溫度誘發霍亂弧菌O139進入活而不長狀態和其致病力   全文下載

Induction of Viable but Non-culturable State in Vibrio cholerae O139 by Temperature and Its Pathogenicity

本研究測定溫度對致病性霍亂弧菌O139成為活而不長狀態的影響，並進一步決定在可被培養、不可被培養和恢復生長三種不同狀態下，菌株的毒力和細胞蛋白質的表現。當O139菌株在含有0.5%食鹽的河水微環境中，4℃培養35-45天時，培養基的菌落已少於1 CFU/mL，而活菌計數的數值並無明顯變化。然而，培養在15℃或25℃下，並未偵測到菌株進入活而不長狀態。將4℃誘發的活而不長細菌樣本接種至含有分解H2O2化合物的培養基或直接轉到28℃下培養，活而不長的O139菌株均會回復生長。吸附實驗顯示，可被培養的O139細菌吸附到HeLa細胞株的數目明顯高於不被可培養的和恢復生長的細菌。細胞毒殺試驗顯示，可被培養的O139細菌會造成HeLa細胞全部殺死，恢復生長的細菌僅會使32.1 ± 4.6%標的細胞死亡；以不可被培養的細菌與無菌培養基分別培養後比較，標的細胞的死亡百分率並無差異。以可被培養的O139細菌攻擊小鼠後之致死率為80%；但是以不可被培養的細菌和恢復生長的細菌攻擊後，所有的小鼠都活存下來。

In this study, the effect of temperature on the formation of viable but non-culturable (VBNC) pathogenic Vibrio cholerae O139 was examined. Furthermore, both the virulence and cellular protein expression of the bacterial cells under culturable, non-culturable and resuscitated states were determined. Following the incubation of bacterial cells in river water microcosms (RWM) with 0.5% NaCl at 4°C for 35-45 days, plate counts declined to less than 1 CFU/mL; but viable counts dropped to 104-105 cells/mL during the first 2 weeks and maintained at this level till the end of incubation. However, the entry of cells into a VBNC state was not detected after the cells were cultured at either 15 or 25°C. When the 4°C -induced VBNC samples were either inoculated to a plate supplemented with H2O2-degrading compounds or shifted to 28°C, non-culturable O139 cells regained their culturability. The adhesion test showed that the number of O139 cells adhering to HeLa cells was highest in culturable cells and significantly lower in either resuscitated cells and non-culturable cells. As for the cytotoxicity test, all HeLa cells died after incubation with culturable O139 cells, whereas 32.1 ± 4.6% and 100% of target cells died after incubation with resuscitated cells for 3 and 5 hr, respectively. No difference was observed between the percentages of cell death after incubation with non-culturable cells and with sterile culture medium. When mice were challenged with culturable O139 cells, the mortality was 80%. However, all mice survived after challenge with either VBNC or resuscitated cells.