Diagnosis of Lyme borreliosis with western blotting]

The endemic and seasonal nature of cholera depends upon the survival of Vibrio cholerae 01 in a viable but not necessarily culturable state in ecologic niches in aquatic environments during interepidemic periods. To understand the ecology of V. cholerae it is necessary to know which aquatic ecosystems can harbor it and thus contribute to the endemic presence of cholera in Latin America. This article presents a summary of the ecology of V. cholerae 01, organized according to the abiotic and biotic factors that are relevant to the microbe's survival in aquatic environments. This pathogen finds favorable conditions in waters characterized by moderate salinity, high nutrient content, warm temperature, neutral or slightly alkaline pH, and the presence of aquatic macrophages, phytoplankton, zooplankton, fish, mollusks, and crustaceans. These ecologic conditions are typical of estuaries and coastal swamps, and toxigenic V. cholerae 01 is now considered an autochthonous member of the microbial flora of these environments. The microorganism has also shown the ability to colonize freshwater ecosystems in its viable but not necessarily culturable form, if organic or inorganic substrates that favor its survival are available.     

Hydrodynamic Tracking of the Massive Spring 1998 Red Tide in Hong Kong

In subtropical coastal waters around Hong Kong, algal blooms and red tides have been frequently observed over the past two decades. In particular, in March–April 1998, a massive red tide invaded the northeastern and southern coastal waters of Hong Kong. The devastating red tide resulted in the worst fish kill in Hong Kong’s history, the most significant impacts being at the Lo Tik Wan and Sok Kwu Wan fish culture zones on Lamma Island. This work reports the first scientific investigation of the cause of this massive red tide. A calibrated three-dimensional (3D) hydrodynamic model for the Pearl River Estuary, Delft3D, is applied to study the advective transport of red tides. Based on the tidal boundary conditions and the measured wind data for a typical spring season, the 3D flow field is computed and extensive surface drogue tracking performed for releases in different parts of the coastal waters and for different tidal and wind conditions. The results show that a bloom initiated in Mirs Bay (Nan Au or Tap Mun) in the northeastern water would likely be transported to the southern coastal waters under the combined action of tidal current and wind. The computed bloom tracking patterns are generally supported by observations and are consistent with the temporal and spatial patterns of individual fish kill events in the 1998 red tide. We conclude that the major cause of the bloom being transported into the southern waters and East Lamma Channel (and causing the massive fish kill) is the generally strong wind in March–April 1998 and the change in wind direction in early April under almost diurnal tidal conditions. Further, it is most probable that the red tide originated in Mirs Bay rather than from outside Hong Kong. The findings provide a firm basis for environmental and fisheries management.     

RNA polymerase II transcription initiation: a structural view

The emergence of new strains of Vibrio Cholerae has added a new dimension to the variability in pathogenicity and potential virulence of the organisms precipitating diarrhoeal diseases. Considering the shifting patterns of V. cholerae 01 there is a continuous need to monitor the strain characteristics. In this study total 541 stool specimens of acute secretory diarrhoea were investigated between May 1992 and November 1994 for strains of Vibrio Cholerae and anti-microbial susceptibility testing of all the confirmed V. Cholerae strains. In 1992, 50 of the 125 strains (40%) were positive for V. cholerae 01 predominantly biotype El Tor serotype ogawa, and 10 (80%) of non 01 type, with most strains susceptible to tetracycline (100%), chloramphenicol (98%) and Cotrimoxazole (98%), but all resistant to polymyxin B and furazolidine. In 1993, 44 (43.6%) of the 010 strains were positive for V. cholerae 0139 and the rest V. cholerae 01. In 1994, another sero group of V. cholerae 010 emerged, with 42 (13.3%) being positive. Isolates did not agglutinate with any of these antisera and have been labelled as 'other than non-01 vibrio cholerae'.     

Membrane filter procedure for enumeration of Vibrio parahaemolyticus

A membrane filtration procedure has been developed for the enumeration of Vibrio parahaemolyticus in marine waters. Background microbial growth on the primary medium was decreased through the use of sodium cholate and copper sulfate, high pH, 3% NaCl, and an elevated incubation temperature. A series of in situ tests was employed to obviate the picking of colonies for identification; thereby, the enumeration of V. parahaemolyticus was accomplished within 30 h. Confirmation of typical colonies approached 95%. Relative to immediate plating on brain heart infusion agar spread plates, the recovery of V. parahaemolyticus cells suspended in phosphate-buffered saline or in seawater held for 24 h at 4 to 6 degrees C was about 90%. Assay variability did not exceed that expected by chance. Recoveries of V. parahaemolyticus from coastal and estuarine surface waters exceeded those obtainable by other methods examined.     

