viable but non-culturable microorganisms (VBNC). WHAT IS A VBNC STATE? CHARACTERISTICS OF BACTERIA IN VBNC STATE. CONDITIONS STIMULATING VBNC STATE. METHODS FOR DETECTION OF VBNC STATE. Impact on public health. Advantages of VBNC. Disadvantages of VBNC state. Conclusion.

1. PRESENTATION ON
MD IBRAHIMUR RASHID (RAKIB)
Medical Technologiest
Evercare Hospital Chattogram.
Prepared By

2. INTRODUCTION
•The cells those are metabolically active but can’t be
cultured on specific media are the viable but non-
culturable cells (VBNC).
•Microorganisms that do not grow in culture methods, but which
are still metabolically active and capable of causing infections in
animals and plants are said to be in VBNC state.
• VBNC will not resume growth immediately when appropriate
nutrients
and conditions are provided.
•VBNC cells of Vibrio cholerae, enteropathogenic E.coli,
Legionella pnuemophila and various other bacteria have
been shown to regain culturability after they have entered
the intestinal tracts of animals.

3. WHAT IS A VBNC STATE
The VBNC state is defined as a state of dormancy triggered by environmental harsh conditions,
such as nutrient starvation, temperature, osmotic stress, oxygen availability, several food
preservations, heavy metals, exposure to white light and determining processes, as
pasteurization of milk and chlorination of waste water.

4. CHARACTERISTICS OF BACTERIA IN VBNC STATE
Some of the characteristics of bacteria in VBNC state are as follows:-
 Maintain apparent cell integrity.
 Possession of some form of measurable cellular activity.
 Possess apparent capacity to regain culturability.
 Respond to external stimulus by specific gene expression.
 Low metabolic activity.
 Exhibit dwarfing.
 Reduced nutrient transport
 High ATP level and high membrane potential.
 Plasmids are retained.
 Extensive modifications in cytoplasmic membrane fatty acid compositions.
 Higher autolytic capability than exponentially growing cells.
 Changes in outer-membrane protein profile.  Continuous gene expression.

5. CONDITIONS STIMULATING VBNC STATE
In the environment, bacterial cells can enter VBNC state may be due to-
1.Lack of nutrients. 2. Lack of temperatures. 3. High pressure.
4. Damage to or lack of an essential cellular component. 5. Sharp changes in pH or salinity.
8.Nutrient starvation.
9. Incubation outside the normal temperature range of growth. 10. Oxygen concentrations.
6. DNAdamage. 7. Elevated or lower osmotic concentrations.
11. Food preservatives.
14. Chlorination of wastewater.
12. Exposure to white light. 13. Heavy metals.
15. Pasteurization of milk.

6. METHODS FOR DETECTION OF VBNC STATE:
 Bright field microscopy with Nalidixic acid
 Fluorescent microscopy
 Gene probe/ oligonucleotide probe/ hybridization
 Blotting
 Fluorescent in situ hybridization (FISH)
 RT-PCR

7. Bright field microscopy with Nalidixic acid
Nalidixic acid (20–40 mg/L) is used to stop cell division. After
exposure to nalidixic acid, viable cells continue to grow and will
appear elongated, whereas the nonviable metabolically inactive
cells will retain their original shape and size. The cells are then
observed under a microscope. Viable cells will be seen as
elongated, whereas VBNC/dormant cells will be seen as oval
and large.

8. Fluorescent microscopy
Various fluorescent staining procedures can be used to determine VBNC
organisms. Frequently used stains are acridine orange, 4,6-diamino-2-phenyl indole
(DAPI), fluorescein isothiocyanate (FITC), indophenyl-nitrophenyl-phenyltetrazolium
chloride (INT), and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) .
In recent years, a new differential staining assay, the BacLight Live/Dead
assay, has been developed. The assay allows simultaneous counting total and viable
(metabolically active) cells, by using two nucleic acid stains, that is, green-fluorescent
SYTO 9 stain and red-fluorescent propidium iodide stain. SYTO 9 stains both live and
dead bacteria, whereas propidium iodide penetrates only bacteria having damaged
membranes. When used together, propidium iodide reduces SYTO 9 fluorescence in
dead bacteria with damaged membranes resulting in red fluorescent cells, whereas
the live bacteria will fluoresce green

9. Molecular Techniques.
Hybridization probes are nucleic acids (DNA/RNA) which have been
chemically or radioactively labeled and are used to detect complementary
target DNA/RNA. Specific amplification of DNA targets in bulk DNA extracts
from environmental and clinical samples permits detection of specific
organisms or groups of related organisms without the need to cultivate
them, provided the appropriate unique primers are used. These procedures
do not discriminate between culturable and nonculturable forms of the
target organisms. Due to the failure of distinguishing between dead or live
cells by DNA-based methods, RNA-based methods are a more valuable
estimate of gene expression and/or cell viability under different conditions.
This technique is more able to discriminate between culturable and
nonculturable forms of an organism. Furthermore, reverse transcriptase
PCR (RT-PCR) can distinguish between live and dead cells. This is
possible because it is an mRNA-based method and mRNA is short-lived
(half-life less than 1 minute). Messenger RNA is only present in
metabolically active cells and not found in nature after cell death. RT PCR
can detect nonculturable but active or live cells.

