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ERG11 gene polymorphisms and susceptibility to fluconazole in Candida isolates from diabetic and kidney transplant patients

Abstract

INTRODUCTION:

Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function.

METHODS:

Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates.

RESULTS:

A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates.

CONCLUSIONS:

Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.

Keywords:
Candida albicans; Drug Resistance; Fluconazole; Sterol; 14α-demethylase

INTRODUCTION

Candidiasis is the most frequent opportunistic mycosis in humans and is one of the major causes of mortality in immunocompromised patients11. Hung CC, Yang YL, Lauderdale TL, McDonald LC, Hsiao CF, Cheng HH, et al. Colonization of human immunodeficiency virus-infected outpatients in Taiwan with Candida species. J Clin Microbiol. 2005;43(4):1600-03.,22. Chen CG, Yang YL, Tseng KY, Shih HI, Liou CH, Lin CC, et al. Rep1p negatively regulating MDR1 efflux pump involved in drug resistance in Candida albicans. Fungal Genet Biol. 2009;46(9):714-20.. The incidence of systemic fungal infections has increased steadily over recent years, mainly due to the growing number of organ and bone marrow transplants, cancer treatments, and AIDS cases, all of which are detrimental to the body's natural defense system33. Xiang MJ, Liu JY, Ni PH, Wang S, Shi C, Wei B, et al. Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans. FEMS Yeast Res. 2013;13(4):386-93.. In view of the compromised immune system in such cases, the availability of effective antifungal agents is particularly critical to treatment. However, treatment can be hindered by the presence of Candida species displaying multiple drug resistance (MDR), which is becoming more frequently reported44. Kouidhi B, Ben Gaied M, Mhadhebi L, Bakhrouf A, Bouraouia A. A. Les pompes à efflux en mycologie médicale mécanismes moléculaires et perspectives thérapeutiques. J Mycol Med. 2010;20(4):304-14.,55. Bondaryk M, Kurzątkowski W, Staniszewska M. Antifungal agents commonly used in the superficial and mucosal candidiasis treatment: mode of action and resistance development. Postepy Dermatol Alergol. 2013;30(5):293-301..

Treatment for Candida infections normally involves triazole antifungals, of which fluconazole is the most commonly prescribed66. Pinjon E, Jackson CJ, Kelly SL, Sanglard D, Moran G, Coleman DC, et al. Reduced azole susceptibility in genotype 3 Candida dubliniensis isolates associated with increased CdCDR1 and CdCDR2 expression. Antimicrob Agents Chemother. 2005;49(4):1312-18.,77. Schubert S, Rogers PD, Morschhäuser J. Gain-of-Function mutations in the transcription factor MRR1 are responsible for overexpression of the MDR1 efflux pump in fluconazole-resistant Candida dubliniensis strains. Antimicrob Agents Chemother . 2008;52(12):4274-80.. The azolic antifungals act by inactivating lanosterol 14α-demethylase, thereby inhibiting biosynthesis of ergosterol, a compound essential for cell integrity and membrane function88. Akins R. An update on antifungal targets and mechanisms of resistance in Candida albicans. Med Mycol. 2005;43(4):285-318.-99. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother. 2008;61(4):798-04.. This results in a decrease in the level of ergosterol available for membrane function and a concomitant increase in the number of intermediate metabolites1010. Debnath S, Addya S. Structural basis for heterogeneous phenotype of ERG11 dependent Azole resistance in C. albicans clinical isolates. Springerplus. 2014;3:660.. Resistance to azole antifungals is commonly associated with continuous drug use, but can also because by intrinsic factors1111. Orozco AS, Higginbotham LM, Hitchcock CA, Parkinson T, Falconer D, Ibrahim AS, et al. Mechanism of fluconazole resistance in Candida krusei. Antimicrob Agents Chemother . 1998;42(10):2645-49.,1212. Ben-Ami R, Garcia-Effron G, Lewis RE, Gamarra S, Leventakos K, Perlin DS, et al. Fitness and virulence costs of Candida albicans FKS1 hot spot mutations associated with echinocandin resistance. J Infect Dis. 2011;204(4):626-35., such as mutations or alterations in the expression of the CDR1, CDR2, PDR51313. White TC, Holleman S, Dy F, Mirels LF, Stevens DA. Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother . 2002;46(6):1704-13.,1414. Niimi K, Harding DR, Holmes AR, Lamping E, Niimi M, Tyndall JD, et al. Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor. Mol Microbiol. 2002;85(4):747-67., ERG31515. Cowen LE, Steinbach WJ. Stress, drugs, and evolution: the role of cellular signaling in fungal drug resistance. Eukaryot Cell. 2008;7(5):747-64., MDR1, FLU11616. Zhang JY, Liu JH, Liu FD, Xia YH, Wang J, Liu X, et al. Vulvovaginal candidiasis: species distribution, fluconazole resistance and drug efflux pump gene overexpression. Mycoses. 2014;57(10):584-91., and ERG111717. Feng LJ, Wan Z, Wang XH, Li RY, Liu W. Relationship between antifungal resistance of fluconazole resistant Candida albicans and mutations in ERG11 gene. Chin Med J (Engl). 2010;123(5):544-48. genes. The ERG11 and ERG3 genes encode proteins involved in the biosynthesis of ergosterol, and mutations at specific points in these two genes can critically alter the effectiveness of azolic antifungal drugs1818. Martel CM, Parker JE, Bader O, Weig M, Gross U, Warrilow AG, et al. Identification and characterization of four azole-resistant ERG3 mutants of Candida albicans. Antimicrob Agents Chemother . 2010;54(11):4527-33..

