start-ver=1.4 cd-journal=joma no-vol=28 cd-vols= no-issue=4 article-no= start-page=1633 end-page=1639 dt-received= dt-revised= dt-accepted= dt-pub-year=2012 dt-pub=201204 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=An extracellular serine protease produced by Vibrio vulnificus NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel en-subtitle= kn-subtitle= en-abstract= kn-abstract=Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in immunocompromised humans, cultured eels or shrimps. An extracellular metalloprotease VVP/VvpE has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA, but not VVP/VvpE. In the present study, we found that these strains had lost the 80 kb genomic region including the gene encoding VVP/VvpE. We also purified VvsA from the culture supernatant through ammonium sulfate fractionation, gel filtration and ion-exchange column chromatography, and the enzyme was demonstrated to be a chymotrypsin-like protease, as well as those from some vibrios. The gene vvsA was shown to constitute an operon with a downstream gene vvsB, and several Vibrio species were found to have orthologues of vvsAB. These findings indicate that the genes vvp/vvpE and vvsAB might be mobile genetic elements. en-copyright= kn-copyright= en-aut-name=MiyoshiShin-ichi en-aut-sei=Miyoshi en-aut-mei=Shin-ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=JiyouWang en-aut-sei=Jiyou en-aut-mei=Wang kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KatohKeizo en-aut-sei=Katoh en-aut-mei=Keizo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=SenohMitsutoshi en-aut-sei=Senoh en-aut-mei=Mitsutoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MizunoTamaki en-aut-sei=Mizuno en-aut-mei=Tamaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=MaeharaYoko en-aut-sei=Maehara en-aut-mei=Yoko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci affil-num=2 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci affil-num=3 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci affil-num=4 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci affil-num=5 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci affil-num=6 en-affil= kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci en-keyword=Polymerase chain reaction kn-keyword=Polymerase chain reaction en-keyword=Purification kn-keyword=Purification en-keyword=Serine protease kn-keyword=Serine protease en-keyword=Metalloprotease kn-keyword=Metalloprotease en-keyword=Vibrio vulnificus kn-keyword=Vibrio vulnificus END start-ver=1.4 cd-journal=joma no-vol=57 cd-vols= no-issue=6 article-no= start-page=904 end-page=908 dt-received= dt-revised= dt-accepted= dt-pub-year=2011 dt-pub=201105 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Inactivation of Vibrio vulnificus hemolysin through mutation of the N- or C-terminus of the lectin-like domain en-subtitle= kn-subtitle= en-abstract= kn-abstract=Vibrio vulnificus is an etiological agent causing serious systemic infections in the immunocompromised humans or cultured eels. This species commonly produces a hemolytic toxin consisting of the cytolysin domain and the lectin-like domain. For hemolysis, the lectin-like domain specifically binds to cholesterol in the erythrocyte membrane, and to form a hollow oligomer, the toxin is subsequently assembled on the membrane. The cytolysin domain is essential for the process to form the oligomer. Three-dimensional structure model revealed that two domains connected linearly and the C-terminus was located near to the joint of the domains. Insertion of amino acid residues between two domains was found to cause inactivation of the toxin. In the C-terminus, deletion, substitution or addition of an amino acid residue also elicited reduction of the activity. However, the cholesterol-binding ability was not affected by the mutations. These results suggest that mutation of the C- or N-terminus of the lectin-like domain may result in blockage of the toxin assembly. en-copyright= kn-copyright= en-aut-name=MiyoshiShin-ichi en-aut-sei=Miyoshi en-aut-mei=Shin-ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=AbeYuki en-aut-sei=Abe en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=SenohMitsutoshi en-aut-sei=Senoh en-aut-mei=Mitsutoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MizunoTamaki en-aut-sei=Mizuno en-aut-mei=Tamaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MaeharaYoko en-aut-sei=Maehara en-aut-mei=Yoko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=NakaoHiroshi