10. Impact on public health
Many believe that pathogens in the VBNC state are unable to induce infection/disease despite retaining their virulent
properties. However, when VBNC pathogens pass through a host animal, resuscitation and resumption of metabolic activity
have led to infections and diseases. The first evidence of pathogenicity of nonculturable cells was the demonstration of fluid
accumulation in the rabbit ileal loop assay (RICA) by VBNC Vibrio cholerae O1, followed by human volunteer experiments.
VBNC E. coli nonculturable cells were also reisolated after passaging through rabbit ileal loops 4 days after inoculation, and
chick embryos died when injected with nonculturable cells of Legionella pneumophila, leading to the conclusion that VBNC
pathogens remain potentially pathogenic. So, VBNC has a huge significance in public health care.
Many indicator bacteria and pathogenic bacteria which exist in aquatic habitats have been shown to have a VBNC state.
Water is routinely tested for such indicators and pathogens, and if they are not detected or enumerated at a concentration
below guidelines, waters are deemed to be safe for public consumption. Therefore, where circumstances indicate possible
presence of VBNC pathogens, additional molecular methodology needs to be used to reduce the risk of infective disease
outbreaks.
Thus, food and environmental and clinical samples no longer can be considered free from pathogens if culturing yields
negative results. For the general public, the presence of VBNC in water and food may be related to low-grade infections or
the so-called aseptic infections. In many cases, the infections are incorrectly attributed to viruses since no bacteria were
detected. For example, Vibrio cholerae O1 in the surface water have been shown to remain in nonculturable state. These
water sources are used for domestic purpose regularly and pose a risk of infection. When conditions are not favorable for
growth, then it transforms to the nonculturable state in association with crustacean copepods. Persistence of Vibrio cholerae
in water in the VBNC state is an important public health factor, since detection will not be successful if only conventional
cultural methods are used.

11. Advantages of VBNC
VBNC is a survival strategy employed by a wide range of gram-negative
heterotrophic bacteria and also by many nonsporulating gram-positive and gram-
variable bacteria. It appears to be a genetically inducible state (Oliver 2000).
VBNC is an advantage to organisms living in changing environments such as the
aquatic environment where sudden fluctuations in a range of conditions could
potentially threaten survival. The ability to rapidly respond to changing conditions
offers obvious evolutionary benefits. Environmental conditions seen to trigger the
VBNC state include; fluctuating temperature, nutrient levels, salinity, age, oxygen
levels and light levels Which are in fact very similar to many conditions found
during pharmaceutical manufacture. The VBNC response to changes in
environmental conditions is now being considered as a possible explanation as to
why different species dominate the same geographical location at different times
of the year (Oliver 2000).

12. Disadvantages of VBNC state
 The ability to enter the VBNC state may be advantageous for bacteria, but
poses a risk to human health. If VBNC cells are present, the total number of
viable bacteria in a sample will be underestimated by the CFU count method
due to the inherent non-culturability of VBNC cells. Even worse, if all bacteria in
the sample are in VBNC state, the sample may be regarded as gram-free due to
non-detection.
 For bacterial species causing human infections, the underestimation or non-
detection of viable cells in quality control samples from the food industry and
water distribution systems, or clinical samples may pose serious risks to the
public. The risk emerge from the fact that pathogenic bacteria can be avirulent
in the VBNC state but virulence after resuscitation into culturable cells under
suitable conditions.
 Apart from this , the identification of conditions that can induce bacteria to enter
VBNC state and the underlying mechanisms, as well as the understanding of
resuscitation conditions and mechanisms are necessary to effectively prevent
bacterial infections and cure infected patients.

13. Conclusion
• The knowledge about the VBNC state comes from research on a variety of bacteria and highlights
the complexity of this mechanism of adaptation. What seems clear is that induction and
resuscitation of the VBNC state are highly variable across bacterial species and in some cases,
strains.
• However, the basic genetic mechanisms may share a common theme and further research into this
field will help tie up the loose ends that exist in this area. The ability to avoid conditions that lead to
resuscitation, or the development of drugs that induce resuscitation during antibiotherapy could
have a major impact on the consequence of the VBNC state in chronic infectious diseases.
• Development of new, inexpensive methods to easily detect cells in the VBNC state is needed to
increase food safety. In conclusion, the potential applications of VBNC research are significant to
prevent food- and water-borne infections, and find new treatments to cure chronic bacterial
infections.
• --THE END--