Candida infections of orthopedic implants are one of the most detrimental orthopedic implant-related complications with unsuccessful treatment and a poor prognosis. Most orthopedic Candida infections form biofilms and have resistance to the commonly used antifungal agents. This study aimed to develop a novel combination of normally prescribed drugs against Candida biofilm on orthopedic implants.

We cultured 26 clinical isolates of Candida strains to form biofilm without titanium sheets or on titanium sheets, which are the most commonly used materials for permanent or orthopedic implants. The checkerboard method was used to evaluate the synergistic effects of chlorhexidine (CHL) and azoles on these Candida biofilms. For the evaluation of synergistic effects, we constructed the cell viability assay by fluorescence staining and CFU reduction hot map of Candida.

Twenty-six clinical isolates of Candida strains formed biofilm in 96-well plates without titanium sheets, and we selected 9 of them to form biofilm on titanium sheets in 24-well plates. In Candida biofilm formed in 96-wells, the synergistic rates of CHL with fluconazole, itraconazole, and voriconazole were 61% (16/26), 65% (17/26), and 23% (6/26), respectively. When compared to the blank control group, CHL monotherapy significantly inhibited biofilm formation on titanium sheets (P < 0.05). We demonstrated 100% synergistic rates of the CHL and fluconazole combination against Candida biofilm formation on titanium sheets, and the minimum inhibitory concentration of CHL and FLU decreased four- to eight-fold.

We concluded that CHL combined with azoles inhibited the Candida biofilm formation 96-wells or on titanium sheets and has the potential to control the infections of orthopedic implants.