CAJ | 학술논문

目的：研究铜绿假单胞菌对碳青霉烯类耐药与外排泵MexAB‐OprM 的相关性。方法琼脂稀释法测定亚胺培南和美罗培南对75株铜绿假单胞菌的最低抑菌浓度（M IC ），联合外排泵抑制剂M C207110进行外排泵表型的筛选；用反转录‐聚合酶链反应（RT‐PCR）扩增MexAB‐OprM融合蛋白结构基因 mexA以及内参基因rpoD ，并测定其mRNA的表达量，用PCR扩增MexAB‐OprM 相对高表达菌株的外排泵调节基因 mexR、nalC和 nalD ，对扩增产物进行DNA 双向测序；结果进行BLAST比对分析。结果75株铜绿假单胞菌中外排泵表型阳性菌株13株，其中10株细菌的MexAB‐OprM 相对表达量增高，此高表达MexAB‐OprM菌株的调节基因 mexR、nalC及nalD均阳性。其中9株菌 nalC均发生第71位氨基酸突变（甘氨酸→谷氨酸）、8株菌同时还有第209位氨基酸突变（丝氨酸→精氨酸），仅发现1株菌 nalD第158位氨基酸突变（苏氨酸→异亮氨酸），8株菌 mexR发生突变。结论外排泵MexAB‐OprM的高表达在铜绿假单胞菌对碳青霉烯类耐药中可能起到重要作用。MexAB‐OprM的高表达与其调节基因突变相关。
목적：연구동록가단포균대탄청매희류내약여외배빙MexAB‐OprM 적상관성。방법경지희석법측정아알배남화미라배남대75주동록가단포균적최저억균농도（M IC ），연합외배빙억제제M C207110진행외배빙표형적사선；용반전록‐취합매련반응（RT‐PCR）확증MexAB‐OprM융합단백결구기인 mexA이급내삼기인rpoD ，병측정기mRNA적표체량，용PCR확증MexAB‐OprM 상대고표체균주적외배빙조절기인 mexR、nalC화 nalD ，대확증산물진행DNA 쌍향측서；결과진행BLAST비대분석。결과75주동록가단포균중외배빙표형양성균주13주，기중10주세균적MexAB‐OprM 상대표체량증고，차고표체MexAB‐OprM균주적조절기인 mexR、nalC급nalD균양성。기중9주균 nalC균발생제71위안기산돌변（감안산→곡안산）、8주균동시환유제209위안기산돌변（사안산→정안산），부발현1주균 nalD제158위안기산돌변（소안산→이량안산），8주균 mexR발생돌변。결론외배빙MexAB‐OprM적고표체재동록가단포균대탄청매희류내약중가능기도중요작용。MexAB‐OprM적고표체여기조절기인돌변상관。
Objective To study the relationship between efflux pump MexAB‐OprM and carbapenem resistance of Pseudomonas aerginosa strains .Methods The minimum inhibitory concentrations of imipenem and meropenem were determined by agar dilution method for 75 strains of P .aerginosa in the absence or presence of MC207110 to screen the phenotypes of active efflux pump .Reverse transcriptase‐polymerase chain reaction (RT‐PCR) method was used to determine the mRNA expression level of mexA which encodes the membrane fusion protein in active efflux pump MexAB‐OprM and the reference (housekeeping) gene rpoD .PCR method was used to amplify the regulatory genes mexR ,nalC ,and nalD of active efflux pump MexAB‐OprM in the strains overexpressing the efflux pump . The PCR products were subject to DNA sequencing and BLAST analysis . Results Of the 75 P .aeruginosa strains ,13 (17 .3% ) were positive for efflux pump MexAB‐OprM .Overexpression of the efflux pump was identified in 10 of the 13 strains and associated with positive regulatory genes mexR ,nalC and nalD .A Gly71→Glu mutation in nalC was found in 9 strains ,and a Ser209→Arg mutation in nalC was identified in 8 strains .Only one strain had a Thr158→Ile mutation in nalD .Eight strains had mutation in mexR .Conclusions Overexpression of multidrug efflux pump MexAB‐OprM plays an important role in carbapenem resistance of P .aeruginosa .High level expression of MexAB‐OprM is related to the mutations of its regulatory genes .