Viable but Non-Culturable state is a unique survival strategy of many bacteria in environment in response to adverse environmental conditions. VBNC bacteria can't be cultured on routine microbiological media but they remain viable and retain their virulent capacity too. VBNC bacteria can be resuscitated when provided with appropriate conditions. A good number of bacteria including many human pathogens have been reported to enter VBNC state. Though, there was disputes on the existence of VBNC in past, extensive molecular studies have resolved most of them and VBNC has been accepted as distinct survival state by all. VBNC bacteria are considered as threats to public health and food safety due to their non-detectability and virulence as food and water have been reported to be contaminated with pathogens at VBNC state though conventional methods declared them as safe and clean. A number of outbreaks have also been reported where VBNC bacteria has been implicated as causative agent. Further molecular and combinatorial research in conjunction with predictive modeling are needed to elucidate the mechanisms and to identify the critical points to tackle the threat posed by VBNC bacteria with regard to public health and food safety.
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Key words: VBNC, Pathogen, public health, Food safety, Detection
The cells that form colony in specific media are the culturable cells. Viable means metabolically or physiologically active. So the cells those are metabolically or physiologically active but can't be cultured on specific media are the viable but non-culturable cells (VBNC) (Bogosian & Bourneuf, 2001). Most microorganisms growing in nature have yet to be cultured in the laboratory. In fact less than 1% of the microorganisms in natural water and soil samples are cultured in viable count procedures (Barcian & Arana, 2009).
In 1982, Prof. Rita Colwell and co-workers introduced the term "Viable But Non-Culturable Bacterial Cells (VBNC)" to distinguish particular cells that could not form colonies on solid media but obtained metabolic activity and the ability to elongate after the administration of nutrients (Xu et al., 1982). According to Oliver (1995), "VBNC can be defined as a metabolically active bacterial cell that crossed a threshold in this way, for known or unknown reasons and become unable to multiply in or on a medium normally supporting its growth". Most of the bacteria that enter VBNC state are gram negative species belonging to the gamma subclass of the Proteobacteria branch, except for Rhizobium, Agrobacterium and Helicobacter-Campylobacter species (Oliver, 2000).
Debra Bashford and colleagues announced that they had recovered Vibrio cholerae from streams and drainage ditches, including sites with negligible chance of sewage contamination. Around the same lime, Rita Colwell was also finding Vibrio cholerae in Maryland. She and her coworkers showed that both this bacterium and E. coli, incubated in artificial sea water remained viable but lost the capacity to form colonies on culture media (Colwell & Grimes, 2000). Soon Salmonella enteritidis, Shigella sonnie and Legionella pneumophila joined the list of organisms known to be capable of entering a state in which they failed to show up on nutrient agar yet took up substrates and signaled in other ways that they were certainly not dead. The use of laboratory media to recover and enumerate bacteria and lo link them with or absolve them from pathological and other activities became obsolete by the new discoveries and a term VBNC (viable but non-culturable) came (McDougald et al., 1998).
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. The conditions for these organisms to resume growth are not being met using the normal laboratory culture conditions (Yamamoto, 2000). Bacteria that have been semi-starved will immediately resume growth when provided with the appropriate nutrients and conditions. Viable but non-culturable cells will not resume growth even when nutrients are provided (NystrÃ-m, 2001). VBNC cells exhibit active metabolism in the form of respiration or fermentation, incorporate radioactive substrates, and have active protein synthesis but cannot be cultured or grown on conventional laboratory media. They have been detected by observing discrepancies between plate count enumeration of bacterial population and direct staining and microscopic counts (Sachidanandham & Gin, 2009). These cells may be of particular problems in the environment if they are pathogens, for example, viable but non-culturable cells of Vibrio cholerae, Enteropathogenic E. coli, Legionella pneumophila and various other bacteria have been shown to regain culturability after they have entered the intestinal tracts of animals (Colwell et al., 1996).
