土壤来源抗MRSA放线菌的筛选及次级代谢潜力评估
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摘要
该研究采用4种分离培养基对32份土壤样本中的放线菌进行初步分离纯化,通过抗耐甲氧西林金黄色葡萄球菌(MRSA)抑菌活性实验进行菌株初筛,并采用聚合酶链式反应(PCR)扩增菌株16S r RNA和次级代谢相关基因,对活性菌株的代谢潜力进行评估。结果表明,从32份样本中共分离纯化到169株放线菌,其中68株放线菌具有抗MRSA活性。活性菌株主要为链霉菌属(Streptomyces sp.),且16S r RNA基因序列相似性<98%的菌株有10株,卤化酶(Hal)基因阳性菌株占17.65%,聚酮合酶I型(PKS I)基因阳性菌株占36.47%,聚酮合酶II(PKS II)基因阳性菌株占61.77%,非核糖体多肽合成酶(NRPS)基因阳性菌株占32.35%。表明黔北地区土样中放线菌资源丰富,具有从中发现放线菌新种和抗生素的潜力。
The Actinomycetes were primary isolated and purified from 32 soil samples using four separation media. The strains were screened out by anti-methicillin-resistant Staphylococcus aureus(MRSA) bacteriostatic test, and the 16 S rRNA and secondary metabolism-related genes were amplified by polymerase chain reaction to evaluate the metabolic potential of the active strain. The results showed that 169 strains of Actinomycetes were isolated and purified from 32 samples. Among them, 68 strains of Actinomycetes had anti-MRSA activity. The active strains were mainly Streptomyces sp., and there were 10 strains with 16 S rRNA gene sequence similarity less than 98%; halogenase(Hal) gene-positive strains accounted for17.65%, polyketide synthase type Ⅰ(PKS Ⅰ) gene-positive strains accounted for 36.47%, polyketide synthase Ⅱ(PKS Ⅱ) gene-positive strains accounted for 61.77%, and non-ribosomal polypeptide synthetase(NRPS) gene-positive strains accounted for 32.35%. Ⅰt was indicated that the Actinomycetes were rich in soil samples in the northern Guizhou province of China, and had the potential to discover new Actinomycetes and antibiotics with novel structure.
The Actinomycetes were primary isolated and purified from 32 soil samples using four separation media. The strains were screened out by anti-methicillin-resistant Staphylococcus aureus(MRSA) bacteriostatic test, and the 16 S rRNA and secondary metabolism-related genes were amplified by polymerase chain reaction to evaluate the metabolic potential of the active strain. The results showed that 169 strains of Actinomycetes were isolated and purified from 32 samples. Among them, 68 strains of Actinomycetes had anti-MRSA activity. The active strains were mainly Streptomyces sp., and there were 10 strains with 16 S rRNA gene sequence similarity less than 98%; halogenase(Hal) gene-positive strains accounted for17.65%, polyketide synthase type Ⅰ(PKS Ⅰ) gene-positive strains accounted for 36.47%, polyketide synthase Ⅱ(PKS Ⅱ) gene-positive strains accounted for 61.77%, and non-ribosomal polypeptide synthetase(NRPS) gene-positive strains accounted for 32.35%. Ⅰt was indicated that the Actinomycetes were rich in soil samples in the northern Guizhou province of China, and had the potential to discover new Actinomycetes and antibiotics with novel structure.
引文
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[2]SHI W,LI J,DONG F,et al.Serotype distribution,antibiotic resistance pattern and multilocus sequence types of invasive Streptococcus pneumoniae isolates in two tertiary pediatric hospitals in Beijing prior to PCV13availability[J].Expert Rev Vaccines,2019,18:89-94.
[3]ZHU H,SWIERSTRA J,WU C,et al.Eliciting antibiotics active against the ESKAPE pathogens in a collection of actinomycetes isolated from mountain soils[J].Microbiology,2014,160(8):1714-1725.
[4]PAYNE K D,DAS A,NDIULOR M,et al.Dosing strategies to optimize currently available anti-MRSA treatment options(Part 2:PO options)[J].Expert Rev Clin Pharmaco,2018,11(2):139-149.
