The Analysis Of Drug-resistance Bacteria Of Holothurian Cultures And Polyphasic Taxonomy Analysis Of Three Novel Marine Strains

Samples were collected from holothurian culture ponds of Weihai, China (36°54’36"N,122°14’33"E) at different time. Then, we counted the total number of culturable bacteria and levofloxacin-resistance bacteria of different samples collected from different environments  different time. The results showed that the total number of culturable bacteria peaked in October, in contrast, the number of the levofloxacin-resistance bacteria was not. This might result from the use of Microecologics, exponential growth of the probiotics reduced the number of levofloxacin-resistance bacteria. More than 500 strains who showed significant difference in morphology were isolated and stored at-80 ℃. Antibiotic sensitivity was assessed using Disk-diffusion method as described by Clinical and Laboratory Standards Institute. The results indicated that resistance level peaked in August, and the antibacterial effect of tetracycline, nalidixic acid and kanamycin were poor. At last, we detected the class I integron of 166 resistance strains using PCR technique,49 of them collected from aquaculture region and 117 from environment, detection rate was 4.1% and 8.5%, respectively.We identified the taxonomic position of some strains by sequencing the 16S rRNA gene. The method of polyphasic taxonomy was also used to identify the accurate taxonomic position of strains HS6T, FA350T and XK5T.A novel Gram-strain-negative, rods-to-spiral-shaped, oxidase- and catalase-positive and facultatively aerobic bacterium, designated HS6T, was isolated from marine sediment of Yellow Sea, China. It can reduce nitrate to nitrite and grow well in marine broth 2216 (MB; HopeBio) with an optimal temperature for growth of 30-33 ℃ (range 12-45 ℃) and in the presence of 2-3%(w/v) NaCl (range 0.5-7%, w/v). The pH range for growth was pH 6.2-9.0, with an optimum at 6.5-7.0. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that the novel isolate was 93.3% similar to the type strain of Neptunomonas antarctica,93.2% to Neptunomonas japonicum and 93.1% to Marinobacterium rhizophilum, the closest identified relatives. The polar lipid profile of the novel strain consisted of phosphatidylethanolamine, phosphatidylglycerol and some other unknown lipids. Major cellular fatty acids were summed feature 3 (C16:1 ω7c/iso-C15:0 2-OH), C18:1 ω7c and C16:0 and the main respiratory quinone was Q-8. The DNA G+C content of strain HS6T was 61.2 mol%. Based on the phylogenetic, physiological and biochemical characteristics, strain HS6T represents a novel genus and species and the name Motiliproteus sediminis gen. nov., sp. nov. is proposed. The type strain is HS6T (=ATCC BAA-2613T= CICC 10858T).A novel Gram-stain-negative, rod-shaped, gliding, facultatively anaerobic, oxidase-negative and catalase-positive bacterium, designated FA350T, was isolated from coastal sediment from Xiaoshi Island, Weihai, China. Strain FA350T showed growth on modified nutrient agar supplemented with 0.1% D-(+)-trehalose and with distilled water replaced by seawater. Optimal growth occurred at 33 ℃ and pH 8.5 with 4%(w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain FA350T belongs to a novel bacterial order in the class Deltaproteobacteria and the most closely related type strains belong to the order Desulfuromonadales, with 85.1-85.6% 16S rRNA gene sequence similarities. The polar lipid profile of the novel strain consisted of phosphatidylethanolamine, phosphatidylglycerol and two unknown phospholipids. Major cellular fatty acids were iso-C15:0, iso-C17:0 and iso-C17:1 ω10c and menaquinone MK-7 was the sole respiratory quinone. The DNA G+C content of strain FA350T was 60.3 mol %. The isolate and its closely related environmental clones formed a novel order-level clade in the class Deltaproteobacteria. Comparative analysis of 16S rRNA gene sequences and characterization indicated that strain FA350T may represent a novel order of Deltaproteobacteria. Here, we propose the name Bradymonas sediminis gen. nov., sp. nov. for strain FA350T (=DSM 28820T=CICC 10904T); Bradymonadales ord. nov. and Bradymonadaceae fam. nov. are also proposed and represented by strain FA350T.A novel Gram-stain-negative, rods or bent rods, facultatively anaerobic, oxidase- negative and catalase-positive bacterium, designated XK5T, was isolated from coastal sediment from Xiaoshi Island, Weihai, China. Strain XK5T was highly adaptable, able to tolerate poor conditions. Optimal growth occurred at 28-35℃ (range 8-42℃) and pH 7.0-8.0 (range 6.0-9.0) with 1-3%(w/v) NaCl (range 0.5-8%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain XK5T was 92.1% similar to the type strain of Thioalkalivibrio thiocyanodenitrificans,91.9% to T. sulfidiphilus and 91.8% to T. denitrificans, these are the only three strains show>91% sequence similarity. The polar lipid profile of the novel isolate consisted of phosphatidylethanolamine, phosphatidylglycerol and some other unknown phospholipids, aminolipids and lipids. Major cellular fatty acids were iso-C17:1 ω9c and iso-C15:0 and the main respiratory lipoquinone was Q-8. The DNA G+C content of strain XK5T was 59.3 mol%. Comparative analysis of 16S rRNA gene sequences and characterization indicated that strain XK5T may represent a novel genus of the family Ectothiorhodospiraceae. Here we propose the name Woeseia oceani gen. nov., sp. nov. to accommodate strain XK5T. The type strain of Woeseia oceani is XK5T (=ATCC BAA-2615T=CICC 10905T).