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Tuesday, 17 February 2009 16:39


  • V. cholerae is a strictly waterborne pathogen.
  • Among about 200 identified serogroups only two are known to cause epidemic cholera: O1 and O139.
  • Occurrence of disease is typically seasonal due climatic factors, physical, and biological factor.
  • V. cholerae has an extraordinary capability to adapt to different environments and can be found in fresh- and brackish water.
  • V. cholerae has the ability to attach to surfaces with amazingly wide substrate specificity.
  • V. cholerae has a planktonic and a biofilm- or aggregate-associated lifestyle and can persist intracellularly in amoebae.
  • V. cholerae can reversibly adopt a non-culturable state.
  • Chlorine disinfection at normal levels results in efficient inactivation.
  • When cultivated on plates, two morphologically distinct colony types can be differentiated: smooth and rugose.
Vibrio cholerae is the most significant member of the gram-negative rod-shaped Vibrio family. Among about 200 identified serogroups only two species are known to cause epidemic cholera: O1 and O139 ( Bitton G 2005). The difference between toxigenic and non-toxigenic strains might be the presence of virulence genes like ctx, zot and ace ( Singh et al. 2001). The O1 strains, which were known first, comprise the classical biotype (which caused the first six cholera pandemics) and the El Tor biotype (which caused the seventh cholera pandemic). In 1993 a new serogroup, V. cholerae O139, was associated with a cholera epidemic in India and subsequently in Bangladesh. The emergence of serogroup O139 was considered a turning point in the microbiological perspective of cholera. This serogroup, although nearly identical with V. cholerae O1 El Tor, possesses a capsule whose layer is distinct from the lipopolysaccharide antigen found in the O1 serogroup ( Colwell RR 1996). The genetic differences are assumed to be a result of recombination events, horizontal gene transfer, and the acquisition of unique DNA ( Colwell RR 1996).

A database of cholera outbreaks is maintained by WHO, but data is considered to be underestimated due to underreporting and the lack of a surveillance system in endemic areas. A study reviewing  data compiled between 1995 and 2005, stated that of the 632 reports, 66% originated in Sub-Saharan Africa, 16.8% in Southeast Asia, 7.1% in South and Central America, and 5.7% in North Africa and West Asia ( Griffith et al. 2006). The authors concluded that the most common risk factor for cholerae outbreaks were water source contamination, heavy rainfall (for example after the monsoon in India and Bangladesh), and flooding, and population dislocation. Other risk factors given in the study are lack of appropriate water infrastructure, a high population density, low education, and lack of previous exposure. In general areas with frequent cholerae outbreaks lack clean water and have a poor sanitation standard. The occurrence of disease is typically seasonal as is easily understood in case of climatic factors like the monsoon in India. Apart from climatic factors, physical and biological factors contribute to the pronounced seasonality.

V. cholerae has the ability to attach to surfaces with an amazingly wide specificity. It can attach not only to the mammalian mucosa and brush border cells in the mammalian gut, but also to hindgut mucosa of chitin-containing blue crabs, to shellfish, crustacean copepods and other zooplankton and invertebrates, and even algae and aquatic vegetation like water hyacinths ( Colwell et al. 1996). The strong tendency of Vibrio cholerae to adhere to surfaces allows efficient reduction of pathogen numbers by methods like water treatment methods like slow and rapid sand filtration, coagulation, flocculation, or sedimentation (Nair GB, WHO document; http://www.who.int/water_sanitation_health/dwq/admicrob6.pdf). Lacking more sophisticated water treatment facilities, the surface attachment properties allow crude removal of V. cholerae from raw water by simple filtration. When using nylon nets or one of several different types of sari cloth, 99% of plankton-attached V. cholerae could be removed ( Huq et al. 1996).

When cultivated on plates, two morphologically distinct colony types can be differentiated: smooth and rugose. The phenotypes result from phase variation. Both variants are pathogenic. The rugose phenotype shows an increased production of polysaccharides, greater resistance to chlorine and other stresses, and an enhanced biofilm-forming capacity ( Yildiz and Schoolnik 1999). The rugose variant might also be less susceptible to phage infection ( Beyhan and Yildiz 2007). Switching between the two variants is genetically driven and likely to increase population diversity and the ability to adapt to different environments to maximize evolutionary success ( Beyhan and Yildiz 2007).

