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