Thus, this study aims to investigate polymorphisms in the ERG11 gene, and correlate this genetic data to the fluconazole susceptibility profile of different Candida species isolated from diabetic and kidney transplant patients.

METHODS

Biological material

Samples of saliva were collected from 90 outpatients, including 40 diabetic patients (12 male and 28 female), 19 kidney transplant recipients (9 male and 10 female), and 31 immunocompetent patients (3 male and 28 female; control group). The 40 diabetic patients were between 40 and 70 years old and had been diagnosed with type II diabetes over five years prior. All had hypertension but were not using insulin, and 40 were diagnosed with hyperglycemia. All transplant patients were between 30 and 60 years old, and had received a kidney transplant over one year before the study. In addition, 19 of the transplant patients were under treatment with prednisone, azathioprine, and cyclosporine. The control group was composed of individuals between the ages of 18 and 30, who had not been diagnosed with any disease, and were not using drugs with antimicrobial or anti-inflammatory activities. The patients within the kidney transplant, diabetic and control groups were not paired by gender or age. A total of 94 Candida isolates were obtained, and the species were identified by Benedetti et al.1919. Benedetti VB, Savi DC, Aluizio R, Adamoski D, Kava-Cordeiro V, Galli-Terasawa LV, et al. Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients. Mem Inst Oswaldo Cruz. 2016; doi:10.1590/0074-0276016004
https://doi.org/10.1590/0074-0276016004...
. Among the 70 isolates used in this study, 58 were classified as Candida albicans, 6 as Candida glabrata and 8 as Candida tropicalis.

Ethical standards

This study was performed in accordance with the ethical standards of the 1964 Helsinki declaration, and was approved by the Ethics Committee (Conselho Nacional de Ética em Pesquisa - CONEP) under registration no. 19885/2011, Universidade Paranaense (Parana-Brazil). Informed consent was obtained from all individual participants involved in the study.

Fluconazole minimum inhibitory concentration

The fluconazole minimum inhibitory concentration (MIC) of Candida isolates was determined using an E-Test Kit (Biomerieux, France) in Mueller-Hinton media (Difco, USA), supplemented with 2% glucose and 0.5 μg/mL methylene blue, according to the protocol of the Clinical and Laboratory Standards Institute2020. CLSI. M44-A2. Reference method for antifungal disk diffusion susceptibility testing of yeasts: Wayne, PA: Clinical and Laboratory Standards Institute, 2010.. The isolates were classified as susceptible to fluconazole if their MIC was ≤8 μg/mL; dose-dependent MIC values were from 8 to 32 μg/mL; and resistant isolates had MIC ≤ 32 μg/mL2121. CLSI. M27-A3. Reference method for broth dilution antifungal susceptibility testing of yeasts: 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute , 2008.. As controls, three reference strains were used: C. albicans ATCC - 44858 (MIC - 1 μg/mL), C. glabrata ATCC - 2001(MIC - 4 μg/mL), and C. tropicalis ATCC - 28707 (MIC - 2 μg/mL). Analysis of variance (ANOVA), was used to compare MIC values of individual isolates, as well as isolate groups (diabetic, kidney transplant, and immunocompetent control) using GraphPad Prism software, version 5 (GraphPad, USA) with a significance level of 0.05. When the MIC value was higher than that reported for each species, the differences between reference strains and the isolates were confirmed with a t-test.

