Zerumbone Combined with Hydrolytic Enzymes: A Promising Strategy to Control Mixed Biofilms of Streptococcus mutans and Fluconazole-Resistant Candida albicans

Abstract

This in vitro study evaluated the efficacy of Zerumbone (ZER), a sesquiterpene phytochemical extracted from the rhizomes of Zingiber zerumbet (L.) Smith with reported antimicrobial properties, alone or combined with Dextranase (DEX) and DNase I (DNase), against polymicrobial biofilms of fluconazole-susceptible and fluconazole-resistant Candida albicans and Streptococcus mutans. Mixed-species biofilms were formed with C. albicans ATCC 90028 (CaS), C. albicans ATCC 96901 (CaR), and S. mutans UA159 and assessed in inhibition and disruption assays. Treatments included ZER, DEX, and DNase, individually or in combination. Efficacy was analyzed by total biofilm mass, metabolic activity (CFU/mm2), and live/dead cell assessment using CLSM. Data were analyzed by one- and two-way ANOVA followed by Tukey’s posthoc test (α = 0.05). In the biofilm inhibition assay, ZER and its combinations, particularly ZER + DEX, demonstrated enhanced activity against both C. albicans and S. mutans strains. For CaS + Sm biofilms, ZER, ZER + DEX, and DNase + DEX achieved similar and the greatest reduction in CaS viability (>2.7 log10), whereas, for S. mutans, DNase + DEX, ZER, and ZER + DEX were the most effective (>2.1 log10). ZER + DEX also significantly reduced total microbial load in CaR + Sm biofilm. Notably, ZER + Dextranase was the only treatment that significantly reduced dry weight in susceptible biofilms and produced the highest biomass reduction in both susceptible and resistant biofilms as measured by crystal violet staining. In the biofilm disruption assay, ZER + DNase caused the greatest reduction in CaS viability (2.08 log10), whereas ZER and ZER + DNase were most effective against S. mutans (>2.5 log10). For CaR + Sm biofilms, ZER + DEX and ZER + DNase were among the most effective treatments. The confocal microscopy results corroborated these findings, showing that ZER and all treatments combined with ZER significantly increased the proportion of dead cells under both tested conditions. Within the limitations of this in vitro study, ZER alone or in combination with matrix-degrading enzymes (DEX and DNase), showed enhanced activity against both inhibition and disruption of mixed-species biofilms of C. albicans and S. mutans, including fluconazole-resistant strains, suggesting potential for further investigation as an adjunctive antimicrobial approach.