Ecology ofVibrio cholerae in the freshwater environs of Calcutta,India

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater accumulations, located in Calcutta.V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August–September and a secondary peak in May–June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities ofV. cholerae. In the lake, sediment samples harbored high densities ofV. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts ofV. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures ofV. cholerae, indicating that the rabbit intestine selects outV. cholerae from a mixed flora. Uniformly high isolation rates ofV. cholerae were observed from brackish water and freshwater species of export quality prawns.V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded thatV. cholerae non-01 is common in the freshwater environs of Calcutta.     

Isolation of Non-O1 Vibrio cholerae Serovars from Oregon Coastal Environments

Water, sediment, and shellfish from three Oregon estuaries were cultured for pathogenic Vibrio species. Non-O1 serovars of V. cholerae were the most common pathogenic Vibrio species recovered. Non-O1 V. cholerae were isolated from all three estuaries sampled, covering an area of about 170 miles along the Oregon coast. Non-O1 V. cholerae were isolated from water and sediment, but not shellfish, at temperatures ranging from 11 to 19°C and salinities of 2.3 to 26‰. Sixteen isolates representing 12 different non-O1 serovars were identified, while four non-O1 V. cholerae isolates failed to react with any of the 54 antisera tested. These results indicate that non-O1 V. cholerae serovars can be found over a large geographic area and under a variety of environmental conditions. These organisms are apparently an autochthonous component of these estuarine microbial communities.     

Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.     

Cross‐protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv

Gram‐negative Vibrio species secrete multifunctional autoprocessing repeats‐in‐toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross‐reactivity and cross‐protectivity of mAbs against V. vulnificus RtxA1/MARTX_Vv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTX_Vc, inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Ethidium and propidium monoazide: comparison of potential toxicity on Vibrio sp. viability

Vibrio sp., ubiquitous in the aquatic ecosystem, are bacteria of interest because of their involvement in human health, causing gastroenteritis after ingestion of seafood, as well as their role in vibriosis leading to severe losses in aquaculture production. Their ability to enter a viable but non-culturable (VBNC) state under stressful environmental conditions may lead to underestimation of the Vibrio population by traditional microbiological enumeration methods. As a result, using molecular methods in combination with EMA or PMA allows the detection of viable (VBNC and culturable viable) cells. In this study, the impact of the EMA and PMA was tested at different concentrations on the viability of several Vibrio species. We compared the toxicity of these two DNA-binding dyes to determine the best pretreatment to use with qPCR to discriminate between viable and dead Vibrio cells. Our results showed that EMA displayed lethal effects for each strain of V. cholerae and V. vulnificus tested. In contrast, the concentrations of PMA tested had no toxic effect on the viability of Vibrio cells studied. These results may help to achieve optimal PMA-qPCR methods to detect viable Vibrio sp. cells in food and environmental samples.     

Alkaline protease from a non-toxigenic mangrove isolate of Vibrio sp. V26 with potential application in animal cell culture

Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae, V. mimicus, V. albensis and uncultured clones of Vibrio. Phenotypically also it resembled both V. cholerae and V. mimicus. Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administering APV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios.     

Evolutionary relationships in Vibrio and Photobacterium as determined by immunological studies of superoxide dismutase

