Seasonal Abundance and Distribution of Vibrio cholerae in Coastal Waters Quantified by a 16S-23S Intergenic Spacer Probe

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera and is indigenous to brackish waters. To advance our understanding of the ecology of this bacterium, we have developed a molecular probing method for detection of V. cholerae in coastal waters. Water samples from 7 locations in the Newport Bay watershed, California were sampled monthly for a whole year. V. cholerae concentrations were determined by membrane filtration-colony hybridization using an oligonucleotide probe targeting the 16S-23S intergenic spacer (ITS) region. In addition to V. cholerae concentrations, environmental parameters, including temperature, salinity, total bacterial direct counts, total viable counts, and chlorophyll a concentrations, were determined for each site. V. cholerae was detected year-round throughout the watershed. Regression analysis indicated that the concentration of V. cholerae inversely correlated with salinity (p <0.001). The sampling sites located nearest to the Pacific Ocean had lower concentrations, whereas sites located along the brackish San Diego Creek (salinity 0-12 per thousand) routinely had higher concentrations. V. cholerae concentrations also correlated with temperature (p <0.01) in the watershed, with concentrations ranging from less than 1 CFU mL-1 to 2,930 CFU mL-1 of water. The results of this study indicate that the dynamics of V. cholerae is mainly influenced, out of the parameters measured, by the temperature and salinity of the environment. This information is valuable for understanding the ecology of V. cholerae.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.     

Human Adenoviruses and Coliphages in Urban Runoff-Impacted Coastal Waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

Transfer of cholera toxin genes from O1 to non‐O1/O139 strains by vibriophages from California coastal waters

Aims: Vibrio cholerae is an important bacterial pathogen that causes global cholera epidemic. Although they are commonly found in coastal waters around the world, most environmental isolates do not contain cholera toxin genes. This study investigates vibriophages in southern California coastal waters and their ability to transfer cholera toxin genes. Methods and Results: Lytic phages infecting V. cholerae were isolated from Newport Bay, California, between May and November, while none was found in winter. Some of the phage isolates can infect multiple environmental V. cholerae strains and El Tor strains. All phages contained double‐stranded DNA. Transduction experiments using kanamycin‐resistant gene marked CTXΦ demonstrated that some environmental vibriophages can transfer CTXΦ genes from O1 El Tor strain to environmental non‐O1/O139 V. cholerae via generalized transduction. Conclusions: Vibriophages are important components of the natural aquatic ecosystem. They play an important role in influencing the dynamics and evolution of V. cholerae in the environment. Significance and Impact of the Study: This study demonstrates the significance of vibriophages in the coastal environment and transduction as one of the mechanisms of pathogenicity evolution among environmental V. cholerae.     

Demonstration of viable but nonculturable Vibrio cholerae O1 in fresh water environment of India using ciprofloxacin DFA-DVC method

Aim: To demonstrate the presence of culturable and nonculturable viable pathogenic Vibrio cholerae O1 in fresh water environments of a cholera‐endemic region in India. Methods and Results: Conventional culture and ciprofloxacin DFA–DVC were utilized to investigate the existence of V. cholerae O1. We isolated pathogenic culturable V. cholerae O1 from water samples collected from cholera‐affected areas. No culturable V. cholerae O1 was isolated from water and plankton samples from natural fresh water bodies. Ciprofloxacin was used for DFA–DVC as V. cholerae O1 are 100% resistant to nalidixic acid in our region. The viable but nonculturable O1 cells were demonstrated in 2·21 and 40·69% samples from natural water bodies and cholera‐affected areas, respectively. Conclusion: Vibrio cholerae O1 VBNC could be demonstrated using modified DFA–DVC technique. Ciprofloxacin is preferable to nalidixic acid for DVC in view of existing high‐level resistance to nalidixic acid in cholera‐endemic areas. Significance and Impact of the study: We endorse that for public health surveillance, cholera outbreak investigation and disease control water samples in addition to culture should be tested for V. cholerae using DFA–DVC.     