DNA isolation, amplification, and partial sequencing of the ERG11 gene

Total genomic DNA was purified from 2 day-old single colony cultures growing on Mueller-Hinton media (Difco, USA) using the Ultraclean Microbial DNA Isolation Kit(MoBio, USA) according to the manufacturer’s instructions. Partial sequences of the ERG11 gene were amplified using the following primer pairs: C. albicans99. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother. 2008;61(4):798-04., Sec1A (forward); 5’-TTAGTGTTTTATTGGATTCCTTGGTT-3’, Sec1B (reverse); 5’-TCTCATTTCATCACCAAATAAAGATC-3’,C. tropicalis2222. Vandeputte P, Larcher G, Bergès T, Renier G, Chabasse D, Bouchara JP. Mechanisms of azole resistance in a clinical isolate of Candida tropicalis. Antimicrob Agents Chemother .2005;49(11):4608-15., CtERGr1 - F (forward); 5’-TCTTTTGTCAACACAGTAATGGC-3’, CtERGr1 - R (reverse); 5’-AACACCTTTACCAAAAACAGGAG-3’, C. glabrata2323. Carvalho VO, Okay TS, Melhem MS, Szeszs MW, Del Negro G.The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance. Rev Iberoam Micol. 2013;30(3):209-12., CgERGr1 - F (forward); 5’-ACTACAATAACATGTCCACTGA-3’, CgERGr1 - R (reverse); 5’-GGGTGGTCAAGTGGGAGTAA-3’. The amplification was performed as described by Xu et al.99. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother. 2008;61(4):798-04. in a final volume of 12.5 μL, containing 1 X Tris Base buffer solution, dNTPs (0.2 µM, Invitrogen, USA), MgCl2 (1.6 mM), primers (15 pmol each), Taq DNA polymerase (0.5 U) (Invitrogen, USA), and template DNA (20 ng). The PCR products were purified using 7.5 M ammonium acetate (15 µL) and absolute ethanol (74 µL). Samples were incubated on ice for one hour, followed by centrifugation for 45 min at 23,100 g. The pellet was re-suspended in 12 µL of Milli-Q water. Sequencing of the PCR products was performed using a Big Dye Kit (Applied Biosystems, USA), followed by purification using Sephadex G-50 fine DNA grade resin (GE Healthcare, UK) in a MultiScreen Column Loader (Merck Millipore, USA), and analysis by electrophoresis in an ABI3500 Automatic Sequencer (Applied Biosystems, USA). The obtained sequences were visually inspected using the BioEdit program version 7.2.52424. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95-8., aligned using ClustalW2525. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25(24):4876-82., and manually adjusted using MEGA software, version 62626. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA.5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 2011;28(10):2731-39.. Additional sequences were obtained from GenBank (Table 1) and alignments were generated in ClustalW. Amino acid prediction was performed using MEGA software, version 62626. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA.5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 2011;28(10):2731-39..

TABLE 1:
Polymorphic sites in sequences of the ERG11 gene from isolated species of C. albicans, C. tropicalis and C. glabrata, and associated amino acid prediction, fluconazole MIC values and GenBank codes.

RESULTS

Fluconazole minimum inhibitory concentration

All isolates with MIC values ≤ 8 μg/mL2121. CLSI. M27-A3. Reference method for broth dilution antifungal susceptibility testing of yeasts: 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute , 2008.were classified as susceptible to fluconazole (Table 1). The mean MIC values was higher for the species C. glabrata (p=0.02), the mean MIC were 1.43 μg/mL for C. albicans, 1.71 μg/mL for C. tropicalis, and 3.0 μg/mL for C. glabrata. Interestingly, although all C. albicans isolates were characterized as susceptible to fluconazole (MIC ≤ 8 μg/mL)2121. CLSI. M27-A3. Reference method for broth dilution antifungal susceptibility testing of yeasts: 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute , 2008., strains isolated from transplanted (MIC = 1.86 μg/mL) and diabetic (MIC =1.63 μg/mL) patients had higher fluconazole MIC values than the control group (MIC =0.94 μg/mL) (p=0.001) (Table 1).

Analysis of polymorphic sites in the ERG11 gene of Candida isolates

The GenBank codes for the partial sequences of the ERG11 gene from Candida isolates are listed in Table 1. The alignment of the ERG11 sequence of the C. albicans isolates was 482 bp-long, with 134 polymorphic sites. The 407 bp-long alignment of the C. tropicalis isolates to the ERG11 genus contained 12 polymorphic sites. No polymorphisms were found in the partial sequence alignment of the ERG11 gene of the six C. glabrata isolates.