A number of Vibrio cholerae non-O1 isolated from aquatic environments

To estimated existence of Vibrio cholerae non-O1 in aquatic environments, the organisms isolated from river, estuary and sea water. V. cholerae non-O1 isolated form midstream and estuary water could be counted from 1.6 to 2400 CFU/100 ml by the membrane filtrated method (MF). V. cholerae non-O1 existed in midstream water more than in estuary water. However, the isolated organisms from estuary rate by MF (37.5%) was lower than it by alkaline peptone enrichment medium method (AP) (75.0%), as a result of halophilic bacteria grow an selected medium of MF. And the number of V. cholerae non-O1 isolated from aquatic environment did not correlate environmental parameters. The number of V. cholerae non-O1 isolated from river water varied, it suggested that the organism collectively adhere a floating matter. V. cholerae non-O1 was not detected in 500 ml sea water by AP and MF method. These results conclude that V. cholerae non-O1 exist in river water more than in sea.     

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either EI Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the EI Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.     

Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae for susceptibility to CTXPhi: molecular basis for origination of new strains with epidemic potential

Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin. The receptor for CTXPhi for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V. cholerae. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTXPhi. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXPhi, leading to the origination of potential new epidemic clones.     

Isolation and characterization of melanin-producing (mel) mutants of Vibrio cholerae

Vibrio cholerae strain Htx-3, a hypertoxinogenic mutant of V. cholerae 569B Inaba, produces a dark brown pigment under certain growth conditions, whereas strain 569B does not. We investigated the biochemical basis for this pigment production and the possible relationships between pigmentation and other phenotypic properties in V. cholerae. After mutagenesis of V. cholerae 569B with N-methyl-N'-nitro-N-nitrosoguanidine, 28 independently derived pigment-forming (mel) mutants were isolated and characterized. The mel mutants frequently differed from wild type in toxinogenicity or motility and occasionally differed in other phenotypic traits. Individual mel mutants differed from wild type both in the amount of toxin produced and in the growth conditions optimal for toxin production. It has not yet been established whether multiple phenotypic changes in individual mel mutants represent pleiotropic effects of single mutations or induction of multiple mutations by N-methyl-N'-nitro-N-nitrosoguanidine or both. Production of pigment by mel mutants occurred at temperatures from 22 to 40 degrees C, was inhibited by anaerobiosis, and was stimulated by supplementation of growth media with the amino acid precursors of melanin (l-phenylalanine, l-tyrosine, or l-tyrosine plus l-cysteine). The pigment possessed several other properties reported for microbial melanins. We conclude that a biochemical pathway for melanin production is present in V. cholerae, that this pathway cannot be fully expressed in wild-type strain 569B, and that mutations in the gene(s) which we have designated mel can permit hyperproduction of melanin under appropriate conditions.     

Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus

A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer. The RAPD-PCR results were scanned, and the images were analyzed with a computer program. Ribotype membranes were evaluated visually. Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous. Ribotyping enabled us to differentiate U.S. and Danish strains and V. vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V. vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes. Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V. vulnificus biotype 2 is an opportunistic pathogen for humans. One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains. This is, to our knowledge, the first time the isolation of V. vulnificus biotype 2 from coastal waters has been described.     

New evidence for an inflammatory component in diarrhea caused by selected new, live attenuated cholera vaccines and by El Tor and Q139 Vibrio cholerae

Using a lactoferrin latex agglutination assay, we have compared the inflammatory responses to a cholera vaccine candidate, CVD 110, in which all known toxin genes have been deleted or mutated yet still produced significant diarrhea, with a less reactive vaccine strain and wild-type El Tor and 0139 Vibrio cholerae strains. Data suggest that diarrhea due to attenuated and wild-type El Tor V. cholerae, and to a lesser extent 0139 V. cholerae, involves an inflammatory response. Further study is required to further elucidate the mechanism of the process(es) involved.     

Frequency and timing of coital orgasm in women desirous of becoming pregnant

Thirty-seven Vibrio cholerae and four non-cholera Vibrio isolates from Ukraine, including strains from the epidemic of 1994-5, were analysed by molecular methods. Results from PFGE and ribotyping indicated that all Ukrainian toxigenic V. cholerae were closely related to each other and to an isolate from a patient from Pakistan. A non-toxigenic river water strain obtained during the height of the epidemic was more distantly related to these V. cholerae strains, while the Vibrio parahaemolyticus isolates and Vibrio alginolyticus isolate were not closely related to V. cholerae or each other. ERIC- and REP-PCR allowed the differentiation of strains identical by other methods. The results obtained confirm that the epidemic Ukrainian strains are most closely related to seventh pandemic strains from Asia and support a hypothesis that the Ukrainian epidemic of 1994-5 was caused by toxigenic environmental strains surviving since the time of the 1991 Ukrainian epidemic or before.     