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index = 0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.     

Predictability of Vibrio cholerae in Chesapeake Bay

Vibrio cholerae is autochthonous to natural waters and can pose a health risk when it is consumed via untreated water or contaminated shellfish. The correlation between the occurrence of V. cholerae in Chesapeake Bay and environmental factors was investigated over a 3-year period. Water and plankton samples were collected monthly from five shore sampling sites in northern Chesapeake Bay (January 1998 to February 2000) and from research cruise stations on a north-south transect (summers of 1999 and 2000). Enrichment was used to detect culturable V. cholerae, and 21.1% (n = 427) of the samples were positive. As determined by serology tests, the isolates, did not belong to serogroup O1 or O139 associated with cholera epidemics. A direct fluorescent-antibody assay was used to detect V. cholerae O1, and 23.8% (n = 412) of the samples were positive. V. cholerae was more frequently detected during the warmer months and in northern Chesapeake Bay, where the salinity is lower. Statistical models successfully predicted the presence of V. cholerae as a function of water temperature and salinity. Temperatures above 19°C and salinities between 2 and 14 ppt yielded at least a fourfold increase in the number of detectable V. cholerae. The results suggest that salinity variation in Chesapeake Bay or other parameters associated with Susquehanna River inflow contribute to the variability in the occurrence of V. cholerae and that salinity is a useful indicator. Under scenarios of global climate change, increased climate variability, accompanied by higher stream flow rates and warmer temperatures, could favor conditions that increase the occurrence of V. cholerae in Chesapeake Bay.     

Vibrios in the Louisiana gulf coast environment

A polyphasic approach, using bacteriological, immunological, and molecular biological techniques was used to elucidate the distribution of pathogenicVibrio species in the Louisiana coastal environment. A variety ofVibrio species pathogenic for man, includingV. cholerae, V. parahaemolyticus, V. fluvialis, andV. vulnificus, were found to be ubiquitous in Louisiana.Vibrio species monitored were shown to fluctuate in response to environmental factors of temperature, salinity, and nutrient level, and to vary independently of fecal coliform counts. A comprehensive serological screening system, based on species specific H antigens, was developed to identify pathogenicVibrio sp. 1 step after primary isolation.Vibrio sp. were correctly identified with accuracies ranging from 93–100%, depending on the specific H antiserum. Over 2,500V. cholerae isolates were rapidly screened for production of cholera toxin by DNA hybridization of specific toxin gene probes to colonies inoculated on nitrocellulose filter paper. The toxin gene probes, together with O antigen analysis, revealed that enterotoxigenicV. cholerae 01 serovars were recovered only from sewage stations or human disease, whereas enterotoxigenicV. cholerae non 01 serovars were recovered from environmental samples in addition to clinical and sewage samples. The results of this study indicate that techniques of immunology and molecular biology are very valuable supplements to conventional bacteriological techniques in studying the epidemiology and ecology of pathogenicVibrio sp.     

Critical Factors Influencing the Occurrence of Vibrio cholerae in the Environment of Bangladesh

The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal communities, and the appearance of the new serotype Vibrio cholerae O139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors influencing the growth and geographic distribution of epidemic Vibrio cholerae serotypes. Because of the severity of recent epidemics, cholera is now being considered by some infectious disease investigators as a “reemerging” disease, prompting new work on the ecology of vibrios. Epidemiological and ecological surveillance for cholera has been under way in four rural, geographically separated locations in Bangladesh for the past 4 years, during which both clinical and environmental samples were collected at biweekly intervals. The clinical epidemiology portion of the research has been published (Sack et al., J. Infect. Dis. 187:96-101, 2003). The results of environmental sampling and analysis of the environmental and clinical data have revealed significant correlations of water temperature, water depth, rainfall, conductivity, and copepod counts with the occurrence of cholera toxin-producing bacteria (presumably V. cholerae). The lag periods between increases or decreases in units of factors, such as temperature and salinity, and occurrence of cholera correlate with biological parameters, e.g., plankton population blooms. The new information on the ecology of V. cholerae is proving useful in developing environmental models for the prediction of cholera epidemics.     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