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The VBNC state is defined as a state of dormancy triggered by environmental harsh conditions, such as nutrient starvation (Cook & Bolster, 2007), temperature (Besnard et al., 2002), osmotic stress (Asakura et al., 2008), oxygen availability (Kana et al., 2008), several food preservatives (Quirós et al., 2009), heavy metals (Ghezzi & Steck, 1999), exposure to white light (Gourmelon et al., 1994) and decontaminating processes, as pasteurization of milk (Gunasekera et al., 2002) and chlorination of wastewater (Oliver, 2005).
VBNC state is believed to be a unique survival strategy of bacteria in response to environmental stresses (Oliver, 2010). It is also considered as an important reservoir of many human pathogens in the environment (Lleo et al., 2007).
VBNC state has been a matter of dispute for ling since its inception, due to the difficulty of differentiation of VBNC cells & dormant cells through resuscitation & phenotypic studies, recent molecular studies, data of which supported the existence of VBNC state, the dispute has mostly been put to rest (Barer and Harwood, 1999).
Following list includes but not limited to pathogenic bacteria that can enter VBNC state (Oliver, 2010)- Aeromonas hydrophila, Agrobacterium tumefaciens, Burkholderia cepacia, Campylobacter jejuni, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli (including EHEC), Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Streptococcus faecalis, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus (types 1 and 2)
Characteristics of Bacteria in VBNC state
1. Maintain apparent cell integrity; 2. possession of some form of measurable cellular activity (Lai et al., 2009); 3. possess apparent capacity to regain culturability (Anuchin et al., 2009); 4. respond to external stimulus by specific gene expression (kell et al., 1998); 5. low metabolic activity (oliver, 2005); 6. Exhibit dwarfing (Costa et al., 1999); 7. reduced nutrient transport; 8. High ATP level and high membrane potential (Signoretto et al., 2000); 9. extensive modifications in cytoplasmic membrane fatty acid compositions (Day & Oliver, 2004); 10. Changes in cell wall peptidoglycan such as increasing cross linking, increasing muropeptides bearing covalently bound lipoprotein & shortening of average length of glycan strands (signoretto et al., 2002); 11. Higher autolytic capability than exponentially growing cells; 12. Plasmids are retained; 13. changed antibiotic sensitivity & as metabolic activity is lower, most bacteria at VBNC state demonstrate high antibiotic resistance (Oliver, 2010); 14. Changes in outer-membrane protein profile (Muela et al., 2008); 15. Continuous gene expression (Maalej et al., 2004) etc.
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. Sharp changes in pH or salinity (Cunningham et al., 2009); 5.damage to or lack of an essential cellular component; 6. DNA damage; 7. activation of lysogenic phages or suicide genes such as sok/hak or autolysins (Aizenman et al., 1996); 8. Nutrient starvation; 9. incubation outside the normal temperature range of growth; 10. elevated or lower osmotic concentrations; 11. oxygen concentrations (Mascher et al., 2000); 12. food preservatives; 13. Heavy metals (Del Campo et al., 2009); 14. exposure to white light; 15. pasteurization of milk (Gunasekara et al., 2002); 16. chlorination of wastewater (Oliver, 2005) etc.
Public health significance of VBNC
Though virulence of bacteria in VBNC state is still not very clear, many believed that pathogens in VBNC state are unable to induce infection/disease but still retain their virulent properties & has potential to cause disease & infection following resuscitation and resume of active metabolic state, which occurs when they pass through host animal (Baffone et al., 2003).
The VBNC state appears to be the common to many bacteria especially those which have aquatic habitats, and may represent a mechanism to survive adverse environmental factors as temperature, salinity etc. or have a means of inducing "cross protection" against other adverse factors (Du et al., 2007). Among these bacteria entering this state are many significant human pathogens and indicator bacteria of these pathogens; such cells may represent a public health hazard and may be a factor in human health and/or disease (Rivers & Steck, 2001).
