評估台灣地區fosfomycin對碳青黴烯類抗藥克雷伯氏肺炎桿菌的殺菌效果及抗藥機轉分析

Characterization of plasmid-mediated fosfomycin resistance determinants in carbapenem-resistant Klebsiella pneumoniae isolates in Taiwan

王亭茵(Ting-Yin Wang)

指導教授 ： 曾嵩斌

高雄醫學大學/健康科學院/醫學檢驗生物技術學系碩士班/碩士(2016年)

摘要

臨床上，多重抗藥性的腸道菌屬 (Enterobacteriaceae)特別是克雷伯氏肺炎桿菌伴隨一系列碳青黴烯酶 (carbapenemase)的產生以往並不常見，隨著藥物頻繁使用之下，短短的幾年間卻已演變為現今醫療所面臨的重要議題，更也備受國際間的高度關注。Carbapenem類抗生素 (包括ertapenem、imipenem、doripenem及meropenem)屬於強力的廣效性β-lactam類藥物，視為腸道菌嚴重感染症的首選用藥。但近幾年來在不同國家中，持續發現對carbapenem產生抗藥性的Klebsiella pneumoniae (carbapenem-resistant klebsiella pneumoniae)比例與日俱增，並有蔓延至全球之情形。克雷伯氏肺炎桿菌是臨床上重要的病原菌，其除了加重醫療處置之困擾，也增加病人死亡的機率。在治療方面，尤其針對多重抗藥性細菌的感染，在臨床用藥的選擇性因抗藥性而受到限制，加上缺乏合適的抗生素做有效之治療，而新藥的研發更趕不上臨床的需求，因此國外有研究學者嘗試回頭尋找早期開發而較少應用的抗生素，其中磷黴素 (fosfomycin)以其獨特的化學結構與殺菌機製，已被證實為具有臨床實效的舊藥之一，此外，在藥物的合併治療上，磷黴素亦被推薦能與meropenem及colistin合併使用，能達到藥物協同效果 (synergistic effect)以治療克雷伯氏肺炎桿菌所造成的感染，甚至於對目前現存後線抗生素完全無效的全抗藥型 (pan-drugresistant)細菌皆具有不錯之療效。相較於國外，台灣僅少數學者於此方面進行探討，因此本研究主要目的為欲評估磷黴素 (fosfomycin)治療台灣地區對carbapenem產生抗藥性的克雷伯氏肺炎桿菌之療效，更進一步針對質體介導的fosfomycin抗藥基因結構與分子流行病學做深入分析。
本論文所研究的臨床菌株其收集時間為2012到2013年來自全臺灣不同地區的16家醫院，總共有642株對carbapenem 產生抗藥性的克雷伯氏肺炎桿菌。所有臨床菌株經藥物敏感試驗確認，其中對於磷黴素 (fosfomycin)的最小抑菌濃度 (MIC) ≥ 256μg/ml的比例為36.4% (234/642)。透過PCR擴增分析對carbapenem 產生抗藥性的克雷伯氏肺炎桿菌攜帶常見的磷黴素抗藥基因情況，偵測結果顯示在磷黴素抗藥菌中其fosA3基因檢出率佔有15% (35/234)及foskp96基因佔有11.5% (27/234)。同時也偵測carbapenemase的抗藥基因存在之情形，結果顯示主要為KPC 27.8% (65/234)基因出現比例較高。進一步以脈衝電泳法 (pulsed-field gel electrophoresis-PFGE)及多位基因序列分析法 (multilocus sequence typing-MLST)分析臨床菌株的親源關係，結果顯示共有81個phenotype，並發現phenotype第23群有群聚現象，因此以第23群為主要的major group，從中亦觀察到帶有fosA3及KPC-2基因之臨床菌株主要聚集於PFGE phenotype第23群，此外，透過MLST分析結果顯示PFGE phenotype第23群同時具有相同的序列型別 (即sequence type 11)。在磷黴素抗藥基因周邊序列分析中，使用PCR mapping技術確認磷黴素抗藥基因fosA3的上下游序列發現具有IS26序列結構的存在，並依序可分成A型：5.7％ (2/35)、B型：65.7％ (23/35)、C型：2.9％ (1/35)、D型：11.4％ (4/35)、E型：14％ (5/35)五型，從而推測IS26序列結構可能是傳遞fosA3的主因。最後，透過fosfomycin與carbapenem類抗生素meropenem的組合治療後發現具有顯著的藥物協同作用之效果，以降低藥物的最小抑菌濃度同時預防抗藥菌株的產生。綜上研究調查及分析之實驗數據值得再深入探討，並藉此了解磷黴素抗藥菌株及其所攜帶的抗藥基因在台灣不同醫院的分佈情況，更盼由以上結果能對於現今流行病學的分析研究及感控措施上有所貢獻。

關鍵字

磷黴素 ； 碳青黴烯類抗藥克雷伯氏肺炎桿菌

並列摘要

Carbapenem-resistant Klebsiella pneumoniae which are highly resistant to many antibiotics have been recognized as emerging problems worldwide. Recently, there has been a renewed interest in reviving older antimicrobial agents, such as fosfomycin, for multidrug-resistant pathogens. Fosfomycin is a bactericidal antibiotic with broad-spectrum activity against wide range of Gram-positive and Gram-negative bacteria. Fosfomycin has been used for the successful treatment of urinary tract infections for a long time. It could be considered as medical treatment of multidrug-resistant pathogens or carbapenem-resistant K. pneumoniae.
In order to evaluate the effectiveness of fosfomycin against carbapenem resistant K. pneumoniae isolates and prevalence of plasmid-mediated fosfomycin resistance determinants in Taiwan, 642 clinical carbapenem resistant K. pneumoniae isolates were collected from 16 hospitals during 2012 to 2013. Antimicrobial susceptibility testing were determined. Plasmid-mediated fosfomycin resistance genes (fosA, fosA3, fosKP96 and fosC2) and carbapenemase resistance genes (KPC, NDM, OXA48, VIM, IMP) were detected by PCR. Flanking regions of fosA3 were determined by PCR mapping and sequencing. Phylogenetic clonal patterns were analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE). Synergistic activity of fosfomycin combined with meropenem was assessed by chequerboard method.
The fosfomycin resistant ratio was 36.4% (234/642) in Taiwan. Percentages of fosA3 and foskp96 were 15% (35/234) and 11.5% (27/234). Flanking regions of fosA3 were arbitrarily designated as type A: 5.7% (2/35), type B: 65.7% (23/35), type C: 2.9% (1/35), type D: 11.4% (4/35), type E: 14% (5/35). Phylogenetic analysis revealed 81 pulsotypes whereas pulsotype XXIII (n=63) was major group containing 39.7% (25/63) fosA3 and 95.2% (60/63) KPC-2. The combination of fosfomycin and meropenem exhibited synergy against 25 (100%) of the 25 isolates. The combinations studied showed improved bactericidal activity compared to fosfomycin alone and prevented the development of fosfomycin resistance in the majority of susceptible isolates. This finding indicated that a disseminative clone (XXIII) carrying fosA3 and KPC-2 is difficult to treat and warrant further monitoring in Taiwan.

並列關鍵字

fosfomycin ； carbapenem-resistant Klebsiella pneumoniae

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