Cholerae In Water Research Articles

Rapid and Visualized Detection of Virulence-Related Genes of Vibrio cholerae in Water and Aquatic Products by Loop-Mediated Isothermal Amplification

Vibrio cholerae can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Continuous testing of V. cholerae contamination in water and aquatic products is imperative for food safety control and human health. In this study, a rapid and visualized method was developed for the first time based on loop-mediated isothermal amplification (LAMP) for detection of the important virulence-related genes ace, zot, cri, and nanH for toxins and the infectious process of V. cholerae. Three pairs of molecular probes targeting each of these genes were designed and synthesized. The one-step LAMP reaction was conducted at 65°C for 40 min. Positive results were inspected by the production of a light green color under visible light or green fluorescence under UV light (302 nm). Limit of detection of the LAMP method ranged from 1.85 to 2.06 pg per reaction of genomic DNA or 2.50 × 100 to 4.00 × 102 CFU per reaction for target genes of cell culture of V. cholerae, which was more sensitive than standard PCR. Inclusivity and exclusivity of the LAMP method were 100% for all target genes. The method showed similar high efficiency to a certain extent in rapid testing of spiked or collected specimens of water and aquatic products. Target genes were detected by absence from all water samples from various sources. However, high occurrences of the nanH gene were observed in intestinal samples derived from four species of fish and one species of shellfish, indicating a risk of potentially toxic V. cholerae in commonly consumed aquatic products. The results in this study provide a potential tool for rapid and visualized detection of V. cholerae in water and aquatic products.

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

In cholera-endemic areas, toxigenic Vibrio cholerae persist in the aquatic ecosystem mostly in a biofilm-associated state in which the bacteria remain embedded in an exopolysaccharide matrix. The biofilm-associated cells often enter into a dormant form referred to as conditionally viable environmental cells (CVEC), which resist cultivation on routine bacteriological media. However, these cells can naturally resuscitate into the active planktonic form through various mechanisms, multiply, and cause epidemics of cholera. This study was conducted to study possible effects of environmental bacteriophages on the prevalence and distribution of the pathogen between the biofilm associated state, and the planktonic form.MethodsPhages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored.ResultsA phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms.SignificanceBesides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways.

Vibrio ecology, pathogenesis, and evolution.

EDITORIAL article Front. Microbiol., 28 May 2014Sec. Aquatic Microbiology Volume 5 - 2014 | https://doi.org/10.3389/fmicb.2014.00256

Impact of solar irradiation on cholera toxin secretion by different strains of Vibrio cholerae

Cholera toxin is the aetiological agent of cholera – a deadly waterborne disease acquired through the consumption of untreated water contaminated with CTXФ bacteriophage harbouring strains of V. cholerae. Solar disinfection is a re-emerging technique that relies on the ultraviolet component of sunlight to inactivate the growth of Vibrio cholerae in water, rendering the water microbiologically safe for consumption. However, studies have shown that DNA damaging agents, such as ultraviolet light, induce the replication of the CTXФ bacteriophage with subsequent expression of the cholera toxin. In this study we investigated the impact of solar irradiation on the secretion of cholera toxin by toxigenic strains of V. cholerae in water. The cholera toxin ELISA assay, qualitative and quantitative real-time PCR as well as growth on solid media were used to determine cholera toxin secretion, DNA integrity and growth of the bacteria after 7 h and 31 h of solar irradiation. Solar irradiation in water reduced the integrity of DNA, inactivated the growth of V. cholerae and, most importantly, prevented the secretion of detectable levels of cholera toxin. This finding is encouraging for resource-poor communities that may rely on solar disinfection to alleviate the burden of cholera-related fatalities.

Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples.

Cholera epidemics have long been known to spread through water contaminated with human fecal material containing the toxigenic bacterium Vibrio cholerae. However, detection of V. cholerae in water is complicated by the existence of a dormant state in which the organism remains viable, but resists cultivation on routine bacteriological media. Growth in the mammalian intestine has been reported to trigger "resuscitation" of such dormant cells, and these studies have prompted the search for resuscitation factors. Although some positive reports have emerged from these investigations, the precise molecular signals that activate dormant V. cholerae have remained elusive. Quorum-sensing autoinducers are small molecules that ordinarily regulate bacterial gene expression in response to cell density or interspecies bacterial interactions. We have found that isolation of pathogenic clones of V. cholerae from surface waters in Bangladesh is dramatically improved by using enrichment media containing autoinducers either expressed from cloned synthase genes or prepared by chemical synthesis. These results may contribute to averting future disasters by providing a strategy for early detection of V. cholerae in surface waters that have been contaminated with the stools of cholera patients or asymptomatic infected human carriers.

