Anti-Biofilm Activity of Phenyllactic Acid against Clinical Isolates of Fluconazole-Resistant Candida albicans

Abstract

Commonly found colonizing the human microbiota, Candida albicans is a microorganism known for its ability to cause infections, mainly in the vulvovaginal region, and is responsible for 85% to 90% of vulvovaginal candidiasis (VVC) cases. The development of drug resistance in C. albicans isolates after long-term therapy with fluconazole is an important complication to solve and new therapeutic strategies are required to target this organism and its pathogenicity. In the present study, phenyllactic acid (PLA) an important broad-spectrum antimicrobial compound was investigated for its antifungal and antivirulence activities against clinical isolates of C. albicans. Previously characterized strains of C. albicans isolates from women with VVC and C. albicans ATCC90028 were used to evaluate the antimicrobial and time dependent killing assay activity of PLA showing a MIC 7.5 mg mL^-1 and a complete reduction of viable Candida cells detected by killing kinetics after 4 h of treatment with PLA. Additionally, PLA significantly reduced the biomass and the metabolic activity of C. albicans biofilms and impaired biofilm formation also with changes in ERG11, ALS3, and HWP1 genes expression as detected by qPCR. PLA eradicated pre-formed biofilms as showed also with confocal laser scanning microscopy (CLSM) observations. Furthermore, the compound prolonged the survival rate of Galleria mellonella infected by C. albicans isolates. These results indicate that PLA is a promising candidate as novel and safe antifungal agents for the treatment of vulvovaginal candidiasis.

Keywords: Candida albicans; Galleria mellonella; biofilm; fluconazole resistance; phenyllactic acid.

Figure 1

Panel (A): Comparison between the total biomass of biofilms of C. albicans ATCC90028 and C. albicans clinical isolates (C7, C14, C17, and C19) at 24 and 48 h. Red line represents ODcut (ODcut = mean of negative control with addition of 3 times). Panel (B): Comparison between the metabolic activities of the same strains at 24 and 48 h. Statistical significance: * p < 0.05, **** p < 0.0001 (Tukey’s test).

Figure 2

Time-kill kinetics of PLA against C. albicans ATCC90028 (panel A) and fluconazole-resistant C. albicans clinical isolates (panel B–E).

Figure 3

Inhibition of biofilm formation by PLA at concentrations of 0.6, 1.2, 2.5, and 5 mg mL⁻¹ against C. albicans ATCC90028 and clinical isolates C7, C14, C17, and C19. Two different methodologies were used to quantify biofilm: crystal violet assay (panel A), which measures biofilm total biomass, and XTT assay (panel B), which measures metabolic activity. Data represent the mean (± standard deviation, SD) of three independent experiments, and each one was carried out with triplicate determinations. For all experimental points, * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001 were obtained for treated samples versus untreated samples (Tukey’s test).

Figure 4

Eradication of pre-formed biofilm by PLA at concentrations of 0.6, 1.25, 2.5, and 5 mg mL⁻¹ against C. albicans ATCC90028 and clinical isolates C7, C14, C17, and C19. Two different methodologies were used to quantify biofilm: crystal violet assay (panel A), which measures biofilm total biomass, and XTT assay (panel B), which measures metabolic activity. Data represent the mean (±standard deviation, SD) of three independent experiments, and each one was carried out with triplicate determinations. For all experimental points, ** p < 0.01, or **** p < 0.0001 were obtained for treated samples versus untreated samples (Tukey’s test).

Figure 5

Effect of PLA on biofilm morphology. C. albicans 90028 biofilm formed for 48 h in the absence of PLA (A) and treated with 2.5 mg mL 1-PLA (B). The morphology of biofilms was visualized using a Zeiss LSM700 confocal microscope at 10× magnification.

Figure 6

Expression analysis of selected genes of Candida albicans using real-time qPCR (ERG11, ALS3, and HWP1) in response to PLA. Histograms represent the fold differences in the expression levels of the genes selected during inhibition of biofilm with PLA at concentration of 2.5 mg mL⁻¹. Red lines show fold change thresholds of −1 and +1, respectively. * p < 0.05, ** p < 0.01, and **** p < 0.0001 vs. C. albicans (Tukey’s test).

Figure 7

PLA toxicity on G. mellonella larvae treated with concentrations of 1.25 mg mL⁻¹, 2.5 mg mL⁻¹, 5 mg mL⁻¹, 10 mg mL⁻¹, and 20 mg mL⁻¹.

Figure 8

Kaplan–Meier plots of survival curves of G. mellonella larvae infected with C. albicans ATCC90028 (A), C7 (B), C14 (C), C17 (D), and C19 (E). The concentration of microorganisms was 1 × 10⁶ CFU/larva. All groups were treated with 2.5 mg mL⁻¹ PLA before or after infection. All groups were compared with control (infected larvae). In all panels, survival curves of intact larvae and larvae injected with PBS are reported. **** represents p-value < 0.001 (Tukey’s).

Figure 8

Kaplan–Meier plots of survival curves of G. mellonella larvae infected with C. albicans ATCC90028 (A), C7 (B), C14 (C), C17 (D), and C19 (E). The concentration of microorganisms was 1 × 10⁶ CFU/larva. All groups were treated with 2.5 mg mL⁻¹ PLA before or after infection. All groups were compared with control (infected larvae). In all panels, survival curves of intact larvae and larvae injected with PBS are reported. **** represents p-value < 0.001 (Tukey’s).