In vitro efficacy of eugenol in inhibiting single and mixed-biofilms of drug-resistant strains of Candida albicans and Streptococcus mutans.

Background: Candida albicans is frequently associated with mixed infections of Streptococcus mutans in plaque biofilms. These pathogens under chemical interactions resulting in mixed biofilm development have turned it into a prevalent and costly oral disease, which is not successfully being treated by existing chemotherapeutics.
Hypothesis: Considering the need for newer drugs to overcome this challenge, the present study was aimed to investigate the efficacy of eugenol in inhibiting single and mixed biofilms of C. albicans and S. mutans.
Methods: The broth dilution assay was used to determine drug resistance in the test strains. Biofilm formation on polystyrene microtiter plate was studied by XTT reduction assay whereas biofilm development on glass coverslips was assessed using 0.1% crystal violet and visualised under light microscope. Single and mixed biofilms formed on glass coverslips in the presence and absence of eugenol was analysed by scanning electron microscopy.
Results: In our study, all the thirteen strains of C. albicans were resistant to fluconazole, itraconazole, ketoconazole, amphotericin B except C. albicans (CAJ-01) and C. albicans MTCC3017 which were sensitive to fluconazole. S. mutans MTCC497 was resistant to ampicillin, azithromycin, ceftriaxone and vancomycin. Among all the strains of C. albicans, CAJ-01, C. albicans ATCC90028 and C. albicans MTCC3017 formed strong biofilms and rest of the strains considered as moderate to weak biofilm formers. S. mutans MTCC497 was also formed strong biofilms. Eugenol showed concentration dependent anti-biofilm activity against single and mixed biofilms of C. albicans (CAJ-01) and S. mutans MTCC497. At sub-MIC of eugenol (100 μg/ml), the biofilm formation was 36.37% and 29.72% in CAJ-01 and S. mutans MTCC497, respectively, whereas 52.65% in mixed biofilms. The cell viability assay showed significant reduction (p < 0.05) in the log ₁₀ CFU/ml from 6.3 to 4.8 at 200 μg/ml of eugenol for CAJ-01, whereas, from 6.4 to 3.8 and 5.3 for S. mutans MTCC497 strains in single and mixed biofilms, respectively. Scanning electron microscopy showed the disruption of cell membrane and matrix structure in both single and mixed biofilms.
Conclusions: Eugenol at sub-MICs effectively inhibited single and mixed biofilms formed by the drug resistant strains of two oral pathogens, C. albicans and S. mutans through multiple mode of action.
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