Extraction from prawn shells of substances cryoprotective for Vibrio cholerae.

Substances cryoprotective for Vibrio cholerae were detected from prawn shells immersed in phosphate-buffered saline. This cryoprotective activity was heat resistant and sensitive to treatment with trypsin. For the exhibition of its full activity, the presence of Mg ion was indispensable. The cryoprotective activity of this substance was more active than that of other known cryoprotectants, like glycerol or serum.     

Extraction from prawn shells of substances cryoprotective for Vibrio cholerae

Substances cryoprotective for Vibrio cholerae were detected from prawn shells immersed in phosphate-buffered saline. This cryoprotective activity was heat resistant and sensitive to treatment with trypsin. For the exhibition of its full activity, the presence of Mg ion was indispensable. The cryoprotective activity of this substance was more active than that of other known cryoprotectants, like glycerol or serum.     

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     

Isolation,identification, and characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from the Danube River in Slovakia

The occurrence of Vibrio cholerae, an important aquatic pathogen, was assessed in the surface water of the Danube River near Bratislava. The isolates were distinguished by biochemical tests and grouped by ARDRA to three clusters corresponding to three species (V. cholerae, Vibrio metschnikovii, and Aeromonas spp.). The identification of V. cholerae was confirmed by multiplex PCR using primer pairs targeted to ompW gene (membrane protein), ctxA gene (toxicity gene), and toxR gene (regulatory gene). None from the isolated V. cholerae from surface water contained ctxA gene; seven of them possessed toxR gene. Serotyping of V. cholerae isolates with polyvalent O antiserum and O/139 antiserum was negative. All isolates of V. cholerae were susceptible to chloramphenicol, rifampicin, tetracycline, variable to ampicillin, and resistant to kanamycin and streptomycin.     

Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae

The redox‐regulated chaperone Hsp33 protects bacteria specifically against stress conditions that cause oxidative protein unfolding, such as treatment with bleach or exposure to peroxide at elevated temperatures. To gain insight into the mechanism by which expression of Hsp33 confers resistance to oxidative protein unfolding conditions, we made use of Vibrio cholerae strain O395 lacking the Hsp33 gene hslO. We found that this strain, which is exquisitely bleach‐sensitive, displays a temperature‐sensitive (ts) phenotype during aerobic growth, implying that V. cholerae suffers from oxidative heat stress when cultivated at 43°C. We utilized this phenotype to select for Escherichia coli genes that rescue the ts phenotype of V. cholerae ΔhslO when overexpressed. We discovered that expression of a single protein, the elongation factor EF‐Tu, was sufficient to rescue both the ts and bleach‐sensitive phenotypes of V. cholerae ΔhslO. In vivo studies revealed that V. cholerae EF‐Tu is highly sensitive to oxidative protein degradation in the absence of Hsp33, indicating that EF‐Tu is a vital chaperone substrate of Hsp33 in V. cholerae. These results suggest an ‘essential client protein’ model for Hsp33's chaperone action in Vibrio in which stabilization of a single oxidative stress‐sensitive protein is sufficient to enhance the oxidative stress resistance of the whole organism.     

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.     

Evaluating the Potential of an Antibody Against Recombinant OmpW Antigen in Detection of Vibrio cholerae by Surface Plasmon Resonance (SPR) Biosensor

Surface plasmon resonance (SPR), a highly sensitive and label-free optical biosensing technique, is a powerful tool for studying biomolecular interactions. An immunosensor for rapid, sensitive, and selective detection of Vibrio cholerae on the basis of SPR is reported. Recombinant OmpW antigen (a bacterial outer-membrane protein) of V. cholerae was expressed and purified and raising of polyclonal rabbit anti-OmpW was done. Antibodies were immobilized on a sensor surface and interactions between OmpW protein and the whole cell of V. cholerae with immobilized antibodies were studied in different experiments. The aim of this study was to evaluate the potential of anti-OmpW in detection of V. cholerae by developing an immunosensor based on SPR. The results showed high affinity interaction between OmpW and anti-OmpW (K _D = 2.4 ± 0.07 × 10⁻⁹ M) and SPR signals had a linear relationship with the number of V. cholerae ranging from 1 × 10² to 1 × 10⁷ cells/mL with limit of detection of 50 cells/mL. The specificity of the developed immunoassay was examined using some non-V. cholerae bacteria which did not produce any significant responses. This method is rapid, sensitive, and specific to target V. cholerae with a total analysis time of less than 60 min.

