A Study of the Disruptive Effect of the Acetate Fraction of Punica granatum Extract on Cryptococcus Biofilms

Abstract

Cryptococcosis, caused by yeasts of the genus Cryptococcus, is an infectious disease with a worldwide distribution. Cryptococcus neoformans and Cryptococcus gattii are the species that commonly cause this disease in humans; however, infections caused by Cryptococcus laurentii, especially in immunocompromised patients, are increasingly being reported. Owing to the increase in the resistance of fungi to antifungals, and a lack of treatment options, it is important to seek new therapeutic alternatives such as natural products. Among these are plant species such as Punica granatum, which is used in folk medicine to treat various diseases. This study aimed to evaluate the activity of the acetate fraction of P. granatum leaf extract against environmental and clinical isolates of Cryptococcus. Three environmental isolates of C. laurentii, PMN, PMA, and PJL II, isolated from soils of different municipalities in the state of Maranhão, a clinical isolate, C. gattii, from a patient with neurocryptococcosis, and a standard strain of C. gattii (ATCC 32068) were used. The minimum and fractional inhibitory concentrations (MIC and FIC, respectively) and time-kill curve of the extract and fluconazole were determined to assess the susceptibility profile of the fungal isolates. Larvae of Tenebrio molitor were infected with Cryptococcus strains, and the effects of acetate fraction of P. granatum extract and fluconazole on the survival and fungal burden were determined. The extract activity was tested against pre-formed biofilms. The acetate fraction of P. granatum extract showed promising antifungal activity against all the species of Cryptococcus evaluated in this study, with an MIC value lower than that of fluconazole. The indices obtained in the FIC test indicated that the antimicrobial effect of the combination of the extract and antifungal was indifferent for 80% of the isolates. The P. granatum acetate fraction reduced the pre-formed biofilm of some isolates, showing better activity than fluconazole, which is consistent with results from fluorescence microscopy. This is the first study on the use of P. granatum and its ability to inhibit Cryptococcus biofilms; therefore, further studies and tests are needed to investigate the components and mechanism of action of P. granatum against cryptococcosis agents.

Keywords: Cryptococcus gattii; Cryptococcus laurentii; Punica granatum; biofilm; cryptococcosis.

FIGURE 1

Time-kill curves of fluconazole and acetate fraction of P. granatum extract alone or in combination against Cryptococcus strains. Time-kill curve were performed with fluconazole or acetate fraction of P. granatum extract at 1× MIC, 2× the MIC, and 4× the MIC. (A) Time-kill curve performed against C. gattii CASA. (B) Time-kill curve performed against C. gattii ATCC 36068. (C) Time-kill curve performed against C. laurentii PMA. (D) Time-kill curve performed against C. laurentii PMN. (E) Time-kill curve performed against C. laurentii PJL.

FIGURE 2

Activity of acetate fraction of P. granatum extract and fluconazole against pre-formed biofilm of C. laurentii PMN. *p < 0.05. Assays were performed in duplicate.

FIGURE 3

Activity of acetate fraction of P. granatum extract and fluconazole against pre-formed biofilm of C. laurentii PMA. Assays were performed in duplicate.

FIGURE 4

Activity of acetate fraction of P. granatum extract and fluconazole against pre-formed biofilm of C. laurentii PJL II. Assays were performed in duplicate.

FIGURE 5

Activity of acetate fraction of P. granatum extract and fluconazole against pre-formed biofilm of C. gattii. Assays were performed in duplicate.

FIGURE 6

Activity of acetate fraction of P. granatum extract and fluconazole against pre-formed biofilm of C. gattii (ATCC 32068). *p < 0.05. Assays were performed in duplicate.

FIGURE 7

Aspects of biofilms of Cryptococcus spp. after treatment with acetate fraction of P. granatum extract and/or fluconazole. Yeast cells were stained with 10 μg/mL acridine orange and visualized at 50× and 400× magnification (within red rectangles). Disturbances on biofilms occurred at equal or greater MIC when compared with non-treated cells, as follow: C. laurentii PMA at 4× MIC for Ext or Flu, and 4× MIC for Flu + Ext; C. gattii ATCC 32068 at 1× MIC for Ext or Flu, and 1× MIC for Flu + Ext; and C. gattii CASA at 2× MIC for Ext or Flu. The sum of fluorescences was used to evaluate disturbances on biofilm. Assays were performed in triplicate. “NT,” “Ext,” “Flu,” and “Flu + Ext” mean non-treated; acetate fraction of P. granatum extract, fluconazole and combination between them, respectively. MIC mean minimal inhibitory concentration. White (50 μm) and bright blue (5 μm) bars.

FIGURE 8

Larvae (n = 10) were monitored daily for the survival curve (A). Fungal burden in larvae infected with the C. gattii CASA strain and treated with fluconazole, acetate fraction of P. granatum extract or combination (B). Fungal burden in larvae infected with the C. gattii ATCC 32068 strain and treated with fluconazole, acetate fraction of P. granatum extract or combination (C). Fungal burden in larvae infected with the C. laurentii PMA strain and treated with fluconazole, acetate fraction of P. granatum extract or combination (D). NT, non-treated group; Flu, fluconazole; Ext, extract. *p < 0.05.

FIGURE 9

New insights into the potential antibiofilm activity of acetate fraction of Punica granatum against clinical and environmental isolates of Cryptococcus.