Natural aquatic habitats are the environmental reservoirs of V. cholerae. The pathogen can be found both in freshwater and, due to its salt tolerance, also in brackish estuarine water and the marine environment. The ability to utilize chitin as a nutrient source is very beneficial in such ecosystems.  Occurrence of disease has been linked to coastal ecosystem dynamics ( Mouriño-Pérez et al. 2003). A temporal association between phytoplankton blooms and the subsequent zooplankton blooms has been observed. Such blooms are caused by global and local aquatic conditions. One of the factors for V. cholerae proliferation might be the increase in dissolved organic matter. Mouriño-Pérez et al. could show that increased DOM levels have the potential to support explosive V. cholerae growth in seawater microcosms. The climatic phenomenon El Niño can create ideal conditions by causing warm sea surface temperatures and bringing rain which again washes nutrients and fecal material from the land into the water ( Colwell et al. 1996). A study of different natural waters used as a source of drinking and household water in rural areas in Bangladesh, correlated higher water temperature, shallow water, increased rainfall, water conductivity, and increased copepod counts with higher numbers of V. cholerae, although often with a lag period in the range of several weeks ( Huq et al. 2005).

Although the major habitat of V. cholerae is the aquatic environment, little is known about concentrations of the pathogen in different water types. Although disease is clearly known to be caused in most cases by consumption of contaminated water, apparent concentrations in surface water are well below the infectious dose ( Faruque et al. 2006). During epidemic periods, toxigenic V. cholerae have been isolated by cultivation only infrequently ( Islam et al. 1994) and in interepidemic periods isolation is extremely rare ( Huq et al. 1990). The problem of detection seems to lie in the standard culture approach: The fact that fluorescence antibody-based and molecular techniques can successfully detect the pathogen throughout the year, suggests the presence of a non-culturable state ( Alam et al. 2006). The adoption of such a state helped V. cholerae to escape detection for a long time. Huq et al. summarized that counts of culturable V. cholerae do ‘not provide useful information for prediction of cholera’ ( Huq et al. 2005). The true prevalence of V. cholerae might be considerably higher than the one observed by cultivation methods ( Faruque et al. 2006). Faruque et al. referred to V. cholerae cells that cannot be cultured as ‘conditionally viable environmental cells’ ( Faruque et al. 2006). The authors suggested that these cells are derived from ‘biofilm-like multicellular clumps’ shed in human stools. These clumps were reported to contain metabolically impeded cells (with partially impaired protein synthesis) that can be resuscitated under appropriate conditions. These in-vivo formed cell aggregates showed greater infectivity than planktonic cells. Interestingly, an independent research group reported the induction of genes at a late stage in infection, which might help V. cholerae to adopt a phenotype that is more persistent in the environment ( Schild et al. 2007). This hypothesis that the intestinal passage increases the fitness of the pathogen before it is shed into the more adverse aqueous environment deserves further evaluation.

Biofilms might be an important reservoir in interepidemic periods. There are indications that V. cholerae can survive in a NC state for extended times in aquatic environmental biofilms ( Alam et al. 2007). Evidence was presented that these nonculturable cells can be resuscitated and are still infectious ( Alam et al. 2007). This is in correlation with an earlier observation that environmental water that was apparently free of V. cholerae by traditional culture methods, yielded virulent pathogens when introduced into rabbit intestines ( Faruque et al. 2006).

Another factor making detection of V. cholerae difficult is the wide spectrum of ecological niches. The pathogen has an extraordinary capability to adapt to different environments and lives in association with crustacean zooplankton, algae, marine macrophytes, benthic animals, and insect egg masses. As mentioned before, it has in addition to a planktonic also a biofilm- or aggregate-associated lifestyle and can persist intracellularly in amoebae. There is an increasing number of studies reporting growth of V. cholerae in water under appropriate conditions (see section ‘survival in water and preferred environment’).
 
Selected studies on occurrence and prevalence of V. cholerae are summarized in the following:

  • In South Africa toxigenic V. cholerae were identified with PCR both in surface and groundwater (Momba et al. 2006).
  • Due to its fecal origin V. cholerae is present in raw wastewater where it was found in elevated concentrations: During a cholera epidemic, levels of 10-104 cells/100 ml were reported (Kott and Betzer 1972). Concentrations up to 105 cells/ml were observed in raw sewage in Peru ( AWWA 2006).
  • Water samples from village wells, ponds, and a river in an cholera-plagued area in Bangladesh were tested for V. cholerae O1 using fluorescent antibody (FA) detection and FA in combination with direct viable counts (DVC). Whereas FA results were reported negative for well samples, river and pond water samples showed typical V. cholerae O1 concentrations of around 103 cells ml-1. Of the V. cholerae population, 16-85% of the cells were elongated and were judged viable when using FA-DVC. Cultivation estimates, on the other hand, were typically <0.3 cells/100 ml-1 for the same samples (with one exception, where 2 cells per 100 ml were obtained). V. cholerae O1 was estimated to constitute 0.01 to 0.1% of the total microbial population ( Brayton et al. 1987).
  • A direct fluorescent antibody assay showed that nearly 24% of 412 water and plankton samples taken from five shore sites in Chesapeake Bay over a three year period were positive for V. cholerae O1. The pathogen was detected more frequently in summer months and in regions with lower salinity. Temperatures above 19°C and salinities between 2 and 14 ppt were reported to show an at least fourfold increase in the number of pathogens. The authors suggested that salinity might be a useful indicator to predict the abundance of this pathogen ( Louis et al. 2003).
  • Among 32 potable water samples from hand pumps and wells in a cholera-plagued area in India, 12 tested positive for V. cholerae O1 using both an immunofluorescence assay and conventional culture methods ( Goel et al. 2005).
Common chemical disinfectants like chlorine at normal levels can effectively inactivate V. cholerae provided that the treated water is clear (i.e. free of particles (Nair GB, WHO document, http://www.who.int/water_sanitation_health/dwq/admicrob6.pdf; AWWA 2006). The same holds true for physical disinfection (like UV treatment). Organic matter can strongly interfere with disinfection efficacy. It also has to be considered that microorganisms are more resistant to disinfection when attached to surfaces or when internalized by amoebae compared to their planktonic counterparts ( Holah et al. 1990). Ideally, disinfection studies would consider those environmental factors as efficient disinfectant doses in such protective environments can vary significantly from data obtained by exposure of pure cultures.

Exposure of V. cholerae to stress can induce a morphological shift to a ‘rugose’ colony phenotype ( Morris et al. 1996). The rugose phenotype shows an increased production of polysaccharides, greater resistance to chlorine and other stresses, and an enhanced biofilm-forming capacity ( Yildiz and Schoolnik 1999). The expression of amorphous exopolysaccharide in the rugose phenotype promotes cell aggregation as shown by flow cytometry ( Morris et al. 1996). Aggregation by itself results in higher resistance to adverse conditions.

Selected studies on disinfection are summarized in the following: 