en-aut-sei=Nakao en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University affil-num=2 en-affil= kn-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University affil-num=3 en-affil= kn-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University affil-num=4 en-affil= kn-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University affil-num=5 en-affil= kn-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University affil-num=6 en-affil= kn-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University en-keyword=Vibrio vulnificus kn-keyword=Vibrio vulnificus en-keyword=Hemolysin kn-keyword=Hemolysin en-keyword=Cell-free translation kn-keyword=Cell-free translation en-keyword=Site-directed mutagenesis kn-keyword=Site-directed mutagenesis END start-ver=1.4 cd-journal=joma no-vol=43 cd-vols= no-issue=8 article-no= start-page=1288 end-page=1291 dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=202008 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Low Viability of Cholera Toxin-Producing Vibrio cholerae O1 in the Artificial Low Ionic Strength Aquatic Solution en-subtitle= kn-subtitle= en-abstract= kn-abstract=It has been well known that Vibrio cholerae inhabit in environmental water. As many patients infected with cholera toxin-producing V. cholerae O1 (toxigenic V. cholerae O1) emerge in Kolkata, India, it has been thought that toxigenic V. cholerae O1 is easily detected in environmental water in Kolkata. However, we could not isolate toxigenic V. cholerae O1 from environmental water in Kolkata, though NAG Vibrio (generic name of V. cholerae non-O1/non-O139) is constantly detected. To clear the reason for the non-isolation of toxigenic V. cholerae O1, we examined the viability of V. cholera O1 and NAG Vibrios in the artificial low ionic strength aquatic solution. We found that the viability of toxigenic V. cholerae O1 in the solution is low, but that of NAG Vibrios is high. Subsequently, we examined the viability of NAG Vibrios possessing cholera toxin gene (ctx) in the same condition and found that the viability of these NAG Vibrios is low. These results indicate that the existence of ctx in V. cholerae affects the viability of V. cholerae in the aquatic solution used in this experiment. We thought that there was closely relation between the low viability of toxigenic V. cholerae O1 in the artificial low ionic strength aquatic solution and the low frequency of isolation of the strain from environmental water. en-copyright= kn-copyright= en-aut-name=PaulSubha Sankar en-aut-sei=Paul en-aut-mei=Subha Sankar kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=TakahashiEizo en-aut-sei=Takahashi en-aut-mei=Eizo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=ChowdhuryGoutam en-aut-sei=Chowdhury en-aut-mei=Goutam kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MiyoshiShin-ichi en-aut-sei=Miyoshi en-aut-mei=Shin-ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MizunoTamaki en-aut-sei=Mizuno en-aut-mei=Tamaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=MukhopadhyayAsish K. en-aut-sei=Mukhopadhyay en-aut-mei=Asish K. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=DuttaShanta en-aut-sei=Dutta en-aut-mei=Shanta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=OkamotoKeinosuke en-aut-sei=Okamoto en-aut-mei=Keinosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Collaborative Research Center of Okayama University for Infectious Diseases in India kn-affil= affil-num=2 en-affil=Department of Health Pharmacy, Yokohama University of Pharmacy kn-affil= affil-num=3 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama University kn-affil= affil-num=6 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=7 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=8 en-affil=Collaborative Research Center of Okayama University for Infectious Diseases in India kn-affil= en-keyword=Vibrio cholerae kn-keyword=Vibrio cholerae en-keyword=cholera toxin kn-keyword=cholera toxin en-keyword=aquatic solution kn-keyword=aquatic solution en-keyword=viability kn-keyword=viability END start-ver=1.4 cd-journal=joma no-vol=8 cd-vols= no-issue=9 article-no= start-page=1303 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200826 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Regulation of Chitin-Dependent Growth and Natural Competence in Vibrio parahaemolyticus en-subtitle= kn-subtitle= en-abstract= kn-abstract=Vibrios can degrade chitin surfaces to soluble N-acetyl glucosamine oligosaccharides (GlcNAc(n)) that