納豆菌培養液於細胞模擬之腸道微環境對糞腸球菌生長之影響

Abstract

糞腸球菌 (Enterococcus faecalis) 為一常見於脊椎動物腸道之革蘭氏陽性菌，一般對人體無害。近年來發現，因糞腸球菌對多種抗生素具抗性，當病人服用抗生素以治療細菌感染時，糞腸球菌為少數可倖存並於腸胃道形成優勢之菌種。當其產生群體感應效應 (quorum-sensing) 並發展出成熟生物膜 (biofilm) 之後，會表達與致病性及多種抗藥性相關之基因。且糞腸球菌可藉由接合式質體轉移 (conjugative plasmid transfer)，將其抗藥性基因轉移予不具抗藥性之菌株，使得抗藥性快速擴散，為院內感染 (nosocomial infection) 之主要病原菌之一。本實驗室先前研究發現，納豆菌 (Bacillus subtilis natto NTU18) 之培養上清液可有效抑制接合式質體 pCF10 轉移，具有應用於抑制糞腸球菌抗藥性基因傳遞之潛力。糞腸球菌多由傷口處或腸胃道感染人體，本研究欲於腸道表皮細胞及免疫細胞等胞外環境模擬人體腸胃道，探討納豆菌培養上清液對糞腸球菌之影響。首先，評估該上清液對細胞存活率及特定細胞激素之影響。結果顯示，納豆菌培養上清液對人體腸道上皮細胞株 Caco-2、大鼠嗜鹼性白血病細胞株 RBL-2H3 及小鼠巨噬細胞株 Raw264.7 之細胞存活率皆無明顯影響，且可顯著提升小鼠免疫細胞 TNF- 之分泌量達 3375 pg/ml，顯示其可提升免疫力。接著探討該上清液對糞腸球菌貼附於腸道上皮細胞之影響，並觀察糞腸球菌之生長情形及其形成生物膜之能力。實驗結果發現，隨著納豆菌培養上清液的添加，雖然糞腸球菌之生長無明顯變化，然而其對於腸道表皮細胞之貼附率相較於未添加之組別大幅降低超過 50%，且於 24 小時內形成生物膜之總量亦減少約 11.4%，推測其可預防糞腸球菌於腸道上皮細胞表面形成菌落並且抑制其生物膜之生成。接下來的實驗，我們將進一步探討納豆菌培養上清液是透過何種機制以達到上述功效，期許未來能發展為預防糞腸球菌感染及抗藥性傳遞之新興成分。

Enterococcus faecalis is a commensal bacterium in human gastrointestinal tract that is generally regarded as harmless. Recently, it is found that when antibiotics are used to treat bacterial infections, due to the resistance of E. faecalis to multiple antibiotics, E. faecalis is the small minority of bacterium that can survive in the gastrointestinal tract after patients take antibiotics. Once E. faecalis become the dominant bacterium in the gastrointestinal tract, they would generate a quorum-sensing effect and develop into a mature biofilm. At this time, E. faecalis would express more virulence and antibiotic resistance genes. What makes it trickier is that E. faecalis could transfer antibiotic resistance genes to resistance-free strains via their conjugative plasmid transfer systems. This event further spreads the antibiotic resistance and let E. faecalis gradually becomes one of the main pathogens of nosocomial infections. In previous study of our laboratory, it was found that the culture supernatant of Bacillus subtilis natto NTU18 could inhibit transfer of the antibiotic resistance plasmid pCF10 between E. faecalis. E. faecalis mostly infects the human body from the wound or gastrointestinal tract; therefore, the purpose of this study is to evaluate whether the culture supernatant of B. subtilis natto NTU18 has the similar effect we had observed in simulated intestinal model created by intestinal epithelial cells and immune cells. We first evaluated the cell viability of cells, and explore whether the culture supernatant has the effect of cytokines production. The results showed that the culture supernatant of B. subtilis natto had no obvious effect on the cell survival rate of Caco-2 (human intestinal epithelial cell), RBL-2H3 (rat basophilic leukemia cell) and Raw264.7 (mouse macrophage cell). The supernatant can also significantly increase the secretion of TNF-α in mice immune cells (up to 3375 pg / ml), which is the symbol for immunity improvement. In addition, we further explored that with the treatment of culture supernatant of B. subtilis natto, the adhesion rate of E. faecalis to intestinal epithelial cells would significantly decrease by more than 50% compared with the untreated group, which indicated that it could prevent E. faecalis from forming colonies on the intestinal cells’ surface. Besides, the supernatant could also decrease 11.4% of the total biomass of biofilm that E. faecalis formed within 24 hours. In the next step, we would collect the cells and E. faecalis that have been treated with the supernatant to look into their gene expression. We hope to find the mechanisms that natto culture supernatant used to achieve the inhibition effects on bioactivities on E. faecalis we have observed, and expect that it can be developed into a new drug for preventing E. faecalis infection and drug resistance transmission in the future.