台灣克雷伯氏肺炎桿菌抗克痢黴素機制之研究

Abstract

克痢黴素為治療多重抗藥性克雷伯氏肺炎桿菌感染的最後一線抗生素。為了研究台灣抗克痢黴素克雷伯氏肺炎桿菌的抗藥機制，本研究分析了26株抗克痢黴素臨床菌株。結果顯示K64和ST11分別為台灣抗克痢黴素菌株最主要的莢膜型和序列型。前人研究指出，細菌可以利用pmrHIJKLM和pmrC增加脂多醣Ara4N和PEtN兩種修飾，進而得到克痢黴素抗藥性。因此我們檢驗臨床菌株pmrH和pmrC的基因表現量，結果發現pmrHIJKLM和pmrC分別於96.2% (25/26) 和38.5% (10/26) 的抗藥性菌株中有顯著的上升。進一步研究pmrHIJKLM和pmrC之相關調控分子，在26株菌株中有8株之mgrB的編碼區或啟動子有外來插入序列 IS5-like和IS10R，並且有2株之mgrB序列被剔除。而後續也於其他15株中分別發現MgrB、PhoP、PhoQ、PmrB以及CrrB中有胺基酸的改變。將MgrB C28Y、Stop48Y和PhoQ L26P以及CrrB Q10L、Y31H、W140R、N141I、P151S、S195N轉移至感受性菌株後，使感受性菌株變成克痢黴素抗藥性菌株。由此可知台灣克痢黴素之抗藥性主要是藉由mgrB改變所導致 (14/26, 53.8%) ，其次則為crrB的點突變所造成 (8/26, 30.8%) 。而僅有菌株Col5既無pmrH和pmrC的表現量增加，且不帶有已知的突變位點，因此抗藥性產生的原因尚不清楚。由於crrAB為新發現之基因，因此我們進一步研究crrAB如何導致抗藥性的產生。結果顯示crrB的突變會導致下游基因H239_3062的表現量升高。而將H239_3062剔除後，克痢黴素抗藥性即大幅度下降，因此將H239_3062命名為crrC。我們也發現crrC是藉由提升下游pmrAB的表現量，使pmrHIJKLM和pmrC表現量升高，進而產生抗藥性。此外crrB的突變不僅會增加crrC的表現，也會增加crrC下游基因H239_3063、H239_3064和H239_3065的表現量，而剔除H239_3063和H239_3064使得菌株對克痢黴素的抗藥性下降8倍。總結來說，台灣克痢黴素的抗藥性主要由調控子 (mgrB、phoQ、pmrB和crrB) 的突變，進而增加脂多醣的修飾所造成。除此之外，crrB的突變會使H239_3063和H239_3064的表現量增加，進而使細菌增加對克痢黴素的耐受性。

Colistin is an important antibiotic for the treatment of multidrug-resistant Klebsiella pneumoniae infection. To study the resistance mechanism(s) to colistin, 26 colistin-resistant K. pneumoniae strains in Taiwan were collected. The K64 (50%) and ST11 (53.9%) are the prevalent capsular and ST types in colistin-resistant strains of Taiwan. Previous studies indicated modification of lipid A with Ara4N or PEtN by pmrHFIJKLM or pmrC reduce susceptibility to colistin. In our 26 strains, expressions of pmrHIJKLM and pmrC were significantly increased in 96.2% (25/26) and 38.5% (10/26) strains. Moreover, regulations of pmrHIJKLM and pmrC were studied. Results showed IS5-like or IS10R element were inserted in promoter or coding region of mgrB in 8 strains, and mgrB was absence in 2 strains. Amino acid substitutions of MgrB, PhoP, PhoQ, PmrB and CrrB were detected in remaining 15 strains. Site-directed mutations in a colistin-susceptible strain demonstrated MgrB (C28Y and Stop48Y), PhoQ (L26P) and CrrB (Q10L, Y31H, W140R, N141I, P151S and S195N) contributed to colistin resistance. These data indicated mgrB-mediated resistance is a major mechanism (14/26) contributing to colistin resistance in Taiwan, and amino acid substitutions in CrrB (8/26) is secondary. Regulation of crrAB was further studied. The crrB mutants showed increased accumulation of H239_3062. Deletion of H239_3062 in the A4528 crrB(N141I) strain attenuated colistin resistance, and H239_3062 was accordingly named crrC. Furthermore, expressions of H239_3063, H239_3064 and H239_3065 were increased in crrB(N141I) strain. Deletion of H239_3063 and H239_3064 could decrease colistin resistance in crrB(N141I) strain. In conclusion, elevated modifications of lipopolysaccharide due to mutations of regulators (mgrB, phoQ, pmrB and crrB) is a major mechanism of colistin resistance in Taiwan. Moreover, mutations of crrB induce expressions of H239_3063 and H239_3064, and increased H239_3063 and H239_3064 enhance tolerance of colistin in K. pneumoniae.