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下一代测序辅助的药用植物内生细菌的筛选、分离、分子鉴定及抗菌潜力

Next generation sequencing-aided screening, isolation, molecular identification, and antimicrobial potential for bacterial endophytes from the medicinal plant, .

作者信息

Tlou Matsobane, Ndou Benedict, Mabona Nokufa, Khwathisi Adivhaho, Ateba Collins, Madala Ntakadzeni, Serepa-Dlamini Mahloro Hope

机构信息

Department of Biochemistry, School of Physical and Chemical Sciences, North-West University, Mmabatho, South Africa.

Department of Biochemistry and Microbiology, University of Venda, Thohoyandou, South Africa.

出版信息

Front Microbiol. 2024 May 22;15:1383854. doi: 10.3389/fmicb.2024.1383854. eCollection 2024.

Abstract

, a wild plant in southern Africa, is utilized in traditional medicine for various ailments, leading to its endangerment and listing on the Red List of South African Plants. To date, there have been no reports on bacterial endophytes from this plant, their classes of secondary metabolites, and potential medicinal properties. This study presents (i) taxonomic characterization of bacterial endophytes in leaf and root tissues using 16S rRNA, (ii) bacterial isolation, morphological, and phylogenetic characterization, (iii) bacterial growth, metabolite extraction, and LC-MS-based metabolite fingerprinting, and (iv) antimicrobial testing of bacterial crude extracts. Next-generation sequencing yielded 693 and 2,459 DNA read counts for the rhizomes and leaves, respectively, detecting phyla including Proteobacteria, Bacteroidota, Gemmatimonadota, Actinobacteriota, Verrucomicrobiota, Dependentiae, Firmicutes, and Armatimonodata. At the genus level, , , , and Ralstonia were the most dominant in both leaves and rhizomes. From root tissues, four bacterial isolates were selected, and 16S rRNA-based phylogenetic characterization identified two closely related sp. (strain BNWU4 and 5), BNWU2, and BNWU1. The ethyl acetate:chloroform (1:1 v/v) organic extract from each isolate exhibited antimicrobial activity against all selected bacterial pathogens. Strain BNWU5 displayed the highest activity, with minimum inhibitory concentrations ranging from 62.5 μg/mL to 250 μg/mL against diarrhoeagenic , , , antibiotic-resistant , , , and . LC-MS analysis of the crude extract revealed common antimicrobial metabolites produced by all isolates, including Phenoxomethylpenicilloyl (penicilloyl V), cis-11-Eicosenamide, 3-Hydroxy-3-phenacyloxindole, and 9-Octadecenamide.

摘要

作为南非的一种野生植物,因其在传统医学中用于治疗各种疾病,导致其濒危并被列入《南非植物红皮书》。迄今为止,尚未有关于该植物细菌内生菌、其次级代谢产物类别及潜在药用特性的报道。本研究展示了:(i)使用16S rRNA对叶和根组织中的细菌内生菌进行分类学表征;(ii)细菌分离、形态学及系统发育表征;(iii)细菌生长、代谢产物提取及基于液相色谱-质谱联用的代谢产物指纹图谱分析;(iv)细菌粗提物的抗菌测试。下一代测序分别在根茎和叶片中产生了693个和2459个DNA读数,检测到的门包括变形菌门、拟杆菌门、芽单胞菌门、放线菌门、疣微菌门、依赖菌门、厚壁菌门和装甲菌门。在属水平上,伯克霍尔德氏菌属、假单胞菌属、肠杆菌属和雷尔氏菌属在叶片和根茎中都是最主要的。从根组织中挑选出4株细菌分离株,基于16S rRNA的系统发育表征鉴定出两株密切相关的伯克霍尔德氏菌属菌株(菌株BNWU4和5)、洋葱伯克霍尔德菌BNWU2和皮氏伯克霍尔德菌BNWU1。各分离株的乙酸乙酯:氯仿(1:1 v/v)有机提取物对所有选定的细菌病原体均表现出抗菌活性。菌株BNWU5表现出最高活性,对致腹泻的大肠杆菌、金黄色葡萄球菌、肺炎克雷伯菌、耐抗生素的鲍曼不动杆菌、铜绿假单胞菌和白色念珠菌的最低抑菌浓度范围为62.5 μg/mL至250 μg/mL。粗提物的液相色谱-质谱联用分析揭示了所有分离株产生的常见抗菌代谢产物,包括苯氧甲基青霉素酰基(青霉素酰基V)、顺式-11-二十碳烯酰胺、3-羟基-3-苯甲酰氧基吲哚和9-十八碳烯酰胺。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/92c7/11160484/c9dd6406f7b2/fmicb-15-1383854-g001.jpg

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