临床分离的携带TR34/L98H型的耐唑类药物烟曲霉的耐药机制分析

Mechanism of azole resistance in Aspergillus fumigatus with TR34/L98H mutations

目的对临床分离的耐唑类药物烟曲霉菌株进行耐药机制等相关研究。方法自1例侵袭性曲霉病(IA)患者的肺泡灌洗液(BALF)中分离得到1株烟曲霉,采用美国临床实验室标准化协会(CLSI)M38-A2微量液基稀释法测定伊曲康唑、伏立康唑、泊沙康唑、两性霉素B和卡泊芬净对该菌株的最低抑菌浓度(MIC)/最低有效浓度(MEC),并对该菌株的唑类药物靶酶基因cyp51A进行克隆和序列测定分析,扩增并检测该菌株的9个微卫星标记位点,将其与国际上已经报告的具有相同耐药基因型的烟曲霉菌株进行非加权配对算术平均法(UPGMA)聚类分析。结果伊曲康唑、伏立康唑、泊沙康唑、两性霉素B对该菌株的MIC分别为>16、2、0.5和2μg/m L,卡泊芬净对该菌株的MEC为0.25μg/m L。该菌株的cyp51A基因序列中存在启动子区-288^-322位间34 bp串联序列的插入,以及编码区T364A、T960A、T1554A的点突变,分别导致所编码蛋白98位、297位及495位氨基酸的置换(TR34/L98H/S297T/F495I)。微卫星分析显示,该菌株的微卫星分型与先前欧洲国家、亚洲其他国家分离的TR34/L98H/S297T/F495I型菌株不同。结论我们从1例IA患者BALF中分离到对唑类药物耐药的烟曲霉临床菌株,其耐药性是由cyp51A基因启动子区34 bp的串联序列插入结合基因编码区突变所致98位、297位及495位氨基酸置换(TR34/L98H/S297T/F495I)所介导,该菌株是中国所特有的、不同于国际上已描述的对唑类药物耐药的病原性烟曲霉。

ObjectiveTo investigate the mechanism of azole resistance in Aspergillus fumigatus. Methods An isolate of Aspergillus fumigatus was collected from bronchoalveolar lavage fluid（BALF） of a patient with invasive aspergillosis（IA）. By microbroth dilution,the minimal inhibitory concentrations（MIC）/minimal effective concentration（MEC） of itraconazole,voriconazole,posaconazole and amphotericin B and caspofungin were determined according to the Clinical and Laboratory Standards Institute（CLSI） M38-A2. The full-length region of the gene encoding the target enzyme of azoles（cyp51A gene including its promoter） was cloned and sequenced. Microsatellite genotyping was performed by amplifying and determining the isolate＇s 9 microsatellite loci,and the genetic relationships among isolates with the same genotype were then established by cluster analysis using unweighted pair-group method with arithmetic means（UPGMA）. ResultsFor this isolate,the MIC of itraconazole,voriconazole,posaconazole and amphotericin B were 〉16,2,0.5 and 2 μg/mL. The MEC of caspofungin was 0.25 μg/mL. The sequence analysis of cyp51A gene showed the presence of 2 copies （-288--322）of a 34 bp sequence in tandem in the promoter of cyp51A gene together with the presence of 3 point mutations at T364A,T960A and T1554A,which were accordingly deduced amino acid substitution including TR34/L98H/S297T/F495I. Microsatellite genotyping indicated that this isolate had a unique genotype being different to those reported previously in Europe and Asia. ConclusionsThe azole-resistant isolate of Aspergillus fumigatus from BALF harboring amino acid substitution TR34/L98H/S297T/F495I has been isolated with a unique genotype being different to isolates reported before.