Even today, it is still not possible to cultivate most bacterial species directly from the environmental samples or after exposure of previously culturable cells to environmental conditions unfavorable for growth and multiplication in vitro. The passage of VBNC through an appropriate animal host will induce return of culturability. Even these VBNC bacteria retain their pathogenicity and may trigger life in vivo and thus cause severe disease (Sardessai, 2005).
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Under normal condition it is not possible to culture or detect VBNC. Many diagnostic laboratory set up does not have sufficient molecular facilities to detect VBNC. In case of food and water quality control test, such VBNC may not be detected. Even some indicator of some pathogenic bacteria undergoes VBNC state and may remain undetected (Signoretto et al., 2004). Upon consuming such food or after drinking such water, one may be infected by those VBNC that can trigger life as well as pathogenicity (Adams et al., 2003).
Thus, 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 so called 'aseptic' infection. For example, Vibrio cholerae O1 in the surface water remain as non-culturable state. These water sources are used for domestic purpose regularly and posed a risk of infection (Edwards, 2000). When conditions are not favorable for growth then it transforms to the non-culturable 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 (Barer et al., 1993).
Similarly, Shigella can undergo VBNC state in water but become a threat when enter in human body. Thus it is important to recognize that non-culturable bacteria are capable of producing diseases. The first evidence of pathogenicity of non-culturable cells was the demonstration of fluid accumulation in rabbit ileal loop assay (RICA) by VBNC Vibrio cholerae O1, followed by human volunteer experiments (Amel et al., 2008).
VBNC E. coli non-culturable cells were re-isolated after passage through rabbit ileal loops 4 days post inoculation and chick embryos died when injected with non-culturable cells of Legionella pneumophila, led to the conclusion that VBNC pathogens remain potentially pathogenic. So, VBNC has a huge significance in public health care (Cappelier et al., 2007).
Reports indicate that many potentially harmful bacteria survive treatment and persist in processed food, pasteurized milk, potable water and in the environment (Colwell et al., 2000). Many evidences suggested that recurrent urinary tract infections in many individuals are caused by uropathogenic E. coli cells which remain in VBNC state (Anderson et al., 2004) & thus resistant to antibiotic treatment & cause reinfection when resuscitate back to active metabolic state (Steck, 2001; Mulvey et al., 2001). Studies also showed that uropathogenic E. coli retain enteropathogenicity at VBNC state through continued production of enterotoxin (Pommepuy et al., 1996). Nilsson et al. (2002) showed that VBNC Helicobacter pylori cells can express virulence factors such as cagA, vacA and vreA.
All these above evidence proved that many deathly pathogenic strains not only enter but also persist & survive in VBNC state in environment & most of them remain infectious as well.
VBNC state of foodborne bacteria- a challenge in food safety
Many evidences suggested presence of VBNC bacteria in food (Ordax et al., 2009). For example, in stored wine, acetic acid and lactic acid bacteria entered VBNC state as consequence of lack of oxygen and presence of sulphites, respectfully (Millet and Lonvaud-Funel, 2000).
Food and its surrounding environment is a complex system, in which physic-chemical characterisitcs (pH, aw, chemical composition) and environmental factors (storage temperature and time, decontamination treatments, packaging under modified atmosphere) act simultaneously on contaminating bacteria (Sun et al., 2008).
For example, it has been demonstrated that refrigerated pasteurized grapefruit juice induced VBNC state in E. coli O157:H7 and S. typhimurium within 24 hours of incubation (Nicolo et al., 2011).
Again, Gunasekera et al. (2002) reported that in pasteurized milk which have undergone thermal treatment, contaminating bacteria such as E. coli and Pseudomonas putida enter into VBNC state but retained transcription and translation machineries.
Several foodborne outbreaks has been reported in Japan, where pathogen such as Salmonella enterica subsp. enterica (Asakura et al., 2002) and E. coli O157 (Makino et al., 2000) in food in VBNC state were responsible for the outbreak.