Transmissibility of cholera: In vivo -formed biofilms and their relationship to infectivity and persistence in the environment

The factors that enhance the waterborne spread of bacterial epidemics and sustain the epidemic strain in nature are unclear. Although the epidemic diarrheal disease cholera is known to be transmitted by water contaminated with pathogenic Vibrio cholerae, routine isolation of pathogenic strains from aquatic environments is challenging. Here, we show that conditionally viable environmental cells (CVEC) of pathogenic V. cholerae that resist cultivation by conventional techniques exist in surface water as aggregates (biofilms) of partially dormant cells. Such CVEC can be recovered as fully virulent bacteria by inoculating the water into rabbit intestines. Furthermore, when V. cholerae shed in stools of cholera patients are inoculated in environmental water samples in the laboratory, the cells exhibit characteristics similar to CVEC, suggesting that CVEC are the infectious form of V. cholerae in water and that CVEC in nature may have been derived from human cholera stools. We also observed that stools from cholera patients contain a heterogeneous mixture of biofilm-like aggregates and free-swimming planktonic cells of V. cholerae. Estimation of the relative infectivity of these different forms of V. cholerae cells suggested that the enhanced infectivity of V. cholerae shed in human stools is largely due to the presence of clumps of cells that disperse in vivo, providing a high dose of the pathogen. The results of this study support a model of cholera transmission in which in vivo-formed biofilms contribute to enhanced infectivity and environmental persistence of pathogenic V. cholerae.

Effect of alum on free-living and copepod-associated Vibrio cholerae O1 and O139.

The effects of alum [KAl(SO4)2] on free-living and copepod-associated Vibrio cholerae O1 and O139 were investigated by using plate counts and immunofluorescence direct viable counting (DVC). Growth of alum-treated cells in 0.5/1000 Instant Ocean seawater was inhibited, i.e., no growth was obtained on Luria-Bertani (LB) agar or thiosulfate-citrate-bile salt-sucrose (TCBS) agar. However, a significant number of the inhibited cells maintained viability, as measured by DVC. In comparison, a significant number of V. cholerae organisms associated with zooplankton, most of which were crustacean copepods, were viable but nonculturable, with only a small number of cells retaining culturability on LB and TCBS agar. Both DVC and viable plate counts (CFU) were significantly greater for V. cholerae O1 and O139 associated with zooplankton than for V. cholerae in water alone, i.e., without copepods. It is concluded that alum is an effective coagulant but not an effective killing agent for V. cholerae and that association with copepods offers protection for V. cholerae O1 and O139 against alum and chlorine treatments.

Effect of iron and pH on the survival of Vibrio cholerae in water

Many physicochemical factors affect the survival of Vibrio cholerae in the aquatic environment. An attempt was made to study the combined effect of pH and iron on the survival of V. cholerae in water in a laboratory environment. None of the 6 strains of V. cholerae used survived at pH 5·0; survival of all strains increased with increasing pH. The effect of ferric oxide on survival was significant for V. cholerae O1 only, not for non O1 strains. The longest survival of V. cholerae non O1 was 82 d, of El Tor V. cholerae 68 d, and of classical V. cholerae 56 d.

Distribution of Vibrio cholerae in the Apalachicola (Florida) Bay Estuary

Vibrio cholerae non-O1 was found throughout the Apalachicola, FL, estuary. V. cholerae O1 was isolated primarily at the City of Apalachicola sewage treatment plant, Scipio Creek and the north shore of St. George Island. Highest concentrations of both serogroups occurred in August and November. Concentrations were lowest in February and increased substantially in May. A cholera toxin-like toxin was not detected in any of the V. cholerae cultures by the Y-1 mouse adrenal cell assay or the enzyme-linked immunosorbent assay (ELISA). However, 35% of the V. cholerae O1 and 22% of the V. cholerae non-O1 cultures selected for testing caused diarrhea in infant rabbits. The proportion of V. cholerae O1 and non-O1 isolates pathogenic to infant rabbits increased as water temperature decreased. Fecal coliforms appeared to be more useful than total coliforms as indicators of the numbers of V. cholerae in water, but neither of those National Shellfish Sanitation Program indicator groups ensured against the presence of pathogenic V. cholerae in shellfish-growing areas.

Ecological relationships between Vibrio cholerae and planktonic crustacean copepods.

Strains of Vibrio cholerae, both O1 and non-O1 serovars, were found to attach to the surfaces of live copepods maintained in natural water samples collected from the Chesapeake Bay and Bangladesh environs. The specificity of attachment of V. cholerae to live copepods was confirmed by scanning electron microscopy, which revealed that the oral region and egg sac were the most heavily colonized areas of the copepods. In addition, survival of V. cholerae in water was extended in the presence of live copepods. Attachment of viable V. cholerae cells to copepods killed by exposure to -60 degrees C was not observed. Furthermore, survival of V. cholerae was not as long in the presence of dead copepods as in the live copepod system. A strain of Vibrio parahaemolyticus was also seen to attach to copepod surfaces without effect on survival of the organism in water. The attachment of vibrios to copepods was concluded to be significant since strains of other bacteria, including Pseudomonas sp. and Escherichia coli, did not adhere to live or dead copepods. Attachment of V. cholerae to live copepods is suggested to be an important factor of the ecology of this species in the aquatic environment, as well as in the epidemiology of cholera, for which V. cholerae serovar O1 is the causative agent.