| 摘要: | 腸桿菌科（Enterobacteriaceae）細菌是存在人類消化系統的正常菌群，但現今許多菌株已對多種抗生素，甚至後線的碳青黴烯類（carbapenem）產生抗藥性，稱為碳青黴烯類抗藥性腸桿菌（carbapenem-resistant Enterobacteriaceae, CRE）。部分菌株可藉由產生碳青黴烯酶（carbapenemase）分解carbapenem，稱為產碳青黴烯酶腸內菌（carbapenemase-producing Enterobacteriaceae, CPE），其中最常見且研究較多的抗藥性基因為blaKPC（Klebsiella pneumoniae carbapenemase, KPC）及blaNDM-1（New Delhi metallo-β-lactamase 1, NDM-1）。乳酸桿菌（Lactobacillus spp.）能產生乳酸及醋酸等有機酸降低腸道酸鹼值，或是分泌具抑菌活性之小分子多肽類物質，進而抑制腸道內病原菌的生長，維持腸道菌相，減少細菌感染發生。本研究的目的在於以試管內（in vitro）方式，測試乳酸桿菌是否能有效抑制CPE。
研究結果顯示，用瓊脂挖洞擴散法（well diffusion method）初步評估五株不同乳酸桿菌上清液對十八株CPE的抑制效果，結果都能產生明顯的抑制圈，整體平均約15 mm。接著以微量肉湯稀釋法（broth microdilution method）來評估不同濃度之乳酸桿菌上清液對CPE之抑制能力，當上清液濃度為50% 時具有完全的抑菌能力，而在25% 時抑菌能力略有下降，平均抑制能力在90-100% 之間；但上清液濃度僅6.25% 時，抑菌能力就有明顯的差異，由0% 至100%。最後以時間殺菌曲線法（time killing method）測試LUC0180個別與7株CPE共同培養時的抑菌能力。當乳酸桿菌起始菌量為1x10⁸ CFU/ mL，在24小時皆達完全抑菌效果，且pH值下降至4.0左右，而起始菌量為1x10⁷ CFU/ mL時，對其中5株CPE在48小時也能完全抑菌；起始菌量為1x10⁶ CFU/ mL時，則僅對其中2株CPE於48小時具有有效抑制，觀察其pH值最終約落於4.3，沒有抑制效果之組別的pH值則亦無明顯下降之現象。
本體外研究證實不同乳酸桿菌對產KPC或NDM-1之CPE均有明顯抑制效果，但仍需更進一步以動物實驗及臨床證據來佐證此結果，以期未來能實際運用於醫療保健上。
Enterobacteriaceae are the normal flora of human gut system. However, some of them may develop resistance to many antibiotics, including carbapenems, which would be defined as carbapenem-resistant Enterobacteriaceae (CRE). Some of CRE with gene encoding carbapenemase, which can break down carbapenems, called carbapenemase-producing Enterobacteriaceae (CPE). The most common carbapenemase are blaKPC (Klebsiella pneumoniae carbapenemase, KPC) and blaNDM-1 (New Delhi metallo-β-lactamase 1, NDM-1). Lactobacillus can produce lactic acid, acetic acid and other acids to decrease the intestinal pH value, or to secret some antimicrobial peptide. Therefore, they can inhibit the growth of intestinal pathogens to maintain normal intestinal microflora. Our studies intend to test whether Lactobacillus spp. can inhibit CPE effectively in vitro.
First, we used well diffusion to analyze that antimicrobial effects of supernatant of five selected Lactobacillus strains against eighteen strains of CPE, and the average inhibition zone are about 15 mm. Second, we used broth microdilution method to test the inhibition percentage of CPE by different concentrations of different Lactobacillus supernatant. Fifty percent supernatant can totally inhibit CPE, 25% supernatant can inhibit 90-100%, and 6.25% supernatant can inhibit 0-100%. Third, we used time killing methods to test antibacterial ability of LUC0180. When co-cultured with different seven CPE strains individually, with inoculum 1x10⸠CFU/ mL which can totally inhibit CPEs and the pH value drop to around pH 4.0 at 24 hr. With the starting inoculum of 1x10ⷠCFU/ mL which can also completely inhibit five CPEs. However, with the inoculum of 1x10ⶠCFU/ mL, which can only inhibit two CPEs at 48 hr. The pH finally falls to about 4.3 with the inoculum of 1x10ⷠand 1x10ⶠCFU/ mL in coculture groups. But the pH was not decreased significantly with the group without inhibition effect.
Our study supported that Lactobacillus spp. can significantly inhibit KPC and NDM-1 in vitro. The amount of bacteria number can be reduced as the pH value decreased. However, further in vivo or clinical study is warranted to confirm these findings. |
