Biofilm formation of Tenacibaculum maritimum, a fish pathogenic bacteria, to evaluate the antimicrobial activity of fish skin mucus

Marc TejeroIgnasi SanahujaCarlos BalsalobreAntoni IbarzCristina MadridLaura Fernandez-Alacid^*

• University of Barcelona, Barcelona, Spain

Biofilms, defined as aggregates of microorganisms embedded in a self-produced matrix of extracellular polymeric substances that adhere to each other and/or to a surface, are produced by most bacteria in natural and pathogenic ecosystems. In clinical and veterinary microbiology, the presence of biofilms poses a major challenge due to their recalcitrance to antimicrobial treatments. Biofilms pose a significant concern in aquaculture due to their formation on facility surfaces and culture sediments. Tenacibaculum maritimum is the primary causative agent of tenacibaculosis, a worldwide emerging disease in fish cultures, resulting in significant economic losses. While the antibacterial activity of fish skin mucus against this pathogen has been evaluated in vitro, its effects on T. maritimum biofilms have not yet been determined. We provide a methodology for in vitro formation and quantification of T. maritimum biofilms to monitor antibacterial properties of mucus. Assays in 48-wells microplates comparing different culture volumes and incubation times have been carried out. The optimal condition for biofilm formation was defined as 24hours in MB at 25ºC using 200µL culture volume. These conditions supported the development of a biofilm while allowing to save both time and mucus. The effects of skin mucus on T. maritimum were evaluated in terms of its ability to inhibit planktonic bacterial growth and its impact on biofilm formation and dispersion. Biofilm produced was assessed by measuring biofilm biomass and biofilm-associated metabolic activity. The results obtained demonstrated that mucus significantly impairs the planktonic growth and biofilm formation of T. maritimum in a concentration-dependent manner. Remarkably, non-diluted mucus completely inhibited T. maritimum planktonic growth and biofilm metabolic activity, while caused a reduction of 81.16 ± 2.54 % in biofilm biomass. Conversely, its impact on mature biofilms was limited, resulting in only a 50% reduction in metabolic activity and a 40% decrease in biofilm biomass. This study provides a platform to assess how different fish culture conditions affect the host’s susceptibility to T. maritimum infections. Additionally, it opens the door to studies analyzing the components of mucus responsible for its antibacterial activity, aiming to develop novel therapeutic compounds for targeting biofilms formed by this pathogen.