can be utilized as a carbon source and also induce a state of natural genetic competence. In this study, we characterized chitin-dependent growth and natural competence in Vibrio parahaemolyticus and its regulation. We found that growth on chitin was regulated through chitin sensors ChiS (sensor histidine kinase) and TfoS (transmembrane transcriptional regulator) by predominantly controlling the expression of chitinase VPA0055 (ChiA2) in a TfoX-dependent manner. The reduced growth of Delta chiA2, Delta chiS and Delta tfoS mutants highlighted the critical role played by ChiA2 in chitin breakdown. This growth defect of Delta chiA2 mutant could be recovered when chitin oligosaccharides GlcNAc(2) or GlcNAc(6) were supplied instead of chitin. The Delta tfoS mutant was also able to grow on GlcNAc(2) but the Delta chiS mutant could not, which indicates that GlcNAc(2) catabolic operon is dependent on ChiS and independent of TfoS. However, the Delta tfoS mutant was unable to utilize GlcNAc(6) because the periplasmic enzymes required for the breakdown of GlcNAc(6) were found to be downregulated at the mRNA level. We also showed that natural competence can be induced only by GlcNAc(6), not GlcNAc(2), because the expression of competence genes was significantly higher in the presence of GlcNAc(6) compared to GlcNAc(2). Moreover, this might be an indication that GlcNAc(2) and GlcNAc(6) were detected by different receptors. Therefore, we speculate that GlcNAc(2)-dependent activation of ChiS and GlcNAc(6)-dependent activation of TfoS might be crucial for the induction of natural competence in V. parahaemolyticus through the upregulation of the master competence regulator TfoX. en-copyright= kn-copyright= en-aut-name=DebnathAnusuya en-aut-sei=Debnath en-aut-mei=Anusuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=MizunoTamaki en-aut-sei=Mizuno en-aut-mei=Tamaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MiyoshiShin-Ichi en-aut-sei=Miyoshi en-aut-mei=Shin-Ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=chitin kn-keyword=chitin en-keyword=chitinase kn-keyword=chitinase en-keyword=GlcNAc(6) kn-keyword=GlcNAc(6) en-keyword=natural competence kn-keyword=natural competence en-keyword=ChiA2 kn-keyword=ChiA2 en-keyword=ChiS kn-keyword=ChiS en-keyword=TfoS kn-keyword=TfoS END start-ver=1.4 cd-journal=joma no-vol=38 cd-vols= no-issue=12 article-no= start-page=241 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20221022 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Second extracellular protease mediating maturation of Vibrio mimicus hemolysin en-subtitle= kn-subtitle= en-abstract= kn-abstract=Vibrio mimicus is a bacterium that causes gastroenteritis in humans. This pathogen produces an enterotoxic hemolysin called V. mimicus hemolysin (VMH), which is secreted extracellularly as an inactive 80-kDa protoxin and converted to a 66-kDa mature toxin through cleavage between Arg(151) and Ser(152). The 56-kDa serine protease termed V. mimicus trypsin-like protease (VmtA) is known to mediate this maturating process. However, some strains including strain ES-20 does not possess the vmtA gene. In the present study, the vmtA-negative strains were found to have a replaced gene that encodes a 43-kDa (403 aa) precursor of a serine protease designated by VmtX (V. mimicus trypsin-like protease X). To examine whether VmtX is also involved in the maturation of VMH, VmtX was isolated from the culture supernatant of V. mimicus strain NRE-20, a metalloprotease-negative mutant constructed from strain ES-20. Concretely, the culture supernatant was fractionated with 70% saturated ammonium sulfate and subjected to affinity column chromatography using a HiTrap Benzamidine FF column. The analysis of the N-terminal amino acid sequences of the proteins in the obtained VmtX preparation indicated that the 39-kDa protein was active VmtX consisting of 371 aa (Ile(33)-Ser(403)). The VmtX preparation was found to activate pro-VMH through generation of the 66-kDa protein. Additionally, treatment of the VmtX preparation with serine protease inhibitors, such as leupeptin and phenylmethylsulfonyl fluoride, significantly suppressed the activities to hydrolyze the specific peptide substrate and to synthesize the 66-kDa toxin. These findings indicate that VmtX is the second protease that mediats the maturation of VMH. en-copyright= kn-copyright= en-aut-name=MiyoshiShin-ichi en-aut-sei=Miyoshi en-aut-mei=Shin-ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=TokoNorie