On comparing the obtained ERG11 sequences to the published ERG11 sequences of fluconazole-sensitive Candida species, we observed that 32.9% (n=31) of isolates showed no nucleotide alteration in their partial ERG11 sequences. The remaining 67.1% (n=63) of isolates showed at least one mutation of these, 19.1% (n=18) had a unique polymorphic site, 21.3% (n=20) showed two substitutions, 17.1% (n=16) had three polymorphic sites, and 9.6% (n=9) had more than three variable sites in the partial sequence of the ERG11 gene (Table 1).

Analysis of the predicted amino acid sequence of ERG11 revealed that 82.9% (121 bp) of variable sites resulted in synonymous mutations, with no change in the predicted amino acid sequence in C. albicans or C. tropicalis isolates, while 17.1% (25 bp) resulted in missense mutations, which were observed only in C. albicans isolates. In addition, 41.8% (56 bp) of mutations in C. albicans isolates occurred in the amino acid lysine (Lys) position F105L, 18.7% (25) in arginine (Arg) position K119N, 11.1% (15) in phenylalanine (Phe) position S137L, and 9.7% (13) in leucine (Leu) position D116E. In C. tropicalis, the most frequent mutation (58.3%) was in the amino acid asparagine (Asn) position G263A, followed by threonine (Thr) position T224C (41.7%). The missense nucleotide mutation observed in the ERG11 gene of C. albicans isolates was a substitution of adenine with cytosine at codon 530, which resulted in the amino acid cysteine instead of phenylalanine (Table 1). In addition, of the 25 missense mutations observed, 92% (n=23) were heterozygous, and 8% were homozygous.

DISCUSSION

Candida infections are the most frequent cause of opportunistic diseases in immunocompromised patients2727. Li YY, Chen W, Li X, Li HB, Li HQ, Wang L, et al. Asymptomatic oral yeast carriage and antifungal susceptibility profile of HIV-infected patients in Kunming, Yunnan Province of China. BMC Infect Dis. 2013;13:46., and fluconazole is the first treatment option for such cases. However, several studies have documented the ability of Candida to develop high-level resistance to azole compound2828. Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD. Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front Microbiol. 2017;7:2173., either through efflux pumps or alterations in the sterol 14-α-demethylase caused by mutations in the ERG11 gene2929. Lamping E, Ranchod A, Nakamura K, Tyndall JD, Niimi K, Holmes AR, et al. Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei. Antimicrob Agents Chemother . 2009;53(2):354-69.,3030. Silva DB, Rodrigues LM, Almeida AA, Oliveira KMP, Grisolia AB. Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species. Mem Inst Oswaldo Cruz . 2016;111(3):192-9.. For effective treatment of Candida infections, and selection of the most efficient prophylactic measures, it is necessary to identify the Candida species, evaluate its susceptibility profile to different antifungals, and determine the mechanisms involved in any observed resistance3131. Goląbek K, Strzelczyk JK, Owczarek A, Cuber P, Ślemp-Migiel A, Wiczkowski A. Selected mechanisms of molecular resistance of Candida albicans to azole drugs. Acta Biochim Pol. 2015;62(2):247-51..

