Saturday, March 30, 2019
A Review On VBNC Bacteria
A Review On VBNC b impresseriumViable b bely Non-Culturable (VBNC) put up is a incomparable survival schema of umteen bacterium in milieu in response to perverse environsal conditions. VBNC bacteria jackpott be cultured on second microbiological media alone if they stick executable and go for their virulent capacity too. VBNC bacteria can be resuscitated when provided with tolerate conditions. A good shape of bacteria including many human pathogens have been accounting to enter VBNC pronounce. Though, there was disputes on the existence of VBNC in past, extensive molecular studies have obdurate most of them and VBNC has been accepted as distinct survival deposit by all(prenominal). VBNC bacteria argon considered as threats to public health and forage precaution collectible to their non- bring outability and malignity as provender and pissing have been describe to be contaminated with pathogens at VBNC evince though conventional methods decl be them as safe and clean. A arrive of outbreaks have overly been reported where VBNC bacteria has been implicated as causative agent. Further molecular and combinative re search in conjunction with predictive modeling be postulate to disentangle the appliances and to identify the critical points to tackle the threat posed by VBNC bacteria with regard to public health and food safety.Key linguistic process VBNC, Pathogen, public health, Food safety, DetectionIntroductionThe cadres that form colony in specialised media are the culturable cubicles. Viable means metabolically or physiologically active agent. So the cells those are metabolically or physiologically active plainly cant be cultured on particular(prenominal) media are the workable nonwithstanding non-culturable cells (VBNC) (Bogosian Bourneuf, 2001). Most microorganisms growing in nature have even up to be cultured in the laboratory. In fact less than 1% of the microorganisms in natural water and soil samples a re cultured in feasible count modus operandis (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 non form colonies on solid media however obtained metabolic activity and the ability to elongate by and by the administration of nutrients (Xu et al., 1982). concord to O pull roundr (1995), VBNC can be defined as a metabolically active bacterial cell that subdueed a threshold in this way, for cognise or unknown reasons and become unable to multiply in or on a medium practicely supporting its harvest-home. Most of the bacteria that enter VBNC carry are gram negative species belonging to the da Gamma subclass of the Proteobacteria branch, except for Rhizobium, Agrobacterium and Helicobacter-Campylobacter species (Oliver, 2000).HistoryDebra Bashford and colleagues announced that they had recovered vibrion cholerae from streams and drainpipe ditches, incl uding sites with negligible chance of sewage contamination. Around the same lime, Rita Colwell was also decision Vibrio cholerae in Maryland. She and her coworkers showed that some(prenominal) 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 enounce in which they failed to show up on nutrient agar yet took up substrates and signaled in different ways that they were certainly not utter. The use of laboratory media to recover and count bacteria and lo link them with or absolve them from pathological and former(a) activities became antiquated by the new discoveries and a term VBNC (viable but non-culturable) came (McDougald et al., 1998).VBNCMicroorganisms that do not grow in culture methods, but which are simmer down metabolically active and capable of causing infections in animals and plants are said to be in VBNC state. Traditional laboratory culture conditions methods cannot meet the necessitatements of VBNC organisms to bear on festering (Yamamoto, 2000). Semi- greedy bacteria usually resume growth immediately when take away nutrients conditions are provided. Viable but non-culturable cells leave not resume growth even when nutrients are provided (Nystr-m, 2001). VBNC cells exhibit active metabolism in the form of public discussion or fermentation, incorporate radioactive substrates, and have active protein synthesis but cannot be cultured or grown on conventional laboratory media. They have been give awayed by observing discrepancies among plate count enumeration of bacterial community and direct spotting and microscopic counts (Sachidanandham Gin, 2009). These cells whitethorn be of particular problems in the environment if they are pathogens, for example, viable but non-culturable cells of Vibri o cholerae, Enteropathogenic E. coli, Legionella pneumophila and various other bacteria have been shown to resume culturability after they have entered the intestinal tracts of animals (Colwell et al., 1996).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), type O availability (Kana et al., 2008), some(prenominal) food preservatives (Quirs et al., 2009), heavy metals (Ghezzi Steck, 1999), exposure to white light (Gourmelon et al., 1994) and de unsporting processes, as pasteurization of take out (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 b een a matter of dispute for ling since its inception, due to the difficultness of differentiation of VBNC cells hibernating(prenominal) cells through and through resuscitation phenotypic studies, new-fashioned molecular studies, info of which supported the existence of VBNC state, the dispute has mostly been put to rest (Barer and Harwood, 1999).VBNC Pathogens divert 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, Vibr io parahaemolyticus, Vibrio vulnificus (types 1 and 2)Characteristics of bacteria in VBNC state1. Maintain discernible cell integrity 2. self-will 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. rock-bottom nutrient transport 8. High ATP level and high tissue layer potential (Signoretto et al., 2000) 9. extensive modifications in cytoplasmic membrane fatty biting 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. high 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 pen (Muela et al., 2008) 15. Continuous gene expression (Maalej et al., 2004) etc.Conditions stimulating VBNC stateIn the environment, bacterial cells can enter VBNC state whitethorn 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 character 6. desoxyribonucleic acid 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 principle temperature range of growth 10. elevated or lower osmotic concentrations 11. group O 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 VBNCThough virulence of bacteria in VBNC state is still not rattling clear, many believed that pathogens in VBNC state are unable to induce infection/ sickness but still retain their virulent properties has potential to cause affection infection following resuscitation and resume of active metabolic state, which occurs when they pass through forces 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 components 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 strong 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 distemper (Rivers Steck, 2001).Even today, it is still not possible to form 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 qualifying 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 hence cause severe disease (Sardessai, 2005).