Antimicrobial & Biocontrol Agents Unit (AmBcAU), Laboratory for Phytobiochemistry and Medicinal Plants Studies, Department of Biochemistry, Faculty of Science, University of Yaoundé I, Cameroon, Messa, Yaoundé, Cameroon.
PLoS One. 2022 May 11;17(5):e0267246. doi: 10.1371/journal.pone.0267246. eCollection 2022.
Urinary tract infections (UTIs) are common bacterial infections. The global emergence of multidrug-resistant uropathogens in the last decade underlines the need to search for new antibiotics with novel mechanisms of action. In this regard, exploring endophytic fungi inhabiting medicinal plants used locally against urinary tract infections could be a promising strategy for novel drug discovery. This study investigates crude metabolites from endophytic fungi isolated from Annona muricata as potential sources of antibiotic drugs to fight against uropathogens and reduce related oxidative stress. Crude ethyl acetate extracts from 41 different endophytic fungi were screened against three bacterial strains using the broth microdilution method, and fungi producing active crude extracts were identified using ITS1-5.8S rRNA-ITS2 nucleotide sequences. The antibacterial modes of action of the five most active extracts were evaluated using Staphylococcus aureus ATCC 43300 and Klebsiella oxytoca strains. The DPPH and FRAP assays were used to investigate their antioxidant activity, and their cytotoxicity against the Vero cell line was evaluated using the MTT assay. Out of the 41 crude extracts tested, 17 were active with minimum inhibitory concentrations (MICs) ranging from 3.125 μg/mL to 100 μg/mL and were not cytotoxic against Vero cell lines with a cytotoxic concentration 50 (CC50) >100 μg/mL. The more potent extracts (from Fusarium waltergamsii AMtw3, Aspergillus sp. AMtf15, Penicillium citrinum AMf6, Curvularia sp. AMf4, and Talaromyces annesophieae AMsb23) significantly inhibited bacterial catalase activity, lysed bacterial cells, increased outer membrane permeability, and inhibited biofilm formation, and the time-kill kinetic assay revealed concentration-dependent bactericidal activity. All seventeen extracts showed weak ferric iron-reducing power (1.06 to 12.37 μg equivalent NH2OH/g of extract). In comparison, seven extracts exhibited DPPH free radical scavenging activity, with RSA50 ranging from 146.05 to 799.75 μg/mL. The molecular identification of the seventeen active fungi revealed that they belong to six distinct genera, including Aspergillus, Curvularia, Fusarium, Meyerozyma, Penicillium, and Talaromyces. This investigation demonstrated that fungal endophytes from Cameroonian Annona muricata, a medicinal plant used locally to treat bacterial infections, might contain potent antibacterial metabolites with multiple modes of action. The antibacterial-guided fractionation of these active extracts is currently ongoing to purify and characterise potential antibacterial active ingredients.
尿路感染（UTIs）是常见的细菌感染。在过去十年中，全球出现了对多种药物具有抗药性的尿路病原体，这突显出需要寻找具有新型作用机制的新抗生素。在这方面，探索本地用于治疗尿路感染的药用植物中的内生真菌可能是发现新型药物的一种很有前途的策略。本研究调查了从番荔枝中分离出的内生真菌的粗代谢产物，作为对抗尿路病原体和减轻相关氧化应激的抗生素药物的潜在来源。使用肉汤微量稀释法对 41 种不同内生真菌的粗乙酸乙酯提取物进行了筛选，并用 ITS1-5.8S rRNA-ITS2 核苷酸序列鉴定产生活性粗提取物的真菌。使用金黄色葡萄球菌 ATCC 43300 和产酸克雷伯氏菌菌株评估了五种最活跃提取物的抗菌作用模式。使用 DPPH 和 FRAP 测定法研究了它们的抗氧化活性，并使用 MTT 测定法评估了它们对 Vero 细胞系的细胞毒性。在测试的 41 种粗提物中，有 17 种具有最低抑菌浓度（MIC）范围为 3.125 μg/mL 至 100 μg/mL 的活性，对 Vero 细胞系无细胞毒性，细胞毒性浓度 50（CC50）>100 μg/mL。更有效的提取物（来自于尖孢镰刀菌 AMtw3、曲霉 AMtf15、桔青霉 AMf6、弯孢菌 AMf4 和拟青霉 AMsb23）显著抑制了细菌过氧化氢酶的活性，裂解了细菌细胞，增加了外膜通透性并抑制了生物膜的形成，时间杀伤动力学试验显示出浓度依赖性的杀菌活性。所有 17 种提取物均表现出较弱的铁还原能力（1.06 至 12.37 μg 氨羟乙酸当量/g 提取物）。相比之下，有 7 种提取物显示出 DPPH 自由基清除活性，RSA50 范围为 146.05 至 799.75 μg/mL。对 17 种活性真菌的分子鉴定表明，它们属于六个不同的属，包括曲霉属、弯孢菌属、镰刀菌属、梅耶罗氏酵母属、青霉属和拟青霉属。本研究表明，喀麦隆药用植物番荔枝中的真菌内生菌可能含有具有多种作用模式的潜在抗菌代谢产物。目前正在对这些活性提取物进行抗菌指导的分离，以纯化和表征潜在的抗菌活性成分。
