viable but non-culturable microorganisms (VBNC).

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. DNA damage. 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. C o nclus i on 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--