Effect of Transport at Ambient Temperature on Detection and Isolation of Vibrio cholerae from Environmental Samples

It has long been assumed that prolonged holding of environmental samples at the ambient air temperature prior to bacteriological analysis is detrimental to isolation and detection of Vibrio cholerae, the causative agent of pandemic cholera. The present study was aimed at understanding the effect of transporting environmental samples at the ambient air temperature on isolation and enumeration of V. cholerae. For water and plankton samples held at ambient temperatures ranging from 31°C to 35°C for 20 h, the total counts did not increase significantly but the number of culturable V. cholerae increased significantly compared to samples processed within 1 h of collection, as measured by culture, acridine orange direct count, direct fluorescent-antibody-direct viable count (DFA-DVC), and multiplex PCR analyses. For total coliform counts, total bacterial counts, and DFA-DVC counts, the numbers did not increase significantly, but the culturable plate counts for V. cholerae increased significantly after samples were held at the ambient temperature during transport to the laboratory for analysis. An increase in the recovery of V. cholerae O1 and improved detection of V. cholerae O1 rfb and ctxA also occurred when samples were enriched after they were kept for 20 h at the ambient temperature during transport. Improved detection and isolation of toxigenic V. cholerae from freshwater ecosystems can be achieved by holding samples at the ambient temperature, an observation that has significant implications for tracking this pathogen in diverse aquatic environments.     

FATP4 missense and nonsense mutations cause similar features in Ichthyosis Prematurity Syndrome

Background

Vibrio cholerae O1 and V. cholerae O139 infect humans, causing the diarrheal and waterborne disease cholera, which is a worldwide health problem. V. cholerae and the free-living amoebae Acanthamoeba species are present in aquatic environments, including drinking water and it has shown that Acanthamoebae support bacterial growth and survival. Recently it has shown that Acanthamoeba species enhanced growth and survival of V. cholerae O1 and O139. Water samples from different cholera endemic areas in Sudan were collected with the aim to detect both V. cholerae and Acanthamoeba species from same natural water samples by polymerase chain reaction (PCR).

Findings

For the first time both V. cholerae and Acanthamoeba species were detected in same natural water samples collected from different cholera endemic areas in Sudan. 89% of detected V. cholerae was found with Acanthamoeba in same water samples.

Conclusions

The current findings disclose Acanthamoedae as a biological factor enhancing survival of V. cholerae in nature.     

Quantitative Microbial Risk Assessment of Pathogenic Vibrios in Marine Recreational Waters of Southern California

This study investigated the occurrence of three types of vibrios in Southern California recreational beach waters during the peak marine bathing season in 2007. Over 160 water samples were concentrated and enriched for the detection of vibrios. Four sets of PCR primers, specific for Vibrio cholerae, V. parahaemolyticus, and V. vulnificus species and the V. parahaemolyticus toxin gene, respectively, were used for the amplification of bacterial genomic DNA. Of 66 samples from Doheny State Beach, CA, 40.1% were positive for V. cholerae and 27.3% were positive for V. parahaemolyticus, and 1 sample (1.5%) was positive for the V. parahaemolyticus toxin gene. Of the 96 samples from Avalon Harbor, CA, 18.7% were positive for V. cholerae, 69.8% were positive for V. parahaemolyticus, and 5.2% were positive for the V. parahaemolyticus toxin gene. The detection of the V. cholerae genetic marker was significantly more frequent at Doheny State Beach, while the detection of the V. parahaemolyticus genetic marker was significantly more frequent at Avalon Harbor. A probability-of-illness model for V. parahaemolyticus was applied to the data. The risk for bathers exposed to recreational waters at two beaches was evaluated through Monte Carlo simulation techniques. The results suggest that the microbial risk from vibrios during beach recreation was below the illness benchmark set by the U.S. EPA. However, the risk varied with location and the type of water recreation activities. Surfers and children were exposed to a higher risk of vibrio diseases. Microbial risk assessment can serve as a useful tool for the management of risk related to opportunistic marine pathogens.     

