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A survey of Vibrio cholerae non-O1 from surface water and sediment samples was carried out in the aquatic environment of Hiroshima City. Of 72 water samples, 49 (68.1%) were positive for the organism, while in sediment samples 17 of 47 (36.2%) were positive. The incidence of the organism was higher in summer than in other seasons in both water and sediment samples. Isolation frequency of V. cholerae non-O1 was independent of other marine vibrios. Salinity and pH did not have an obvious influence on the distribution of this organism.All the isolates were hemolytic on sheep blood agar, and quantitative analysis revealed a high potential of hemolysin production of more than 1, 000 hemolytic units in 10 of 130 strains tested. A large number of isolates (60/102) were cytotoxigenic on CHO cell culture. Various cytopathic effects were displayed, including cytolytic, cell elongation, and a combination of these. All the isolates were incapable of producing any detectable cholera-like enterotoxin. These findings suggest that V. cholerae non-O1 is widespread in and around the Hiroshima City aquatic environment throughout the year and possesses the ability to cause infection.

A 67-year-old Thai female with alcoholic cirrhosis presented with fever and abdominal pain for 5 days. On examination, there was marked ascites with generalized abdominal tenderness. The result of ascitic fluid analysis showed yellow turbid fluid, a WBC count of 6,100 cells/mm3 with polymorphonucleocytes predominant. Blood cultures yielded non-O1/ non-O139 Vibrio cholerae. The patient improved gradually and recovered fully after 1 week of parenteral antibiotic.

The serogroups and antimicrobial susceptibility patterns of V. cholerae isolated in Hubli, India during the years 2000 to 2004 were monitored. A total of 256 V. cholerae isolates were obtained during the study period, of which 129 (50.4%) belonged to serogroup O1 while the O139 and non-O1, non-O139 serogroups constituted 61 (23.8%) and 66 (25.8%) isolates, respectively. V. cholerae O1 Ogawa was the predominant isolate during the first 2 years of the study. However, this was replaced by V. cholerae non-O1, non-O139 serogroups in the following years. The V. cholerae, which was susceptible to most enteric antimicrobials in 2000, was found to be multidrug resistant in subsequent years, with the development of fluroquinolone resistance since 2002. Surveillance of the epidemiological and microbiological characteristics of V. cholerae provides useful information for managing cholera cases. The V. cholerae non-O1, non-O139 serogroups coupled with multiple antimicrobial resistance may form a group of emerging diarrheal pathogens in the tropics.

Septicemia of Vibrio spp. such as non-O1 Vibrio cholerae presented with diarrhea, fasciitis, cellulitis or otitis media are common in cirrhotic patients (Lin, C.-J., Chiu, C.-T., Lin, D.-Y., et al., Am. J. Gastroenterol., 91, 336-340, 1996). It may result from a lower C3/C4 level, a lower serum ferritin level or opsonophagocytosis dysfunction. High mortality in such cases has been noted. However, endophthalmitis is rare in such patients, and has never been reported. We present a cirrhotic patient of non-O1 and non-O139 V. cholerae septicemia complicated with endophthalmitis.

Few extraintestinal Vibrio infections have been reported in the African region. We report 3 cases from Mauritius: one case of Vibrio alginolyticus otitis externa; one case of soft tissue infection caused by non-O1 Vibrio cholerae and Vibrio parahaemolyticus; and one fatal case of non-O1 V. cholerae cellulitis and septicaemia.

Vibrio species are ubiquitous in the marine environment and can cause severe infections in cirrhotic patients. Patients with liver disease should be warned about the potential dangers of consuming raw or undercooked seafood, and avoiding exposure of wounds to seawater. We report a case of severe sepsis from Vibrio cholerae non-O1 in a patient with cirrhosis awaiting orthotopic liver transplant. This case is aimed to advise clinicians about the importance of V. cholerae subtypes, and non-cholera Vibrio species infections in cirrhotic patients, highlighting the need to educate these patients to stay away from undercooked seafood.

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR , and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin ( ctxA ), heat-stable toxin ( est ), heat-labile toxin ( elt ), and Shiga toxin variants ( stx ) of Escherichia coli . Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin ( hly ). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae . Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.

Vibrio cholerae non-O1/non-O139 strains have caused several cases of ear, wound, and blood infections, including one lethal case of septicemia in Austria, during recent years. All of these cases had a history of local recreational activities in the large eastern Austrian lake Neusiedler See. Thus, a monitoring program was started to investigate the prevalence of V. cholerae strains in the lake over several years. Genetic analyses of isolated strains revealed the presence of a variety of pathogenic genes, but in no case did we detect the cholera toxin gene or the toxin-coregulated pilus gene, both of which are prerequisites for the pathogen to be able to cause cholera. In addition, experiments were performed to elucidate the preferred ecological niche of this pathogen. As size filtration experiments indicated and laboratory microcosms showed, endemic V. cholerae could rapidly grow in a free-living state in natural lake water at growth rates similar to those of the bulk natural bacterial population. Temperature and the quality of dissolved organic carbon had a highly significant influence on V. cholerae growth. Specific growth rates, growth yield, and enzyme activity decreased markedly with increasing concentrations of high-molecular-weight substances, indicating that the humic substances originating from the extensive reed belt in the lake can inhibit V. cholerae growth.

