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来自巴西潘塔纳尔湿地的海茅蔺内生放线菌产生具有抗菌和抗肿瘤活性的化合物。

Endophytic actinobacteria of Hymenachne amplexicaulis from the Brazilian Pantanal wetland produce compounds with antibacterial and antitumor activities.

机构信息

Postgraduate Program in Genetics, Department of Genetics, Federal University of Paraná (UFPR), Centro Politécnico, Jardim das Américas, 81531-990, Curitiba, Paraná State, Brazil.

Postgraduate Program in Genetics, Department of Genetics, Federal University of Paraná (UFPR), Centro Politécnico, Jardim das Américas, 81531-990, Curitiba, Paraná State, Brazil; University Center - Católica de Santa Catarina, Joinville, SC, Brazil.

出版信息

Microbiol Res. 2021 Jul;248:126768. doi: 10.1016/j.micres.2021.126768. Epub 2021 Apr 6.

Abstract

The increase in the number of deaths from infections caused by multidrug-resistant bacteria and cancer diseases highlights the need for new molecules with biological activity. Actinobacteria represent a potential source of new compounds, as these microorganisms have already produced a great diversity of clinically employed antibiotics. Endophytes from unexplored biomes, such as the Pantanal (the largest wetland in the world), can be a source of new molecules. Hymenachne amplexicaulis is among the unexplored native plants of the Pantanal in terms of its endophytic community. This plant is considered a weed in other countries due to its ability to adapt and compete with native plants, and there is evidence to suggest that the endophytic community of H. amplexicaulis plays an important role in this competitiveness. To explore its therapeutic potential, the present study isolated, identified (using partial sequence of the 16S rDNA) and bioprospected H. amplexicaulis endophytic actinobacteria. Ten isolates belonging to the genera Streptomyces, Microbispora, Leifsonia, and Verrucosispora were obtained from root fragments. The susceptibility profile of the isolates to the different classes of antibiotics was evaluated, with 80 % of the isolates showing resistance to the antibiotics Nalidixic Acid, Ampicillin, Chloramphenicol, Oxacillin, and Rifampicin. To assess antibacterial and antitumor activities, methanolic extracts were obtained by fermentation in SG culture medium at 36 °C at 180 rpm for 10 days. The extract produced from the S. albidoflavus CMRP4854 isolate was the only one to show activity against the Gram-negative bacterium Acinetobacter baumanii. Due to the great clinical importance of this pathogen and the difficulty in obtaining active compounds against it, the CMRP4854 isolate should be further investigated for the identification of active compounds and mode of action. We also emphasize the results obtained by the extract of the isolates Streptomyces albidoflavus CMRP4852 and Verrucosispora sp. CMRP4860 that presented antibacterial effect against Methicilin-resistant Staphylococcus aureus (MRSA) (MIC: 1.5 μg/mL and 13 μg/mL, respectively) and Vancomycin-resistant Enterococcus (VRE) (MIC: 40 μg/mL for both extracts). Extracts (200 μg/mL) of these two endophytes also showed selective cytotoxicity action against murine B16-F10 melanoma cells. However, the CMRP4852 extract also affected the density of normal cells. Due to these results, the crude extract of isolate CMRP4860 Verrucosispora sp., which was the only one that presented cytotoxicity and reduced cell density only in tumor cells, was selected for subsequent analysis involving scale-up fermentation of the CMRP4860 resulting in 9 fractions that were tested against both bacteria and tumor cells, with particular fractions showing promise and meriting further investigation. Taken together, the results of this study not only show for the first time that the endophytic community of H. amplexicaulis actinobacteria can produce secondary metabolites that potentially possess important antibacterial and cytotoxic properties, but also reinforce the pressing need to conserve biomes such as the Brazilian Pantanal.

摘要

由于耐多药细菌和癌症疾病导致的死亡人数增加,突显了需要具有生物活性的新分子。放线菌是新化合物的潜在来源,因为这些微生物已经产生了大量临床上使用的抗生素。潘塔纳尔(世界上最大的湿地)等未开发生物群落的内生菌可能是新分子的来源。在潘塔纳尔未被探索的本地植物中,海茅草(Hymenachne amplexicaulis)的内生群落尚待研究。由于其适应和与本地植物竞争的能力,这种植物在其他国家被认为是一种杂草,有证据表明,海茅草的内生群落在这种竞争力中发挥着重要作用。为了探索其治疗潜力,本研究从根片段中分离、鉴定(使用 16S rDNA 的部分序列)并对海茅草内生放线菌进行了生物勘探。从根片段中获得了属于链霉菌属、微孢菌属、莱氏菌属和疣孢菌属的 10 个分离株。评估了分离株对不同类抗生素的敏感性,80%的分离株对萘啶酸、氨苄西林、氯霉素、苯唑西林和利福平表现出耐药性。为了评估抗菌和抗肿瘤活性,在 36°C、180rpm 的 SG 培养基中通过发酵获得了甲醇提取物,在 10 天内进行。唯一表现出对革兰氏阴性菌鲍曼不动杆菌活性的是 S. albidoflavus CMRP4854 分离株产生的提取物。由于这种病原体具有重要的临床意义,而且很难获得针对它的活性化合物,因此应该进一步研究 CMRP4854 分离株,以鉴定活性化合物和作用模式。我们还强调了从链霉菌属和疣孢菌属分离株 Streptomyces albidoflavus CMRP4852 和 Verrucosispora sp. CMRP4860 的提取物获得的结果,它们对耐甲氧西林金黄色葡萄球菌(MRSA)(MIC:1.5μg/mL 和 13μg/mL)和耐万古霉素肠球菌(VRE)(MIC:两种提取物均为 40μg/mL)均表现出抗菌作用。这两种内生菌的提取物(200μg/mL)也对鼠 B16-F10 黑色素瘤细胞表现出选择性细胞毒性作用。然而,CMRP4852 提取物也影响了正常细胞的密度。鉴于这些结果,选择了分离株 CMRP4860 的粗提取物 Verrucosispora sp. 进行后续分析,该提取物仅在肿瘤细胞中表现出细胞毒性和降低细胞密度,随后进行了放大发酵,得到了 9 个馏分,这些馏分都对抗菌和肿瘤细胞进行了测试,其中一些馏分显示出了希望,并值得进一步研究。总之,本研究的结果不仅首次表明,海茅草内生放线菌的内生群落可以产生可能具有重要抗菌和细胞毒性特性的次生代谢物,而且还强调了保护巴西潘塔纳尔等生物群落的紧迫需要。

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