

• Fresh produce – Often eaten uncooked, relying on decontamination.
• Meat – High microbial load, risk from improper cooking or cross-contamination.
• Listeria-prone foods – Listeria can grow at low temperatures and may resuscitate in the fridge.

Question 5

What is the global burden of foodborne disease? (4)

Answer 5

• 600 million cases of foodborne disease worldwide.
• 420,000 deaths (WHO estimate).
• Listeria monocytogenes – Leading cause of death from foodborne illness in England and Wales (2008).
• E. coli O157 – Fewest deaths among listed pathogens.

Question 6

What are common sources of VBNC pathogen contamination? (5)

Answer 6

• Fertiliser
• Manure
• Slurry
• Roaming animals and overflying birds
• Contaminated water

Question 7

How was E. coli O157 investigated in soil? (6)

Answer 7

1. qPCR – Detected E. coli O157 gene tir (qPCR will detect live, dead, or VBNC).

• Tried to culture O157 from the soil, but could not; source of the gene must be from dead cells or VBNC cells.

2. PNA-FISH – Identified O157 cells in soil.

• A technique that involves a targeted probe to identify a particular bacterial species within a target.
• PNA probe binds to the ribosome of O157 and makes it fluoresce orange, where cells of other species will not.

3. Cell elongation – Confirmed viability of E. coli O157.

• An incubation of bacteria with an antibiotic (nalidixic acid or pipemidic acid).
• The antibiotic inhibits DNA gyrase, an enzyme required for cell division.
• Cells are being incubated but can’t divide, just grow longer, hence cell elongation. (weird coz VBNCs do not divide)

4. Immunomagnetic separation – Isolated the pathogen.

• Uses magnetic beads to isolate cells, microorganisms, or molecules from a variety of samples.

5. Culture on CHROMagar O157 – Confirmed resuscitation.
6. Nematode killing assay – Confirmed infectivity.

• resuscitated soil isolate fed to the nematodes and measured how it affected their lifespan compared to their normal food.

Question 8

What was the key finding of the soil study?

Answer 8

• Sequencing revealed the soil isolate was not E. coli O157 but Citrobacter freundii.
• qPCR, PNA-FISH, and other methods had misidentified the pathogen.

Wasting my time

Question 9

What went wrong in soil study? (6)

Answer 9

• qPCR of genes in soil – could have been free DNA from dead cells.
• PNA-FISH in soil – the probe is not perfect, as time goes on more non-target matches are found.
• Cell elongation in soil – shows that the isolate is viable but not necessarily O157
• Immunomagnetic separation – in the concentrated resuscitated culture, C. freundii may have transferred across on the magnetic bead.
• Culture on CHROMagar O157 - false positive?
• Nematode killing assay – C. freundii is an opportunistic pathogen.

Question 10

What lessons were learned from the soil study? (4)

Answer 10

• VBNCs are difficult to identify in complex samples.
• No single technique is definitive.
• Even multiple techniques together can lead to misidentification.
• The food industry needs better methods to assess VBNC risks.

Question 11

How does chlorine induce the VBNC state in foodborne pathogens? (4)

Answer 11

• Listeria monocytogenes and Salmonella enterica were treated with chlorine.
• Some bacteria became non-culturable but remained detectable via microscopy.
• Chlorine killed up to 90% foodborne pathogens across the range of concentrations tested. The remaining 10% survived as VBNC.
• More chlorine was required to induce complete VBNC when pathogens were attached to spinach leaves compared to free bacteria in water .

Question 12

Are chlorine-induced VBNCs infectious? (2)

Answer 12

• Previous studies (Cappelier) suggested starvation-induced VBNCs were not infectious.
• This study showed chlorine-induced VBNCs are still infectious to nematodes (remained virulent)

Question 13

Why use C. elegans as a VBNC infection model? (8)

Answer 13

Pros:

• Has a gastrointestinal tract.
• Ingests bacteria as food.
• Infection progression is visible.
• Cost-effective, no ethical concerns.

Cons:

• Does not survive at 37°C.
• Lacks human gut microbiota.
• No adaptive immunity.
• Difficult to detect mild infections.

Question 14

What are the implications for chlorinated chicken? (2)

Answer 14

• US regulations – Chicken can be washed in 50 ppm chlorine for 3 minutes.
• This study and futher studies using chicken suggests that this can induce the VBNC state in Salmonella.

Question 15

What should the food industry do about VBNCs? (3)

Answer 15

• Evaluate direct detection techniques vs. culture.
• Determine if all VBNCs are a human health risk.
• Explore alternatives to chlorine for decontamination.