In this study, we report the presence of the SXT element and Class I integron in Vibrio cholerae non-O1, non-O139 strains isolated from Varanasi, India. Isolates were resistant to cotrimoxazole, trimethoprim and/or streptomycin, furazolidone and ampicillin. None contained plasmids. Polymerase chain reaction (PCR) and DNA sequencing revealed the presence of antibiotic resistance gene cassettes, aadA1, aadA2, aadA5 and dfrA15, in the Class I integron and SXT, an integrative conjugative element containing dfr18, sulII and strAB, in three and six of the isolates respectively. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence for the transferable nature of intSXT and associated antibiotic resistance gene cassettes. This is the first report of the occurrence of SXT ICE, dfr18, sulII, strAB and aadA5 genes in environmental V. cholerae non-O1, non-O139 strains from Varanasi, India, that had been isolated before 1992.

Plasmid profiles and factors associated with toxigenicity in 51 strains of Vibrio cholerae non-O1 isolated from water samples collected in Bangladesh were analysed. Eleven (21.5%) strains were found to harbour at least one plasmid of 1.7-115 Mda; seven of these strains shared a 115-Mda plasmid. Six of 13 strains tested gave positive cytotoxic and enterotoxic responses. However, two non-cytotoxic strains were enterotoxigenic. Only three of the six cytotoxic and enterotoxic strains caused haemagglutination of human erythrocytes which indicated that toxin production and haemagglutinating activity were unrelated in these V. cholerae non-O1 strains. Conjugal transfer assays demonstrated that the 115-Mda plasmid harboured by some of the toxigenic V. cholerae non-O1 strains carried genes coding for antibiotic resistance and cytotoxin production but not for enterotoxin production. However, this plasmid was also carried by non-toxigenic strains. Some other strains carrying no plasmids or only small-mol.-wt plasmids, were found to be toxigenic. Therefore, toxin production is not plasmid-mediated in all V. cholerae non-O1 strains. Regardless of their pathogenic potential, V. cholerae non-O1 strains possessed the capacity to grow in conditions of iron limitation and, under these conditions, synthesis of at least two new outer-membrane proteins was induced.

A monoclonal antibody (MAb) against synthetic heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was produced. The MAb, namely, 2F, belonged to the immunoglobulin G1 class. Ascitic fluid drawn from pristane-primed BALB/c mice injected with a 2F-producing clone demonstrated anti-NAG-ST activity which could be detected in enzyme-linked immunosorbent assay even at a dilution of 1:128,000. Fifty-fold-diluted ascitic fluid could completely neutralize the activity of NAG-ST (synthetic and native) and Vibrio mimicus ST (identical to NAG-ST) in suckling mice. In the same assay, 2F could also neutralize Yersinia enterocolitica ST (Y-ST) but did not neutralize Escherichia coli STh and STp. A similar pattern of reactivity occurred in a competitive enzyme-linked immunosorbent assay with homologous and heterologous toxins. Competitive inhibition curves with synthetic peptides representing NAG-ST and its shorter analogs revealed that aspartic acid located at position 2 from the N terminus of NAG-ST was the essential residue of the recognized epitope. Significantly, in Y-ST, to which 2F cross-reacted, aspartic acid is in the corresponding position as that of NAG-ST, thereby confirming our conclusions that the epitope defining this MAb is aspartic acid.

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site. Key words: cholera toxin, epitopes, monoclonal antibodies.

Pili of Vibrio cholerae non-O1 strain S7 were purified and characterized. The pili of S7 were morphologically, electrophoretically, and immunologically (as far as polyclonal antibody was used) indistinguishable from the 16-kilodalton pili of V. cholerae O1 strain 82P7. The purified pili and organisms had D-mannose- and L-fucose-resistant hemagglutinin. The hemagglutinating activity of the purified pili was inhibited by the Fab fraction of antipilus antibody, but the hemagglutinating activity of live organisms was not inhibited completely. The purified pili or Fab fraction of antipilus antibody did not inhibit the adhesion of V. cholerae non-O1 to rabbit intestines. Therefore, the pili were not regarded as a colonization factor of V. cholerae non-O1. A total of 148 V. cholerae non-O1 and O1 clinical isolates were screened for the presence of S7 pili by using an agglutination test with anti-S7 pilus serum; 12 of 49 V. cholerae non-O1 strains and 25 of 99 V. cholerae O1 strains were positive for agglutination. These agglutination reactions were not correlated with adhesion of the organisms to intestines.

The morphology of "free endotoxin" in cell suspensions of Vibrio cholerae non-O1 and in fractions purified from culture filtrate (Fraction 1) was examined at the beginning of the exponential phase of growth in complex medium. The observed structures were compared with those of the lipopolysaccharide obtained by phenol extraction. Blebs, vesicles, and ribbons with a trilaminar, membrane-like structure were observed by electron microscopy of ultrathin sections and negatively stained samples. Endotoxic activity was detected in these samples by the Limulus amoebocyte lysate pyrotest. The free endotoxin appeared as a heterogeneous population of particles in Fraction 1 (20-60 nm in diameter) and in the bacterial suspension (10-40 nm in diameter) while the lipopolysaccharide obtained by phenol extraction of the cells was revealed to be a uniform particle population (45 nm in diameter).

