白色念珠菌对唑类抗真菌药物的耐药机制探讨

目的探讨白色念珠菌对唑类抗真菌药物的耐药机制。方法从复发性外阴阴道念珠菌病(RVVC)患者分离出白色念珠菌,采用NCCLS公布的《酵母菌液基稀释法抗真菌药物敏感试验参考方案》(M-27方案)进行体外药敏试验,分离出氟康唑(FLC)及伊曲康唑(ITC)的耐药株;随机选择4株耐FLC和ITC白色念珠菌耐药株,分别用A1-A2、B1-B2、C1-C2、D1-D2、E1-E2、F1-F2 6对引物进行PCR扩增,将其产物测序,并与NCBI网站提供的白色念珠菌参考株进行比对、分析。结果4株耐FLCI、TC白色念珠菌的CYP 51基因全序列比对、分析,发现有32个碱基发生了突变,但引起氨基酸的突变只有5个,分别是:第116位遗传密码GAT变成GAA,导致该位置的天冬氨酸(D)突变为谷氨酸(E)(D116E 1株);第117位遗传密码GCT变成GGT,导致该位置的丙氨酸(A)突变为甘氨酸(G)(A117G 1株);第266位遗传密码GAA变成GAC,引起该位置的谷氨酸(E)突变为天冬氨酸(D)(E266D 2株);第488位的遗传密码GTT变成ATT,导致该位置的缬氨酸(V)突变为异亮氨酸(I)(V488I 1株);其中有1株菌分别在第266和488位同时发生了突变。结论本研究发现5个有意义的碱基突变,引起其编码的氨基酸发生了突变,其中A117G的突变目前尚未见相关报道,它在耐药机制中的作用尚需进一步探讨。

Resistant Mechanisms of Candida albicans to Azoles

OBJECTIVE To investigate the resistant mechanisms of Candida albicans to azoles at molecular level.METHODS NCCLS M-27 protocols were used to test the in vitro susceptibilities of 102 C.albicans strains isolated from the patients with recurrent vulvovaginal candidiasis(RVVC) against fluconazole(FLC) and itraconazole(ITC) to screen the FLC-and ITC-resistant C.albicans isolates;six pairs of primers,A1-A2,B1-B2,C1-C2,D1-D2,E1-E2 and F1-F2 were respectively to amplify gene CYP51 of 4 strains with FLC-and ITC-resistance.The PCR products were sequenced and analyzed to identify the mutation sites by compared with the sequence of gene CYP51 of referenced C.albicans strain in NCBI site of Internet.RESULTS The analysis of full length sequence of CYP51 from 4 FLC-and ITC-resistant strains showed that from total 32 mutation sites there were 4 significant site mutations,where the mutation of GAT to GAC at 116 caused the substitution of D by E(E266D in two strains);GCC to GGT at 117 caused the substitution of A by G(A117G in 1 strain);GAA to GAC at 266 caused the substitution of E by D(E266D in 2 strains);and GTT to ATT at 488 caused the substitution of I by V(V488I in 1 strain).The site mutations of 266 and 488 were tested in 1 strain of 4 strains.CONCLUSIONS The CYP51 total gene of 4 strains has been checked out.Of FLC and ITC-resistant C.albicans alignment in this time,find out 4 significant bp mutations.Causing its amino acide change,among them,A117G has not be interrelated report still now.The details of mechanism need to be further studied.