摘要
目的探讨白色假丝酵母菌临床株CYP51基因突变与对唑类抗真菌药物耐药的关系,从分子水平了解白色假丝酵母菌的耐药机制。方法纸片扩散法初步筛选呼吸道感染对耐唑类白色假丝酵母菌,按NCCLS公布的M-27方案测定初筛耐药株对氟康唑和伊曲康唑的MIC,设计3对引物,对分离的2株耐唑类白色假丝酵母菌(2007H株,2007T株)进行PCR,扩增CYP51基因;扩增产物纯化后,进行测序并与GenBank序列(X13296)相比较分析。结果PCR扩增产物大小与预期结果一致;测序分析表明,成功扩增到白色假丝酵母菌CYP51基因,与X13296序列相比较,两个耐药株都存在有义突变和无义突变,两株白色假丝酵母菌共有22个碱基突变;突变发生氨基酸替换的有F105L、K128T、Y132H、T199I、R267H、G464S和G467K,其中,两株菌都有Y132H和G467K突变,F71L、W244R、T311N和T352I为新发现的突变,同时,也发现了9个未发生氨基酸替换的突变。结论白色假丝酵母菌对唑类抗真菌药物的耐药与CYP51基因突变有关,且为多位点突变,研究发现了新的突变点,它在耐药机制中的作用需进一步研究。
OBJECTIVE To explore the relationship between the point mutations of gene CYP51 and the azoleresistance mechanism in clinical Candida albicans isolates. METHODS The paper diffusion test and NCCLS M-27 protocols were used to screen the fluconazole-resistant and itraconazole-resistant C. albicans clinical isolates. Gene CYP51 of two azole-resistant C. albicans clinical strains (2007H and 2007T strains) was amplified by three pairs of primers, respectively. The PCR products purified were sequenced, and compared with the nucleotide sequences of C. albicans ( accession No. :X13296 ) to find out the mutation sites. RESULTS The nucleotide sequence analysis showed that there were both significant point mutations and silent mutations in gene CYP51 from two azole-resistant isolates of C. albicans. Seven mutations previously described, F105L, K128T, Y132H, T199I, R267H, G464S, and R467K, were identified in the two strains. The animo acid substitutions of Y132 H and R467K, known to contribution to azole resistance, were detected in both 2007H strain and 2007T strain. Four novel mutations,including F71L, W244R, T311N and T352I, were simultanously identified. Nine silent mutations appeared in two isolates. CONCLUSIONS In this survey, the two azole-resistant C. albicans clinical isolates contained more than one mutation in gene CYP51 that is associated with azole resistance. Four novel mutations of CYP51 may be associated with the resistance of C. albicans to azoles. And the mechanisms need to be further studied in detail.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2009年第5期500-502,共3页
Chinese Journal of Nosocomiology