     

Vibrio cholerae laboratory infection of the adult house fly Musca domestica

The present study was designed to test the hypothesis that house flies may be capable of specifically harbouring ingested Vibrio cholerae in their digestive tracts. Flies were continuously fed green fluorescent protein (GFP)‐labelled, non‐O1/non‐O139 environmental strains of V. cholerae. Bacterial burdens were quantitatively measured using plate counts and localization was directly observed using confocal microscopy. Vibrio cholerae were present in the fly alimentary canal after just 4 h, and reached a plateau of ～10⁷ colony‐forming units (CFU)/fly after 5 days in those flies most tolerant of the pathogen. However, individual flies were resistant to the pathogen: one or more flies were found to carry < 180 V. cholerae CFU at each time‐point examined. In flies carrying V. cholerae, the pathogen was predominantly localized to the midgut rather than the rectal space or crop. The proportion of house flies carrying V. cholerae in the midgut was dose‐dependent: the continuous ingestion of a concentrated, freshly prepared dose of V. cholerae increased the likelihood that fluorescent cells would be observed. However, V. cholerae may be a transient inhabitant of the house fly. This work represents the first demonstration that V. cholerae can inhabit the house fly midgut, and provides a platform for future studies of host, pathogen and environmental mediators of the successful colonization of this disease vector.     

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.     

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.     

wbeT sequence typing and IS1004 profiling of Vibrio cholerae isolates

Aims: To investigate the molecular basis for serotype variation in Vibrio cholerae O1 and the genetic relatedness amongst different serotypes isolated from 2004 to 2008 in Iran. Methods and Results: Despite the presence of all three serotypes of V. cholerae O1 (Ogawa, Inaba and Hikojima) in Iran in the last decade, the Inaba strains have been the dominated serotype. Sequence analysis of wbeT determined only a single substitution of G for A at position 295 in all Inaba strains resulting in a replacement of serine to proline. No difference was found in the copy numbers and profile of IS1004 between the classical and El Tor V. cholerae O1 strains, supporting the clonality amongst the isolates obtained over 5 years in Iran. In addition, Southern blots of HpaII‐digested chromosomal DNAs of our Ogawa and Inaba isolates showed the presence of an incomplete copy of IS1004 for all isolates. Conclusions: IS1004 profiling can be a reliable method for analysis of clonal dissemination of V. cholerae. The results indicated that specific point mutation at a particular position within the wbeT of V. cholerae O1 strains in Iran may occur which, in turn, may result in serotype switching. Significance and Impact of the Study: Understanding the molecular basis for serotype conversion of V. cholerae and their genetic relatedness could give insights for the incoming cholera epidemic prediction and control.     

Predicting the Distribution of Vibrio spp. in the Chesapeake Bay: A Vibrio cholerae Case Study

Vibrio cholerae, the causative agent of cholera, is a naturally occurring inhabitant of the Chesapeake Bay and serves as a predictor for other clinically important vibrios, including Vibrio parahaemolyticus and Vibrio vulnificus. A system was constructed to predict the likelihood of the presence of V. cholerae in surface waters of the Chesapeake Bay, with the goal to provide forecasts of the occurrence of this and related pathogenic Vibrio spp. Prediction was achieved by driving an available multivariate empirical habitat model estimating the probability of V. cholerae within a range of temperatures and salinities in the Bay, with hydrodynamically generated predictions of ambient temperature and salinity. The experimental predictions provided both an improved understanding of the in situ variability of V. cholerae, including identification of potential hotspots of occurrence, and usefulness as an early warning system. With further development of the system, prediction of the probability of the occurrence of related pathogenic vibrios in the Chesapeake Bay, notably V. parahaemolyticus and V. vulnificus, will be possible, as well as its transport to any geographical location where sufficient relevant data are available.     

