抗金黄色葡萄球菌融合魏斯氏菌细菌素的理化特性分析及全基因组测序分析

doi:10.16431/j.cnki.1671-7236.2024.10.036

摘要/Abstract

摘要： 【目的】筛选产抗金黄色葡萄球菌细菌素的乳酸菌，分析其所产细菌素的理化特性并对其进行全基因组测序分析。【方法】本研究以金黄色葡萄球菌为指示菌株，从吉林省延边市的桔子中筛选产抑菌物质的乳酸菌。对筛选得到的菌株进行形态学观察和相似性鉴定，并构建系统发育树。通过排酸、排过氧化氢和蛋白酶敏感试验对抑菌物质进行初步分析，并测定抑菌物质对温度和紫外线的耐受性。对菌株进行全基因组测序及功能分析。【结果】筛选得到1株产抗金黄色葡萄球菌抑菌物质的乳酸菌，命名为LMM-1，在MRS固体培养基上为乳白色、光滑湿润的圆形菌落；革兰染色后在油镜下为短杆阳性菌。核酸序列经16S rRNA鉴定与融合魏斯氏菌相似性为99.86%，确定LMM-1为融合魏斯氏菌。排除有机酸试验结果显示，发酵上清液抑菌活性显著大于对照组；排除过氧化氢试验结果显示，抑菌活性几乎不变，可排除有机酸和过氧化氢的干扰。经蛋白酶处理分离菌抑菌产物对金黄色葡萄球菌的抑菌作用降低；在100 ℃高温、pH 2.0～8.0和2 h的紫外线照射下，仍具有良好抑菌活性。基因测序发现,LMM-1基因组长2 194 218 bp，共有2 045个基因被预测，基因组平均GC含量为44.97%。GO功能富集分析结果显示，分子功能中的催化活性和生物过程中的代谢过程得到的基因组功能注释最多，分别为905和801个。KEGG通路富集分析结果显示，在遗传代谢、遗传信息处理和环境信息处理中有49条代谢通路，共有1 050个基因得到功能注释。【结论】融合魏斯氏菌LMM-1产细菌素对金黄色葡萄球菌具有良好的抑菌活性，且细菌素具有一定的耐酸碱、高温和紫外线的能力，对环境具有良好的适应性，该菌含有较多与催化活性和新陈代谢相关的基因。本研究结果可为抗金黄色葡萄球菌替抗产品的研发奠定基础。

关键词: 融合魏斯氏菌; 抗菌作用; 细菌素; 金黄色葡萄球菌

Abstract: 【Objective】 This study was aimed to screen a lactic acid bacteria producing bacteriocin resistant to Staphylococcus aureus,analyze the physicochemical characteristics of the bacteriocin produced by the strain,and analyze the whole genome sequencing of the strain.【Method】 In this study,lactic acid bacterum producing antibacterial substances was screened from oranges in Yanbian city,Jilin province,using Staphylococcus aureus as an indicator strain.Morphological observation and similarity identification were performed on the selected strain,and a phylogenetic tree was constructed.Preliminary analysis of the antibacterial substances was carried out by acid excretion,hydrogen peroxide excretion and protease sensitivity tests,and the tolerance of the antibacterial substances to temperature and ultraviolet light was determined.Whole genome sequencing and function was performed on the strains.【Result】 One strain of lactic acid bacteria producing antibacterial substances against Staphylococcus aureus was obtained named as LMM-1,which showed milky white,smooth and moist round colonies on MRS solid medium.After Gram staining,the bacteria were short rod positive under the oil microscope.The nucleic acid sequence was identified by 16S rRNA to be 99.86% similar to the Weissella confuse,it was confirmed that LMM-1 was Weissella confuse.The antibacterial activity of the fermentation supernatant was significantly higher than that of control group when the organic acid was excluded,and the antibacterial activity of antibacterial products of the isolate was almost unchanged when the hydrogen peroxide was excluded.The interference of organic acid and hydrogen peroxide could be excluded.And the inhibitory effect of protease treatment of antibacterial products of the isolate on Staphylococcus aureus was reduced.It still had good antibacterial activity under high temperature of 100 ℃,pH 2.0-8.0 and ultraviolet irradiation for 2 h.The length of LMM-1 genome was 2 194 218 bp,2 045 genes were predicted,and the average GC content of the genome was 44.97%.GO functional enrichment analysis results showed that the catalytic activity in molecular functions and the metabolic processes in biological processes obtained the most genomic functional annotations,with 905 and 801,respectively.KEGG pathway enrichment analysis results showed that there were 49 metabolic pathways in genetic metabolism,genetic information processing and environmental information processing,and a total of 1 050 genes were functionally annotated.【Conclusion】 The bacteriocin produced by the Weissella confusa LMM-1 had a good antibacterial activity against Staphylococcus aureus,and the bacteriocin had a certain ability of acid and alkali resistance,high temperature and ultraviolet light,and had a good adaptability to the environment.The isolated strain contained many genes related to catalytic activity and metabolism.The result of this study laid a good foundation for the development of anti-Staphylococcus aureus replacement antibody products.

Key words: Weissella confusa; antibacterial effect; bacteriocin; Staphylococcus aureus

中图分类号:

S816.3

刘咪咪, 赵新凌, 苏惠, 李国江, 董文龙. 抗金黄色葡萄球菌融合魏斯氏菌细菌素的理化特性分析及全基因组测序分析[J]. 中国畜牧兽医, 2024, 51(10): 4559-4567.

LIU Mimi, ZHAO Xinling, SU Hui, LI Guojiang, DONG Wenlong. Analysis of Physicochemical Properties and Whole Genome Sequencing of Anti-Staphylococcus aureus Weissella confusa Bacteriocin[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4559-4567.

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