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Resensitization of Fluconazole-Resistant Urinary Candida spp. Isolates by Amikacin through Downregulation of Efflux Pump Genes

EVA A. EDWARD
EVA A. EDWARD
, 
NELLY M. MOHAMED
NELLY M. MOHAMED
 y 
AZZA S. ZAKARIA
AZZA S. ZAKARIA
   | 11 mar 2020
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Article Category: original-paper
Publicado en línea: 11 mar 2020
Páginas: 73 - 84
Recibido: 12 nov 2019
Aceptado: 06 feb 2020
DOI: https://doi.org/10.33073/pjm-2020-010
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© 2020 Eva A. Edward et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Inhibitory effect of amikacin (4,000 μg/ml) on the efflux of rhodamine in fluconazole-resistant Candida albicans (C6).
Inhibitory effect of amikacin (4,000 μg/ml) on the efflux of rhodamine in fluconazole-resistant Candida albicans (C6).

Fig. 2.

Inhibitory effect of amikacin (4,000 μg/ml) on the efflux of rhodamine in fluconazole-resistant Candida glabrata (C8).
Inhibitory effect of amikacin (4,000 μg/ml) on the efflux of rhodamine in fluconazole-resistant Candida glabrata (C8).

Fig. 3.

Inhibitory effect of amikacin (4,000 μg/ml) on the efflux of rhodamine in fluconazole-resistant Candida albicans (C21).
Inhibitory effect of amikacin (4,000 μg/ml) on the efflux of rhodamine in fluconazole-resistant Candida albicans (C21).

Fig. 4.

Percentage of reduction in the rhodamine fluorescence intensity after 120 minutes of amikacin (4,000 μg/ml) treatment of C6, C21 and C8 Candida isolates.
Percentage of reduction in the rhodamine fluorescence intensity after 120 minutes of amikacin (4,000 μg/ml) treatment of C6, C21 and C8 Candida isolates.

Fig. 5.

Relative gene expression levels of the efflux pump mediated genes MDR1, CDR1, and CDR2 in (A) Candida albicans C6 and (B) Candida albicans C21 as measured by the quantitative real-time PCR. Total RNAs were prepared from both selected isolates after 48-hour treatment with 4,000 μg/ml amikacin. The levels of transcripts were normalized to the level of the ACT1 gene expression and then compared to the level of expression in the untreated control cells. The level of gene transcript in control untreated cells (CO) was set as 1. Results were expressed as the means and standard deviations of three independent determinations. The error bars represent SDs.
Relative gene expression levels of the efflux pump mediated genes MDR1, CDR1, and CDR2 in (A) Candida albicans C6 and (B) Candida albicans C21 as measured by the quantitative real-time PCR. Total RNAs were prepared from both selected isolates after 48-hour treatment with 4,000 μg/ml amikacin. The levels of transcripts were normalized to the level of the ACT1 gene expression and then compared to the level of expression in the untreated control cells. The level of gene transcript in control untreated cells (CO) was set as 1. Results were expressed as the means and standard deviations of three independent determinations. The error bars represent SDs.

Fluconazole susceptibility of different Candida spp.

Candida species Fluconazole susceptibility n (%) MIC50 MIC90 MIC range (μg/ml)
S R
Candida albicans (n = 18) 12 (66.7%) 6 (33.3%) 16 1024 1–1024
Non-albicans Candida isolates (n = 10) 4 (40%) 6 (60%) 128 1024 8–1024
Candida glabrata (n = 7) 4 (57.1%) a 3 (42.9%) 32 1024 8–1024
Candida tropicalis (n = 2) 0 (0%) a 2 (100%) 128 b ND c ND
Candida famata (n = 1) 0 (0%) a 1 (100%) ND ND ND

Resistance-modulating effect of amikacin (4000 μg/ml) on fluconazole-resistant Candida clinical isolates.

Isolate code Candida species MIC of fluconazole alone (μg/ml) MIC of fluconazole (μg/ml) in the presence of amikacin Modulation factor (MF) a
C1 C. tropicalis 128 4 32
C6 C. albicans 1024 4 256
C7 C. tropicalis 128 4 32
C8 C. glabrata 1024 32 32
C9 C. glabrata 1024 32 32
C14 C. albicans 256 4 64
C17 C. albicans 1024 4 256
C20 C. albicans 1024 4 256
C21 C. albicans 1024 4 256
C23 C. albicans 1024 4 256
C26 C. glabrata 256 256 1
C28 C. famata 128 4 32

Identification, virulence determinants, and the minimum inhibitory concentration (MIC) of fluconazole against the Candida spp. isolates tested.

Isolate code Candida species Virulence determinants MIC of Fluconazole (μg/ml) c
Biofilm formation Proteases production Pz value a Phospholipase production Pz value b
C1 C. tropicalis Strong 0.26 0.33 128
C2 C. albicans Strong 0.4 0.48 16
C3 C. albicans Strong 0.16 0.29 8
C4 C. albicans Strong 0.19 0.29 32
C5 C. albicans Strong 0.2 1 8
C6 C. albicans Strong 0.2 0.3 1024
C7 C. tropicalis Strong 0.22 0.23 128
C8 C. glabrata Weak 0.18 0.21 1024
C9 C. glabrata Weak 0.67 0.22 1024
C10 C. glabrata Moderate 0.5 0.2 32
C11 C. albicans Strong 0.26 0.26 16
C12 C. glabrata Moderate 0.43 0.23 32
C13 C. albicans Strong 0.4 0.2 8
C14 C. albicans Strong 0.26 0.3 256
C15 C. albicans Strong 0.24 0.24 16
C16 C. albicans Strong 0.24 0.24 8
C17 C. albicans Strong 0.15 0.27 1024
C18 C. glabrata Moderate 0.25 0.21 8
C19 C. albicans Weak 0.15 0.67 2
C20 C. albicans Strong 0.14 0.31 1024
C21 C. albicans Strong 0.17 0.4 1024
C22 C. albicans Weak 0.15 0.39 1
C23 C. albicans Strong 0.18 0.32 1024
C24 C. albicans Strong 0.17 0.41 8
C25 C. glabrata Weak 0.17 0.23 8
C26 C. glabrata Moderate 0.2 0.16 256
C27 C. albicans Strong 0.16 0.32 16
C28 C. famata Strong 0.16 0.4 128

Demographic profile of candiduric patients.

Demographic variables
Sex Age group (years)
Male Female 30–40 41–50 51–60 61– > 70
n % n % n % n % n % n %
Candida albicans (n = 18) 11 61.1 7 38.9 0 0 6 33.3 1 5.6 11 61.1
Non-albicans Candida isolates (n = 10) 5 50 5 50 1 10 3 30 1 10 5 50
Total (n = 28) 16 57.1 12 42.9 1 3.6 9 32.1 2 7.1 16 57.1

Sequences of primers for the genes selected for transcript analysis using quantitative RT-PCR.

Gene Orientation Sequence (5′–3′) Reference
ACT1 F TTGGTGATGAAGCCCAATCC Chau et al. 2004
R CATATCGTCCCAGTTGGAAACA
MDR1 F TTACCTGAAACTTTTGGCAAAACA Chau et al. 2004
R ACTTGTGATTCTGTCGTTACCG
CDR1 F TTTAGCCAGAACTTTCACTCATGATT Chau et al. 2004
R TATTTATTTCTTCATGTTCATATGGATTGA
CDR2 F GGTATTGGCTGGTCCTAATGTGA Chau et al. 2004
R GCTTGAATCAAATAAGTGAATGGATTAC
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