Therefore, the role of food and treatment for food preservation in induction of VBNC state has to be elucidated. Predicitve models offered by biomathematics and bioinformatics would be very helpful tools, in order to evaluate the possibility that, under certain conditions, pathogen bacteria contaminating a tipology of food may enter the VBNC state (Fakruddin et al., 2012).
Methods of detection of VBNC bacteria
1. Bright Field Microscopy with Nalidixic acid
For detection of Bright-field or light microscopic is usually used. Cell division inhibitor such as nalidixic acid (20-40 mg/L) is used to stop cell division. After such treatment the viable cells, which actively growing, will be appeared as lengthen and the non-viable/ metabolically inactive cell will remain as it is. The cells are then observed under microscope. Viable cells will be seen as elongated whereas VBNC/ dormant cells will be seen as oval and large.
2. Fluorescent Microscopy
Various fluorescent staining procedures are used in combination with other procedure to determine VBNC organisms. Frequently used stains are Acridine orange, 4',6- Diamino-2-phenyl indole (DAPI), Fluorescein isothiocyanante (FITC), Indophenyl-nitrophenyl-phenyl tetrazolium chloride (INT), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) etc (Villarino et al., 2000).
Table: Fluorescent dyes used for detection of VBNC bacteria
Acridine orange stain cells depending on the ratio of DNA to protein in the cells
Actively reproducing cells appear green but slow-grower or non-reproducing cells at time of staining appear orange
Living cells look green under fluorescent microscope
Indophenyl-nitrophenyl-phenyl tetrazolium chloride (INT)
INT deposit red dye in cells that have active dehydrogenase and thus determine which of the observed cells are metabolically active
INT react with dehydrogenase enzyme to produce formazone and red color, thus living cells appear red.
Nalidixic acid (NA)
Lengthen metabolically active cells, VBNC cells remain as it is
Cells that are dividing appear to be longer in size than VBNC
Fluorescein isothiocyanante (FITC)
Enzyme activity in living cell
FITC stain living cells violet or blue
In recent years, a new differential staining assay, the BacLightÂ® Live/Dead assay, has been developed. The assay allows to simultaneously count total and viable (metabolically active) cells, by using two nucleic acid stains, that is green-fluorescent SYTOÂ® 9 stain and red-fluorescent propidium iodide stain. These stains differ in their ability to penetrate intact cell membranes. When used alone, SYTOÂ® 9 stain labels both live and dead bacteria. In contrast, propidium iodide penetrates only bacteria with damaged membranes, reducing SYTOÂ® 9 fluorescence when both dyes are present. Thus, live bacteria with intact membranes fluoresce green, while dead bacteria with damaged membranes fluoresce red (Rowan, 2011).
3. Gene probe / oligonucleotide probe / hybridization:
Molecular analysis can also be used to study non-culturable microorganisms in nature. Oligonucleotide probes of l8-20 nucleotides are proving most useful because they hybridize rapidly to specific DNA sequences of target organisms. These gene probes can reveal closely related organisms or organisms with similar functional capabilities. Especially useful for the analyses of rRNA that demonstrate the presence of diverse microbial populations whose phylogenetic relationships can be ascertained by comparison with rRNA sequences from previously described microorganisms (Josephson et al., 1993).
There are different types of blotting such as colony blot, slot blot, dot blot and southern blot. The principle of blotting is the use of radio- or non-radioactive or fluorescence labeled probe (DNA/RNA/Antibody) to detect VBNC cells directly from the environmental samples.
Fluorescent in situ Hybridization (FISH):
In situ hybridization is an alternative format for hybridization probes in which fluorescence labeled DNA or RNA probes are hybridized with target nucleic acids in whole, permeabilized cells. The application of this method to the detection of single microbial cells by using rRNA-targeted probes in combination with epifluorescent microscopy has been developed. This is done through selective targeting of regions of rRNA, which consist of conserved and variable nucleotide regions. By choosing the appropriate rRNA probe sequence, FISH can be used to detect all bacterial cells (a universal probe) or a single population of cells (a strain specific probe) of VBNC. It has lower sensitivity and cannot distinguish live and dead cells.
