外源硫化氢对氧化应激下铜绿假单胞菌生长和生物膜的调节作用

Regulation of Growth and Biofilm Formation of Pseudomonas aeruginosa by Exogenous Hydrogen Sulfide under Oxidative Stress

摘要:

探究外源硫化氢（H2S）对铜绿假单胞菌（Pseudomonas aeruginosa，PAO1）在氧化应激（H2O2）胁迫条件下的作用。选取硫氢化钠（NaHS）作为外源H2S的供体。将铜绿假单胞菌分为两组；对照组的培养基中不添加NaHS；实验组的培养基中添加0.2 mmol/L NaHS；观察两组菌株在0、1、2、2.5 mmol/L H2O2的压力下存活率、细胞形态、DNA外渗量、生物膜生长的变化情况。研究结果表明，随着时间的推移（从0.5 h到1 h再到1.5 h），与对照组菌株相比，实验组菌株的存活率显著降低（p<0.05），其中在1 mmol/L H2O2的处理下，实验组比对照组显著降低24.83%、15.69%、11.36%；在2 mmol/L H2O2的处理下，实验组比对照组显著降低4.92%、11.16%、5.75%；在2.5 mmol/L H2O2的处理下，实验组比对照组降低6.83%、2.98%、0.39%。实验组较对照组相比，细胞形态受损和DNA外渗更加严重。利用结晶紫染色、扫描电子显微镜和激光共聚焦显微镜检测在不同浓度H2O2胁迫下生物膜形成的变化，结果表明，与对照组菌株的生物膜相比，在含有1 mmol/L和2 mmol/L H2O2的Luria Bertani（LB）培养基中实验组菌株的生物膜形成在培养48 h和72 h后显著减少。结果表明，H2S能增强H2O2对铜绿假单胞菌细胞的损伤。

Abstract:

The effect of exogenous hydrogen sulfide (H2S) on Pseudomonas aeruginosa (PAO1) growth under hydrogen peroxide (H2O2)-induced oxidative stress conditions was investigated using sodium sulfide hydride (NaHS) as the exogenous H2S-releasing agent. Two groups of Pseudomonas aeruginosa were grown: a control and an experimental group with no NaHS and with 0.2 mmol/L NaHS added to the medium, respectively. The survival rate, cell morphology, DNA extravasation volume, and biofilm growth of the strain in both groups were observed under the oxidative stress induced by 0, 1, 2, and 2.5 mmol/L H2O2. The results showed that with the passage of time (i.e., from 0.5 h to 1 h through to 1.5 h), the survival rate of the cells in the experimental group was significantly lower than that in the control group (p<0.05); i.e., at 0.5, 1, and 1.5 h, the survival rates of the experimental group were reduced by 24.83%, 15.69%, and 11.36%, respectively, under 1 mmol/L H2O2 treatment; by 4.92%, 11.16%, and 5.75%, respectively, under 2 mmol/L H2O2 treatment; and by 6.83%, 2.98%, and 0.39%, respectively, under 2.5 mmol/L H2O2 treatment, relative to those of the control group. The experimental group demonstrated more severe cell morphology damage and DNA extravasation than the control group did. Variations in biofilm formation under different H2O2-induced stress conditions were examined using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy. The results showed a significant reduction in biofilm formation by the experimental group compared with that by the control group after 48 and 72 h of culture in Luria-Bertani medium containing 1 or 2 mmol/L H2O2. These results suggest that H2S enhances H2O2-induced PAO1 cell damage in this bacterial strain.