察汗淖放线菌资源探查及抗菌活性筛选

Resource exploration and antibacterial activity screening of actinomycetes from Chahan Nur

摘要:

【背景】细菌耐药形势严峻，寻找新型抗生素迫在眉睫。放线菌是生产抗生素的重要药物资源。盐湖是一种高盐度的内陆水体，既往研究表明其中的放线菌物种多样、新资源丰富、生物活性广泛，极具开发潜力。【目的】探究察汗淖盐湖土壤中放线菌菌种的组成，并筛选抗菌活性菌株，为发现新颖的抗菌化合物储备菌种资源。【方法】采用19种选择培养基，以涂布平板法分离放线菌。基于16S rRNA基因的相似度鉴定菌株，并分析其多样性和新颖性。根据菌属类别及其新颖性，选择代表菌株进行Ⅰ型、Ⅱ型聚酮合酶(polyketide synthases, PKS)和非核糖体肽合成酶(non-ribosomal polypeptide synthases, NRPS)抗生素生物合成基因的PCR扩增；代表菌株的发酵液上清和菌体分别经乙酸乙酯和丙酮提取后，采用纸片扩散法进行抗菌活性检测。【结果】从9个土壤样品中分离获得250株放线菌，隶属于放线菌纲的9个目16个科28个属，链霉菌属(88株，占比35.2%)和拟诺卡氏菌属(68株，占比27.2%)为优势菌属。15株链霉菌与近缘菌株16S rRNA基因的最高相似度低于98.65%，推测其归属于4个链霉菌属新种，同时还发现了拟诺卡氏菌属和类诺卡氏菌属新种。75株代表菌株中，66株放线菌至少检出1种抗生素生物合成基因，20株同时检出3种功能基因。筛选出7株抗菌活性较强的放线菌，包括5株链霉菌、1株拟诺卡氏菌和1株原小单孢菌。【结论】察汗淖盐湖土壤中可培养放线菌资源较丰富，部分菌株产生的活性物质抗菌活性强，值得进一步开展次级代谢产物的研究。

Abstract:

[Background] Due to the wide spread of drug-resistant bacteria, new antibiotics are urgently needed. Actinomycetes are major antibiotic-producing pharmaceutical resources. Saline lakes are special water bodies with high concentrations of dissolved salts. Studies have demonstrated that actinomycetes with high diversity, rich novel metabolites, and a wide range of biological activities from saline lakes are potential resource for drug discovery. [Objective] This study aims to reveal the composition of actinomycete species in the soil samples collected from Chahan Nur and identify the strains with antibacterial activities, which will accumulate strains for the discovery of novel antibacterial compounds. [Methods] The actinomycetes were isolated by dilution coating method in 19 selective culture media. The diversity and novelty of the isolates were analyzed by comparison of 16S rRNA genes. According to the taxa and novelty, representative isolates were selected for detection of the genes involved in the biosynthesis of type Ⅰ, type Ⅱ polyketide synthases (PKS) and non-ribosomal polypeptide synthases (NRPS) by PCR. The fermented broths and mycelia of representative isolates were extracted with ethyl acetate and acetone, respectively, and then antibacterial tests were carried out with the disk diffusion method. [Results] A total of 250 actinomycete strains were isolated from nine soil samples, belonging to 28 genera, 16 families of 9 orders, and Streptomyces (88 isolates, 35.2%) and Nocardiopsis (68 isolates, 27.2%) were the dominant genera. Fifteen Streptomyces isolates showed the highest 16S rRNA gene similarities below 98.65% to relative strains, which implied they might belong to four novel Streptomyces species. Meanwhile, novel species of Nocardiopsis and Nocardioides were discovered. Among the 75 tested strains, 66 strains were detected with at least one gene involved in antibiotic biosynthesis, and 20 strains carried all the three functional genes. Seven actinomycete strains with strong antibacterial activities were screened out, including five isolates of Streptomyces, one isolate of Nocardiopsis, and one isolate of Promicromonospora. [Conclusion] Soil samples from Chahan Nur have abundant culturable actinomycetes, and the active substances from some strains have strong antibacterial activities. Therefore, the secondary metabolites of these strains are worthy of further study.