Two serogroups of Vibrio cholerae non-O1 possessing somatic antigen factors in common with Hakata serogroup of V. cholerae are described. One group was included in the Hakata serogroup and has its own antigen other than all O-antigens of the Hakata group. The other group had its own major antigen, but not factor F specific for the Hakata group.

Non-O1/non-O139 Vibrio cholerae can cause invasive extraintestinal disease as well as enteritis. The pathogenesis of invasive non-O1/non-O139 V. cholerae infections remains to be determined. This study compared the clinical manifestations and predisposing factors between bacteremic and non-bacteremic non-O1/non-O139 V. cholerae infections and examined virulence-associated genes in the pathogenic strains causing invasive disease.We retrospectively investigated clinical characteristics of 18 bacteremic patients and 18 non-bacteremic patients, including demographic, laboratory and clinical data. Fourteen clinical isolates (ten isolated from blood and four from stool specimens) were obtained for polymerase chain reaction tests of the presence of virulence-associated genes ctxA, ctxB and tcpA.There was no difference in age, gender and gastrointestinal symptoms including abdominal pain and diarrhea, laboratory findings including leukocytosis and anemia, or underlying immunocompromised condition, except cirrhosis, between the bacteremic and non-bacteremic groups. Compared to patients with non-bacteremic infections, patients with non-O1/non-O139 V. cholerae bacteremia were significantly more likely to have cirrhosis and thrombocytopenia (0.0% vs 77.8% and 5.9% vs 72.2%, respectively; p0.001). The cholera toxin genes (ctxA and ctxB) were found in only one strain (isolated from the stool specimen of a patient with enteritis) among fourteen clinical strains (7%). The tcpA gene, encoding the toxin-coregulated pilus, was present in thirteen of fourteen isolates (93%) [including ten isolates from blood, and three isolates from stool specimens].Cirrhotic patients with thrombocytopenia were vulnerable to non-O1/non-O139 V. cholerae bloodstream invasion. The low prevalence of ctxA and ctxB genes in stool specimens indicates other toxins could have contributed to diarrhea. The fact that the tcpA gene was highly prevalent in clinical isolates in this study could imply an important role of tcpA in the pathogenesis of invasive disease caused by non-O1/non-O139 V. cholerae.

To evaluate the effect of six culture media (five complex and one mineral) on biofilm formation and response to oxidative stress in Pseudomonas aeruginosa (3 strains) and Vibrio cholerae non-O1 (3 strains).Biofilm formation was quantitatively determined by a crystal violet absorption assay. The bacterial response to oxidative stress evoked by hydrogen peroxide was visualized as a zone of clearing around the disc after 24 h incubation at 37 degrees C.For both of the bacterial species studied, biofilm formation was the highest after cultivation in tryptone soya broth (TSM) or in TSM supplemented with 8% glucose (TSM+GL), being the lowest in mineral medium (MM). V. cholerae non O1 strains were 1.4 to 4.3 times more responsive on average to oxidative stress depending on culture medium as compared with P. aeruginosa strains. The culture medium had no significant effect on H2O2 evoked by response to oxidative stress in vibrios in contrast to P. aeruginosa. In P. aeruginosa, the highest mean resistance to H2O2 was observed after cultivation in peptone water while the most sensitive cells were found after incubation in TSM+GL and MM.The culture medium composition influnced biofilm formation in both of the bacterial species tested and had a considerable effect on response to oxidative stress in P. aeruginosa.

Study of molecular-epidemiological characteristics of Vibrio cholerae non O1/non O139 serogroup with complete and limited set of virulence genes was performed. Differences of their genes composition as compared to these of O1 serogroup (classic and El Tor biovars) were revealed, which points to their origin from avirulent environmental cholera vibrios.

The emergence of multiple resistance to antimicrobials in Vibrio cholerae isolated in the state of Ceará, Brazil, alerted researchers in this area to the sensitivity to antimicrobials of strains isolated in Rio Grande do Norte (RN), Brazil.One hundred and four strains of V. cholerae of human origin, isolated by Laboratório Central de Saúde Pública Dr. Almino Fernandes, were serologically typified and evaluated for in vitro sensitivity to eight antibiotics belonging to different groups (polymyxine, tetracycline, chloramphenicol, nitrofurantoin, sulphazotrin, pefloxacine, erythromycine, ampicillin). The strains were collected from patients suspected of contracting choleric diarrhea in the year 1999, in Natal/RN/Brazil.From the sample total, 100 were identified as V. cholerae, serogroup O:1, biotype El Tor, with 99 (95.3%) belonging to serovar Ogawa and only 1 (0.9%) to serovar Inaba. The 4 remaining were characterized as non O:1 V. cholerae, with 3 (2.9%) biochemically identified as Heiberg type I and 1 (0.9%) as type II. All the V. cholerae serogroup O:1 strains were sensitive to tetracycline, chloramphenicol, sulphazotrin, pefloxacine, erythromycine and resistant to polymyxine. In relation to nitrofurantoin, only 1 was sensitive. Only 1 was resistant to ampicillin. The non O:1 V. cholerae strains were resistant to polymyxine.The results showed sensitivity in 100% of the V. cholerae serogroup O:1 strains to tetracycline, an elective drug in the treatment of cholera, and an absence of multiple resistant strains in our environment. An interesting finding was the frequency of serovar Ogawa in 1999, considering the greater incidence of serovar Inaba in other years of cholera outbreaks in RN.