  • In a study comparing the susceptibilities of different enteric bacteria to solar disinfection (SODIS), V. cholerae 01 Ogawa biotype El Tor was less resistant than Salmonella typhimurium, E. coli, and Shigella flexneri. As solar disinfection is based on a synergistic action of UVA light and increased water temperatures, these two factors were studied separately. Resistance to sunlight at a constant water temperature of 37°C reduced culturability of V. cholerae by app. 6 log10 units at a fluence of around 900 kJ m-2, whereas a fluence of more than 2400 kJ m-2 was needed to achieve the same inactivation with E. coli or Shigella. A fluence of 2400 kJ m-2 resulted in only an app. 2 log10 inactivation of Salmonella (a fluence of 2400 kJ m-2 at 350 – 450 nm corresponds to 6-7 h of sunlight exposure in Switzerland from June to August). V. cholerae was also more susceptible to mild heat: Temperature sensitivity was about the same for E. coli, Salmonella, and Shigella and was observed above 45°C, whereas V. cholerae was already sensitive to temperatures above 40°C ( Berney et al. 2006).
  • Solar disinfection proved useful in an African study involving 131 households with children under 6 years of age. Disinfection was achieved by exposing water in clear plastic bottles to sunlight on house roofs. Only 3 out of 155 children (aged under 6) drinking solar disinfected water developed cholera compared to 20 out of 144 controls drinking water kept indoors (Conroy et al. 2001).
  • Sousa et al. studied the chlorine concentrations necessary to eliminate culturability of freshly grown V. cholerae O1 cells. Cells were grown in TSB for 18-20 h before adding 1 ml aliquots to 100 ml of NaCl solution containing 5–10 ppm of chlorine. The exposure time was 5 min. For chlorine concentrations up to 7 ppm viable cells were recovered, whereas chlorine concentrations of 8 ppm and higher completely eliminated culturability starting from an initial cell concentration of 107 cfu mL-1. Only a 68% reduction was observed when dipping artificially contaminated shrimps into 10 ppm chlorine solution for 5 min. The authors acknowledged that the presence of organic material would result in lower chlorine efficiency (due to the chlorine demand of the organic matter), but also mentioned that surface attachment might contribute to higher resistance ( Sousa et al. 2001).
  • When comparing the response of different pathogenic Vibrio species to high hydrostatic pressure (200-300 mPa, 5-15 min, 25°C, initial cell concentrations of 107 cfu mL-1), culturability was lost without triggering a NC state. There was evidence that NC cells showed greater pressure resistance. Non-culturability was induced by storage in artificial sea water at 4°C ( Berlin et al. 1999).
  • Iodine has proven a good disinfectant with two drops of 2% iodine tincture being sufficient for 1 liter of water (http://www.who.int/water_sanitation_health/dwq/admicrob6.pdf).
  • When comparing two serotypes of V. cholerae O1 El Tor with E. coli in regard to their susceptibilities to disinfectants used in food processing, kitchen, and personal hygiene, V. cholerae was slightly more sensitive than E. coli. Experiments were performed with pure cultures. Disinfectants tested were chlorine, quaternary ammonium compound SU 319, Amphobac 4, isopropanol, and chlorhexidine. Peracetic acid was found to be the most effective compound. Hypochlorite efficiency was reported to be lower in the presence of protein ( Jones et al. 1992).
  • Rugose variants of V. cholerae O1 El Tor showed higher resistance to chlorine than the smooth variants. Viable cells persisted for >30 min to 2 mg L-1 free chlorine. Increase resistance was also reported for complement-mediated serum bactericidal activity ( Morris et al. 1996).
  • Ferrate was suggested to be an effective disinfectant for ballast water. Cultures of V. cholerae and other microorganisms typically found in ballast water were grown in saline solution to simulate ballast water. A ferrate dose of 5 mg L-1 was reported to result in complete disinfection of the organisms tested ( Jessen et al. 2008).
V. cholerae is a strictly waterborne pathogen ( Bitton G 2005). Reservoirs of V. cholerae during periods between outbreaks are unknown ( Alam et al. 2006; Islam et al. 1990). Although often associated with fecal pollution, a relation to sewage contamination is not always given ( Kaper et al. 1979). In water V. cholerae can occur in the free-living planktonic state or attached to a variety of abiotic surfaces or associated with phytoplankton (e.g. Volvox), zooplankton (e.g., copepods, amoebae) or cyanobacteria (e.g. Anabaena) ( Bitton G 2005; Islam et al. 2007; Huq et al. 1990; Huq et al. 1983; Worden et al. 2006). The identification of marine plankton as a vector for V. cholerae has greatly helped to understand its persistence in the environment. The concentration of Vibrio species has been reported to increase during phytoplankton blooms and the following zooplankton blooms ( Colwell et al. 1996). Attachment might increase survival. Interestingly, V. cholerae seems to attach only to live copepods but not to copepods killed by freezing ( Huq et al. 1983). Survival in the presence of live copepods was longer than in the presence of dead copepods. Plankton-associated cells might occur in a viable-but nonculturable state: V. cholerae O1 in plankton samples in Bangladesh was detectable in high numbers by FA, but not by conventional culturing ( Huq et al. 1990). The VBNC form was found to be able to convert under favorable conditions into the pathogenic transmissible state and can cause disease in the human intestines ( Binsztein et al. 2004; Colwell RR 1996). Persistence of more than 15 months has been reported inside the mucilaginous sheath of the aquatic alga Anabaena variabilis using microscopy ( Islam et al. 1990a). Outbreaks have been temporally related to phytoplankton blooms by remote sensing (Lobitz et al. 2000).

Warmer temperatures favor growth ( Vital et al. 2007). Seawater conditions under some circumstances seem to be beneficial for survival: 90 % of V. cholerae cells seeded into freshwater were inactivated after 18 hours, while survival was increased fivefold in seawater ( AWWA 2006). This statement has to be detailed, however: Other studies reported that high salinities and low temperatures as encountered in marine habitats might induce a dormant state ( Singleton et al. 1982). V. cholerae in a microcosm with chemically defined sea salt solution (25 g/l salinity, 10°C) was culturable for less than 4 days ( Singleton et al. 1982). Although Na+ ions were demonstrated to be required for growth, excessive concentrations might inhibit ( Singleton et al. 1982). Testing different salinity concentrations Vital et al. found best growth rates were seen at moderate NaCl levels (5 g/l) ( Vital et al. 2007). These findings contribute to the view that estuarine conditions with brackish water might be more beneficial than the marine environment ( Singleton et al. 1982). It is part of the naturally occurring microbial flora there ( Kaper et al. 1979; Mouriño-Pérez et al. 2003). Also other ions probably affect V. cholerae survival: It was observed that cells survive longer when water was stored in metal drums compared to storage in clay pots or plastic drums. Increasing iron concentrations in dechlorinated tap water was shown to be beneficial for the survival of V. cholerae O1 ( Patel et al. 1995). V. cholerae O1 survived in dechlorinated tap water from < 24 h to 10 days with very low numbers of surviving bacteria. In the presence of Fe2O3, however, bacteria survived from 4 to 12 d with very high numbers of surviving bacteria ( Patel et al. 1995). This was supported in a later study which found that V. cholerae O1 survived in plain water for 8 days, whereas in the presence of Fe2O3 survival was prolonged to 15 days ( Joseph and Bhat 2000). The presence of organic material resulted in a further promotion of bacterial survival for another 4 days. Regarding pH, different V. cholerae strains did not survive at pH 5.0, but increasing pH positively affected survival ( Patel et al. 1995). Also the availability of phosphorous might be important: Internally stored inorganic polyphosphate was suggested to enhance the ability of V. cholerae to overcome environmental stresses in a low-phosphate environment ( Jahid et al. 2006).  

