Screening Of Marine Antagonistic Bacteria And Preliminary Study On Antibiotic Material Of Strain RF106

For the past few years,Antimicrobial resistance is increasing severity,and becoming one of the greatest challenges worldwide.However,the exploitation of new antibiotic was slow down,and dig antibiotic active substance from soil source microbe was bogged down.As the most important part of marine ecosystem,marine microbe evolved special physiology and biochemistry function,because of expose in high pressure,high concentration of salt,low temperature and low oxygen and other extreme environment condition,which particular different from land source microbe.Marine microbe has become important resource of antibiotic active substance,and shown enormous potential.In this research,four samples were collected from the sea shore of Weihai in China,and several different enrichment and medium were used to isolate antagonistic bacteria.After first selected,the purified bacterial 16S rDNA were amplified by PCR,and the sequence used to identify by blast in NCBI.After that,109 strains of marine potential antagonistic bacteria were collected,including 36 strains of actinomycetes,and 3 strains of novel species.For the verification research,we choose 4 pathogenic bacteria as standard stains to select active antagonistic bacteria,and 41 strains were affirmed.Strain RF106 is conducted a preliminary study,according to 16S rRNA gene sequence,it showed 99%similarity to Alcaligenes aquatilis.And stain RF106 still have antibiotic active after been fermented.We analysed the strain RF106 including phenotypic characteristics,physiological characteristics,exploring of antibacterial spectrum,preliminary optimization of fermentation conditions,analysis of genomic information and prediction of antagonistic genes by antiSMASH.In order to develop the database of marine microbial resources,2 strains isolated from different samples are identified by polyphasic taxonomy.Comparative sequence analysis of 16S rRNA genes led to placement of strain HA2012T in the genera Vibrio,and showed that strain HA2012T is closely related to Vibrio.gazogenes ATCC 29988T(95.35%).Strain HA2012T Gram-stain-negative,facultative anaerobic and slightly curved rods,without flagella.Colonies on MA are pale yellow,smooth,circular,entire,and less than 1 mm in diameter after 48 h of incubation at 30?.Growth occurs at 20-37 °C,pH 6.0-8.0,and in the presence of 1-7%(w/v)NaCl.Positive for catalase and oxidase activity.Starch and Tween 20 are hydrolyzed;agar,gelatin,alginate,carboxymethylcellulose,and Tweens 40,60,and 80 are not hydrolyzed.Positive for reduction of nitrate to N2 and production of H2S.Positive for alkaline phosphatase,esterase(C4),esterase lipase(C8),leucine arylamidase,valine arylamidase,cystine arylamidase,a-chymotrypsin,acid phosphatase,naphthol-AS-BI-phosphohydrolase,?-glucosidase and ?-glucosidase;The predominant fatty acids are C16;1 ?7c and/or iso-C15:0 2-OH and C16:0.Based on phenotypic and genetic characterization,a novel species Vibrio sediminis sp.nov.is proposed.The type strain is HA2012T(=KCTC 52272T =MCCC 1H00160T),which was isolated from a marine sediment obtained of Weihai,China.The DNA G+C content of the type strain is 45.4 mol%.Comparative sequence analysis of 16S rRNA genes led to placement of strains SD302T in the family Lewinellaceae,and showed that strain SD302T is closely related to L.aquimaris(92.62%).Strain SD302T Gram-negative,aerobic,flexible rods or filaments(sized 0.4-0.5x 5.8-4.5 ?m),without flagella.light orange yellow,smooth,opaque,circular and entire after 5 days of incubation at 30 ?.Growth occurs at 25-33 ?,pH 6.0-8.0,and in the presence of 1-4%(w/v)NaC1.Positive for oxidase and catalase activity.Tween 40,60,and 80 are hydrolyzed;starch,agar,alginate,carboxymethylcellulose,DNA and Tweens 20 are not hydrolyzed.Positive for reduction of nitrate to N2 and production of H2S.Positive for alkaline phosphatase,esterase(C4),esterase lipase(C8),leucine arylamidase,valine arylamidase,cystine and trypsin;negative for lipase(C14),a-chymotryp sin,a-galactosidase,?-galactosidase,?-glucuronidase,?-glucosidase,?-glucosidase,a-mannosidase,?-fucosidase.The sole respiratory quinone is MK-7 and the main cellular polar lipids are phosphatidylethanolamine,glycolipid,one unidentified phospholipid and one unidentified glyco-phospholipid.The predominant fatty acids are C16:1 ?7c and/or iso-C15:0 2-OH and iso-C15:0.The DNA G+C content is 50.4 mol%.Based on phenotypic and genetic characterization,a novel genus Bradybacter gen.nov.is proposed.The type strain is SD302T(=KCTC 52634T=MCCC 1H00222T),which was isolated from a marine sediment obtained of Weihai,China.