Broaden properties of ambroxol hydrochloride as an antibiofilm compound

Biofilm-associated microorganisms can cause many infections and are an important cause of resistance to several antimicrobials. The antibiotic crisis has led to a pressing need for new therapeutic tools. Ambroxol is frequently used as a mucolytic agent in respiratory diseases with increased mucus production. In addition, a wide range of properties has been described, including the effect on biofilms. In this work, we evaluate the anti-biofilm effect of ambroxol on four strains with clinical relevance: Proteus mirabilis, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii. In vitro, biofilm formation was assessed using the crystal violet quantification technique in microplate and glass coverslip. The inhibition of biofilm formation was evaluated by adding ambroxol at the initial time. Ambroxol hydrochloride was evaluated over the preformed biofilm and live/dead bacteria were quantified. The effect of ambroxol in the ethidium bromide efflux assay and the relative expression of the five major P. mirabilis efflux pump family genes were analyzed. Ambroxol inhibited biofilm formation in all the bacteria tested. Moreover, ambroxol significantly reduces both biofilm biomass and viable bacteria. Ambroxol was able to affect P. mirabilis efflux pumps depending on the concentration used and induced the overexpression of several efflux pump genes. In summary, ambroxol kills planktonic cells, reduce biofilm biomass as it increases cell death, and affect the expression of efflux pumps. Furthermore, it presents a viable alternative for the treatment of biofilm infection alone or in combination with antibiotic therapy.