Vibrio spp. are Gram-negative, rod-shaped bacteria that are widely distributed in freshwater, estuarine, and marine environments. They have the potential to cause infections in animals and are frequently isolated from aquaculture farms, resulting in mss mortalities and significant economic losses. Vibrio bacterial pathogens may persist in aquaculture ponds in a biofilm form, a complex microbial structure, attached to a surface and integrated into an adhesive extracellular matrix. This matrix provides protection against environmental stressors, including cleaning and disinfection procedures used in aquaculture industry. Therefore, it is crucial for professionals to use effective disinfectants to eliminate these biofilms and prevent the transfer of Vibrio spp. cells from surfaces to food.
The aim of the present study was to assess the effectiveness of benzalkonium chloride, a commonly used disinfectant in aquaculture farms, against biofilms of Vibrio species that are frequently encountered in aquaculture and pose significant economic challenges.
Two strains of V. parahaemolyticus , two strains of V. alginolyticus , and one strain each of V. harveyi and V. cholerae were studied. Biofilms were grown in a 96-well microtiter plate at 23°C for 24 hours to simulate conditions encountered in aquaculture farms. Treatments with disinfectants or water (as control) were applied either before biofilm formation or on pre-formed 24-hour biofilms. The total biomass of the biofilms (matrix and bacterial population) was evaluated by crystal violet staining, the metabolic activity of cells within the biofilms was measured by 2,3,5-triphenyl tetrazolium chloride, a metabolic dye used as a mitochondrial redox potential indicator staining. The viability status of these bacterial populations was then evaluated by flow cytometry and epifluorescence microscopy coupled with a live/dead staining.
Surprisingly, in four out of six strains of Vibrio studied, crystal violet quantification showed that benzalkonium chloride induced overproduction of Vibrio biofilm biomass. Metabolic activity was not always correlated with the overproduction of biofilm. Flow cytometry and microscopy data indicated that injured cells were responsible for this overproduction of biofilm.
This study demonstrates for the first time the ineffectiveness of benzalkonium chloride in removing biofilms formed by Vibrio species that pose significant economic problems in aquaculture. Worse still, this study shows that benzalkonium chloride induces overproduction of Vibrio biofilms, which represents a major risk in the resistance, dissemination and persistence of this pathogenic organism.