A novel mechanism of action of ketoconazole: inhibition of the NorA efflux pump system and biofilm formation in multidrug-resistant Staphylococcus aureus

Figures & data

Table 1 Resistance pattern of the tested Staphylococcus aureusstrains

Strain	Resisance pattern
S. aureus ATCC6538	OX, AK, DA, CRO, CAZ, CIP, LEV, CN, NOR, FOX, SAM, AMC, CFR, TE, AM, AZM
S. aureus(quinolone-sensitive strain)	AM, FOX, CN, CRO, CAZ, E, CFR, CEC, CAZ, CL, AMC, SXT
S. aureus R	OX, AK, DA, FOX, CN, CRO, CAZ, CIP, LEV, NOR, SAM, AMC, CFR, TE, E, NA, S, OFX, PEF
S. aureus 5ʹ	OX, DA, CRO, CAZ, CIP, LEV, NOR, CFP, SAM, AMC, E, DO, CN, FOX

Abbreviations: AK, amikacin; AM, ampicillin; AMC, amoxicillin/clavulanic; AZM, azithromycin; CAZ, ceftazidime; CEC, cefaclor; CFP, cefoperazone; CIP, ciprofloxacin; CN, gentamicin; CRO, ceftriaxone; CTX, cefotaxime; DA, clindamycin; DO, doxycycline; FOX, cefoxitin; LEV, levofloxacin; NA, nalidixic acid; NOR, norfloxacin; OFX, ofloxacin; OX, oxacillin; PEF, pefloxacin; S, streptomycin; SAM, ampicillin/sulbactam; SXT, sulfamethoxazole/trimethoprim; TE, tetracycline.

Table 2 Primer sequence of the tested genes

Primer	Sequences 5ʹ→3ʹ	Product size (bp)
NorA_Fw NorA_Rv	TTCACCAAGCCATCAAAAAG CTTGCCTTTCTCCAGCAATA	620
NorB_Fw NorB_Rv	GCAGGTGGTCTTGCTGATAA AGCGCGTTGTCTATCTTTCC	213
NorC_Fw NorC_Rv	ATACCTGAAGCAACGCCAAC AATGGGTTCTAAGCGACCAA	216

Table 3 MICs/MBCs (µg/mL) of norfloxacin, levofloxacin, and ciprofloxacin alone and in combination with CCCP or ketoconazole for the tested bacterial isolates

Strain	Norfloxacin (MIC/MBC) (∆)	Levofloxacin (MIC/MBC) (∆)	Ciprofloxacin (MIC/MBC) (∆)	Ethidium bromide (MIC/MBC) (∆)
S. aureus sensitive strain ● Antibiotic alone ● +CCCP ● +ketoconazole	1/1 0.5/0.5 1/1	0.5/1 0.25/0.5 0.5/1	0.25/0.5 0.25/0.25 0.25/0.25	2/4 2/2 2/4
S. aureus ATCC6538 ● Antibiotic alone ● +CCCP ● +ketoconazole	16/32 1/1 (16) 1/1 (16)	16/32 0.25/0.5 (64) 0.25/0.25 (64)	64/64 0.25/0.5 (256) 0.25/0.5 (256)	512/1024 0.5/0.5 (1024) 0.5/0.5 (1024)
S. aureus R ● Antibiotic alone ● +CCCP ● +ketoconazole	16/32 0.5/1 (32) 0.125/0.25 (128)	16/16 1/2 (16) 0.5/1 (32)	16/32 2/4 (8) 1/2 (16)	64/128 0.125/0.25 (512) 0.125/0.25 (512)
S. aureus 5ʹ ● Antibiotic alone ● +CCCP ● +ketoconazole	16/32 8/16 (2) 2/4 (8)	16/32 8/16 (2) 2/2 (8)	16/32 2/4 (8) 0.5/1 (32)	8/16 4/8 (2) 0.25/0.5 (32)

Notes: (∆) MIC fold change between antibiotic alone and antibiotic+CCCP, ie ratio of MIC of antibiotic alone to that of antibiotic+CCCP.

Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; MBC, lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by ≥99.9%; MIC, minimum inhibitory concentration; S. aureus, Staphylococcus aureus.

Table 4 Average of zone inhibition diameters of the tested antibiotic discs with and without ketoconazole or CCCP

Strain	Inhibition Zone diameter in mm ± S.D
	CIP	TE	DA	OX	FOX	CAZ	LEV	AMC	CN	CRO
S. aureus ATCC6538  Ab  Ab+ketoconazole^$$  Ab+CCCP^$	6±0.5 26±0.3 36±0.21	18±0.4 30±0.6 35±0.62	10±0.7 26±0.45 30±0.32	5±0.44 28±0.63 35±0.46	6±0.46 20±0.53 23±34	5±0.72 20±0.76 50±0.82	10±0.34 27±0.52 35±0.33	11±0.42 29±0.35 36±0.7	8±0.28 22±0.45 34±0.37	6±0.64 27±0.74 34±0.82
S. aureus R  Ab  Ab+ketoconazole^$$  Ab+CCCP^$	6±0.42 20±0.61 22±0.48	28±0.65(s) 28±0.33 30±0.8	30±0.52(s) 30±0.73 34±0.62	5±0.21 17±0.31 26±0.5	5±0.66 28±0.47 28±0.55	5±0.42 14±0.52 28±0.49	13±0.7 21±0.5 25±0.7	10±0.67 26±0.81 32±0.34	8±0.35 20±0.46 24±0.77	6±0.52 24±0.62 28±0.43
S. aureus 5ʹ  Ab  Ab+ketoconazole^$$  Ab+CCCP^$	12±0.52 23±0.42 28±0.38	12±0.66 20±0.34 30±0.69	28±0.52(s) 28±0.64 31±0.82	22±0.43(s) 22±0.4 24±0.71	5±0.37 22±0.42 40±0.61	6±0.34 26±0.88 34±0.6	10±0.3 22±0.6 26±0.32	6±0.54 24±0.6 34±0.34	10±0.6 23±0.4 30±0.4	8±0.51 25±0.34 29±0.62
S. aureus sensitive strain ● Antibiotic alone ● +CCCP ● +ketoconazole	30±0.25 32±0.3 30±0.21	24±0.29 25±0.16 24±0.62	21±0.7 24±0.45 21±0.31	29±0.42 28±0.63 29±0.46	26±0.5 26±0.3 27±0.21	25±0.22 25±0.37 25±0.62	26±0.76 26±0.45 26±0.32	31±0.44 33±0.23 31±0.46	16±0.5 16±0.3 16±0.21	28±0.4 30±0.6 28±0.12

