碳青黴烯類抗生素抗藥性克雷伯氏肺炎桿菌莢膜型分析和抗藥性機制之探討

Capsular type and carbapenem resistance of Klebsiella pneumoniae

陳俊堂(Chun-Tang Chen)

指導教授 ： 王錦堂

國立臺灣大學/醫學院/微生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10656

摘要

克雷伯氏肺炎桿菌具有由多醣體所組成的莢膜，目前已知至少有79種莢膜型，不同莢膜型的克雷伯氏肺炎桿菌的致病力和其造成的疾病也會有所不同，因此在臨床上鑑定其莢膜型具有一定的重要性。近年來克雷伯氏肺炎桿菌造成院內感染增加，且多為抗藥性菌株，甚至已有菌株對於用來治療多重抗藥性細菌最後防線的碳青黴烯類抗生素產生抗藥性，但目前對於其莢膜分型的相關研究鮮少有報告。本實驗室先前研究來自台大、長庚、成大、榮總四家醫院的85株碳青黴烯類抗生素抗藥性的克雷伯氏肺炎桿菌之菌株，發現其莢膜型以K64的比例最高32/85 (38%)，而後依序是K62為11/85 (13%)、K24為7/85 (8%)、KN2為6/85 (7%)和K28為5/85 (6%)，而其它莢膜型之菌株皆少於或等於3株，顯示特定莢膜型似乎和細菌獲得抗藥性有關聯。本研究由接合實驗結果，發現不論是碳青黴烯類抗生素抗藥性菌株或是藥物敏感性菌株，抗藥盛行莢膜型(K64、K62、K24、KN2和K28)菌株之平均接合效率，相較非抗藥盛行莢膜型之菌株皆來得高，且在統計上有顯著差異。利用聚合酶鏈鎖反應，發現抗藥盛行莢膜型之碳青黴烯類抗生素抗藥性菌株，其具有第1類整合子的比例，相較非抗藥盛行莢膜型之菌株有較高的趨勢，但在統計上並無顯著差異。另一方面，因為85株碳青黴烯類抗生素抗藥性菌株只有12株具有carbapenemase基因(~14%)，因此進一步探討其它可能的抗藥機制。以EtBr- agar cartwheel和RT-qPCR方法，發現碳青黴烯類抗生素抗藥性菌株，其外排幫浦活性和外排幫浦蛋白AcrA的RNA表現量，相較藥物敏感性菌株有較高的趨勢，但在統計上無顯著差異。除此之外，以質體補回完整的外膜蛋白OmpK35或OmpK36，發現有11/12 (91%)和3/12 (25%)碳青黴烯類抗生素抗藥性菌株的最小抑制濃度下降2-8倍。本研究結果顯示，造成碳青黴烯類抗生素抗藥性菌株莢膜型集中的現象，可能是因為抗藥盛行莢膜型之菌株較容易獲取抗藥基因；此外，外膜蛋白在克雷伯氏肺炎桿菌對碳青黴烯類抗生素之抗藥性也扮演重要的角色。

關鍵字

莢膜 ； 碳青黴烯類抗藥性克雷伯氏肺炎桿菌 ； 外排幫浦 ； 外膜蛋白

並列摘要

Klebsiella pneumoniae is enveloped by a polysaccharide capsule. At least 79 capsular types were defined in K. pneumoniae so far. Capsular types were associated with different clinical settings and severity of illness caused by K. pneumoniae. Therefore, to determine the capsular type of clinical K. pneumoniae isolates will be important. Recently, nosocomial infections caused by K. pneumoniae were increasing, and a great number of them developed drug resistance. K. pneumoniae has been reported to exhibit resistance to carbapenem which is one of last-line defense for multiple drug resistant strains, but the capsular type of multi-drug-resistant K. pneumoniae was poorly understood. Previously we determined the capsular types of 85 carbapenem-resistant K. pneumoniae (CRKP) strains from NTUH, CGMH, NCKUH and VGH, and found that K64 (38%) was the dominant type among these strains, followed by K62 (13%), K24 (8%), KN2 (7%) and K28 (6%), suggesting that specific capsular types may be associated with drug resistance. Conjugation experiments revealed that conjugation efficiency of prevalent capsular types (K64, K62, K24, KN2 and K28) strains was higher than non-prevalent capsular types strains in CRKP or drug-sensitive strains. The prevalence rate of class 1 integron among prevalent capsular types strains was slight higher than non-prevalent capsular types strains (48/61, 79% vs. 14/24, 58%) in CRKP by PCR. Because carbapenemase were only present in 12 of 85 strains (~14%), we further to explore other possible carbapenem resistance mechanisms. Detection of efflux pump activity by EtBr-agar cartwheel method and determination of RNA expression levels of efflux pump protein AcrA by RT-qPCR showed no significantly different among CRKP and drug-sensitive strains. Restoration of plasmid carrying intact OmpK35 or OmpK36 significantly decreased the MICs of imipenem in 11/12 (91%) and 3/12 (25%) CRKP strains, respectively. In summary, K. pneumoniae strains with prevalent capsular types are more easily to acquire drug resistance genes and result capsular types clustering among CRKP strains. Besides, outer membrane proteins, OmpK35 and OmpK36, play important roles in carbapenem resistance of CRKP.

並列關鍵字

capsule ； carbapenem-resistant Klebsiella pneumoniae ； efflux pump ； outer membrane protein

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