Collection, isolation and classification of marine vibrios from coastal waters in Taiwan

• Main Authors: Hsiu-Hui Chiu, 邱秀慧
• Other Authors: Wung Yang Shieh
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/58867212098044200279

博士 === 國立臺灣大學 === 海洋研究所 === 94 === Marine vibrios are heterotrophic, halophilic, facultatively anaerobic, Gram-negative, rod-shaped bacteria capable of fermenting glucose and other carbohydrates. They are ubiquitous in estuarine, coastal and oceanic waters and in marine sediments. They are closely associated with many kinds of marine organisms from plankton to fish. According to 16S rRNA, marine vibrios belongs to γ-Proteobacteria. Eighty Vibrionaceae species are classified into 5 genii including Enterovibrio, Grimontia, Photobacterium, Salinovibrio and Vibrio. Enterovibrio and Grimontia. This paper described 16S rDNA- and recA-RFLP (restriction fragment length polymorphism) of known Vibrionaceae species and identification and classification of vibrio-like strains, luminous, high-NaCl tolerant vibrios. Two unidentified strains of glucose-fermentation and gas-producting bacteria, FG1T and VR1T, reported by us previously were proven to represent two novel Vibrio species, respectively. The 16S rDNA sequences of eighty-two Vibrionaceae species were simulated by four restriction endonuclease enzymes, DdeI, HhaI, RsaI and ScaI, and were classified into seven, thirteen, ten and four restriction types. These Vibrionaceae species were separated into forty-six genotypes. Eighteen species in first and fourth genotypes could be classified into eighteen types by the restriction patterns of recA. The RFLP analysis of 16S rDNA and recA was a simple method to classify vibrios. Forty-one strains were chosen by the Gram-negative reaction, NaCl-requirement and glucose-fermentation from 587 vibrio strains in our laboratory. The selected strains were characterized into twenty-three groups by the features of swarming, agarase, oxidase, fermentation with gas-production, arginine dihydrolase, lysine decarboxylase, 42˚C, growth in 10% NaCl and utilization of mannose as sole carbon source. These isolates were classified into 14 genotypes by 16S rRNA-RFLP digested with the four enzymes, DdeI, HhaI, RsaI and ScaI. Ten genotypes were new and different from standard strains in Vibrionaceae. The analysis of recA-RFLP had distinguished into different strains in the same 16S rRNA genotype. All of them were halophilic, facultatively anaerobic, Gram-negative rods, and could be identified as the family of Vibrionaceae. According to the growth at 4 and 40˚C, in 8% NaCl and utilization of mannitol and sucrose as sole carbon source, these isolates were divided into five groups. The first group had the highest similarity with Photobacterium leiognathi (99.4%). The other 4 groups had higher than 98% similarity with Vibrio harveyi or its closely related species. However, they could be clustered into only two genotypes according to the analysis of 16S rRNA-RFLP digested with various restriction enzymes (DdeI, HhaI, RsaI and ScaI). The characterization data together with a 16S rRNA gene-based phylogenetic analysis revealed that the isolates included in phenotype I (seven isolates) could be members of Photobacterium leiognathi subsp. mandapamensis (ATCC 25521T), while the strains included in phenotypes II-V (twenty isolates) might be classified as V. harveyi (ATCC 14126T). However, phylogeny based on gyrB sequences indicated that phenotypes II-V could be classified into two species, Vibrio harveyi and Vibrio campbellii. Culturable luminous bacteria in the shallow coastal waters of Taiwan during the sampling period are dominated by V. harveyi/campbellii and P. leiognathi, and the former species appeared to be more prevalent and numerous than the latter species in general. Besides Salinovibrio, we want to find out if another vibrios could tolerate more than 10 or 12% NaCl. The coastal water samples were collected along the northern Taiwan. The count of high-NaCl tolerate bacteria was between 0-1500 CFU/ml. From the counting plates, twenty-five bacterial strains were isolated. According to the swarming, agarase, fermentation with gas-production, ornithine decarboxylase, lysine decarboxylase, growth at 42˚C and utilization β-hydroxybutyrate as sole carbon source, the twenty-five isolates were classified into fourteen groups. The isolates were further classified into eight genotypes by 16S rRNA-RFLP digested with 4 enzymes described above. Three strains, L3AN-7, H3AN-1 and 2HAN4-3, could grow in PY broth with 15% NaCl. L3AN-7 was identified as Vibrio natriegens (ATCC 14048T) with similarity of 99.7%. H3AN-1 and 2HAN4-3 were classified as Salinivibrio costicola (NCIMB 701T) with similarities 98.9 and 98.4%. Two strains, FG1T and VR1T, could both ferment glucose with gas production and were isolated from a seagrass bed sediment sample collected from Nanwan Bay and a sea-water sample collected in the shallow coastal region of Keelung, Taiwan. FG1T and VR1T were motile by means of two flagella at on pole of the cell and one polar sheathed flagellum, respectively. Strain FG1T required Na+ for growth, and exhibited optimal growth at 30-35°C, pH 6-7 and about 4% NaCl. The strain VR1T grew optimally at 25-30°C and pH 6-7 and required the presence of NaCl, the optimal concentration being about 2%. Both strains, FG1T and VR1T, grew anaerobically by fermenting glucose and other carbohydrates and producing acids and gases. The DNA G+C content of FG1T and VR1T were 45.9 and 45.8 mol%. The major cellular fatty acids of FG1T and VR1T were 2-OH-16:0 & 3-OH-14:0 and C12:0. Characterization data, together with the results of a 16S rDNA-based phylogenetic analysis, indicate that strain FG1T represents a new species of the genus Vibrio. Strain VR1T is phenotypically similar to Vibrio gazogenes. However, the reduction of nitrate to nitrite, the ability to utilize D-arabinose, melibiose and L-glycine as sole carbon sources, the inability to utilize sorbitol as a sole carbon source, resistance to O/129 and susceptibility to erythromycin and novobiocin allow differentiation between V. gazogenes and strain VR1T. The names Vibrio aerogenes sp. nov. and Vibrio ruber sp. nov. are proposed for the novel species, with strains FG1T and VR1T as the type strains.