Loss of culturability and association with biofilms. A potential survival strategy might be the adoption of a viable- but nonculturable (VBNC) state (also referred to as non-culturable (NC) state). The loss of culturability goes along with the transformation from curved rods into coccoid cells and a reduction in size ( Alam et al. 2007). Such nonculturable cells can be found in biofilms. Biofilms were shown to provide a sheltered microenvironment for V. cholerae ( Alam et al. 2007; Islam et al. 2007). They were reported to be a potential source of culturable and VBNC V. cholerae ( Alam et al. 2006). Large numbers of V. cholerae O1 and O139 in thin biofilms in regions where cholera is endemic were revealed by FA counting ( Alam et al. 2006). Biofilm-borne V. cholerae in a VBNC state could be resuscitated after animal-passage in contrast to free planktonic cells ( Alam et al. 2007). Biofilms were suspected to be one of the reservoirs between seasonal epidemics. V. cholerae populations in such an attached form might be controlled by grazing by protozoan predators as suggested for marine waters ( Worden et al. 2006). The close proximity in biofilms as found for example on the chitinous exoskeletons of copepods might also facilitate the exchange of genetic material and therefore result in increased fitness in terms of the potential to adapt and survive in their environmental niches and in terms of pathogenicity. The transfer of large DNA fragments between V. cholerae serogroups O1 and O139 has been experimentally observed in a chitin-based biofilm ( Blokesch and Schoolnik 2007).

In summary of the above, warm waters with moderate salinities and high apparent assimilable organic carbon (AOCapp) levels in the presence of attachable surfaces and plankton provide good conditions for survival and replication of V. cholerae.

Selected studies on survival in water are summarized in the following: 