[5]HASSOUN A,LINDEN P K,FRIEDMAN B.Incidence,prevalence,and management of MRSA bacteremia across patient populations-a review of recent developments in MRSA management and treatment[J].Crit Care,2017,21:211.
[6]RAN C,HICKS K,ALEXIEV B,et al.Cervicofacial necrotising fasciitis by clindamycin-resistant and methicillin-resistant Staphylococcus aureus(MRSA)in a young healthy man[J/OL].BMJ Case Rep,2018,http://dx.doi.org/10.1136/bcr-2018-226975.
[7]BERDY J.Thoughts and facts about antibiotics:Where we are now and where we are heading[J].J Antibiot,2012,65(6):385-395.
[8]MINGYAR E,NOVAKOVA R,KNISCHOVA R,et al.Unusual features of the large linear plasmid p SA3239 from Streptomyces aureofaciens CCM3239[J].Gene,2018,642:313-323.
[9]AGARWAL V,MILES Z D,WINTER J M,et al.Enzymatic halogenation and dehalogenation reactions:Pervasive and mechanistically diverse[J].Chem Rev,2017,117(8):5619-5674.
[10]LI X G,TANG X M,XIAO J,et al.Harnessing the potential of halogenated natural product biosynthesis by mangrove-derived actinomycetes[J].Mar Drugs,2013,11:3875-3890.
[11]LU Y,YUE C,SHAO M,et al.Molecular genetic characterization of an anthrabenzoxocinones gene cluster in Streptomyces sp.FJS31-2 for the biosynthesis of BE-24566B and zunyimycin ale[J].Molecules,2016,21(6):E711.
[12]LU Y,SHAO M,WANG Y,et al.Zunyimycins B and C,new chloroanthrabenzoxocinones antibiotics against methicillin-resistant Staphylococcus aureus and Enterococci from Streptomyces sp.FJS31-2[J].Molecules,2017,22(2):E251.
[13]杨小燕,金晶,周梦洁,等.利用靶向Ⅱ型聚酮合酶的PCR技术从海洋放线菌中发现angucyclinone聚酮类天然产物[J].中国药学,2017(3):173-179.
[14]李园园,保玉心,吕玉红,等.梵净山保护区土壤放线菌分离及其次级代谢产物初步研究[J].长江大学学报,2013(8):60-65.
[15]王苗,李园园,邵美云,等.赤水丹霞来源抗白色念珠菌放线菌分离及进化分析[J].遵义医学院学报,2014,37(4):404-408.
[16]岳昌武,李园园,黄兵,等.赤水丹霞山土壤来源抗菌活性链霉菌的分离及放线菌素产生链霉菌Streptomyces sp.CSDX001发酵产物分析[J].中国酿造,2014,33(11):41-46.
[17]王希玮,刘超兰,郭义东,等.湖泊放线菌SIIA-A16124的分类鉴定及基因组挖掘[J].中国抗生素杂志,2018,43(4):424-428.
[18]KUMAR S,STECHER G,LI M,et al.MEGA X:molecular evolutionary genetics analysis across computing platforms[J].Mol Biol Evol,2018,35(6):1547-1549.
[19]SAITOU N,NEI M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Mol Biol Evol,1987,4(4):406-425.
[20]LIAO L,CHEN R,JIANG M,et al.Bioprospecting potential of halogenases from Arctic marine actinomycetes[J].BMC Microbiol,2016,16:34.
[21]HOMUNG A,BERTAZZO M,DZIARNOWSKI A,et al.A genomic screening approach to the structure-guided identification of drug candidates from natural sources[J].Chem Bio Chem,2007,8(7):757-766.
[22]刘发旺,周梦洁,金晶,等.利用靶向非核糖体肽合成酶的PCR技术从链霉菌HMU0027中发现thiazostatin嗜铁素[J].中国药学,2017(10):47-56.
[23]程少军,刘鸿雁,龙云川,等.黔东北喀斯特土壤放线菌多样性研究[J].贵州大学学报,2017(3):35-40.
[24]CHRISTIANSEN G,DITTMANN E,VIA ORDORIKA L,et al.Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC[J].Arch Microbiol,2001,176(6):452-458.