運輸蛋白質在抗碳青黴烯類抗生素之克雷伯氏肺炎桿菌對於磷黴素高抗藥性之研究

Abstract

磷黴素（fosfomycin）是早期用以治療泌尿道感染的抗生素，近年來本來已較少使用。但由於現今多重抗藥性（Mμltidrμg-resistant；MDR）菌株的肆虐，在後線藥物逐漸失效的情況下，便將磷黴素合併使用其他抗生素作為多重抗藥性菌株治療。然而臨床上也漸漸出現對磷黴素產生抗藥性的菌株，本研究針對台北榮民總醫院臨床分離出56 株碳青黴烯類類抗生素之克雷伯氏肺炎桿菌（carbapenem-resistant Klebsiella pneumoniae；CRKP）先以磷黴素濃度為256 μg/ml做初步篩選後，再進一步以濃度0-2048 μg/ml進行最小抑菌濃度（Minimal inhibitory concentration；MIC）分析，結果發現其中有19 株為對磷黴素高抗藥性（MIC≧1024 μg/ml）。將此19株臨床菌株做磷黴素抗藥性相關基因之定序分析，結果發現共有12株具有甘油-3-磷酸運輸蛋白質（glycerol-3-phosphate transporter；glpT） 基因突變；9株具有葡萄糖-6-磷酸運輸蛋白質（glucose-6-phosphate transporter；uhpT）基因突變；4株菌株於調節蛋白質（glpR, uhpA, uhpB, uhpC,ptsI, cyaA）相關基因上帶有突變或缺失。在運輸蛋白質功能測試（Transporter functional assay）有11 株在甘油-3-磷酸（glycerol-3-phosphate；G3P）M9 培養基生長較差，10 株在葡萄糖-6-磷酸運輸蛋白質（glucose-6-phosphate；G6P）M9 培養基生長較差。而在使用次世代定序（Next generation sequencing , NGS）技術分析後，得知CP02在glpT上距轉錄起始位置（Transcription start site）第+865個位置被一跳躍子（Transporson）插入，在uhpT上距轉錄起始位置第+287個位置有跳躍子插入；CP15在glpT第388-420個核苷酸有缺失情形；在uhpA、uhpB、uhpC、uhpT上皆有缺失。另外將抗藥性菌株上所帶有表現GlpT D274V的glpT及表現UhpC A393V的uhpC置換於磷黴素感受性菌株NTUH-K2044上測定MIC變化，發現NTUH-K2044菌株MIC從64 μg/ml上升至1024 μg/ml，上升了約16倍，顯示此兩基因可能為造成磷黴素抗藥性的機制。

Fosfomycin was used to treat urinary tract infection in early time, and it has been rarely used recently. Nowadays due to the ravages of multidrug resistant bacteria and the third-line antibiotic become less effect, fosfomycin combined with other antibiotics was chosen as new treatments against multidrug resistant bacteria. However, it has been reported that the fosfomycin-resistant bacteria were isolated on clinical research, including Klebsiella pneumoniae. Our study focused on the 56 strains of carbapenem-resistant Klebsiella pneumoniae（CRKP） derived from Taipei Veterans General Hospital. At first, we screened the 56 strains with 256 μg/ml fosfomycin MH agar plate（Base on CLSI guideline, fosfomycin MIC≧256 μg/ml are resistant）. Minimal inhibitory concentration（MIC） test from 0 to 2048 μg/ml fosfomycin MH agar plate was performed. 19 high fosfomycin- resistant strain（MIC≧1024 μg/ml）were found. Next, we analyzed the gene related with fosfomycin resistance of the 19 high fosfomycin-resistant CRKP. The results showed that 12 strains carried glycerol-3-phosphate transporter gene （glpT） mutation；9 strains carried glucose-6-phosphate transporter gene（uhpT） mutation；4 strains carried defect in genes encoding GlpT-related or UhpT-related regulatory protein defect. Furthermore, we evaluated the transporter function of those strains carrying transporter gene mutation by performing the GlpT/UhpT transporter functional assay and found 11 strains were poorly grown on glycerol-3-phosphate M9 agar plate and 10 strains were poorly grown on glucose-6-phosphate M9 agar plate. And the next generation sequencing results showed that CP02 had a transposon inserted at 865th position from the transcription start site on glpT. A transposon inserted at 287th position from the transcription start site on uhpT was also found ; 30 mer deletion on glpT and deletion on uhpA, uhpB, uhpC and uhpT was found in CP15. On the other hand, fosfomycin-susceptible strain-NTUH-K2044 transformed with gene expressing GlpT D274V and UhpC A393V carried on fosfomycin-resistant CRKP strains showed an increase of MIC to 1024 μg/ml, about 16-fold higher than non-transformants. The results indicated that both genes were involved in the mechanism mediating the fosfomycin resistance.