  • Alam et al. provided evidence that biofilms harbor V. cholerae O1 cells in a VBNC state and that biofilm formation might be a survival strategy. Microcosms with autoclaved pond water from Mathbaria, Bangladesh, were seeded with V. cholerae O1 cells and maintained at room temperature or at 4°C. Culturability was gradually lost over time with different rates using different cultivation media. The time needed for colony formation became longer over time. Starting from an initial count of around 107 cells per ml (using TTGA or Luria agar), no colonies could be obtained any more after 40 and 84 days from the room temperature and the 4°C microcosms, respectively. Loss in culturability was preceded by a transformation of the initially curved rod-shaped cells into coccoid cells. Nonculturable cells underwent a reduction in size. The biofilm formation rate was negatively correlated to culturability and was also temperature-dependent. Biofilm formation was observed for the room temperature microcosm after 2 days of incubation with large numbers of cells in clusters after 7 days. Much larger biofilms were formed after 26 days. At 4°C, biofilm formation occurred slower with direct fluorescent antibody counts unchanged until day 26. Biofilm formation was not obvious after 40 days, but began at this time and increased up to day 99. Biofilm-borne pathogens from these microcosms could be resuscitated by animal passage up to 495 days. However, although culturability could be regained, they did not produce significant fluid accumulation after 18 hours in the ileum of infected rabbits. In contrast, V. cholerae cells from biofilms in microcosms inoculated with stools could only be revived up to 59 days indicating that cells from environmental biofilms are more robust than freshly discharged cells. The authors concluded that biofilms can act as a reservoir for this pathogen in interepidemic periods ( Alam et al. 2007).
  • Abd et al. could show that V. cholerae O1 classical and El Tor strains can survive intracellularly in Acanthamoeba castellanii and grow in their hosts. Amoebal growth was not compromised by the presence of the bacteria in co-culture. Numbers of both amoebae and intracellular bacteria increased over the cultivation time with a strong positive correlation. Confocal microscopy showed that the intracellular bacteria localized in the cytoplasm. Multiplication occurred in the trophozoites. Bacteria were also found in cysts after one day of cocultivation. After 7 days cysts were heavily loaded with intracellular V. cholerae. Release of the bacteria into the environment could have resulted in pathogen concentrations high enough to infect humans ( Abd et al. 2007). An earlier publication showed similar data for V. cholerae O139 ( Abd et al. 2005). Presence of the bacterium did not inhibit growth of A. castellanii. The interaction with the amoebae seemed to be beneficial for survival of V. cholerae. Whereas the counts of bacteria decreased from 2 x 106 CFU ml-1 to non-detectable levels within 4 days in the absence of amoebae, the presence of A. castellanii enhanced survival of culturable pathogens until the end of the experiment of 16 days. Final bacterial counts after 16 days of co-culture were higher than the initial counts. Intracellular pathogens were found after the first day of co-culture.
  • A study could prove extensive growth of V. cholerae O1 in autoclaved and filtered freshwater ( Vital et al. 2007). The initial cell density after seeding was 5 x 103 cells ml-1 and growth was monitored using flow cytometry. Replication was seen in river and lake water and in wastewater plant effluent reaching densities of up to 1.5 x 106 cells ml-1. Attachment to particles or higher organisms was not seen compulsory for survival and growth. Seeded V. cholerae competed successfully with autochthonous bacterial consortia in direct competition experiments and were able to grow and constitute around 10% of the final cell concentration ( Vital et al. 2007). The study confirmed that freshwater – especially when mesotrophic- can be an environmental reservoir. Apparent assimilable organic carbon (AOCapp) was suggested to be the key parameter governing V. cholerae growth in freshwater. A positive correlation was reported between the AOCapp concentration and the final V. cholerae cell density. No growth was observed in tap and bottled drinking water containing less than app. 60 μg AOCapp liter-1. This correlates with the earlier finding that V. cholerae grows rapidly on the dissolved organic matter (DOM) fraction of sea water as given during intense phytoplankton blooms ( Mouriño-Pérez et al. 2003). The conditions led to concentrations of free-living culturable cells exceeding the infectious dose (10-4 cells ml-1) by 3 orders of magnitude ( Mouriño-Pérez et al. 2003).
