益生菌於干擾糞腸球菌抗藥性轉移之研究與應用

Study and application of probiotics in interfering antibiotic resistance transfer of Enterococcus faecalis

林雨頡(Yu-Chieh Lin)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/生化科技學系/博士(2021年)

https://doi.org/10.6342/NTU202103284

摘要

糞腸球菌 (Enterococcus faecalis) 是引發院內感染的重要病原菌，它已被發現對多種臨床使用的抗生素產生抗藥性，包括以往用於治療腸球菌感染的最後一線抗生素–萬古黴素 (Vancomycin)。此外，攜帶抗藥性之糞腸球菌可藉由信息素反應式接合質體轉移 (pheromone-inducible conjugative plasmid transfer) 將抗藥性基因進一步傳遞給不具抗藥性之糞腸球菌，這樣的特性使得防範該類感染變得更加困難。為了減緩糞腸球菌抗藥性基因的散播，本研究探討益生菌對糞腸球菌抗生素抗藥性質體 pCF10 轉移之影響。納豆菌 (Bacillus subtilis subsp. natto) 是自日本納豆發酵食品分離出之益生菌，我們發現其培養液可藉由影響糞腸球菌胜肽信息素 cCF10 之活性而有效抑制質體 pCF10 轉移，且不會影響糞腸球菌之生長。該抑制效果來自納豆菌所分泌介於 30 至 50 kDa 分子量的熱不穩定物質，我們推測可能為其胞外蛋白酶。進一步，我們發現納豆激酶 (nattokinase) 於此抑制效果中扮演著重要角色，其可將 cCF10 (LVTLVFV) 水解為 ”LVTL+VFV” 片段，使其失去活性。此外，納豆激酶所水解出之cCF10片段 “LVTL” (命名為 L 胜肽) 也具有抑制質體 pCF10 轉移的效果。除了cCF10之外，抗萬古黴素糞腸球菌之胜肽信息素 faecalis-cAM373 和 gordonii-cAM373 也會被納豆激酶水解，此結果顯示納豆菌可能具有干擾糞腸球菌傳遞萬古黴素抗藥性的潛力。除了抑制糞腸球菌之抗生素抗藥性質體轉移，我們也發現納豆菌的培養液會抑制糞腸球菌細胞外膜 (cell envelope) 合成相關之基因表現，進而影響其生物膜 (biofilm) 之形成。我們的研究顯示了可將納豆菌發酵產物和 L 胜肽應用於對抗抗生素抗藥性糞腸球菌的可行性。

關鍵字

糞腸球菌 ； 抗生素抗藥性 ； 信息素反應式接合質體轉移 ； 納豆菌 ； 納豆激酶

並列摘要

Enterococcus faecalis, a leading cause of hospital-acquired infections, is resistant to many known antibiotics. More concerningly, resistance-carrying E. faecalis has the propensity to transfer antibiotic resistance genes through its unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. We found that culture fluid of Bacillus subtilis natto potently inhibited pCF10 transfer by degrading peptide pheromone cCF10 without inhibiting E. faecalis growth. The inhibitory effect was attributed to 30-50 kDa extracellular protease present in B. subtilis natto. Nattokinase of B. subtilis natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into “LVTL + VFV” fragments. The cleavage product “LVTL” (L peptide) was found to interfere with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis natto can likely interfere with vancomycin resistance transfer in E. faecalis. We also found that culture fluid of B. subtilis natto down-regulated the transcription of genes related to cell envelope synthesis, thereby impairing the biofilm formation of E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis natto and L peptide to mitigate the threat of antibiotic-resistant E. faecalis.

並列關鍵字

Enterococcus faecalis ； antibiotic resistance ； pheromone-inducible conjugative plasmid transfer ； Bacillus subtilis natto ； nattokinase

參考文獻

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