Bacteriophages Improve the Effectiveness of Rhamnolipids in Combating the Biofilm of Candida albicans

Abstract

Biofilms formed by Candida albicans pose therapeutic challenges due to their resistance to conventional antimicrobials, highlighting the need for more effective treatments. Rhamnolipids (RLs) are biosurfactants with diverse antimicrobial properties. Bacteriophages are viruses that target specific bacterial strains. Recent studies have shown that they may affect biofilm formation by fungi and yeasts. This study investigated the combined antimicrobial effects of RLs and bacteriophages against C. albicans biofilms, focusing on their anti-adhesive and inhibitory effects on biofilm development. RT-PCR assays were used to analyze gene modulation in C. albicans biofilm formation in response to RLs and bacteriophage treatments, while hyphae formation was examined using microscopy. The results showed that RLs-bacteriophage combinations significantly reduced biofilm formation compared to individual treatments. A combination of 200 mg/L RLs with bacteriophage BF9 led to a 94.8% reduction in biofilm formation. In a subsequent model, the same RL concentration with bacteriophage LO5/1f nearly eliminated biofilm formation (~96%). Gene expression analysis revealed downregulation of key biofilm-associated genes when Candida cells were treated with 200 mg/L RLs and four bacteriophages (BF17, LO5/1f, JG004, FD). These results show the potential of RL and bacteriophage combinations in combating C. albicans biofilms, presenting a promising therapeutic approach against resilient infections.

Keywords: Candida albicans; antimicrobial agents; bacteriophages; biofilm assay; combined therapy; rhamnolipids.

Figure 1

Effect of RLs concentration on surface tension: the critical micelle concentration (CMC) was determined by identifying the intersection point of the regression lines describing the two segments of the curve below and above the CMC.

Figure 2

The percentage of biofilm formation by C. albicans on surfaces pretreated with RLs, bacteriophages, and their combinations. The results represent the averages of triplicate experiments. The figure includes a color scale indicating a percentage of biofilm formation.

Figure 3

The percentage of biofilm formation by C. albicans in the presence of RLs, bacteriophages, and their combinations. The results represent the averages of triplicate experiments. The figure includes a color scale indicating a percentage of biofilm formation.

Figure 4

Expression of genes responsible for biofilm formation in Candida yeast treated with RLs, phages (BF17, LO5/1f, JG004, FD), and their combinations. Data are expressed as the means ± SD of the independent assays in triplicate. The same superscript letters designate homogenous groups comparing treatment samples within the same gene at a p-value of < 0.05.

Figure 5

Effect of RLs, phages, and RLs/bacteriophages combinations on C. albicans mature hyphae. Scale bars = 50 µm. The ratio of hyphae (Ratio of hyphae) was determined as the number of hyphal cells divided by the number of yeast-form cells. The percentage of hyphal cells (% Hyphae) was calculated as the number of hyphal cells divided by the total number of cells (hyphal + yeast), multiplied by 100%. * UC—uncountable-cell counting is impossible due to extensive cell overlapping.