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). so , environmental and clinical samples no womb-to-tomb can be considered discontinue from pathogens if culturing yields negative results. For the general public, the front end of VBNC in water and food may be relate 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 apply for domestic purpose regularly and posed a lay on the line 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 perception 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 pro ducing diseases. The first indorse of pathogenicity of non-culturable cells was the demonstration of fluid accumulation in rabbit ileal loop taste (RICA) by VBNC Vibrio cholerae O1, followed by human volunteer experiments (Amel et al., 2008).VBNC E. coli non-culturable cells were re- specifyd after passage through rabbit ileal loops 4 days post inoculation and annulus 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 great significance in public health care (Cappelier et al., 2007).Previous studies indicated that a good number of pathogenic bacteria can survive food water treatment processes persist as well as retain virulence in processed food, change integrity milk, potable water 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 V BNC 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 payoff 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 preceding(prenominal) 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 safetyMany evidences suggested posture 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 sur rounding environment is a composite plant agreement, 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 O157H7 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 caloric 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 credi bothrthy for the outbreak.Therefore , the role of food and treatment for food preservation in introduction 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 bacteria1. Bright Field Microscopy with Nalidixic acidFor 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 sluggish cell will remain as it is. The cells are then find under microscope. Viable cells will be seen as elongated whereas VBNC/ dormant cells will be seen as oval and large.2. light MicroscopyVarious light staining procedures are used in combination w ith other procedure to determine VBNC organisms. Frequently used stains are Acridine chromatic, 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 bacteriaDyeMechanismReactionAcridine orangeAcridine orange stain cells depending on the ratio of DNA to protein in the cellsactively reproducing cells appear green but slow-grower or non-reproducing cells at time of staining appear orangeDi-amino-phenyl-indole (DAPI)Living cells look green under fluorescent microscopeIndophenyl-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 activeINT react with dehydrogenase enzyme to offer formazone and red color, thus living cells appear red.Nalidixic acid (NA)Lengthen metabolic ally active cells, VBNC cells remain as it isCells that are dividing appear to be longer in size than VBNCFluorescein isothiocyanante (FITC)Enzyme activity in living cellFITC stain living cells violet or blueIn recent years, a new differential staining hitch, the BacLight 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 SYTO 9 stain and red-fluorescent propidium iodide stain. SYTO 9 propidium iodide has significant difference in their cell membrane penetration capability. SYTO 9 stains both live and pulseless bacteria whereas propidium iodide penetrates only bacteria having damaged membranes. When used together, propidium iodide reduces SYTO 9 fluorescence in staining dead bacteria with damaged membranes. As a result, viable bacteria with intact membranes fluoresce green while dead bacteria with damaged membranes fluoresce red (Rowan, 2011).3. Ge ne probe / oligonucleotide probe / crossroadMolecular analysis can also be used to line of business non-culturable microorganisms in nature. Oligonucleotide probes of l8-20 nucleotides are proving most useful because they hybridize rapidly to specific DNA sequences of manoeuvre organisms. These gene probes can reveal closely related organisms or organisms with similar functional capabilities. Especially useful for the analyses of rribonucleic acid that demonstrate the presence of diverse microbial populations whose phylogenetic alliances can be ascertained by comparison with rRNA sequences from previously described microorganisms (Josephson et al., 1993).BlottingThere are different types of blotting such as colony blot, slot blot, dot blot and southern blot. The rule 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 int erbreeding 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 do 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 techniquesHybridization probes and DNA/RNA amplificationHybridization 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 stabl e double stranded complex body part with target nucleic acid via H-bonding between complementary bases.Amplification of targetsDNA establish 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 tenuous 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 messenger RNA level may be a valuable estimate of gene expression and/or cell viability under different conditions (Lleo et al., 2000).RT-PCRRT-PCR (Reverse Transcriptase PCR) can distinguis h 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 do the term, VBNC a misnomer are as follow1. VBNC bacteria semi-starved bacteria very often mimic each other. Semi-starved bacteria resume growth if provided with appropriate nutrients conditions. But viable but non-culturable bacteria do not resume growth even though nutrients are provided. VBNC cells become too starved to grow on nutrient rich medium directly. This phenomenon resembles a widely accepted condition termed substrate accelerated death (Heim et al., 2002). These starved VBNC cells require an adjustment period to allow phenotypic adaptation back to normal growth state (Epstein, 2009). Sudden shift to nutrient enriched media imbalances metabolic networks of the cells resulting institution of DNA damage agents such as super-oxide free radicals causing cell death (Barer Harwood, 1997).2. There is y et 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 engage environmental conditions in the laboratory.ConclusionFrom the above discussions, it is evident that a number of non-spore forming human pathogenic bacteria enter VBNC state with maintained cellular structure biology persistent gene expression but remain non-culturable by traditional cultural techniques. Thet can survive revert to culturable conditions when provided with appropriate conditions. It is also evident that VBNC bacteria pose significant threat to both public health and food safety. Further research is needed to elucidate the mechanism to combat the threat of VBN C in future.