Pseudo-outbreak of candidaemia with Candida parapsilosis

Vibrio cholerae was isolated from 1008 of 3496 stool samples (28.8%) examined in Tamil Nadu State, India, between November 1992 and December 1995. During November and December 1992, 363 of the 370 isolates serotyped (98%) were V. cholerae O139 (Bengal). The epidemic predominantly affected adults (91%; 597/656). Both V. cholerae O1 and O139 serotypes were sometimes isolated in the same locality from different individuals. From January 1993 onwards, the rate of isolation of V. cholerae O139 declined, and in 1995 V. cholerae E1 Tor (serotype O1) was isolated from most of the cases (85.6%; 131/153). V. cholerae E1 Tor has clearly not been replaced by serotype O139, but can survive during inter-epidemic periods and reappear at an opportune moment. The decline of serotype O139 may be due to the development of immunity as a result of repeated exposure.     

Taking care of the elderly: the unmentioned abuses

A total of 10,427 diarrhoeal stool specimens were cultured for Vibrio cholerae between 1992 and 1997. The isolation rates were 2%, 2.6%, 6.7%, 7.08%, 0.9% and 2.6% in the years from 1992 to 1997 respectively. Till 1992, Vibrio cholerae 01 ogawa was the predominant strain. In 1993, 81.3% of the isolates were of 0139 Bengal strain and the rest were V. cholerae 01. From 1994 to 1997, V. cholerae 01 ogawa was the predominant strain and there were no isolation of 0139 strain. The predominant phage type in 1992 and 1993 were T2 and T27 thereafter. Most Vibrio cholerae strains were sensitive to tetracycline, gentamycin, netromycin, norfloxacin and furazolidine. Strains were resistant to cotrimaxozole till 1996, but were 100% sensitive in 1997. Strains were sensitive to chloramphenicol till 1993 but acquired resistance thereafter.     

Septicemia due to a non-0:1, non-0:139 Vibrio cholerae

We describe a patient with a non-0:1, non-0:139 Vibrio cholerae septicemia associated with ecythema gangrenosa-like skin lesions. The patient acquired the infection in Puerto Rico. Given the high fatality rate, it is important for the medical community to consider the diagnosis in high risk patients with exposures in Puerto Rico tropical waters.     

Distribution of serogroups of Vibrio cholerae non-O1 non-O139 with specific reference to their ability to produce cholera toxin, and addition of novel serogroups

A total of 1898 strains of Vibrio cholerae non-O1 non-O139, which had been collected worldwide for the past 3 year period of 1994-1996, were serogrouped. The strains were also examined for presence of cholera toxin (CT) gene (ctx) and NAG-ST gene, and strains which carried to ctx were further analyzed for their ability to produce CT. In addition, attempts were made to establish novel serogroups for those serologically untypable strains. Of those examined, 1,774 strains of V. cholerae non-O1 non-O139 was classified into 128 known serogroups while 50 strains were found to belong to R type, and the rest of the 74 strains could not be serotyped. Distribution of the serogroups did not seem to correspond to either the strains geographic distribution or sources of isolation. Of those serologically untypable strains, 38 novel serogroups (O156-O193) were established and added to our reference of V. cholerae antigenic schema. It was also found that antisera raised against many V. cholerae strains included R antibodies. This indicates that any V. cholerae antisera for diagnostic purpose should be absorbed with the reference R strains, CA385, before use. There were luminescence producing strains among those sucrose and VP reaction negative strains. Subsequent DNA/DNA homology analysis revealed that they were identified as V. cholerae. This points to a possibility that strains tentatively identified as Vibrio mimicus by conventional biochemical tests may have included luminescent strains of V. cholerae. It is thus highly recommended that strains in question should be tested for the luminescence production in order to differentiate V. cholerae from V. mimicus. Of those 1989 strains examined, 37 strains (ca. 2%) were found to produce CT. Interestingly, CT producing strains were prevalent in serogroup O141; 10 strains out of 16 strains (63%) were positive for CT. The evidence calls for a caution to possible occurrence of cholera-like diarrhea caused by V. cholerae O141 in the future.     

Purification and characterization of pili of a Vibrio cholerae non-O1 strain

Pili of the Vibrio cholerae non-O1 strain V10 were purified and characterized. The V10 pili were physicochemically and immunologically different from those of the previously reported V. cholerae non-O1 strain S7, although the pili of the two strains had homologous N-terminal amino acid sequences. V10 plus antigen was detected only in V. cholerae non-O1 strains.     

Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene

An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.     

Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae

Vibrio cholerae causes epidemic diarrhea throughout the world. Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics. V. cholerae, like most bacteria, has developed resistance to some antibiotics. In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics. Bengal isolates showed unique trends in antimicrobial resistance. Many clinical laboratories use automated antibiotic susceptibility testing for V. cholerae. It is important to know if automated susceptibility test results for V. cholerae coincide with reported trends in antibiotic susceptibility. In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V. cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates. Vitek susceptibilities for V. cholerae showed a good correlation with preestablished epidemiological data. Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains. Regardless of serogroup, > or = 98% of the V. cholerae isolates tested were susceptible to most antibiotics tested by us. It is important to continue susceptibility testing of all new isolates of V. cholerae because of emerging resistant strains. However, V. cholerae remains susceptible to most of the available antibiotics.     

Role of rpoS in stress survival and virulence of Vibrio cholerae

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.