嗜麦芽寡养单胞菌临床株多重耐药外排泵SmeDEF调控基因smeT的序列分析

Repressor SmeT of Efflux Pump SmeDEF in Stenotrophomonas maltophilia

目的了解嗜麦芽寡养单胞菌的临床株中,SmeDEF外排泵表达调控基因smeT与耐药性有关的序列变化。方法对嗜麦芽寡养单胞菌smeD-smeT基因间区和部分smeT序列进行PCR扩增,并对扩增产物进行序列分析;提取的RNA并加聚A尾,对smeT的3′端进行分析。结果选取耐药、敏感的泵抑制阳性株和耐药、敏感的泵抑制阴性嗜麦芽寡养单胞菌7株;发现对氟喹诺酮类耐药的泵抑制阳性菌的smeD-smeT间区测序基因序列与敏感株明显不同;所检测7株菌无论敏感或耐药在SmeT序列的166位氨基酸均为Leu,泵抑制阳性耐药株的SmeT序列在218位氨基酸为Asp/Glu替换。结论嗜麦芽寡养单胞菌临床株在相应于smeT的-165^-82区序列差异,以及SmeT的Asp218Glu氨基酸替换和多个位点的突变可能与多重外排的耐药有关。

OBJECTIVE To investigate the repressor SmeT and other sequences of efflux pump SmeDEF in the clinic isolates of Stenotrophomonas maltophilia to involve in antibiotic resistance. METHODS The mRNA level of smeD examined the resistance profile of the clinical strains to antimicrobials was by agar dilution and their reaction to pump inhibitor was investigated by RT-PCR, and the smeD-smeT fragment was amplified by PCR, and then sequenced. Adding poly（A） to 3＇ end of RNA, applying ：3＇ RACE, nested PCR, then sequence-analysis. RESULTS The amino acid sequences of the forepart of SmeT were conservative in all the 7 strains. In antibioticresistant strains with positive reaction to pump inhibitor, the intergenic sequences of smeD-smeT were different from those in sensitive ones. The variations in -165 to -82nt of smeT and several nucleic acids before the start codon of smeT, possibly involved in antimicrobial resistance. All isolates didnPt have Leu166Gln mutation within the SmeT protein of D457R, which was reported possibly associated with antibiotic resistance. The resistant strains with efflux positively inhibited had Asp218Glu substitution, different from the negatively-inhibited ones. CONCLUSIONS The expression level of SmeDEF is related with the antibiotic resistance of S. maltophilia. The possible resistance mechanism includes the variations in the intergenie smeD-smeT and/or smeT codon sequence.