The amino acid sequence divergence of superoxide dismutases (SODs) from 22 species and five groups of Vibrio, Photobacterium, and a number of related organisms was determined by means of the microcomplement fixation technique and the Ouchterlony double diffusion procedure. Five reference antisera were used which had been prepared against the purified SODs from V. alginolyticus, V. splendidus II, V. fischeri, V. cholerae, and P. leiognathi. With a few exceptions the results were in agreement with past studies of other informational molecules and provided a comprehensive overview of evolutionary relationships in Vibrio and Photobacterium. The genus Vibrio was found to consist of a major group of primarily marine species which included V. fischeri, V. logei, V. splendidus, V. pelagius, V. nereis, V. campbellii, V. harveyi, V. natriegens, V. alginolyticus, V. parahaemolyticus, V. proteolyticus, V. fluvialis, V. vulnificus, V. nigripulchritudo, and V. anguillarum. On the outskirts of this large and relatively heterogeneous group were the fresh water and estuarine species V. cholerae and V. metschnikovii as well as the marine species V. gazogenes. A considerable distance from Vibrio were the related species of Photobacterium: P. phosphoreum, P. leiognathi, and P. angustum. Both genera were distant from species of Aeromonas as well as from Plesiomonas shigelloides, Escherichia coli, and Alteromonas hanedai, a luminous strict aerobe. The agreement between these and previous studies of evolution of informational molecules in Vibrio and Photobacterium is best explained by vertical evolution (involving no genetic exchange between species) rather than by its opposite — horizontal evolution.Non-Standard Abbreviations Anti-Plei, anti-Valg, anti-Vcho, anti-Vfis, anti-Vspl antisera to the Fe-containing superoxide dismutases from Photobacterium leiognathi strain 480, Vibrio alginolyticus strain 90, V. cholerae strain M 13, V. fischeri strain 61, and v. splendidus biotype II strain 2, respectively - AP alkaline phosphatase - ATCC American Type Culture Collection - GS glutamine synthetase - ImD immunological distance - NCMB National Colleciion of Marine Bacteria - NCTC National Collection of Type Cultures - NRC National Research Council of Canada Culture Collection - SDS sodium dodecyl sulfate - SOD superoxide dismutase     

Toxigenicity and toxin genes of Vibrio cholerae 01 isolated from an artificial aquatic environment

A simple experiment was carried out to examlne the effect of varlous physicochemical conditions on toxigenicity and toxin genes of Vibrio cholerae 01 lsolated from an artificial aquatic environment. All isolated strains, tested by tissue culture assay, DNA-DNA hybridization and ELISA, were cytotoxic to Vero cells, did not lose their toxin genes and continued to produce cholera toxin. These results are consistent with the hypothesis that V. cholerae can survive in the environment without losing potential pathogenicity.     

Power plays: iron transport and energy transduction in pathogenic vibrios

The Vibrios are a unique group of bacteria inhabiting a vast array of aquatic environments. Many Vibrio species are capable of infecting a wide assortment of hosts. Some of these species include V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. anguillarum, and V. cholerae. The ability of these organisms to utilize iron is essential in establishing both an infection in their hosts as well as surviving in the environment. Bacteria are able to sequester iron through the secretion of low molecular weight iron chelators termed siderophores. The iron-siderophore complexes are bound by specific outer membrane receptors and are brought through both the outer and inner membranes of the cell. The energy needed to drive this active transport is achieved through the TonB energy transduction system. When first elucidated in E. coli, the TonB system was shown to be a three protein complex consisting of TonB, ExbB and ExbD. Most Vibrio species carry two TonB systems. The second TonB system includes a fourth protein; TtpC, which is essential for TonB2 mediated iron transport. Some Vibrio species have been shown to carry a third TonB system that also includes a TtpC protein.     

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio “sRNA core.”     

Evaluation of enrichment media for improved PCR-based detection of V. cholerae and V. vulnificus from estuarine water and plankton

Aims: Pathogenic Vibrio spp., including V. cholerae and V. vulnificus, are commonly found along the estuaries of the south‐east United States; however, it is often difficult to recover these species directly from environmental samples. Pre‐enrichment assays are commonly used to improve the detection of pathogenic vibrios from environmental sources. Here, we evaluated a novel enrichment procedure using freshly collected and autoclaved natural estuarine water amended with 1% peptone (designated as estuarine peptone water, EPW) and compared it to traditional alkaline peptone water (APW) for detection by PCR of V. cholerae and V. vulnificus. Methods and Results: Of the 50 samples collected in total, V. cholerae DNA was detected in APW 10% of the time and in EPW 40% of the time. Likewise, the cholera toxin gene (ctxA) was detected in 4 vs 18% of the samples using APW and EPW, respectively. Conversely, APW showed improved recovery for V. vulnificus relative to EPW with respective detection frequencies of 46 and 20%. Results showed similar patterns across different sample types (water and plankton). Conclusions: While enrichment in traditional APW was adequate for the recovery of Vibrio vulnificius, use of sterile estuarine water amended with peptone significantly improved the detection of V. cholerae and the virulence gene ctxA from estuarine sources.     

