Reversal of fluconazole resistance induced by a synergistic effect with Acca sellowiana in Candida glabrata strains

Figures & data

Figure 1. Chromatogram and absorption spectrum of RS catechin and F1, F2 and F3 active fractions in 1000 μg/mL. (a) RS catechin. Retention time: 19.744. (b) F1 active fraction (major peaks 1, 2 and 3) with retention time of peak catechin: 19.699. (c) F2 active fraction (major peaks 1 and 2) with retention time of peak catechin: 19.073. (d) F3 active fraction (major peaks 1 and 2) with retention time of peak catechin: 19.764. Analyzed by high performance liquid chromatography coupled to photodiode array detector (PDA). Wavelength: 280 nm. Ultra C18 pre-column 4.6 mm; 5 μ C18 100A column (250 × 4.6 mm). Mobile phase 0.05% phosphoric acid (solution A) and acetonitrile (solution B). Flow rate: 0.7 mL/min. Injection volume: 20 μL.

Figure 2. Standard calibration curve and regression equation for the catechin.

Table 1. Catechin concentrations present at the F1, F2 and F3 fractions.

Fraction	Catechin concentration (μg/mL)
F1	7.82
F2	1.38
F3	1.30

F1, fraction: acetonic; F2, fraction: acetone:ethanol; F3, fraction: ethanol. Tested concentration for fractions: 1000 μg/mL.

Table 2. Minimum inhibitory concentration (MIC) values expressed in μg/mL of active fractions of A. sellowiana front the strains of non-albicans Candida.

Strains/fractions	F1	F2	F3	F4	F6
CF RL23	250	500	R	R	R
CG RL12m	31.25	250	125	R	R
CG RL34m	250	125	62.5	R	R
CG RL37m	500	62.5	500	R	R
CK CK01	500	R	R	500	R
CP RL11m	R	R	R	R	R
CP RL13m	250	R	R	R	R
CP RL27m	R	R	R	500	500
CT 17A	R	R	R	R	R
CT 57A	R	R	R	R	R
CT 94A	R	R	R	R	R

F1 active fraction: acetonic; F2 active fraction: acetone:ethanol; F3 active fraction: ethanol; F4 active fraction: ethanol:ethanol and water (70:30); F6 active fraction: ethanol and water (70:30):ethanol and water (50:50). R: strains resistant to concentrations ≥ 500 μg/mL. Candida famata (CF RL23); Candida glabrata (CG RL12m, CG RL34m and CG RL37m); Candida krusei (CK CK01); Candida parapsilosis (CP RL11m, CP RL13m and CP RL27m); Candida tropicalis (CT 17A, CT 57A and CT 94A). Reading of the MIC values carried out in 48 h.

Table 3. Minimum inhibitory concentration (MIC) values in 24 and 48 h to F1, F2 and F3 active fractions expressed in μg/mL front of Candida glabrata strains sensitive and resistant to FLZ.

Strains	F1		F2		F3
	24 h	48 h	24 h	48 h	24 h	48 h
CG RL02	7.81	62.5	7.81	62.5	62.5	500
CG RL03	7.81	62.5	7.81	31.25	125	R
CG RL12m	15.62	31.25	3.91	250	15.62	125
CG RL12	15.62	250	15.62	31.25	R	R
CG RL22	R	R	R	R	R	R
CG RL24	15.62	125	31.25	125	250	R
CG RL34m	15.62	250	15.62	125	31.25	62.5
CG RL34	R	R	7.81	31.25	R	62.5
CG RL37m	62.5	R	3.91	62.5	62.5	500
CG RL37	62.5	250	15.62	500	31.25	125

F1 active fraction: acetonic; F2 active fraction: acetone:ethanol; F3 active fraction: ethanol. R: resistant strains to concentrations ≥ 500 μg/mL. CG: Candida glabrata. Sensitive strains: CG RL02, CG RL03, CG RL12, CG RL34 and CG RL37. Resistant strains: CG RL12m, CG RL22, CG RL24, CG RL34m and CG RL37m.

Table 4. Evaluation of the interaction between the F2 active fraction and the FLZ against Candida glabrata strains.

	MIC		MIC combination
Strains	F2	FLZ	F2	FLZ	Cell damage	FICI	Interaction
CG RL02	62.5	8	7.81	4	68.8	0.63	Ind
CG RL03	31.25	32	15.62	8	86.0	0.31	Syn
CG RL12m	250	256	31.25	64	89.9	0.38	Syn
CG RL12	31.25	4	7.81	1	66.1	0.50	Syn
CG RL22	1000	128	125	16	92.1	0.25	Syn
CG RL24	125	64	31.25	16	63.4	0.50	Syn
CG RL34m	125	64	7.81	16	91.7	0.31	Syn
CG RL34S	31.25	64	15.62	16	93.1	0.75	Ind
CG RL37m	62.5	64	15.62	16	94.6	0.38	Syn
CG RL37	500	16	62.5	4	77.0	0.38	Syn

MIC and MIC combination: μg/mL; cell damage: (%); FICI: Fractional Inhibitory Concentration Index. F2 active fraction: acetone:ethanol. Interaction: Synergism (Syn), Indifference (Ind). Sensitive strains: CG RL02, CG RL03, CG RL12, CG RL34 and CG RL37. Resistant strains: CG RL12m, CG RL22, CG RL24, CG RL34m and CG RL37m. Reading of the MIC values carried out in 48 h.

Figure 3. (a) CG RL03. (b) CG RL22. (c) CG RL37m. Percentage of cellular damage found in the three most effective combinations resulting from the interaction between the F2 active fraction and fluconazole (F2 + FLZ). Horizontal axis: percentage of cellular damage obtained when the F2 active fraction was evaluated separately in two minimum inhibitory concentrations different. Depth axis: percentage of cellular damage obtained when the fluconazole (FLZ) was assessed separately in two minimum inhibitory concentrations different. Vertical axis: percentage of cellular damage resulting from the interaction between the F2 active fraction with fluconazole (F2 + FLZ) in different minimum inhibitory concentrations.

F2 active fraction

FLZ

(F2+FLZ)

Figure 4. Morphological representation of C. glabrata strains susceptible to the effect of F2 active fraction through scanning electronic microscopy. (a) CG RL02S (5000 times magnification). (b) CG RL02S (10,000 times magnification). (c) CG RL34m (15,000 times magnification). (d) CG RL12S (10,000 times magnification). (e) CG RL37m (10,000 times magnification). (f) ATCC 40039 in RPMI 1640 (10,000 times magnification). Electric current: 5 kV.

Figure 5. Histopathological evaluation of porcine cells treated with lyophilized aqueous extract of leaves of A. sellowiana and negative control at 100 and 400 times magnification. (1a) Swine epidermal cells treated with lyophilized aqueous extract of leaves of A. sellowiana at 1 mg/mL (100 times magnification). (1b) Swine epidermal cells treated with PBS buffer solution pH 7.0 (100 times magnification). (2a) Swine epidermal cells treated with lyophilized aqueous extract of leaves of A. sellowiana at 1 mg/mL (400 times magnification). (2b) Swine epidermal cells treated with lyophilized aqueous extract of leaves of A. sellowiana at 1 mg/mL (400 times magnification).