抗碳青黴烯類抗生素之克雷伯氏肺炎桿菌對於磷黴素抗藥性研究(II)

Abstract

由於具碳青黴烯類抗生素抗性之克雷伯氏肺炎桿菌(carbapenem-resistant Klebsiella pneumoniae, CRKP)抗藥性問題日益嚴重，而磷黴素(fosfomycin)為最後一線的候選藥物之一，但也有抗藥性菌株發現。本實驗室先前測試52株CRKP，有11株臨床菌株對磷黴素具有抗性(最小抑制濃度, minimal inhibitory concentration, 大於等於256 µg/ml)，且發現其中四株的抗藥機制為glpT基因序列產生突變，使得運輸磷黴素的運輸蛋白質功能喪失。因此，本研究將繼續探討剩餘7株的抗藥機制。 本研究發現七株對磷黴素高抗性菌株，其中一株9921菌株(最小抑制濃度大於等於8192 µg/ml)的uhpA基因序列第226個核苷酸缺失，使UhpA蛋白質合成早期終止，將9921菌株的uhpA基因置換進對磷黴素具感受性的NTUH-K2044菌株中，使其最小抑制濃度由64 µg/ml上升至1024 µg/ml，提升了16倍。以即時聚合酶聚合酶鏈式反應測定，發現NTUH-K2044-9921uhpA菌株相較於NTUH-K2044菌株，其uhpT RNA表現量下降六成 ; 利用運輸蛋白質功能試驗(transporter functional test)，在含有0.2%葡萄糖-6-磷酸(glucose 6-phosphate ,g6p)的基本培養基(M9 minimal salt agar plate)中，NTUH-K2044-9921uhpA菌株相較於NTUH-K2044菌株，無法利用g6p作為碳源生長。證明9921菌株的抗藥機轉為uhpA基因序列缺失而造成對磷黴素具有高抗性。另外萃取這七株高抗性菌株的質體，以電穿孔送入至具磷黴素感受性的大腸桿菌DH10B (最小抑制濃度等於1µg/ml)中，其中CB28和Ear菌株 (最小抑制濃度大於等於8192 µg/ml)的質體可造成DH10B對磷黴素產生高抗性(最小抑制濃度等於2048 µg/ml)，推測其質體上帶有可抵抗磷黴素的相關基因。將這兩株高抗性的菌株CB28和Ear，分別利用Pacbio、illumina進行DNA定序，CB28菌株帶有134Kb的質體pCB28，質體上帶有可修飾磷黴素的酵素FosA3，利用BLAST(Basic Local Alignment Search Tool)資料庫比對後，此質體序列與K. pneumoniae LJ04菌株的質體pCT-KPC核酸序列相似度為84% ; 在Ear質體pEar上也發現帶有修飾磷黴素的酵素FosA3，與K. pneumoniae KP1034的質體相似度為53%。利用插入式突變方法，分別將pCB28、pEar上的fosA3基因破壞，此兩株菌株的最小抑制濃度皆由大於8192 µg/ml下降至256 µg/ml，證實其抗藥機轉為質體上的fosA3基因所導致。 總結來說，本研究發現52株臨床CRKP中，11株對磷黴素具有抗性(最小抑制濃度大於等於256 µg/ml),(11/52,22%)，其中四株抗藥性機轉為GlpT蛋白質功能喪失導致(4/11,36%)，一株為UhpA蛋白質功能喪失使UhpT蛋白質表現量降低 (1/11,9%)，兩株為質體上帶有修飾磷黴素的酵素FosA3 (2/11,18%)，而剩餘4株磷黴素抗性較低(最小抑制濃度等於256-512 µg/ml)的菌株機轉仍未知。

The problem of carbapenem resistant Klebsiella pneumoniae (CRKP) have become serious and fosfomycin is one of the last-line antibiotic candidates. Unfortunately, the fosfomycin resistant strains have been found. Our previous findings tested 52 CRKP strains. Eleven strains of these isolates were resistant to fosfomycin (minimal inhibitory concentration,MIC ≥ 256 µg/ml). Four of these resistant strains were found to have mutation on the glpT gene, resulting in loss of the function of fosfomycin transporter. Resistance of the other 7 strains remained unknown. In this study, we continued to study the 7 remaining strains. Deletion of the 226th nucleotide of uhpA gene resulted in early termination of UhpA protein was observed in 9921 strain (MIC ≥ 8192 µg/ml). The wild-type uhpA gene in NTUH-K2044 was replaced by mutated uhpA gene in the 9921 strain and increased the MIC of this strain from 64 µg/ml to 1024 µg/ml, a 16-fold increase. In addition, the expression of uhpT RNA in NTUH-K2044-9921uhpA was lower than that in NTUH-K2044-wt by the real time polymerase chain reation assay. Using a transporter functional test, NTUH-K2044-9921uhpA was unable to grow on the M9 salt agar plate containing 0.2% g6p compared to NTUH-K2044-wt. These results indicated that the transporter function of UhpT protein was defect in NTUH-K2044-9921uhpA. Meanwhile, plasmids from seven resistant strains were extracted and then electrotransferred to fosfomycin susceptible Escherichia coli DH10B (MIC = 1 µg/ml). pCB28 and pEar bearing recombinant DH10B become resistant to fosfomycin, respectively (MIC=2048 µg/ml). pCB28 and pEar plasmid were sequenced by Pacbio and illumine, respectively. The pCB28 plasmid with size of 134Kb carrys a gene encoding fosfomycin modified enzyme FosA3. By BLAST analysis, pCB28 plasmid showed high sequence similarity (84%) to the pCT-KPC plasmid of K pneumoniae strain LJ04. Fosfomycin modified enzyme FosA3 was also found on pEar plasmid. The sequence similarity of pEar plasmid was 53% to the K. pneumoniae strain KP1034 plasmid. Insertional mutation of fosA3 gene on CB28 and Ear all decreased the MIC of these two strains from ≥ 8192 µg/ml to 256 µg/ml. Taken together, 11 of 52 clinical CRKP strains were resistant to fosfomycin in this study(22%,11/52). Among these, four strains were loss of GlpT protein function (4/11,36%), one strain was loss of UhpA protein function and resulted in decreased the expression of UhpT protein (1/11,9%), and two strains contained fosfomycin modified enzyme FosA3 on their plasmids (2/11,18%). The resistant mechanisms of the remaining 4 strains with low resistance (MIC = 256-512 µg/ml) were unclear (4/11,36%).