In the article was described a case of Vibrio choleare infection non O1 non O139. The Vibrio choleare bacterium caused cholera in 49 years old man and was a cause of death. The man died on July the 12, 2006. The diagnostic proceeding and the way of perform epidemiological investigation were also described in the article.

Evidence indicates that the atmospheric and oceanic processes that occur in response to increased greenhouse gases in the broad-scale climate system may already be changing the ecology of infectious diseases. Recent studies have shown that climate also influences the abundance and ecology of pathogens, and the links between pathogens and changing ocean conditions, including human diseases such as cholera. Vibrio cholerae is well recognized as being responsible for significant mortality and economic loss in developing countries, most often centered in tropical areas of the world. Within the marine environment, V. cholerae is found attached to surfaces provided by plants, filamentous green algae, copepods, crustaceans, and insects. The specific environmental changes that amplified plankton and associated bacterial proliferation and govern the location and timing of plankton blooms have been elucidated. Several studies have demonstrated that environmental non-O1 and non-O139 V. cholerae strains and V. cholerae O1 El Tor and O139 are able to form a three-dimensional biofilm on surfaces which provides a microenvironment, facilitating environmental persistence within natural aquatic habitats during interepidemic periods. Revealing the influence of climatic/environmental factors in seasonal patterns is critical to understanding temporal variability of cholera at longer time scales to improve disease forecasting. From an applied perspective, clarifying the mechanisms that link seasonal environmental changes to diseases’ dynamics will aid in developing strategies for controlling diseases across a range of human and natural systems.

Cholera toxin (CT) gene-negative Vibrio cholerae non-O1, non-O139 strains may cause severe diarrhea though their pathogenic mechanism remains unclear. V. cholerae cytolysin (VCC) is a pore-forming exotoxin encoded in the hlyA gene of V. cholerae whose contribution to the pathogenesis is not fully understood. In this work, the virulence properties of a CT gene-negative V. cholerae non-O1, non-O139 strain causing a cholera-like syndrome were analyzed. Inoculation of rabbit ileal loops with the wild type strain induced extensive fluid accumulation, accompanied by severe histopathological damage characterized by villus shortening, lymphangiectasia and focal areas of necrosis. These pathogenic effects were abrogated by mutation of the hlyA gene thus pointing out the main role of VCC in the virulence of the strain. Interestingly, this toxin was capable of triggering apoptosis in human intestinal cell lines due to its anion channel activity. Moreover, the wild type strain also induced increased apoptosis of the intestinal epithelium cells which was not observed upon inoculation of the VCC null mutant strain, indicating that VCC may trigger apoptotic cell death during infection in vivo. Altogether, these results support a main role of VCC in the pathogenesis of the CT gene-negative V. cholerae non-O1, non-O139 strain and identify apoptosis as a previously unrecognized cell death pathway triggered by VCC.

Environmental V. cholerae (Vc) have the potential for virulence in people and they may also be a reservoir of accessory virulence genes. We infected mice with two non-O1, non-O139 Vc (TP and SIO) that were isolated in San Diego County and compared them to Vc O1 El Tor N16961 using a model of pneumonia in adult mice. Live but not heat killed Vc El Tor and TP caused fatal hemorrhagic pneumonia despite a >90% decrease in CFU in 24 h suggesting the disease was toxin mediated. SIO did not cause pneumonia in normal mice but neutropenic, gp91phox and complement (C3) mice were more susceptible to all three strains. TP and SIO lack ctx but have rtxA, hlyA, and hapA, genes that encode virulence factors in Vc El Tor. The explanation for the enhanced virulence of TP remains to be determined. 2007 Published by Elsevier Masson SAS.

Background In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. Results The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. Conclusion The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations.

Hemolysin of ctx+ Vibrio cholerae strains was obtained and studied. Ctx+ Vibrio cholerae O1 and O139 strains produced the hemolysin during cultivation in triptone medium without FeCl3. Mol.wt. and the spectrum of lytic activities of hemolysins of ctx+ Vibrio cholerae did not differ from hemolysins of ctx- strains.

Two cases of septicaemia caused by Vibrio cholerae non-O1, non-O139 were reported in different regions of Poland during one

Objetivo: Identificar la presencia de Vibrio cholerae en muestras de agua no cloradas para consumo humano en las localidades de Santa y Coishco. Materiales y métodos: Entre julio de 2003 a junio de 2004 se tomaron muestras de agua, en forma semanal, provenientes de siete pozos con bomba manuable y de seis pozos con reservorio. A cada muestra de agua se le midió in situ el cloro residual mediante un comparador de cloro Hatch, método colorimétrico, usando para ello las pastillas DPD 1. En las muestras con cloro

The first case of Vibrio cholerae infection to be reported in Slovenia since 1977 was detected in July 2005

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin (ST), as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary (CHO) cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin (CT). Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen (CAMP) method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