Development of a DNA probe using differential hybridization to detect the fish pathogenVibrio anguillarum

The fish pathogenVibrio anguillarum causes significant economic losses in commercially cultured fish species worldwide. At present, identification ofV. anguillarum requires conventional isolation and culturing techniques. Using differential hybridization, a 310 base pairV. anguillarum-specific DNA fragment was isolated for use as a probe. In specificity studies against 19 different bacterial species, including twoVibrio sp. and fish pathogens, and 223 marine bacterial isolates, the probe hybridized exclusively toV. anguillarum strains. The probe also strongly hybridizes to 7 of 9 serotypes tested, with serotype 09 giving a weak probe reaction and serotype O7 negative. The probe allows rapid and accurate detection of both pathogenic and environmental strains ofV. anguillarum.     

Vibrio trachuri Sp. Nov., a New Species Isolated from Diseased Japanese Horse Mackerel

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4-diamino-6,7-diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.     

Identification and Initial Characterization of Elastase Activity Associated with Vibrio cholerae

Strains of Vibrio cholerae O1 (Ogawa, Inaba) and non-O1 serogroups have been found to produce an elastolytic protease that can be detected on 0.3% elastin agar plates or in broth cultures. The elastase enzyme appears to be maximally expressed in late log phase (14–18 h postinoculation) and has optimum activity at a pH range between 7 and 8. Comparative studies indicate that more than 60% of V. cholerae strains analyzed quantitatively produce more elastase in broth (two- to fourfold higher) than other elastase-positive Vibrio species such as Vibrio vulnificus. The V. cholerae elastase enzyme was not inhibited by trypsin, serine-protease, or thiol-protease inhibitors, but was inhibited by phosphoramidon. Ultrafiltration studies indicate the V. cholerae elastase enzyme has a molecular weight >30,000, and a 34K protein with possible elastase activity has been detected by SDS-PAGE for one non-O1 isolate (strain 2396). Cumulative results suggest that the V. cholerae elastase is probably a member of the N-type metalloprotease family and shares similar properties with other elastase enzymes described for pathogenic and nonpathogenic species in this genus. Received: 26 February 1999 / Accepted: 29 March 1999     

Comparative immunogenicity of outer membrane protein K and whole-cell antigens of Vibrio parahaemolyticus for diagnosis

The immunogenicity of soluble outer membrane protein K (OmpK)- small ubiquitin-like modifier, OmpK inclusion bodies, formalin, and heat-killed Vibrio parahaemolyticus cells were prepared and studied in a mouse model. The results of whole-cell ELISA and Western blot (WB) revealed that the serum against soluble OmpK and OmpK inclusion bodies reacted only with homologous V. parahaemolyticus. Furthermore, recombinant OmpK proteins were not recognized by the serum against whole-cell V. parahaemolyticus antigens. Unexpectedly, the serum against formalin and heat-killed V. parahaemolyticus reacted broadly with homologous (an immunization strain) and heterologous (non-immunization strains) V. parahaemolyticus and Vibrio species. The WB results revealed that the serum against the two V. parahaemolyticus whole-cell antigens primarily reacted with proteins that were approximately 100, 70, 36, 28, and 22 kDa in the cell lysates from different Vibrio strains, rather than the recombinant OmpK. The 70 and 28 kDa proteins exhibited specificity to Vibrio species, while the 22 kDa protein was more specific to V. parahaemolyticus. This study showed the limitation of recombinant OmpK to prepare diagnostic antibodies and revealed several specific Omps of Vibrio sp. and V. parahaemolyticus that were promising in diagnosis and vaccine development.     

