Complex Electromagnetic Fields Reduce Candida albicans Planktonic Growth and Its Adhesion to Titanium Surfaces

Abstract

This study evaluates the effects of different programs of complex electromagnetic fields (C.M.F.s) on Candida albicans, in planktonic and sessile phase and on human gingival fibroblasts (HGF cells). In vitro cultures of C. albicans ATCC 10231 and HGF cells were exposed to different cycles of C.M.F.s defined as: oxidative stress, oxidative stress/antibacterial, antibacterial, antibacterial/oxidative stress. Colony forming units (CFUs), metabolic activity, cells viability (live/dead), cell morphology, filamentation analysis, and cytotoxicity assay were performed. The broth cultures, exposed to the different C.M.F.s, were grown on titanium discs for 48 h. The quantity comparisons of adhered C. albicans on surfaces were determined by CFUs and scanning electron microscopy. The C. albicans growth could be readily controlled with C.M.F.s reducing the number of cultivable planktonic cells vs. controls, independently by the treatment applied. In particular, the antibacterial program was associated with lower levels of CFUs. The quantification of the metabolic activity was significantly lower by using the oxidative stress program. Live/dead images showed that C.M.F.s significantly decreased the viability of C. albicans. C.M.F.s inhibited C. albicans virulence traits reducing hyphal morphogenesis, adhesion, and biofilm formation on titanium discs. The MTS assay showed no negative effects on the viability of HGF. Independent of the adopted protocol, C.M.F.s exert antifungal and anti-virulence action against C. albicans, no cytotoxicity effects on HGF and can be useful in the prevention and treatment of yeast biofilm infections.

Keywords: CFU; Candida albicans; complex electro-magnetic fields; germ tube test; titanium.

Figure 1

Culture samples of Candida albicans ATTC 10,231 during the treatment with complex magnetic fields (C.M.F.s).

Figure 2

Colony forming units of Candida albicans ATCC 10231 after exposure to C.M.F.s. A = Oxidative Stress, B = Oxidative Stress + Antibacterial, C = Antibacterial, D = Antibacterial +Oxidative Stress, UE = control, unexposed C. albicans ATCC 10231. * p-value < 0.05.

Figure 3

Live/dead staining of Candida albicans ATCC 10231 exposed to different programs of C.M.F.s. Fluorescent representative images show viable (green) and dead (red) cells after exposure to C.M.F.s with respect to the unexposed ones. Histograms show the percentages of viable and dead cells for each exposed group vs. the unexposed samples obtained with the identical methodologies in every way except for the use of C.M.F.s. The control group UE was used to evaluate the effect of each exposition (* p-value < 0.05). See the legend of Figure 2 for treatment groups.

Figure 4

Metabolic activity of Candida albicans ATCC 10231 through the INT assay. See the legend of Figure 2 for treatment groups *** p-value < 0.01, ** p-value = 0.017, * p-value = 0.008.

Figure 5

Representative images of the effect of C.M.F.s on the germ tube formation (arrows) in Candida albicans ATCC 10231 (microscopic examination under a magnification of 100×). Histograms show the percentage of germ tube reduction after the C.M.F.s exposure. See the legend of Figure 2 for treatment groups. * p-value < 0.01, ** p-value = 0.06, *** p-value = 0.002.

Figure 6

Candida albicans ATCC 10231 biofilm growth on saliva-conditioned titanium discs assessed by plate count agar. UE corresponds to saliva conditioned titanium disc covered by unexposed Candida albicans ATCC 10231. The unexposed sample was used to evaluate the effect of each exposition (* = p-value < 0.001; ** p-value = 0.001; *** p-value = 0.003; **** p-value = 0.009; ***** p-value = 0.005). Error bars = +/− standard deviation. See the legend of Figure 2 for treatment groups.

Figure 7

SEM images of machined titanium discs covered by adherent Candida albicans ATCC 10231, previously exposed to different C.M.F.s protocols, at different original magnifications, 820× (up) and 5000× (down). UE were titanium discs covered by unexposed Candida albicans ATCC 10231. Arrows display the germ tube presence. See the legend of Figure 2 for treatment groups.

Figure 8

Effect of magnetic fields on HGFs viability after 24 h of C.M.F.s exposure. No cytotoxicity effects were observed within all groups. There was no significant difference between each group and unexposed cells (UE). See the legend of Figure 2 for treatment groups.