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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