Life-history variation related to the first adult instar in daphnids derived from diapausing and subitaneous eggs

Vibrio cholerae is the causative agent of the severe dehydrating diarrheal disease cholera. This bacterium has been detected in many estuaries around the world and the United States. In this study we examine the abundance and distribution of V. cholerae in recreational beach waters and tributaries of Southern California. Water samples were taken from 11 beach locations adjacent to freshwater runoff sources between February 8th and March 1st, 1999. Water samples were also taken from rivers, creeks and coastal wetlands along the Southern California coast between May 19th and June 28th, 1999. In addition to the detection of V. cholerae, environmental parameters including temperature, salinity, coliphage counts, viable heterotrophic plate counts and total bacterial direct counts were also determined to understand the relationships between the presence of V. cholerae and environmental conditions. A direct colony hybridization method using an oligonucleotide probe specific for the 16S–23S intergenic spacer region of V. cholerae, detected V. cholerae in 3 of the 11 beach samples with the highest concentration (60.9 per liter) at the mouth of Malibu Lagoon. V. cholerae and coliphage were not correlated for beach samples, indicating that the presence of V. cholerae is independent of sewage pollution. V. cholerae were detected in all samples taken from rivers, creeks and wetlands of coastal Southern California where salinities were between 1 to 34 parts per thousand (ppt), but was not found at a freshwater sampling site in upper San Juan Creek. The highest density of V. cholerae was found in San Diego Creek with a concentration of 4.25×10⁵ CFU/L. The geographical distribution of V. cholerae was inversely correlated with salinity. High concentrations of V. cholerae were more frequently detected in waters with lower (but above 0) salinity. The results of this study provide insight into the ecology of this aquatic species and are potentially important to the understanding of the epidemiology of cholera on a global scale.     

Incidence of Non-01 Vibrio cholerae and Aeromonas spp. in Fresh Water in Araraquara, Brazil

The occurrence of Aeromonas spp., Vibrio cholerae, and Plesiomonas shigelloides in fresh water from various sources in Araraquara, State of S?o Paulo, Brazil was determined. Samples from ten distinct irrigation systems used in vegetable cultivation, from five distinct streams, from two reservoirs, from one artificial lake, and from three distinct springs were analyzed. All isolates were serotyped and tested for hemolysin, cytotoxin, heat-stable (ST) and heat-labile (LT) enterotoxins production; presence of plasmid; autoagglutination and drug resistance. V. cholerae isolates were also tested for cholera enterotoxin (CT) production, and Aeromonas isolates for suicide phenomenon. No P. shigelloides was found. V. cholerae non 01 was found in five irrigation water samples and in three stream samples. Aeromonas sp. were isolated in two samples of irrigation water, in three streams, and in one reservoir. All the V. cholerae and Aeromonas isolates were positive for β-hemolysin production, and all Aeromonas isolates were positive for suicide phenomenon; cytotoxic activities were observed in two Aeromonas strains. Cholera enterotoxin was not found in eight V. cholerae non-01 isolates tested by the Y-1 mouse adrenal cell. All isolates were also negative for the other virulence markers. V. cholerae isolates were found to be sensitive to the majority of drugs tested, while Aeromonas strains presented multiple drug resistance. Received: 4 November 1997 / Accepted: 23 January 1998     

Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes

Aims: To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation. Methods and Results: Four pairs of primers targeting gyrB gene of Vibrios at genus level and pntA gene of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus were designed. This PCR method precisely identified 250 Vibrio species and demonstrated sensitivity in the range of 4 × 10⁴ CFU ml^?1 (c. 200 CFU per PCR) to 2 × 10³ CFU ml^?1 (c. 10 CFU per PCR). Overall, the gyrB gene marker showed a higher specificity than the dnaJ gene marker for Vibrio detection and was able to distinguish Aeromonas from Vibrio species. Conclusions: The multiplex PCR based on combined gyrB and pntA provides a high discriminatory power in the differentiation between Vibrio alginolyticus and V. parahaemolyticus, and between V. cholerae and Vibrio mimicus. Significance and Impact of the Study: This assay will be useful for rapid differentiation of various Vibrio species from clinical and environmental sources and significantly overcomes the limitations of the conventional methods.