Notes: ^$Combination of antibiotic and CCCP or ketoconazole (as efflux inhibitor). A difference of >5 mm between the antibiotic discs and Ab–CCCP zone diameters was interpreted as positive efflux-mediated resistance. ^$$Ab–ketoconazole disc combinations which give a difference of >5 mm between the antibiotic discs.

Abbreviations: Ab, antibiotic; AMC, amoxicillin/clavulanic acid; CAZ, ceftazidime; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CIP, ciprofloxacin; CN, gentamicin; CRO, ceftriaxone; DA, clindamycin; FOX, cefoxitin; LEV, levofloxacin; OX, oxacillin; (s), susceptible; S. aureus, Staphylococcus aureus; TE, tetracycline.

Figure 1 Antibiotic–EPI disc synergy test using antibiotic discs combined with CCCP or ketoconazole as EPIs. (A) Antibacterial activity of methanol (used for dissolving CCCP), of dimethyl sulfoxide (DMSO) used as a solvent for ketoconazole, of CCCP, and of ketoconazole (each alone). (B) Norfloxacin (NOR)-sensitive Staphylococcus aureusshowing no change in zone diameter in combination with ketoconazole. Addition of CCCP to antibiotic disc showed an increase in zone diameter due to its antibacterial activity. (C–E) CCCP and ketoconazole increase the activity of the tested antibiotics. (F) Effect of ketoconazole on the addition of ceftriaxone (CRO), oxacillin (OX), and amoxicillin/clavulanic acid (AMC) discs.

Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; FOX, cefoxitin; KET, ketoconazole; LEV, levofloxacin.

Figure 2 Effect of the tested antibiotic (levofloxacin) alone or in combination with ketoconazole or CCCP on the accumulation of ethidium bromide by Staphylococcus. aureus ATCC 6538 and S. aureus R strain. Data represent the mean±SD of two different experiments performed in triplicate. **Highly significant, P<0.001 (Student’s t-test).

Abbreviation: CCCP, carbonyl cyanide m-chlorophenylhydrazone.

Figure 3 Time-kill curves of levofloxacin against two MDR Staphylococcus aureus strains: (A) R strain and (B) 5ʹ strain. (G) The sensitive strain levofloxacin+CCCP (½×MIC) against R strain (C) and 5ʹ strain (D). (H) The sensitive strain and levofloxacin+ketoconazole (500 µg/ml) against (E) R strain and (F) 5ʹ strain.

Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; CFU, colony forming unit; LEV, levofloxacin; MIC, minimum inhibitory concentration.

Figure 4 Inhibitory effect of ketoconazole and levofloxacin each alone and in combination (using different concentrations of levofloxacin) on biofilm formation by the tested Staphylococcus aureus strains. (A) Effect of ketoconazole, ¼×MIC of levofloxacin each alone and in combination. (B) Effect of ketoconazole, ½×MIC levofloxacin each alone and in combination. (C) Effect of ketoconazole, 1×MIC levofloxacin each alone and in combination. (D) Effect of ketoconazole, 2×MIC levofloxacin each alone and in combination. Data represent the mean±SD of two different experiments performed in triplicate. **Highly significant, P<0.001 (Student’s t-test).

Abbreviations: LEV, levofloxacin; MIC, minimum inhibitory concentration.

Figure 5 Effect of levofloxacin, ketoconazole, CCCP, and levofloxacin combination with ketoconazole or CCCP on the relative expression of efflux pump genes of Staphylococcus aureus (A) 5’ strain and (B) R strain. Error bars indicate the SD of three independent experiments. *Significant, P<0.05; **highly significant, P<0.01.

Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; LEV, levofloxacin; Nor, norfloxacin.

Figure 6 Effect of ethidium bromide, ketoconazole, CCCP, and ethidium bromide combination with ketoconazole or CCCP on the relative expression of efflux pump genes of Staphylococcus aureus (A) 5’ strain and (B) R strain. Error bars indicate the SD of three independent experiments. *Significant, P<0.05; **highly significant, P<0.01.

Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; EtBr, ethidium bromide; Nor, norfloxacin.

Figure 7 (A) 2D and (B) 3D molecular interaction of 4ʹ,5ʹ-O-dicaffeoylquinic acid into the active site (Protein Data Bank ID: 1PW4).

Abbreviation: 1PW4, glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily.

Figure 8 (A) 2D and (B) 3D molecular interaction of 2S,4R and 2R,4S diastereoisomers into the active site (Protein Data Bank ID: 1PW4).

Abbreviation: 1PW4, glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily.