Study on the mechanism of toxin production by Clostridium difficile

从营养需要的角度对艰难梭菌的产毒机制进行了研究。通过对18个氨基酸组成的基础培养基单氨基酸删除法和加入法的研究，发现了缬氨酸、半胱氨酸、色氨酸、脯氨酸、蛋氨酸、异亮氨酸、亮氨酸、甘氨酸和苏氨酸是产毒所必需的，通过定性分析，确定了由色氨酸0.6、蛋氨酸1.2、缬氨酸1.8、异亮氨酸1.8、亮氨酸2.4、甘氨酸0.2、苏氨酸0.4和半胱氨酸0.5组成的6xMADM对产毒有较好的刺激作用。在6xMADM和m-BHI的比较中，20株供试菌株中有13株在两种培养基产毒素的量相同，表明上述9种氨基酸对产毒特别重要。通过6×MADM的单一维生素缺失法发现，吡哆醇、泛酸酯和生物素是细菌良好生长和产毒所必需的，生物素缺乏可能会刺激毒素的产生。以菌株KZ1647为研究对象，详细分析了生物素的作用效果。毒素产生与生物素浓度的关系研究发现，随着生物素浓度的降低，细菌生长减少，但毒素产生显著增加，尤其是在0.05 nM生物素浓度下，生物素限制条件下毒素产生的时间进程与生物素浓度增加条件下毒素产生的时间进程相似。在测试的19株菌株中，有18株也观察到生物素对毒素产生的影响，这表明这种影响在艰难梭菌中是普遍存在的。这些发现表明生物素在艰难梭菌结肠炎的发展中起着重要作用。

The mechanism of toxin production by Clostridium difficile was investigated from the stand poit of nutritional requirements. By the single-amino acid omission and addition methods with a basal defined medium consisting of 18 amino acids, it was found that valine, cysteine, tryptophan, proline, methionine, isoleucine, leucine, glycine and threonine were essential for good toxin production.Further, by qualitative analysis, a defined medium (6xMADM) consisting (g/L) of tryptophan 0.6, methionine 1.2, valine 1.8, isoleucine 1.8, proline 1.8, leucine 2.4, glycine 0.2, threonine 0.4 and cysteine 0.5 was found to be fairly effective for stimulating toxin production. In a comparison of 6xMADM and m-BHI,which is excellent comple medium for toxin production, 13 of 20 strains tested produced the same amounts of toxin in both media, suggesting that the 9 amino acids mentioned above were particularly important for toxin production. By single-vitamin omission method with 6xMADM,it was found that pyridoxine, pantothenate and biotin were essential for good growth and toxin production and that biotin deficiency might stimulate toxin production. The effect of biotin was analyzed in detail with a strain KZ 1647. When toxin production was examined in relation to biotin concentration, it was found that with decreasing concentration of biotin bacterial growth decreased, but toxin production was remarkably increased, particularly with 0.05 nM biotin.The time course of toxin production in biotin-limited conditions was similar to that in biotin-enriched conditions. The biotin effect on toxin production was also observed in 18 out of 19 strains tested, suggesting that the effect is a general phenomenon amongst toxigenic C.difficile strains.These findings suggest that biotin play an important role in the development of C.difficile colitis.