该研究从150份健康人粪便样品中分离出9株丁酸梭菌,对其进行生物学特性研究并结合基因层面分析,包括菌株特有和同源基因、系统进化、碳源利用能力、抗生素耐受性以及模拟胃肠液耐受性。非靶向泛基因组分析表明,丁酸梭菌中菌株特异性基因为4~1 501个基因/菌株,占菌株总基因数6%~29%,靶向分析表明碳水化合物利用酶谱以及抗生素抗性基因谱呈现菌株特异性的特征;并且分离自同一样本的菌株基因组大小在4.48~4.65 Mb,基因数目在4 006~4 203个,也存在差异,这些结果均表明丁酸梭菌存在菌株水平上的基因组多样性;系统进化分析发现,分离源与丁酸梭菌的进化关系无明显相关性;碳源利用实验表明,9株丁酸梭菌可利用甘露糖、纤维二糖和低聚果糖,不能利用菊粉、赤藓糖醇和鼠李糖,与功能基因分析结果一致;菌株耐受氨基糖苷类抗生素,并与抗性基因预测一致,对其他抗生素普遍不耐受,存在基因型与表型不匹配的情况;模拟胃肠液实验表明,芽孢形成可显著提高菌株的耐酸、耐胆盐特性。以上对丁酸梭菌基因层面和生理特性的研究可为其优良菌株的筛选和应用开发奠定基础。
In this study, biological characteristics and genetic diversity of nine strains of Clostridium butyricum, previously isolated from 150 healthy human stool samples, were studied. The analyses included the study of strain-specific and homologous genes, phylogenetic evolution, carbon source utilization, antibiotic resistance and simulated gastrointestinal fluid tolerance. Non-targeted pan-genomic analysis showed that the number of strain-specific genes in C. butyricum was 4-1 501 genes/strain, accounting for 6% to 29% of the total genes. The profiles of carbohydrate utilization and antibiotic resistance genes exhibited strain-specific characteristics. The genome sizes of the strains isolated from the same sample ranged from 4.48 Mb to 4.65 Mb, whereas the number of genes ranged from 4006 to 4203. These results indicated a significant strain level genomic diversity in C. butyricum. Phylogenetic analysis found that there was no significant correlation between strains clustering and their origin. Carbon utilization experiments showed that these C. butyricum strains were able to use mannose, cellobiose, and fructooligosaccharides, whereas, they failed to utilize inulin, erythritol, and rhamnose, and these findings were consistent with the results of functional gene analysis. The strains showed high resistance towards aminoglycosides, which confirmed the functional role of antibiotic resistance genes. These nine strains were generally non-resistant to other antibiotics, and there was a mismatch between the genotype and phenotype. Simulated gastrointestinal fluid experiments showed that the spore formation could significantly improve the acid and bile salt resistance. The above studies at the gene level including physiological characteristics of C. butyricum strains could lay the foundation for the screening and application of its excellent strains.
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