Concomitant Inhibition and Collaring of Dual-Species Biofilms Formed by Candida auris and Staphylococcus aureus by Triazole Based Small Molecule Inhibitors

Abstract

Background/objectives: Biofilm-associated infections, particularly those involving Candida auris and Staphylococcus aureus, pose significant challenges in clinical settings due to their resilience and resistance to conventional treatments. This study aimed to synthesize novel triazole derivatives containing a piperazine ring via click chemistry and evaluate their efficacy in disrupting biofilms formed by these pathogens.

Methods: Triazole derivatives were synthesized using click chemistry techniques. The antimicrobial activity of the compounds was tested against planktonic cells of C. auris and S. aureus in single and dual-species culture conditions. Biofilm disruption efficacy was assessed, alongside the evaluation of physicochemical properties, oral bioavailability potential, and toxicity profiles.

Results: The compound T3 demonstrated potent antimicrobial activity against planktonic cells of C. auris and S. aureus in both single and dual-species cultures. T3 exhibited significant efficacy in reducing microbial viability within biofilms formed by these pathogens. Physicochemical analyses revealed favorable solubility and permeability profiles, supporting its potential for oral bioavailability. Toxicity assessments showed a non-toxic profile, highlighting a promising safety margin for further development.

Conclusions: This study underscores the anti-biofilm properties of novel triazole-piperazine derivatives, particularly T3, against single and dual-species biofilms of C. auris and S. aureus. These findings position T3 as a promising candidate for developing therapies targeting polymicrobial infections and provide a foundation for future research into alternative strategies for combating biofilm-associated infections.

Keywords: biofilms; click chemistry; drug resistance; physicochemical properties; triazole.

Figure 1

Different antimicrobial drugs featuring a triazole (red) and piperazine ring (blue).

Scheme 1

Synthesis of piperazine based triazole acetamide derivatives (T1–T12). Reagents and conditions. (i) Dry acetone, r.t (ii) Sodium ascorbate, CuSO₄, DMF (iii) HATU/DIPEA, DMF, 60 °C.

Figure 2

Bioavailability radar plot of the most active compound T3 and the reference drug fluconazole obtained using ADMETLab 3.0. Green represents the lower limit, while as blue represents the upper limit. The compound properties are depicted in orange.

Figure 3

XAI heatmaps and molecular diagrams of T3 and Fluconazole (FLZ), highlighting key structural features that contribute to their classification as non-toxic compounds. The visualizations depict the influence of individual atoms on the overall toxicity profile, with green indicating toxicity-reducing features and red representing toxicity-enhancing features. Images were obtained using the cytosafe tool from LabMol.

Figure 3

XAI heatmaps and molecular diagrams of T3 and Fluconazole (FLZ), highlighting key structural features that contribute to their classification as non-toxic compounds. The visualizations depict the influence of individual atoms on the overall toxicity profile, with green indicating toxicity-reducing features and red representing toxicity-enhancing features. Images were obtained using the cytosafe tool from LabMol.

Figure 4

XTT reduction assay to evaluate biofilm inhibitory potential of T3 under single and dual—species condition. The absorbance was measured at 490 nm. Effect on biofilm formation (A) and on 24 h preformed biofilms (B) of individual pathogens and in combination was studies in untreated (control) and BIC₅₀ and BIC₉₀ of T3. Data were expressed as mean ± SEM and were analyzed for statistical significance using two-way ANOVA followed by Tukey’s multiple comparisons test. Significance levels are denoted by *** p < 0.001; **** p < 0.0001.

Figure 5

Viability assay of single and dual—species biofilms at different concentrations of T3. Cells embedded in the dual—species biofilm of C. auris and S. aureus (C. auris +, viable cells of C. auris in mixed biofilms; S. aureus +, viable cells of S. aureus in mixed biofilms). (A) Cellular viability during biofilm formation. (B) Cellular viability in 24 h preformed biofilm. Data were expressed as mean ± SEM and were analyzed for statistical significance using two-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are denoted by ** p < 0.01, *** p < 0.001; **** p < 0.0001.

Figure 6

Percent inhibition in total biomass of formation and 24 h preformed dual—species biofilm of C. auris and S. aureus. Violet crystal (VC) staining method was adopted to evaluate the biofilm disruptive property of T3 at various concentrations. Percent inhibition in total biomass in untreated control was considered 0%.**, p < 0.005; ***, p < 0.0005.

Figure 7

Light micrograph of biofilm formed by C. auris and S. aureus under monomicrobial and mixed condition. The figure displays, (A) the inhibitory effect of T3 on biofilm formation and (B) effect on 24 h preformed biofilm. White and black arrows denote the presence of C. auris in the dual—species biofilm. Scale bar = 50 µm.

Figure 8

CLSM analysis of dual—species biofilm stained with Con-A and FUN-1. The figure represents the anti-biofilm potential of T3 against biofilm formation, and 24 h mature polymicrobial biofilms formed by C. auris and S. aureus. The images show yeast and bacterial cells embedded in the dual–species biofilms that is captured at 63× oil immersion objective at ×2.0 magnification. Scale bar = 10 µm.

Figure 9

SEM micrographs dual-species biofilms formed by C. auris and S. aureus. (A) Untreated mixed biofilms; (B) treated with T3 at BIC₉₀.

Figure 10

Hemolytic activity of T3. Hemolysis of RBCs by T3 at various concentrations. p < 0.0001.

Figure 11

Evaluation of the cytotoxicity of T3 against RAW 264.7 cells using the MTT assay. The cytotoxicity analysis of various concentrations of T3 against RAW 264.7 cells.