Increasing the Efficacy of Treatment of Staphylococcus aureus- Candida albicans Mixed Infections with Myrtenol

Abstract

Infectious diseases caused by various nosocomial microorganisms affect worldwide both immunocompromised and relatively healthy persons. Bacteria and fungi have different tools to evade antimicrobials, such as hydrolysis damaging the drug, efflux systems, and the formation of biofilm that significantly complicates the treatment of the infection. Here, we show that myrtenol potentiates the antimicrobial and biofilm-preventing activity of conventional drugs against S. aureus and C. albicans mono- and dual-species cultures. In our study, the two optical isomers, (-)-myrtenol and (+)-myrtenol, have been tested as either antibacterials, antifungals, or enhancers of conventional drugs. (+)-Myrtenol demonstrated a synergistic effect with amikacin, fluconazole, and benzalkonium chloride on 64-81% of the clinical isolates of S. aureus and C. albicans, including MRSA and fluconazole-resistant fungi, while (-)-myrtenol increased the properties of amikacin and fluconazole to repress biofilm formation in half of the S. aureus and C. albicans isolates. Furthermore, myrtenol was able to potentiate benzalkonium chloride up to sixteen-fold against planktonic cells in an S. aureus-C. albicans mixed culture and repressed the adhesion of S. aureus. The mechanism of both (-)-myrtenol and (+)-myrtenol synergy with conventional drugs was apparently driven by membrane damage since the treatment with both terpenes led to a significant drop in membrane potential similar to the action of benzalkonium chloride. Thus, due to the low toxicity of myrtenol, it seems to be a promising agent to increase the efficiency of the treatment of infections caused by bacteria and be fungi of the genus Candida as well as mixed fungal-bacterial infections, including resistant strains.

Keywords: Candida albicans; Staphylcoccus aureus; benzalkonium chloride; drug synergism; mixed infections; myrtenol.

Figure 1

Viability of S. aureus and C. albicans in mixed culture in presence of benzalkonium chloride with concentrations 0, 0.25, 0.5, 1, 4, and 8 µg/mL separately and in combination with (−)-myrtenol and (+)-myrtenol at a concentration of 256 µg/mL. The viability of bacterial and fungal cells was assessed after 24 h growth in culture liquid and after 48 for adherent cells. The viable cells were counted after a series of ten-fold dilutions followed by plating on selective media for differentiation of S. aureus and C. albicans.

Figure 2

Relative membrane potential of S. aureus cells expressed in fluorescence units of DioC2(3) reduced on an intact cell membrane resulting in green fluorescence. Cells were grown for 18 h in LB broth, washed with PBS, and resuspended until a final density of 10⁶ CFU/mL in PBS supplemented with DioC2(3) to a final concentration of 10 μM/mL. After a 30 min incubation at 25 °C, compounds of interest were added to the samples. Fluorescence detection was performed for 30 min with 5 min intervals with excitation and emission wavelengths of 497 and 520 nm, respectively.

Figure 3

Relative membrane potential of C. albicans cells expressed in fluorescence units of DioC2(3) reduced on an intact cell membrane resulting in green fluorescence. Cells were grown for 18 h in Sabouraud broth, washed with PBS, and resuspended until a final density of 10⁵ CFU/mL in PBS supplemented with DioC2(3) to a final concentration of 10 μM/mL. After a 30 min incubation at 25 °C, compounds of interest were added to the samples. Fluorescence detection was performed for 30 min with 5 min intervals with excitation and emission wavelengths of 497 and 520 nm, respectively.

Figure 4

Myrtenol-lum penetration into S. aureus cells. (a) The localization of either (−)-myrtenol or (+)-myrtenol carrying the fluorophore BODIPY (Myrtenol-lum) assessed by confocal laser scanning microscopy. The solely fluorophore (lum) and Calcofluor-White (CFW) membrane dyes served as references. (b) Penetration of myrtenol-lum into S. aureus cells. Myrtenol-lum was added to S. aureus cells, and the sole fluorophore (lum) was used as a control. After 4, 8, 16, 32, and 64 min of incubation, cells were harvested, washed with PBS, and residual fluorescence was measured. The half-time of penetration (t½) was 24 ± 1.3 min and 26 ± 1.5 min for (−)-myrtenol and (+)-myrtenol, respectively, while for the fluorophore solely (lum) was t½ > 5000 min.

Figure 5

Myrtenol-lum penetration into C. albicans cells. (a) The localization of either (−)-myrtenol or (+)-myrtenol carrying the fluorophore BODIPY (myrtenol-lum) assessed by confocal laser scanning microscopy. The solely fluorophore (lum) and Calcofluor-White (CFW) membrane dyes served as references. (b) Penetration of myrtenol-lum into C. albicans cells. Myrtenol-lum was added to C. albicans cells, and the sole fluorophore (lum) was used as a control. After 4, 8, 16, 32, and 64 min of incubation, cells were harvested, washed with PBS, and residual fluorescence was measured. The half-time of penetration (t½) was 24 ± 1.3 min and 18 ± 1.2 min for (−)-myrtenol and (+)-myrtenol, respectively, while for the fluorophore solely (lum) was t½ > 5000 min.