| Summary: | Biofilms consist of microbial communities enclosed in a self-produced extracellular matrix which is mainly responsible of biofilm virulence. Targeting this matrix could be an effective strategy to control biofilms. In this work, we examined the efficacy of two proteolytic enzymes, pepsin and trypsin, to degrade <i>P. aeruginosa</i> and <i>E. faecalis</i> biofilms and their synergistic effect when combined with carvacrol. The minimum dispersive concentrations (MDCs) and the contact times of enzymes, as well as the minimal inhibitory concentrations (MICs) and contact times of carvacrol, were determined against biofilms grown on polystyrene surfaces. For biofilms grown on stainless steel surfaces, the combined pepsin or trypsin with carvacrol treatment showed more significant reduction of both biofilms compared with carvacrol treatment alone. This reduction was more substantial after sequential treatment of both enzymes, followed by carvacrol with the greatest reduction of 4.7 log CFU mL<sup>−1</sup> (<i>p</i> < 0.05) for <i>P. aeruginosa</i> biofilm and 3.3 log CFU mL<sup>−1</sup> (<i>p</i> < 0.05) for <i>E. faecalis</i> biofilm. Such improved efficiency was also obvious in the epifluorescence microscopy analysis. These findings demonstrate that the combined effect of the protease-dispersing activity and the carvacrol antimicrobial activity could be a prospective approach for controlling <i>P. aeruginosa</i> and <i>E. faecalis</i> biofilms.
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