4. Molecular techniques:
Hybridization probes and DNA/RNA amplification:
Hybridization probes are nucleic acids (DNA/RNA) which have been (a) chemically or radioactively labeled and are used to detect complementary target DNA/RNA. Hybridization assay DNA/ RNA probes form a stable double stranded structure with target nucleic acid via H-bonding between complementary bases.
Amplification of targets:
DNA Based methods: 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. DNA recovery procedures do not discriminate between culturable and non-culturable forms of the target organisms- all cells with intact amplification targets will be detected. Confocal laser microscopy in combination with fluorescence-based hybridization assays, also provide a more sensitive method for detecting and identifying VBNC organisms.
RNA based methods: Due to the failure of distinguishing between dead or live cells by DNA-based methods, the mRNA level may be a valuable estimate of gene expression and/or cell viability under different conditions (Lleo et al., 2000).
RT-PCR (Reverse Transcriptase PCR) can distinguish between Live and dead cell. This is possible because this is an mRNA based method and mRNA is short lived (half-life less than 1 minute), mRNA is only present in metabolically active cells, not found in nature after the cell death. By this method we can study community relationship and can also detect non-culturable but active or live cells. DNase enzyme is used during the isolation of RNA from environmental samples. Reverse transcriptase and random primers are added to the reaction mixture and the RNA in the sample (both RNA and rRNA) is transcribed into DNA. PCR is then use to amplify the specific sequence of interest (Pai et al., 2000).
Is the concept of VBNC is a misnomer?
By extending the concept of bacterial self-suicide scientists tried to explain what happens when cells are exposed to chemical and physical injury (Forsman et al., 2000). Thus VBNC organisms came alongside with those, which do not grow in ordinary media but which do grow when offered selective or enrichment media. They said, "Such cells are not un-culturable" they wrote "We are simply failing to provide appropriate conditions to support culture" (Sinton, 2006).
The reasons, which made the term, VBNC a misnomer are as follow:
1. Bacteria that have been semi-starved will immediately resume growth when provided with the appropriate nutrients and conditions. But viable but non-culturable bacteria do not resume growth when nutrients are provided. Evidence suggests that these cells have become too starved to be able to grow on rich medium initially; this phenomenon was observed over three decades ago and was known then as "substrate accelerated death" (Heim et al., 2002). It was found that substrate that normally supported growth of certain Gram-negative bacteria hastened their death when introduced into starved populations of these bacteria. These substrates were considered "lethal" substrates. These starved cells require an adjustment period to allow phenotypic adaptation back to the normal growth state (Epstein, 2009). A sudden shift to nutrient rich agar creates a metabolic imbalance that results in the formation of super-oxide and free radicals, causing DNA damage that can result in cell death (Barer & Harwood, 1997).
2. There is yet no complete and perfect media to isolate arid culture all the organisms from environment.
3. Cells are usually-injured or stressed or starved condition in natural environment. So complete system has been devised to enrich or resuscitated the VBNC cells.
Culture condition that can be applied in laboratory is not sufficient to recover all microorganisms i.e. yet it is not possible to provide or stimulate exact environmental conditions in the laboratory.
Regardless of the role that the VBNC state plays, it is clear that a large number of non-spore-forming bacteria, most notably a large number of human pathogens, are capable of entering this state, maintaining cellular structure and biology and continuing significant gene expression while otherwise non-culturable by 'standard' laboratory methods. That they can exit from this state, and become culturable again, is also undeniable. Finally, it can no longer be questioned that the VBNC state plays a critical role in the survival of important human (and other) pathogens, and possibly in their ability to produce disease.