en-aut-sei=Toko en-aut-mei=Norie kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=DodoTetsuya en-aut-sei=Dodo en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=NankoAyako en-aut-sei=Nanko en-aut-mei=Ayako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MizunoTamaki en-aut-sei=Mizuno en-aut-mei=Tamaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=Vibrio mimicus kn-keyword=Vibrio mimicus en-keyword=Serine protease kn-keyword=Serine protease en-keyword=Hemolysin kn-keyword=Hemolysin en-keyword=Maturation kn-keyword=Maturation END start-ver=1.4 cd-journal=joma no-vol=12 cd-vols= no-issue= article-no= start-page=726273 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210820 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Virulence of Cholera Toxin Gene-Positive Vibrio cholerae Non-O1/non-O139 Strains Isolated From Environmental Water in Kolkata, India en-subtitle= kn-subtitle= en-abstract= kn-abstract=Cholera toxin (CT)-producing Vibrio cholerae O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, V. cholerae non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (ctx). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with ctx from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains in vivo was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium in vitro at two temperatures, 25 degrees C and 37 degrees C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25 degrees C, but that was low when cultured at 37 degrees C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of ctx. However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of V. cholerae O1, may emerge in various parts of the world and cause epidemics in the future. en-copyright= kn-copyright= en-aut-name=TakahashiEizo en-aut-sei=Takahashi en-aut-mei=Eizo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=OchiSadayuki en-aut-sei=Ochi en-aut-mei=Sadayuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MizunoTamaki en-aut-sei=Mizuno en-aut-mei=Tamaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MoritaDaichi en-aut-sei=Morita en-aut-mei=Daichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MoritaMasatomo en-aut-sei=Morita en-aut-mei=Masatomo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=OhnishiMakoto en-aut-sei=Ohnishi en-aut-mei=Makoto kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=KoleyHemanta en-aut-sei=Koley en-aut-mei=Hemanta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=DuttaMoumita en-aut-sei=Dutta en-aut-mei=Moumita kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=ChowdhuryGoutam en-aut-sei=Chowdhury en-aut-mei=Goutam kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=MukhopadhyayAsish K. en-aut-sei=Mukhopadhyay en-aut-mei=Asish K. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=DuttaShanta en-aut-sei=Dutta en-aut-mei=Shanta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=MiyoshiShin-Ichi en-aut-sei=Miyoshi en-aut-mei=Shin-Ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=OkamotoKeinosuke en-aut-sei=Okamoto en-aut-mei=Keinosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= affil-num=1 en-affil=Collaborative Research Center of Okayama University for Infectious Diseases in India kn-affil= affil-num=2 en-affil=Department of Health Pharmacy, Yokohama University of Pharmacy kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama University kn-affil= affil-num=4 en-affil=Collaborative Research Center of Okayama University for Infectious Diseases in India kn-affil= affil-num=5 en-affil=Department of Bacteriology I, National Institute of Infectious Diseases kn-affil= affil-num=6 en-affil=Department of Bacteriology I, National Institute of Infectious Diseases kn-affil= affil-num=7 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=8 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=9 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=10 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=11 en-affil=National Institute of Cholera and Enteric Diseases kn-affil= affil-num=12 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama University kn-affil= affil-num=13 en-affil=Collaborative Research Center of Okayama University for Infectious Diseases in India kn-affil= en-keyword=Vibrio cholerae kn-keyword=Vibrio cholerae en-keyword=NAG Vibrio kn-keyword=NAG Vibrio en-keyword=cholera toxin kn-keyword=cholera toxin en-keyword=virulence kn-keyword=virulence en-keyword=environmental water kn-keyword=environmental water en-keyword=gene analysis kn-keyword=gene analysis END