One hundred seventy nine Vibrio cholerae non-O1/non-O139 strains from clinical and different environmental sources isolated in Brazil from 1991 to 2000 were serogrouped and screened for the presence of four different virulence factors. The Random Amplification of Polymorphic DNA (RAPD) technique was used to evaluate the genetic relatedness among strains. Fifty-four different serogroups were identified and V. cholerae O26 was the most common (7.8%). PCR analysis for three genes (ctxA, zot, ace) located of the CTX genetic element and one gene (tcpA) located on the VPI pathogenicity island showed that 27 strains harbored one or more of these genes. Eight (4.5%) strains possessed the complete set of CTX element genes and all but one of these belonged to the O26 serogroup suggesting that V. cholerae O26 has the potential to be an epidemic strain. The RAPD profiles revealed a wide variability among strains and no genetic correlation was observed. Cento e setenta e nove amostras de V. cholerae não O1/não O139, isoladas de casos clínicos (139) e de meio ambiente (40), no período de 1991 a 2000 no Brasil, foram caracterizadas antigenicamente pelo National Institute of Health (Japão) e investigadas quanto ao seu potencial genético de virulência, representado pelos genes ctxA, zot, ace e tcpA. As análises fenotípicas revelaram extraordinária diversidade antigênica, com a ocorrência de 54 diferentes sorogrupos, com prevalência para O26 (7,8%). A técnica de PCR, empregada na detecção dos genes localizados no elemento genético CTX (ctxA, zot, ace) e na Ilha de Patogenicidade de Vibrio-VPI (tcpA), possibilitou a identificação de 27 cepas contendo qualquer um desses genes. O gene ctxA (codificador da sub-unidade A de CT), só foi evidenciado no sorogrupo O26, sendo também o único capaz de se apresentar com o cassete de virulência de forma intacta. Com base nos resultados obtidos deste estudo preliminar, admite-se a hipótese da potencialidade destas cepas, evoluir para raças epidêmicas.

An epidemic of cholera in Iraq was anticipated for the year 1999 and a plan of notification and treatment of cases of diarrhoea was made. This paper documents the clinical and bacteriological profile of cholera cases admitted to the 6 hospitals in Baghdad during the epidemic. The number of stool culture-positive cases was 874. The peak incidence of cases (June 1999) was earlier than previous epidemics with no cases registered in November/December 1999. All age groups were affected and the case fatality rate was 1.3%. Strains isolated were Vibrio cholerae El-Tor O1, serotypes Ogawa (79.6%) and Inaba (12.1%), V. parahaemolyticus (2.0%) and non-agglutinable vibrios (6.1%). V. cholerae O139 was isolated from 2 cases (0.2%) for the first time in Iraq. Antibiotic resistance was noted, especially to tetracycline.

The virulence of a pathogen is reliant on the presence of a discrete set of genetic determinants and their expression in the host. The virulence of Vibrio spp. is regulated by the ctxAB and tcpA genes. These genes are alleged to be exclusively associated with clinical strains of O1 and O139 serogroups. In the present study, we examined the presence of virulence genes viz. stn, OmpW , ctxA and tcpA of classical and ElTor variants, in environmental strains of non-O1 Vibrio cholerae cultured seasonally from four sampling stations of the river Narmada at Jabalpur (MP), India. Unexpectedly, the PCR analysis of the strains revealed the presence of these genes among environmental V. cholerae . The strains harboring the tcpA gene also carried the ctxA gene. Sequencing of the tcpA gene and ctxA gene carried by an environmental strain showed ∼97% homology with the previously sequenced genes submitted in the GenBank. We report here the prevalence of cholera toxin gene and the gene for toxin co-regulated pilus among non-O1 V. cholerae strains isolated from fresh water environment. This study supports the idea that cholera toxin has an environmental derivation and that the intricate aquatic environment can give rise to pathogenic Vibrio organisms.

Six hundred and ninety colonies from 11 water samples were tested for V. cholerae and other vibrio species. Four isolates of V. cholerae non-O1 were isolated besides a few other vibrios. Isolates of V. cholerae were found to be toxin-negative and resistance to most antibiotics.

The most common clinical manifestation of Vibrio cholerae non-O1 non-O139 is gastroenteritis. This vibrion may also cause bacteremia, soft tissue infection, and other extraintestinal invasive disease, especially in immunocompromised patients. This study evaluated the current status of antimicrobial resistance in clinical isolates of V. cholerae non-O1 non-O139 in Taiwan as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. Minimal inhibitory concentrations (MICs) of 9 antimicrobial agents were determined by the agar dilution method. All of the isolates were susceptible to minocycline (MIC at which 90% of the isolates were inhibited [MIC(90)], 0.12 microg/mL), cefotaxime (MIC(90), 0.06 microg/mL), lomefloxacin (MIC(90), 0.12 microg/mL), levofloxacin (MIC(90), 0.03 microg/mL), ciprofloxacin (MIC(90), 0.03 microg/mL), moxifloxacin (MIC(90), 0.06 microg/mL), sparfloxacin (MIC(90), 0.06 microg/mL), gatifloxacin (MIC(90), 0.03 microg/mL), and cefazolin (MIC(90), 8 microg/mL). We conducted time-kill studies to evaluate the inhibitory activities of either cefazolin or minocycline alone or in combination against V. cholerae non-O1 non-O139 (Vc2). We also evaluated the inhibitory activity of cefazolin or cefotaxime combined with minocycline. The individual MICs of cefazolin, cefotaxime, and minocycline were 4 microg/mL, 0.0075 microg/mL, and 0.12 microg/mL, respectively, when approximately 5 x 105 colony-forming units/mL of V. cholerae non-O1 non-O139 was incubated. Bacterial growth was inhibited initially but resumed later when cefazolin, cefotaxime, or minocycline was used alone. When cefazoline or cefotaxime was combined with minocycline, V. cholerae non-O1 non-O139 was inhibited over 48 h and no regrowth was noted. We conclude that the combination of cefazolin or cefotaxime with minocycline has a synergistic inhibitory effect on V. cholerae non-O1 non-O139 in vitro.

