金黄色葡萄球菌拮抗菌株的筛选鉴定及其抗菌物质分析

Screening and identification of antagonistic strains against Staphylococcus aureus

摘要:

【背景】植物内生菌的次生代谢产物是新型天然活性物质的重要来源。【目的】从芍药内生细菌中筛选对金黄色葡萄球菌有抑菌活性的菌株和次生代谢产物。【方法】采用平板对峙法筛选拮抗菌株，根据形态学特征和分子生物学的方法鉴定菌株，PCR扩增检测合成脂肽类物质的功能基因；运用牛津杯法依次测定内生细菌发酵液和脂肽类粗提物的抑菌活性，利用Sephadex LH-20凝胶层析分离脂肽类物质，利用基质辅助激光解吸电离飞行时间质谱分析具有抑菌作用的分离组分。【结果】共筛选出13株对金黄色葡萄球菌具有不同程度抑制作用的内生菌株，其中菌株SY11的抑菌作用最为显著，其发酵液和脂肽类粗提物均具有较强的抑制作用。结合形态学鉴定以及16S rRNA基因序列分析，鉴定其为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。PCR扩增检测表明菌株SY11含有3个合成脂肽类物质的功能基因fenA、ituD和srfkn，推测该菌株可能具有合成脂肽类物质的能力。根据具有抑菌活性分离组分的质谱分析结果，推测其有效物质的主要成分为Bacillomycin D。【结论】解淀粉芽孢杆菌SY11对金黄色葡萄球菌有良好抑制效果，其脂肽类粗提物也具有较强的体外抑菌活性。本研究为芍药内生细菌的开发应用奠定了基础。

Abstract:

[Background] The secondary metabolites of endophytic bacteria are the important sources of new natural active substances. [objective] We aimed to isolate the bacterial strains and their secondary metabolites with antibacterial activity against Staphylococcus aureus from Paeonia lactiflora. [Methods] The antagonistic strains were screened against Staphylococcus aureus through the method of dual culture. The strains were indentified based on characteristics in morphology and DNA identification. The biosynthetic genes of lipopeptide were amplified using polymerase chain reaction, and the antibacterial activity of their fermentation and crude lipopeptides were detected using the method of Oxford cup. The antibacterial activity of the crude lipopeptides was isolated through Sephadex LH-20 gel chromatography, which was analyzed by MALDI-TOF mass spectrometry. [Results] A total of 13 antagonistic strains were screened from the endophytic bacteria isolates, which had different degrees of inhibition against Staphylococcus aureus. The isolates SY11 had the most obvious inhibition ability, and their fermentation and crude lipopeptides had strong inhibitory effect. The isolates SY11 was identified as Bacillus amyloliquefaciens based on characteristics in morphology and phylogenetic analysis of 16S rRNA gene sequences. Three biosynthetic genes of lipopeptide, fenA, ituD and srfkn were detected using polymerase chain reaction. The results suggested that the strain had the ability to synthesize lipopeptide. By further analysis of MALDI-TOF mass spectrometry. The main active substance was presumed as Bacillomycin D. [Conclusion] Bacillus amyloliquefaciens SY11 had significant inhibitory effect on Staphylococcus aureus, and their crude lipopeptides also showed inhibitory activity against Staphylococcus aureus in vitro, which laid the foundation for the further development and application of endophytes from Paeonia lactiflora.