Immunological Biosensor for Detection of Vibrio cholerae O1in Environmental Water Samples

Environmental surveillance for the presence of Vibrio cholerae O1 is of utmost importance for the effective public health protection of cholera. In the present study, an amperometric immunosensor was developed for detection of Vibrio cholerae in environmental samples by using disposable screen-printed electrodes (SPEs). For this purpose, the experiments done include fabrication of SPEs by using carbon ink, electrochemical characterization of electrodes, optimization of dilutions of antibodies and immobilization of antibody. V. cholerae O1 bacteria were spiked in various environmental water samples in known number. The seeded samples were filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly for detection of V. cholerae using the immunosensor. The immunosensor could detect as few as 8 c.f.u./ml in hand-pump water (ground water) and seawater, and 80 c.f.u./ml in sewer water and tap water. The total time taken in this detection assay was 55 min. Thus, the proposed method is simple and can be used for environmental monitoring of V.␣cholerae.     

Immunogenicity of Vibrio cholerae O1 Fimbriae in Animal and Human Cholera

Parenteral immunization with either formalin-fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non-immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.     

Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Detection of Vibrio cholerae O1 in the aquatic environment in Brazil employing direct immunofluorescence microscopy

Culturing and immunofluorescence (FA) methods for detection of Vibrio cholerae O1 in samples collected from the aquatic environment at selected sites in Brazil were compared. Of the samples examined, 90% were positive for V. cholerae O1 by FA but none was positive by culture, although strains of V. cholerae other than O1 strains were readily isolated. Evidence for V. cholerae O1 being autochthonous to the aquatic environment of Brazil is presented. Furthermore, FA methods are recommended for cholera surveillance programmes directed at the natural environment.M.T. Martins is with the Department of Microbiology, I.C.B. II, University of Sao Paulo, Sao Paulo, S.P. CEP 05508, Brazil. P.S. Sanchez and M.I.Z. Sato are with the State Agency for Environmental Control-CETESB, Sao Paulo, S.P. CEP 05459, Brazil. P.R. Brayton and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD 20742, USA; R.R. Colwell is also with the Center of Marine Biotechnology, Maryland Biotechnology Institute, 600 East Lombard St, Baltimore, MD 21202, USA.     

Toxigenic Vibrio cholerae from environmental sources associated with the cholera outbreak after ‘AILA’ cyclone in West Bengal,India

Aims: West Bengal experienced a devastating storm named ‘AILA’ in its coastal and southern districts. We attempted to understand the transmission dynamics emphasizing on potable water to detect the presence of toxigenic strains of Vibrio cholerae, followed by the natural devastation. Methods and Results: A total of 33 water samples (from tap, tube well and ponds) were analysed. From them, 11 (33·3%) samples were found to be contaminated with V. cholerae, among which 5 (45%) isolates were V. cholerae O1 biotype Ogawa. Antibiogram profile reveals that most of the V. cholerae O1 isolates were highly sensitive against fluroquinolone group of antibiotics, but less sensitive against cefuroxime (50%), cefotaxime (40·9%), ceftriaxone (38·63%), trimethoprim (37·3%), streptomycin (29·2%) and furazolidon (4·54%). Three (36%) V. cholerae isolates were found to be ctxB positive (2 ctxB classical). Conclusions: Potable water plays a crucial role in cholera transmission. Natural disasters like the reported one aided with feacal–oral contamination enhance the possibilities of drinking water contamination. Significance and Impact of the Study: The application of the modified technique, making use of the enrichment subsequently followed by culture and PCR, will help us to detect the presence of toxigenic V. cholerae contamination in different aquatic environment. Moreover, natural extremes have a direct role in increase of salinity level, followed by higher predominance of V. cholerae along with their toxicity development in terms of genetic modification.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.