N-Butylphthalide Potentiates the Effect of Fluconazole Against Drug-Resistant Candida glabrata and Candida tropicalis. Evidence for Its Mechanism of Action

Figures & data

Table 1 Primers Used in This Study

Gene	Primers Sequence (5’-3’)
CG-CDR1	F: GAACATACTCGCTATACCACCCA R: GACATGGCAAATACTGCAACAA
CG-CDR2	F: CGTGGGTTCGGTGTTGGTAT R: GTTGGTCGCATTGGAGGTTAT
CG-CYTB	F: CTCAGCTATTCCTTTCGTTGGT R: CATTCCAATTCTATCCATATTACCT
CG-COX1	F: TGATTGGTGCATCAGATATGGC R: CACTAGGTCCTGAATGAGCTTGA
CG-COX2	F: TTCAGCTACACCTAATCAAGAAGGT R: CATAGCTGGTGAAATAACTTCATCAC
CG-COX3	F: TTCATTCAGCAATGAGTCCAGA R: CAGTACCAGCATAGAATACTGATCC
CT-ERG11	F: GCCATTCGGTGGTGGTAGAC R: TCCCAAACGATTTCAGCAGGT
CT-CYTB	F: CGTTCCCTTTATTTGAGGTGGT R: AATCGGTCGATGTTACCTGTGA
CT-COX1	F: ACCAGCTCCAGTTTCAATGAGT R: TTCCTATTTGTTATGCCCTTCC
CT-COX2	F: GCCTTCCCATCATTCATCCTG R: TACCTGGCTCAAGCATGTCGT
CT-COX3	F: CGTTCCATTTAGTTGGTCCTTC R: AGAATCCCTGATTAAGCCCTCTT

Table 2 MIC of FLC and NBP in Candida Glabrata and Candida Tropicalis

Candida strains^a	MIC (μg·mL^−1)		Spearman
	FLC	NBP	r	P
CG 1	4	128	−0.239	0.240
CG 2	4	4
CG 3	2	128
CG 4	2	128
CG 5	16	256
CG 6	32	64
CG 7	2	32
CG 8	1	128
CG 9	2	128
CG 10	2	128
CG 11	2	128
CG 12	32	32
CG 13	1	256
CG 14	2	64
CG 15	64	64
CG 16	64	64
CG 17	1	64
CG 18	64	16
CG 19	512	64
CG 28	0.5	64
CG 29	16	32
CG 30	1	32
CG 31	2	32
CG 32	1	32
CG 33	16	64
CG 34	16	8
CT 20	1024	64	−0.096	0.704
CT 21	256	128
CT 22	128	32
CT 23	128	32
CT 24	64	64
CT 25	64	64
CT 26	512	64
CT 27	256	64
CT 34	256	32
CT 35	128	16
CT 36	2	32
CT 37	1	128
CT 38	1	16
CT 39	1	64
CT 40	256	8
CT 41	1	64
CT 42	128	16
CT 43	1	64

Abbreviations: ^aCG, Candida glabrata; CT, Candida tropicalis.

Table 3 Static Antifungal Activity of NBP and FLC

Strains^a	MICFLC/μg·mL^−1	MICNBP/μg·mL^−1	MICFLCcomb/μg·mL^−1	MICNBPcomb/μg·mL^−1	FICI^b	Interpretation
CG15	64	64	1	32	0.5156	Partial synergism
CG16	64	64	2	32	0.5315	Partial synergism
CG18	64	16	1	8	0.5156	Partial synergism
CG19	512	64	1	32	0.5020	Partial synergism
CT20	1024	64	1	32	0.5001	Partial synergism
CT26	512	64	1	32	0.5020	Partial synergism
CT27	256	64	1	32	0.5039	Partial synergism

Abbreviations: ^aCG, Candida glabrata; CT, Candida tropicalis; ^bFICI, fractional inhibitory concentration index, FICI=MIC_FLCcomb/MIC_FLC + MIC_NBPcomb/MIC_NBP.

Figure 1 The time-kill curve of NBP on CG19 and CT20((A) CG19; (B) CT20).

Figure 2 The time-kill curve of NBP and FLC on CG19 and CT20((A) 64 μg·mL⁻¹ NBP+1 μg·mL⁻¹ FLC on CG19; (B) 128 μg·mL⁻¹ NBP+1 μg·mL⁻¹ FLC on CG19; (C) 64 μg·mL⁻¹ NBP+1 μg·mL⁻¹ FLC on CT20; (D) 128 μg·mL⁻¹ NBP+1 μg·mL⁻¹ FLC on CT20).

Figure 3 Effect of NBP on the gene expression of the mitochondrial respiratory chain enzyme of CG19(**P<0.01, ***P<0.001, compared to control group).

Figure 4 Effect of NBP on the gene expression of the mitochondrial respiratory chain enzyme of CT20(**P<0.01, ***P<0.001, compared to control group).

Figure 5 Effect of NBP on the expression of the CDR1 and CDR2 genes of CG19(*P<0.05, **P<0.01, ***P<0.001, compared to the susceptible group; ^###P<0.001, compared to the resistance group).

Figure 6 Effect of NBP on the expression of CT20 ERG11 gene (*P<0.05, compared to the susceptible group).