超声和绿原酸联用对杀鲑气单胞菌生物膜的协同杀菌作用

Synergistic Bactericidal Effect of the Combined Use of Ultrasound and Chlorogenic Acid against Aeromonas salmonicida Biofilms

摘要:

为了探究超声联合绿原酸处理对杀鲑气单胞菌生物膜的杀菌效果及可能的作用机制，分析了超声（ultrasound，US）、绿原酸（chlorogenic acid，CA，0.5%、1%、2%）以及超声联合绿原酸（US+0.5% CA、US+1% CA、US+2% CA）处理后的杀鲑气单胞菌生物膜。结果表明US+1% CA可在60 min内使得杀鲑气单胞菌生物膜细胞降低8 logCFU/mL，这与2% CA单独处理的杀菌效果一致。胞外多糖含量分析发现US+1% CA能使得生物膜内可溶性多糖与不可溶性多糖含量分别降低到13.01和16.52 μg/mL，扫描电镜、激光共聚焦电镜和胞外物质含量分析发现联合处理明显导致生物膜空间结构的瓦解，造成了生物膜内菌体细胞的细胞膜通透性急剧增加，导致胞外核酸和ATP的浓度显著高于对照组（p<0.05）。此外，联合处理显著降低了菌体细胞内呼吸链脱氢酶活性（p<0.05）。综上可知，US+CA处理能通过协同降低生物膜胞外多糖的含量，破坏菌体细胞的完整性及胞内酶的活性来达到对杀鲑气单胞菌生物膜的控制效果。

Abstract:

The bactericidal effect of ultrasound (US) combined with chlorogenic acid (CA) on Aeromonas salmonicida biofilms was investigated, and the mechanism underlying the combined bactericidal effect was explored. A. salmonicida biofilms were treated with US, chlorogenic acid (0.5%, 1%, 2% CA), or US combined with chlorogenic acid (US plus 0.5% CA, US plus 1% CA, US plus 2% CA). Results showed that treatment with US combined with 1% CA for 60 min reduced the formation of A. salmonicida biofilms by approximately 8 logCFU/mL, similar to that in treatment using 2% CA alone. Analysis of the extracellular polysaccharide content revealed that the concentrations of soluble and insoluble polysaccharides in A. salmonicida biofilms were reduced to 13.01 and 16.52 μg/mL, respectively, after treatment with US combed with 1% CA. Analysis of extracellular contents using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) revealed that the combined treatment resulted to a significant disintegration of the spatial structures of biofilms. Subsequently, membrane permeability of the bacterial cells in the biofilms sharply increased, resulting in significantly higher extracellular nucleic acid and ATP concentrations than the control group (p<0.05). Moreover, combined treatment significantly lowered the activity of respiratory chain dehydrogenase (RCD) within the bacterial cells (p<0.05). These results suggest that the combined use of US and CA exerted synergistic bactericidal effects on biofilms by reducing extracellular polysaccharide content and intracellular enzymatic activity of biofilms and disrupting the bacterial cell integrity.