The purpose of this work was to characterize the toxin profile and the presence of other virulence factors involved in the pathogenesis and biology of 13 V. cholerae O1 (11 clinical cases and 2 waters) and 6 V. cholerae non O1 strains (4 clinical cases and 2 waters) using genetic (PCR), immunological (RPLA), biochemical (NAD degradation, haemolysis, Kanagawa phenomenon, caseinase, lecithinase, mucinase, amylase, esculine hydrolysis) and cell culture (Vero E6, HEp-2) assays. The results indicated a concordance between PCR-RPLA (84%), PCR-NAD (73%) and RPLA-NAD (84%) methods. The sensitivity of RPLA and NAD degradation methods were comparable to PCR in detecting CT in Vibrio cholerae O1 strains. Although NAD degradation method was not exclusively specific for the CT detection, it proved its usefulness in screening certain virulent, CT-negative clones of V. cholerae. The cytotoxic effect on Vero E6 cells, enzyme production (Kanagawa haemolysins, lecithinase, caseinase, esculine hydrolysis) as well as adherence ability on inert substrate proved to be much more constant in V. cholerae non O1 (CT- negative) than in V. cholerae O1 (CT-positive). All V. cholerae non O1 strains isolated in diarrheal cases were Kanagawa positive. This complex of virulence factors detected in V. cholerae non O1 strains could probably contribute during interepidemic periods to human-to-human transmission and to greater resistance as compared to O1 strains in the environment.

The results of the serotyping of 244 V. cholerae non O1/O139 cultures isolated from patients in Uzbekistan in 2000 and 2001 are presented. All isolates were studied by the method of molecular probing and in the polymerase chain reaction for the presence of virulence genes and for sensitivity to phages ctx+, ctx- and hemolytic activity. The use of monoreceptor O-sera O2-O83 made it possible to determine vibrios of 32 serogroups with the dominating role in the etiology of acute enteric diseases belonging to serogroups O18, O62, O82, O37. Genes ctx AB were detected in none of the isolates, 5 of them contained gene tcp A. A group of cultures, sensitive to phage ctx+ and belonging mainly to enteropathogenic serogroups, was detected.

Four hundred ninety seven strains of Vibrio cholerae selected from isolates in Romania in the last decade 1990-1999 were investigated for antibiotic resistance and for classical and putative virulence factors. V. cholerae O1 strains predominated in clinical cases and non O1 strains in the environment, excepting in 1992 when non O1 strains were frequent in clinical and environmental sources. V. cholerae O1 strains previously susceptible to tetracycline acquired clinically significant resistance to this drug during 1993-1994, but this trend was reversed in 1995, following the introduction of nalidixic acid in cholera treatment in 1994. V. cholerae O1 and non O1 clinical isolates acquired simultaneous resistance to the vibriostatic agent O/129 and cotrimoxazole during 1994-1995. High levels of intrinsic resistance to multiple antibiotics were exhibited by all strains examined. The presence of cholera toxin (CT) was concentrated in clinical V. cholerae O1 strains and was substituted in clinical non O1 strains by four putative virulence markers (Kanagawa haemolysin, slime, lipase, and colonial opacity). Colonial opacity (30%) was present only in clinical isolates of V. cholerae non O1. Pigmentogenesis (11.7%) has present only in environmental sources. Antibioresistance profiles differ for V. cholerae O1 and non O1 strains with respect to their source of isolation. This aspect may imply a role in virulence and survival of V. cholerae in the natural environment where they may serve as a reservoir of virulence and multiple drug resistance genes.

We have developed a novel typing method based on Vibrio cholerae repeat sequences (VCR) using primers directed out of the VCR sequences. To evaluate the VCR–polymerase chain reaction (PCR) as a typing system, 2 categories, efficacy and efficiency, were analyzed in 69 strains of human and environmental V. cholerae O1 toxigenic and nontoxigenic, and non-O1 strains isolated since 1992–2000 from Argentina. The discriminatory power (0.91), stability (0.95), reproducibility (1), typeability (1), rapidity, accessibility, as well ease of use, indicated that the VCR-PCR method provides an alternative useful tool for molecular epidemiology of V. cholerae. The VCR-PCR of V. cholerae isolates showed 29 patterns, of which pattern 1 represented 68% of the V. cholerae O1 isolates, supporting the hypothesis that a clone with epidemic behavior was responsible for the epidemic in Latin America. These results showed a good correlation and a better epidemiologic analysis when the results were compared in parallel with repetitive extragenic palindromic sequences–PCR. In conclusion, VCR-PCR showed excellent performance as a typing method for cholera surveillance programs.