  • An example of the beneficial effect of plants was the finding that V. cholerae O1 survived better in sea-salt solutions with the plant Lemna minor (duckweed) compared to a control without the plant using viable counts ( Islam et al. 1990b). A commensal relationship had been proposed.
  • A study by Faruque et al. suggested that environmental cholera phages might play a role in ending cholera epidemics. Water samples were collected from different points of a lake and two major rivers in Bangladesh over a 3-year period. Significantly more samples were found to contain either a phage or a phage-susceptible V. cholerae strain than both together at the same time. The incidence of cholera varied seasonally with more patients during times when samples showed pathogenic V. cholerae strains but lacked detectable cholera phages. Cholera phages, on the other hand, were found frequently in interepidemic periods, with simultaneously low numbers of viable V. cholerae cells. The inverse correlation between bacteria and phages might be an important factor to explain cholera seasonality. The authors pointed out that phages might also play a role in the emergence of new pandemic serogroups ( Faruque et al. 2005a). In a later publication it was reported that host-mediated phage amplification might contribute to the self-limiting nature of seasonal cholera epidemics ( Faruque et al. 2005b). It was observed that the peak of a cholera outbreak in Bangladesh was preceded by high concentration of V. cholerae in the environment, whereas it was followed by high levels of lytic cholera phages towards the end of the epidemic. It was suggested that the increase in phage numbers was a result of both an amplification in the environment and in human patients. At the end of the epidemic, patients ingested both the disease-causing V. cholerae strain and increasingly also the phage which could replicate in the host. The buildup of the phage peak coincided with greater phage excretion in stools of cholera patients. Such an in-patient in vivo phage amplification could have contributed to the environmental phage amplification and to overall higher phage numbers resulting in a decline of pathogenic bacteria and therefore a collapse of the epidemic.
  • A mutant of V. cholerae with a deletion in the polyphosphate kinase gene lacking the ability to synthesize polyphosphate was found to be more susceptible to environmental stresses under phosphate limited conditions than the wild type strain. The deletion did not significantly impact cholera toxin production, biofilm formation, growth rates, motility, or colonization of mouse intestines. The mutant was, however, more sensitive to low pH, high salinity, and oxidative stress in low-phosphate minimal medium and failed to induce catalase. It was suggested that the ability of V. cholerae to build up large polyphosphate stores could be beneficial for survival in estuaries and brackish waters in which phosphorous can be a limiting nutrient ( Jahid et al. 2006).
  • Survival of V. cholerae O1 was studied in microcosms of 3.5% salinity. Cells were grown in M9 minimal medium with 0.5 M sucrose at 37°C and used to inoculate artificial seawater microcosms to a final cell concentration of 1 to 5 x 106 CFU/ml. Culturability dropped approx. 2 log10 units within 10-12 days and then remained nearly unchanged during the next 50 days. When the cells were grown in M9 medium alone or M9 medium with 0.5 M NaCl, culturability was reduced by 3 and 5 log10 units, respectively within the initial time period, before recovering in the following 40 to 50 days to levels near the ones with cells grown in the presence of sucrose. The authors concluded that V. cholerae cells has the potential to remain culturable in seawater sufficiently long to be carried by ocean currents to distant geographical locations ( Munro and Colwell 1996).
The infective dose of V. cholerae is between 103-106 cells if ingested with water and between 102-104 cells if ingested with food (http://www.emedicine.com/med/topic351.htm). Interestingly, a individual colonized copepod can potentially contain up to 104 cells of V. cholerae ( Colwell RR 1996). The ingestion of a glass of untreated water with a few infected copepods can therefore be sufficient to cause disease. The concentration of copepods can be high in cholera endemic countries at times of plankton blooms.
Both phase variants (regular smooth and stress-induced rugose) are pathogenic: Six volunteers fed with 106 cfu of a rugose variant developed cholera symptoms ( Morris et al. 1996). Isolates recovered from the stool of the infected individuals retained the rugose phenotype.
The occurrence of non-culturable (NC) or dormant V. cholerae makes data obtained by microscopy or molecular methods appear more relevant in terms of microbiological water quality monitoring. Brayton et al. observed dramatically higher counts applying fluorescence microscopy compared to cultivation ( Brayton et al. 1987).

