纳米氧化铝在多重耐药质粒RP4接合转移过程中对细菌形态学的影响

The Mechanism of Al_2O_3 Nanoparticle Influence on RP4-mediated Conjugation:a Preliminary Research in Bacterium Morphology

目的探讨纳米氧化铝(平均粒径为20nm)对液相条件下多重耐药质粒RP4接合转移的影响,并从形态学角度初步探讨其影响机制。方法接合供体菌为大肠杆菌HB101(RP4),受体菌为Salmonella aberdeen Kauffmann 50312(strR),供、受体菌液均为109cfu/ml(浓度比为1∶3),纳米Al2O3浓度分别为0.005、0.05、0.5、5、50mmol/L,25℃静止接合8h后计算转接合子数,然后采用透射电镜和激光扫描共聚焦显微镜进行形态学研究。结果RP4的接合转移随着纳米Al2O3浓度的升高具有上升趋势。5mmol/L和50mmol/L纳米Al2O3组接合率分别为空白对照组的150倍和40倍,差异均有统计学意义。透射电镜观察结果表明,空白对照组只能观察到单个供、受体菌接合,5mmol/L和50mmol/L纳米Al2O3组能观察到多个细菌同时发生接合的情况;50mmol/LAl2O3组一些细菌观察不到完整的细胞膜结构,该损伤可能是引起50mmol/L纳米Al2O3组接合率低于5mmol/L纳米Al2O3组的原因。激光扫描共聚焦显微镜观察结果提示纳米Al2O3能损伤细菌的细胞膜,细胞膜的损伤程度与进入细菌的纳米Al2O3的浓度呈正相关。结论纳米Al2O3能够促进接合子的产生,其影响机制可能是因为纳米Al2O3能够改变细胞膜通透性,间接影响接合过程,或者直接促进接合基因的表达所致。

Objective To explore the influence of Al2O3 nanoparticle （20nm in average） on RP4-mediated conjugation and its mechanism was explored further. Methods Mating was conducted between E.coli HB101（RP4） and Salmonella aberdeen Kauffmann 50312（strR） in saline at 25 ℃ without stirring for 8 h,the concentrations of Al2O3 nanoparticle were 0.005,0.05,0.5,5,50 mmol/L respectively. The initial concentration of donor and recipient were both about 109 cfu/ml（donor per recipient ratio was 1∶3）.Later transmission electron microscope（TEM） and laser scan confocal microscope （LSCM） were exploited to investigate cell morphology and structure. Results 5 mmol/L and 50 mmol/L Al2O3 nanoparticle could increase the conjugal transfer frequency by 150-fold and 40-fold respectively. TEM observation on thin section indicated several bacterial were prone to form conjugational junction together in 5 mmol/L and 50 mmol/L Al2O3 nanoparticle treated group,which was rarely seen in control group. Meanwhile the cell envelope of bacterial was significantly damaged upon the treatment by 50 mmol/L Al2O3,which might be the reason why transfer frequency of 50 mmol/L Al2O3 group was less than 5 mmol/L Al2O3 group. LSCM result indicated that Al2O3 nanoparticle might damage cell envelope and the damage was positively related to the accumulation of Al2O3 in bacterial. Conclusion Al2O3 nanoparticle can stimulate RP4 conjugal transfer. Conjugative gene is highly regulated. Al2O3 nanoparticle might modulate conjugative gene expression directly or influence cell membrane permeability and thus modulate the process of conjugation indirectly .