Temperate phages of Vibrio cholerae are the major force in the emergence of newer pathogenic clones. PS166, a temperate vibriophage, has the ability to convert pathogenic biotypes of the V. Cholerae O1 serogroup. During the conversion process, PS166 also tends to produce a group of lysogens neither reported nor characterized so far. Characterization of those lysogens may provide insight into the emergence of newer pathogenic clones of V. Cholerae.The V. cholerae eltor strain MAK757 was used to generate PS166 lysogens which were serotyped by using antisera specific to V. Cholerae O1 and O139 strains. Multiplex PCR was used to compare the status of various pathogenic markers of those lysogens. The outer membrane proteins (OMP) of the lysogens were determined by SDS-PAGE.Temperate phage PS166 infection of V. cholerae eltor MAK757 gave rise to a new kind of lysogens termed unserotypable due to their lack of reactivity to both O1 and O139 antisera. Moreover, these lysogens displayed a new phage sensitivity profile different from both classical and parental eltor biotypes. The genetic profiles of the CTX element and VPI region of these lysogens were found to be unaltered, together with the biotype-specific markers mshA and hlyA. Though the genetic loci of major outer membrane proteins did not change after lysogenization, the outer membrane protein profile of these lysogens showed the absence of a 40-kDa band corresponding to the toxR-controlled porin, ompT.We provide evidence for the vibriophage PS166-mediated lysogenic conversion of pathogenic cholera strains.

The uptakes of Escherichia coli, Vibrio cholerae non-O1 and Enterococcus durans by mussels (Mytilus galloprovincialis) and the times for depuration were investigated in order to determine the most useful indicator of vibrio contamination. The mussels were maintained in tanks of static seawater contaminated with bacteria at 5 log10 CFU/ml for bioaccumulation. Depuration was carried out by circulating fresh seawater through the tanks. Each organism was presented alone and with others to mussels, at temperatures of 14 and 21 degrees C. In water contaminated with either single or mixed organisms, the bacteria accumulated rapidly in the mussels reaching high concentrations after 1 h. With both single and mixed organisms, the maximum numbers of E. coli in mussels were 6.6 log10 CFU/g at 14 degrees C and 5.4 log10 CFU/g at 21 degrees C. Both V. cholerae non-O1 and E. durans alone or with other organisms reached a number ranging from 6.5 to 7 log10 CFU/g at both temperatures. During depuration the numbers of all the organisms slowly decreased, with E. coli alone, numbers ranged from 2.8 to 2 log10 CFU/g after 72 h at both 14 and 21 degrees C, and the organisms were undetectable after 144 h. With mixed organisms at 14 degrees C E. coli became undetectable after 168 h but at 21 degrees C no E. coli were recovered after 72 h. At 14 degrees C V. cholerae non-O1 alone also was undetectable after 168 h, but at 21 degrees C and with mixed organisms at both temperatures. V. cholerae was recovered after 168 h at numbers about 1 log10 CFU/g. After 168 h numbers of E. durans alone ranged from 2.6 log10 CFU/g at 14 degrees C to 1.5 log10 CFU/g at 21 degrees C, and with mixed organisms the numbers ranged from 2.3 to 2.0 log10 CFU/g at both temperatures. Of the three bacteria of faecal origin, E. durans is quickly acquired by mussels and released more slowly than the others, while E. coli quickly becomes undetectable. The results suggest that, for this kind of seafood, enterococci may be a more appropriate indicator than E. coli of risks to consumers from vibrios.

Bacteremia due to Vibrio cholerae non-O1 and non-O139 is a rare condition and potentially fatal. We report a case of bacteremia due to V. cholerae non-O1 and non-O139 in a Portuguese male with Hepatitis C cirrhosis, admitted due to acute diarrhea, after consuming shrimp. He had no recent travels. To our knowledge, this is the first reported case of bacteremia due to V. cholerae non-O1 and non-O139 in Portugal.

O estudo foi desenvolvido com o objetivo de analisar o perfil plasmidial, pesquisar genes de virulência e identificar os perfis genéticos de 31 cepas de Vibrio cholerae não O1 isoladas de zooplâncton dos estuários dos rios Anil e Bacanga em São Luis MA. O estudo do DNA plasmidial revelou a presença de 2 a 3 plasmídeos em 10 cepas, com pesos moleculares variando de 5,5 a 40 kilobases. A ribotipagem revelou um perfil comum a todas as cepas. A amplificação do DNA genômico por PCR não revelou os genes ctxA, ace e zot, mostrando tratar-se de cepas não patogênicas, enquanto a RAPD-PCR identificou múltiplos perfis genéticos, achado compatível com o grande potencial de variabilidade desta espécie. The study was developed to analyze the plasmidial DNA, research virulence genes and identify genetic diversity of 31 strains of Vibrio cholerae non-O1 isolated from zooplankton of the Bacanga and Anil rivers in São Luis-MA. The study of plasmidial DNA showed 2 or 3 plasmids from 10 strains between 5.5 and 40 kilobasis. There was only single ribotype pattern. PCR methods did not show the genes ctxA, ace and zot, while RADP-PCR identified genetic diversity in the strains, showing the potential for variability in this species.

