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目的体外培养铜绿假单胞菌(pseudomonas aeruginosa,PA),观察低浓度红霉素对铜绿假单胞菌蹭行运动(twitching motility)的影响。方法将铜绿假单胞菌 ATCC1244菌株接种于3组不同浓度红霉素(2.5 mg/L、0.5 mg/L、0.25 mg/L)的细菌培养基中,同时接种于未加红霉素的对照培养基中,采用肉眼观察、免疫荧光显微镜检查、免疫印迹实验、点杂交实验、电镜观察等方法观察铜绿假单胞菌蹭行运动的变化及鉴别其蹭行运动的分子组成。结果经过18～36 h 的孵育,LB 平板上不同浓度的红霉素对 PA 蹭行运动有抑制作用,细菌光晕(halation)直径与药物浓度呈反比;18 h 时细菌的平均光晕直径2.5 ms/L 组为(0.48±0.14)cm,0.5 mg/L 组为(0.64±0.20)cm,0.25 mg/L 组为(0.95±0.18)cm,对照组为(1.40±0.21)cm,组间比较差异有统计学意义(F=123.15,P<0.01);24 h时2.5 mg/L 组为(0.67±0.12)cm,0.5 mg/L 组为(0.82±0.23)cm,0.25 mg/L 组为(1.18±0.24)cm,对照组为(1.58±0.28)cm,组间比较差异有统计学意义(F=76.37,P<0.01);36 h 时2.5 mg/L 组为(0.91±0.17)cm,0.5 mg/L 组为(1.04±0.32)cm,0.25 mg/L 组为(1.49±0.31)cm,对照组为(2.07±0.38)cm,组间比较差异有统计学意义(F=54.75,P<0.01)。免疫荧光显微镜观察结果可见细菌菌体一端呈簇状突起,为Ⅳ型菌毛的位置所在,簇状突起的多少与药物浓度呈反比。免疫印迹实验结果显示,随着药物浓度的减少,Ⅳ型菌毛的主要成分 PilA 蛋白表达增加。点杂交实验结果显示在蹭行运动中起作用的 PilA 蛋白主要表达在光晕的最外周,但组间未见明显区别。电镜下可见红霉素作用后 PA 菌体一端的Ⅳ型菌毛较对照组减少,部分脱离菌体。结论低浓度红霉素对 PA 的蹭行运动有抑制作用,且与红霉素浓度呈正比。
Objective To study the effect of low concentration erythromycin on the twitching motility of Pseudomonas aeruginosa in vitro culture of Pseudomonas aeruginosa (PA). Methods Pseudomonas aeruginosa ATCC1244 strain was inoculated into three different concentrations of erythromycin (2.5 mg / L, 0.5 mg / L, 0.25 mg / L) in bacterial culture medium and inoculated into the control without erythromycin Culture medium was observed by macroscopic observation, immunofluorescence microscopy, Western blotting, dot hybridization, electron microscopy and other methods to observe the changes of Pseudomonas aeruginosa rub rub exercise and identify the molecular composition of rub rub exercise. Results After 18-36 h of incubation, different concentrations of erythromycin on LB plates inhibited the PA rubbing motion. The diameter of the bacteria halation was inversely proportional to the drug concentration. The average halo diameter of the bacteria at 18 h was 2.5 (0.48 ± 0.14) cm in MS / L group, (0.64 ± 0.20) cm in 0.5 mg / L group and (0.95 ± 0.18) cm in 0.25 mg / L group and 1.40 ± 0.21 cm in control group (0.67 ± 0.12) cm at 24 h and (0.82 ± 0.23) cm at 0.25 mg / L and 0.25 mg / L at 0.5 mg / L, respectively (F = 123.15, P <0.01) (1.18 ± 0.24) cm in the control group and (1.58 ± 0.28) cm in the control group. The difference between the two groups was statistically significant (F = 76.37, P <0.01) (1.04 ± 0.32) cm in 0.5 mg / L group, (1.49 ± 0.31) cm in 0.25 mg / L group and (2.07 ± 0.38) cm in control group, the difference was statistically significant (F = 54.75 , P <0.01). The results of immunofluorescence microscopy showed that one end of the bacterial cell body was in the form of tufted protrusions, which was the location of type IV pili. The number of tufted protrusions was inversely proportional to the drug concentration. The results of Western blotting showed that the expression of PilA protein, which is the main component of type IV pili, increased with the decrease of drug concentration. The result of spot hybridization showed that the PilA protein that plays a role in the rubbing motions is mainly expressed in the outermost periphery of the halo, but no significant difference was found between the two groups. Electron microscope shows erythromycin-type PA strain at the end of the type IV pili reduced compared with the control group, part of the detached cells. Conclusions Low concentration erythromycin can inhibit the rubbing of PA and is proportional to the concentration of erythromycin.