Selected molecular detection methods include:

  • FA staining and FA-DVC: Monoclonal antibodies against the V. cholerae O1 antigen were used in this fluorescent-antibody approach ( Brayton et al. 1987; Huq et al. 1990). Approx. 3 h were required to complete the staining process for FA enumeration. An additional 6 to 24 h were needed to allow the cells to elongate during the direct viable count incubation part. Polyclonal antibodies against outer membrane proteins of V. cholerae O1 were used by Goel et al. ( Goel et al. 2005). No cross-reactivity with other bacteria was observed. The technique was able to detect 240 CFU/ml of V. cholerae O1.
  • PCR: End-point PCR reactions were developed for non-quantitative species-specific identification. Targeted sequences include the 16S-23S ITS region, wbeO, rfb, ctxA, rtxA, rtxB, rtxC, ompW, and toxR ( Lipp et al. 2003; Chow et al. 2001; Nandi et al. 2000).
  • Seminested PCR: A PCR method developed originally for stool samples (Varela P 1994) and targeting a region spanning the ctxA and ctxB genes proved useful for water samples when V. cholerae concentrations were very low (2-3 CFU/ml) ( Binsztein et al. 2004).
  • Enrichment-pit-stop semi nested PCR: A pit-stop seminested PCR assay targeting the ctxAB operon of V. cholerae was reported to have a detection limit as low as 1 CFU/ml-1 of seeded water after enrichment. The assay was validated with treated sewage, surface, ground and drinking water. The analysis can be completed within 10 h ( Theron et al. 2000).
  • Multiplex nested PCR: A single-tube assay targeting ctxA and rfbN with subsequent gel visualization was designed with a detection limit of 1 pg of V. cholerae O1 DNA ( Mendes et al. 2008).
  • Multiplex PCR: Multiplex PCR assays with subsequent gel visualization of PCR products were developed targeting ompW and ctxAB ( Nandi et al. 2000) and rfb and ctxA ( Hoshino et al. 1998). The first aimed at species identification and screening of toxigenic and nontoxigenic strains of clinical and environmental origin, the second at detection of toxigenic strains in stool samples with detection limits of 65 CFU (V. cholerae O1) and 200 CFU (V. cholerae O139) per reaction . The assays were not tested with water samples in contrast to a multiplex PCR assay described by Goel et al. ( Goel et al. 2007). The sensitivity of this assay targeting ompW, ctxB, rfbG, zot, and tcpB, was 5 x 104 V. cholerae cells per PCR reaction. When applied to different water samples artificially spiked with V. cholerae, as few as 8 cells per ml of water could be detected after a filtration and enrichment procedure. Another relatively recent duplex-PCR assay targeting tcpA and ctxA was reported to detect 100 fg of V. cholerae DNA template per reaction corresponding to 23 cells ( Chomvarin et al. 2007). The assay was successfully tested with different water samples. Filtration and subsequent enrichment for 3 hours significantly increased the efficiency of detection.
  • qPCR: A TaqMan qPCR assay targeting the hlyA gene was designed for detection of V. cholerae O1, O139, non-O1, and non-O139 ( Lyon WJ 2001). The method was successfully validated with pure cultures, raw oysters, and synthetic seawater. The sensitivity was reported to be 6-8 CFU per g of spiked raw oysters and 10 CFU per ml of spiked synthetic seawater. The total time demand was 3 h. A linearity over more than 6 log units was obtained with pure cultures as for a qPCR assay published by Blackstone et al. ( Blackstone et al. 2007). The latter assay targets ctxA and could detect down to <10 CFU per reaction with pure culture. It was applied together with an internal amplification control and tested with different water enrichments which were found negative.
  • Multiplex qPCR: A molecular beacon-based qPCR assay targeting four virulence and regulatory genes (rtxA, epsM, tcpA, ompW) was designed and tested with pure cultures and spiked water samples ( Gubala and Proll 2006). As few as 10 CFU of V. cholerae per reaction were detected when a DNA purification step was employed to remove inhibitory substances. This assay improved a SYBR-based multiplex qPCR assay with higher detection thresholds of 103 CFU per reaction for spiked water samples ( Gubala AJ 2006).
  • Multiplex PCR and DNA microarray: sensitivity 102-103 CFU/ml and specificity 100% without enrichment, 1 CFU/g oyster homogenate after 5 hours enrichment ( Panicker et al. 2004).
  • Microarray: Vibrio cholerae was one of 18 pathogenic species detected by a Multi-Pathogen Identification (MPID) microarray. The array was designed to simultaneously detect eighteen pathogenic prokaryotes, eukaryotes and viruses. Species-specific primers were used to amplify multiple diagnostic regions unique to each individual pathogen and amplification products were hybridized to the microarray. The array was tested with spiked air samples (Wilson WJ 2002).
  • Real-time NASBA: A multitarget molecular beacon-based real-time NASBA assay was developed for specific detection of V. cholerae. Target transcript sequences comprised toxR, hlyA, and groE,  ctxA, and tcpA. The first three targets were chosen to identify all V. cholerae isolates, the last two to specifically detect clinical isolates. The assay was successful in detecting mRNA from environmental water samples spiked with V. cholerae. A detection threshold 50 CFU/ml (determined by mRNA dilution) was reported with a time demand of 3 hours for the entire assay ( Fykse et al. 2007).
  • Immunochromatographic dipsticks: A dipstick assay was developed for detection of V. cholerae O1 and O139 detection based on LPS detection. Detection thresholds were reported to be 10-50 ng LPS/ml, the sensitivity compared to culture was between 94 – 100% and the specificity between 84-100%. ( Nato et al. 2003)
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.

Figure 1:

                                      Vibrio_photo 1                

This gelatin agar medium was used in the identification of the bacteria Vibrio cholerae, the causal agent of cholera.

Source: http://phil.cdc.gov/phil/home.asp
Content provider(s): Centers for Disease Control and Prevention
Photo ID: 3907

Figure 2:

                                      Vibrio_photo 2             

This photograph depicts a close-up of a ”string test” being performed on a droplet of sample that was harvested from a Petri dish growing suspected colonies of Vibrio cholerae bacteria. Note the mucoid “string” still clinging to the wire inoculating loop.

Source: http://phil.cdc.gov/phil/home.asp
Content provider(s): Centers for Disease Control and Prevention
Photo ID:  3909

Figure 3:

                                      Vibrio_photo 3                   

This scanning electron micrograph (SEM) depicted a number of Vibrio cholerae bacteria of the serogroup 01; Magnified 22371x.

Source: http://phil.cdc.gov/phil/home.asp
Content provider(s): Centers for Disease Control and Prevention/ Janice Carr
Credit: Janice Carr
Photo ID: 7819

Links to useful external websites can be found in the following.
Please note that this list does not endorse the content of any of these sites.

CDC
http://www.cdc.gov/nczved/dfbmd/disease_listing/cholera_gi.html

WHO
http://www.who.int/topics/cholera/en/
http://www.who.int/water_sanitation_health/dwq/admicrob6.pdf

Last Updated on Tuesday, 18 May 2010 14:57
 

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