Bacterial adherence to eukariotic cells represents an important step of tissue colonization and is mediated by specific molecules called adhesins. Bacterial adherence to cellular substrate is a very complex process consisting in specific interactions between the surface of host cell and bacterial cell surface respectively. Adherence to cellular substrate confers selective advantages to bacterial cells, as: rapid growth rate by shorter lag period and protection against antibodies and lysozime. Adherence and colonization of small bowel represent the early steps of cholera infection (1, 2). The purposes of this study were to characterize the adherence ability of 46 Vibrio cholerae O1 and non O1 strains with different sources of isolation (acute diarrhea, water sources) to HEp-2 cell; to determine the influence of different factors (culture media, bacterial culture growth phase, proteolytic enzymes, carbohydrates and polyvalent agglutinant anti V. cholerae O1 serum) on the bacterial adherence capacity. Adherence capacity was assayed using the qualitative Cravioto's method. The adherence ability was appreciated by semi quantitative ("+", "++" and "+++") and quantitative assays. The adherence pattern of the tested strains was predominantly a diffuse one. The agar medium proved to be the most appropriate for the early and maximal expression of adhesion molecules, by comparison with nutritive broth and alkaline peptone water. Manose in different concentrations (1% and 3%) inhibited the adherence ability, demonstrating the role of manose-sensitive haemagglutinating fimbriae (MSHA) in mediating the adherence of V. cholerae strains to cellular substrate. Trypsine has no notable effect on the adherence ability, suggesting that the major V. cholerae adhesion molecules are not essentially of protein nature, so that the afimbrial adhesins could also play an important role in bacterial adhesion to eukariotic cells. The agglutinant polyvalent anti-V. cholerae O1 serum had the most significant inhibitory effect on the adherence ability, which was completely abolished in the presence of sub-agglutinant dilutions of serum titer (1/60-1/120) and partially reduced at titers ranging from 1/240 to 1/920. This inhibitory effect could be explained by bacterial agglutination, but also by the specific blocking of some surface structure implicated in the adherence process (i.e. lipopolysaccharides, as demonstrated by the inhibitory effect of sub-agglutinant serum titers). The inhibitory effect of polyvalent anti-V. cholerae O1 serum was limited to O1, but was not evident for the non O1 serogroups, demonstrating that the serum antibodies are acting on serogroup specific antigenic fractions.

The comparative analysis of the hybridization patterns of DNA restricts for 20 V. cholerae, groups 01 and non-01 (non-0139), containing the incomplete CTX element (ctxAB-) was carried out with the use of probes, complementary to the genes of the proximal part of the virulence cassettle and flanking its RS1 sequences. This group was found to be heterogeneous both in the number of copies of "truncated" CTX prophage and their localizations in the genome, as well as in the position of the sites of restriction endonucleases HindlII and BglII. Among 17 clinically noncholerigenic isolates, 5 etiologically dangerous clones were found, each of them characterized by the definite time and place of isolation. At least one of them proved to be the causative agent of the local outbreak of diarrheal diseases in Uzbekistan.

Decrease in adherence of Vibrio cholerae to rabbit small intestine was observed following treatment with antisera against outer membrane (OM), lipopolysaccharide (LPS) and flagella. Anti LPS antibodies were more efficient than the other two antibodies in inducing adherence inhibition and promoting in vivo protection.

To study the distribution of serotype and the positive rate of toxins among vibrio cholerae non-O(1) isolated from environmental waters in Foshan city.Water specimens were collected from river and cultured for vibrio cholerae non-O(1). The PCR method was used to detect cholerae enterotoxin (CT) gene; the ELISA method was used to detect heat-stable toxin (ST) and heat-labile toxin (LT).478 vibrio cholerae non-O(1) strains were isolated from 1 644 water specimens, with a positive rate of 29.07%. Serological assay showed that the main serotype of vibrio cholerae non-O(1) in Foshan city is VBO(7). Positive rate of CT, ST and LT were 1.91%, 13.14% and 12.17%, respectively.A few non-O(1) strains were found to have several virulent factors simultaneously, and the results suggest that vibrio cholerae non-O(1) in environmental waters is potentially pathogenic and may affect people's health. It is necessary to pay attention to the prevention of diarrhoea caused by vibrio cholerae.

The gene blaCARB-9 was located in the Vibrio cholerae super-integron, but in a different location relative to blaCARB-7. CARB-9 (pI 5.2) conferred -lactam MICs four to eight times lower than those conferred by CARB-7, differing at Ambler’s positions V97I, L124F, and T228K. Comparison of the genetic environments of all reported blaCARB genes indicated that the CARB enzymes constitute a family of cassette-encoded -lactamases. Fil: Petroni, Alejandro. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Melano, Roberto. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Saka, Héctor A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Garutti, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Mange, Laura. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Pasterán, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Rapoport, Melina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Miranda, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Faccone, Diego. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Rossi, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Hoffman, Paul S. Dalhousie University. Department of Microbiology and Immunology; Estados Unidos. Fil: Galas, Marcelo F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.