Cloning and expression in Escherichia coli of a recA-like gene from Vibrio cholerae

Summary A library containing more than 80% of the Vibrio cholerae genome was constructed by cloning BamH1 restriction fragments into pBR322. Using interspecific complementation of an Escherichia coli recA mutant with plasmids containing the gene bank of V. cholerae, a recA-like gene was identified. The recombinant plasmid, designated as pDP145, contained a 1.45 kb segment of V. cholerae DNA which codes for a protein of molecular weight 39,000. The product of this gene confers methyl methane sulphonate resistance on the E. coli recA mutant, suppresses its ultraviolet (UV) light sensitive phenotype and has proteolytic activity on the phage repressor. Induction of a 39,000 dalton protein in UV-irradiated V. cholerae cells was demonstrated.     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild-type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS-PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem-iron utilization genes allow transport of the entire haem moiety into the cell.     

Characterization of a 20-kDa pilus protein expressed by a diarrheogenic strain of non-O1/non-O139 Vibrio cholerae

A diarrheogenic strain of non-O1/non-O139 Vibrio cholerae (10325) belonging to serogroup O34 was earlier shown to express a new type of pilus composed of a 20-kDa subunit protein. Amino-terminal sequence data (determined up to 20 amino acid residues) of this protein showed it to be different from the subunit proteins of other known types of pili of V. cholerae. On the other hand, it showed complete homology with the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V. cholerae. Expression of 10325 pili was favored in AKI rather than in NB medium and at 30°C rather than at 37°C. Further, cultural conditions favoring pilus expression also enhanced autoagglutination and adherence properties of strain 10325. An antiserum to the 20-kDa protein induced passive protection against challenge with the parent organism 10325, but not against V. cholerae O1 strains. Such protection was shown to be mediated by inhibition of intestinal colonization in vivo.     

Specificity of cobamide remodeling,uptake and utilization in Vibrio cholerae

Cobamides are a group of compounds including vitamin B₁₂ that can vary at the lower base position of the nucleotide loop. They are synthesized de novo by only a subset of prokaryotes, but some organisms encode partial biosynthesis pathways for converting one variant to another (remodeling) or completing biosynthesis from an intermediate (corrinoid salvaging). Here, we explore the cobamide specificity in Vibrio cholerae through examination of three natural variants representing major cobamide groups: commercially available cobalamin, and isolated pseudocobalamin and p-cresolylcobamide. We show that BtuB, the outer membrane corrinoid transporter, mediates the uptake of all three variants and the intermediate cobinamide. Our previous work suggested that V. cholerae could convert pseudocobalamin produced by cyanobacteria into cobalamin. In this work, cobamide specificity in V. cholerae is demonstrated by remodeling of pseudocobalamin and salvaging of cobinamide to produce cobalamin. Cobamide remodeling in V. cholerae is distinct from the canonical pathway requiring amidohydrolase CbiZ, and heterologous expression of V. cholerae CobS was sufficient for remodeling. Furthermore, function of V. cholerae cobamide-dependent methionine synthase MetH was robustly supported by cobalamin and p-cresolylcobamide, but not pseudocobalamin. Notably, the inability of V. cholerae to produce and utilize pseudocobalamin contrasts with enteric bacteria like Salmonella.     

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.     

Role of the Vibrio cholerae Matrix Protein Bap1 in Cross-Resistance to Antimicrobial Peptides

Outer membrane vesicles (OMVs) that are released from Gram-negative pathogenic bacteria can serve as vehicles for the translocation of effectors involved in infectious processes. In this study we have investigated the role of OMVs of the Vibrio cholerae O1 El Tor A1552 strain in resistance to antimicrobial peptides (AMPs). To assess this potential role, we grew V. cholerae with sub-lethal concentrations of Polymyxin B (PmB) or the AMP LL-37 and analyzed the OMVs produced and their effects on AMP resistance. Our results show that growing V. cholerae in the presence of AMPs modifies the protein content of the OMVs. In the presence of PmB, bacteria release OMVs that are larger in size and contain a biofilm-associated extracellular matrix protein (Bap1). We demonstrated that Bap1 binds to the OmpT porin on the OMVs through the LDV domain of OmpT. In addition, OMVs from cultures incubated in presence of PmB also provide better protection for V. cholerae against LL-37 compared to OMVs from V. cholerae cultures grown without AMPs or in presence of LL-37. Using a bap1 mutant we showed that cross-resistance between PmB and LL-37 involved the Bap1 protein, whereby Bap1 on OMVs traps LL-37 with no subsequent degradation of the AMP.