Elsevier BVActa Medica Okayama0013-93512222023Environmental water in Kolkata is suitable for the survival of Vibrio cholerae O1115374ENEizoTakahashiCollaborative Research Center of Okayama University for Infectious Diseases in IndiaKeiKitaharaCollaborative Research Center of Okayama University for Infectious Diseases in IndiaShin-ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama UniversityGoutamChowdhuryNational Institute of Cholera and Enteric DiseasesAsish K.MukhopadhyayNational Institute of Cholera and Enteric DiseasesShantaDuttaNational Institute of Cholera and Enteric DiseasesSadayukiOchiDepartment of Health Pharmacy, Yokohama University of PharmacyKeinosukeOkamotoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama UniversityMany patients with cholera emerge in Kolkata, India throughout the year. Such emergency indicates that cholera toxin-producing Vibrio cholerae O1 (toxigenic V. cholerae O1) are widespread in Kolkata. This suggests that the suitable conditions for replication of toxigenic V. cholerae O1 is provided in Kolkata. In previous studies, we found that the replication rate of toxigenic V. cholerae O1 is low in the low ionic aqueous solution. Then we measured the ion concentration in the environmental water of Kolkata. As a control, we measured them in Japanese environmental water. The ion concentration in the environmental water of Kolkata was significantly high. Then, we examined the survival of toxigenic V. cholerae O1 in groundwater from Kolkata and found that V. cholerae O1 survive for long time in the solution but not in the solution diluted with Milli Q water. In addition, we found that V. cholerae O1 proliferated in environmental water of Kolkata to which a small amount of nutrient was added, but did not grow in the environmental water diluted with water to which the same amount of nutrient was added. These results indicate that the environmental water from Kolkata is suitable for survival of V. cholerae O1.No potential conflict of interest relevant to this article was reported.Frontiers Media SAActa Medica Okayama1664-302X122021Virulence of Cholera Toxin Gene-Positive Vibrio cholerae Non-O1/non-O139 Strains Isolated From Environmental Water in Kolkata, India726273ENEizoTakahashiCollaborative Research Center of Okayama University for Infectious Diseases in IndiaSadayukiOchiDepartment of Health Pharmacy, Yokohama University of PharmacyTamakiMizunoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama UniversityDaichiMoritaCollaborative Research Center of Okayama University for Infectious Diseases in IndiaMasatomoMoritaDepartment of Bacteriology I, National Institute of Infectious DiseasesMakotoOhnishiDepartment of Bacteriology I, National Institute of Infectious DiseasesHemantaKoleyNational Institute of Cholera and Enteric DiseasesMoumitaDuttaNational Institute of Cholera and Enteric DiseasesGoutamChowdhuryNational Institute of Cholera and Enteric DiseasesAsish K.MukhopadhyayNational Institute of Cholera and Enteric DiseasesShantaDuttaNational Institute of Cholera and Enteric DiseasesShin-IchiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama UniversityKeinosukeOkamotoCollaborative Research Center of Okayama University for Infectious Diseases in IndiaCholera 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.No potential conflict of interest relevant to this article was reported.WileyActa Medica Okayama038556006462020Genomic characterization of antibiotic resistance‐encoding genes in clinical isolates of Vibrio cholerae non‐O1/non‐O139 strains from Kolkata, India: generation of novel types of genomic islands containing plural antibiotic resistance genes435444ENDaichiMoritaCollaborative Research Center of Okayama University for Infectious Diseases in India, Okayama UniversityEizoTakahashiCollaborative Research Center of Okayama University for Infectious Diseases in India, Okayama UniversityMasatomoMoritaDepartment of Bacteriology I, National Institute of Infectious DiseasesMakotoOhnishiDepartment of Bacteriology I, National Institute of Infectious DiseasesTamakiMizunoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shin‐ichiMiyoshiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityDevaratiDuttaDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesThandavarayanRamamurthyCenter for Human Microbial Ecology, Translational Health Science and Technology InstituteGoutamChowdhuryDivision of Bacteriology, National Institute of Cholera and Enteric Diseases Asish K.MukhopadhyayDivision of Bacteriology, National Institute of Cholera and Enteric DiseasesKeinosukeOkamotoCollaborative Research Center of Okayama University for Infectious Diseases in India, Okayama UniversityNon‐O1/non‐O139 nontoxigenic Vibrio cholerae associated with cholera‐like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non‐O1/non‐O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole‐genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance‐determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non‐O1/non‐O139 strains through the action of newly generated genomic islands.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama1880-70463612014Effects of Oral Administration of Non-genotoxic Hepato-hypertrophic Compounds on Metabolic Potency of Rat Liver19ENXingFangTatsuoNunoshibaMidoriYoshidaAkiyoshiNishikawaKiyomitsuNemotoMasakuniDegawaSakaeArimotoKeinosukeOkamotoEizoTakahashiTomoeNegishiIt remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6262008Toxin Production by Aeromonas sobria in Natural Environments: River Water vs. Seawater363371ENRaselKhanEizoTakahashiHironoriNakuraMohammadAnsaruzzamanSukalyaniBanikThandavarayanRamamurthyKeinosukeOkamotoOriginal Article10.18926/AMO/30947<p>Aeromonas are water-borne pathogens. They are halotolerant, which means that they can survive in environments whose salt content corresponds to that of seawater (3.0% NaCl). However, the presence of Aeromonas in seawater is extremely rare compared with that in river water. In this study, we tested the ability of Aeromonas sobria to produce toxins in river water and seawater. First, we cultured
A. sobria on skim milk agar plates supplemented with either river water (SARW) or seawater (SASW). The bacteria grew on both plates. A clear zone around the bacteria was generated in SARW. However, such a zone was not observed in SASW, suggesting that proteases were not generated in SASW. Subsequently, we cultured A. sobria in a nutrient broth supplemented with either river water (NRW) or with seawater (NSW), and examined the protease activity of their culture supernatants. The protease activity of the culture supernatant from NSW was extremely low compared to that from NRW. The immunoblotting analysis showed that serine protease (ASP) was not produced by the culture
in NSW. By contrast, aerolysin-like hemolysin was produced in all conditions examined in this study. This indicates that the salinity of water is deeply involved in the production of ASP by A. sobria.</p>No potential conflict of interest relevant to this article was reported.Acta Medica Okayama2005変異原物質高感度検出株の作製と突然変異の抑制に関する研究ENEizoTakahashiNo potential conflict of interest relevant to this article was reported.