The Candida isolates used in this study had been classified in a previous study2121. CLSI. M27-A3. Reference method for broth dilution antifungal susceptibility testing of yeasts: 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute , 2008.as the three different species; C. albicans, C. glabrata, and C. tropicalis. All isolates (70) were classified as susceptible to fluconazole, with MIC ≤ 8 μg/mL2020. CLSI. M44-A2. Reference method for antifungal disk diffusion susceptibility testing of yeasts: Wayne, PA: Clinical and Laboratory Standards Institute, 2010.. However, there were significant differences in the susceptibility ranges, with isolates from the control group displaying higher susceptibility to fluconazole than isolates from diabetic and kidney transplant patients (Table 1). This higher susceptibility of isolates from the control group maybe related to the absence of continuous fluconazole use, which has been suggested as one of the principal causes of fluconazole resistance in Candida1212. Ben-Ami R, Garcia-Effron G, Lewis RE, Gamarra S, Leventakos K, Perlin DS, et al. Fitness and virulence costs of Candida albicans FKS1 hot spot mutations associated with echinocandin resistance. J Infect Dis. 2011;204(4):626-35.,3232. Qiao J, Liu W, Li R. Antifungal resistance mechanisms of Aspergillus. Nihon Inshinkin Gakkai Zasshi. 2008;49(3):157-63.,3333. Berkow EL, Lockhart SR. Fluconazole resistance in Candida species: a current perspective. Infect Drug Resist. 2017;10;237-245.. In contrast, the higher fluconazole MIC values of strains isolated from transplant patients may be related to frequent contact with hospital patients, which facilitates oral colonization by less susceptible strains. Another contributor to this lower susceptibility may be the high cellular stress level of this group, caused by exposure to other drugs such as chemotherapeutics, corticosteroids, and antibiotics, which have been shown to activate resistance mechanisms in yeast3434. Yan L, Li M, Cao Y, Gao P, Cao Y, Wang Y, et al. The alternative oxidase of Candida albicans causes reduced fluconazole susceptibility. J Antimicrob Chemother . 2009;64(4):764-73.,3535. Morschhäuser J. Regulation of multidrug resistance in pathogenic fungi. Fungal Genet Biol . 2010;47(2):94-106..

In addition, we observed a difference in the mean fluconazole MIC values between different Candida species (Table 1). The lowest fluconazole MIC values were observed in C. albicans, followed by C. tropicalis, then C. glabrata. These differences may be related to intrinsic factors specific to each species3636. Pfaller MA, Diekema DJ, Jones RN, Sader HS, Fluit AC, Hollis RJ, et al. International surveillance of bloodstream infections due to Candida species: frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole and voriconazole of isolates collected from 1997 through 1999 in the SENTRY antimicrobial surveillance program. J Clin Microbiol . 2001;39(9):3254-9.,3737. Barbedo LS, Sgarbi DBG. Candidíases. J Bras Doenças Sex Transm. 2010;22(1):22-38.(Table 1). In the case of C. glabrata isolates, the relatively high tolerance to fluconazole is supported by the absence of any mutations in the ERG11 gene (Table 1), suggesting that the higher tolerance may be a common characteristic of this species. Consistent with this notion, previous studies have demonstrated that after exposure to fluconazole, C. glabrata isolates showed lower susceptibility profiles compared to other Candida species3636. Pfaller MA, Diekema DJ, Jones RN, Sader HS, Fluit AC, Hollis RJ, et al. International surveillance of bloodstream infections due to Candida species: frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole and voriconazole of isolates collected from 1997 through 1999 in the SENTRY antimicrobial surveillance program. J Clin Microbiol . 2001;39(9):3254-9.,3737. Barbedo LS, Sgarbi DBG. Candidíases. J Bras Doenças Sex Transm. 2010;22(1):22-38..

Analysis of the ERG11 gene sequence showed that, despite the high number of polymorphic sites observed in C. albicans and C. tropicalis, none occurred in the region coding for the binding site of the antifungal, the enzyme sterol 14-α-demethylase3838. Carrillo-Muñoz AJ, Giusiano G, Ezkurra PA, Quindós G. Antifungal agents: mode of action in yeast cells. Rev Esp Quimioter. 2006;19(2):130-9.,3939. Maccallum DM, Coste A, Ischer F, Jacobsen MD, Odds FC, Sanglard D. Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection. Antimicrob Agents Chemother . 2010;54(4):1476-83.. Although specific ERG11 amino acid substitutions are known to be responsible for resistance to azolic compounds4040. Marichal P, Koymans L, Willemsens S, Bellens D, Verhasselt P, Luyten W, et al. Contribution of mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans. Microbiology. 1999;145(Pt 10):2701-13.,4141. Grossman NT, Pham CD, Cleveland AA, Lockhart SR. Molecular mechanisms of fluconazole resistance in Candida parapsilosis isolates from a US surveillance system. Antimicrob Agents Chemother . 2015;59(2):1030-7., the mutations characterized as F105L, D116E, K119N, K128T and S137L in C. albicans isolates, and T224C and G263A in C. tropicalis (Table 1) were not associated with antifungal resistance99. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother. 2008;61(4):798-04.,2323. Carvalho VO, Okay TS, Melhem MS, Szeszs MW, Del Negro G.The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance. Rev Iberoam Micol. 2013;30(3):209-12.,4242. Morio F, Loge C, Besse B, Hennequin C, Le Pape P. Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature. Diagn Microbiol Infect Dis. 2010;66(4):373-84.,4343. Sanglard D, Ischer F, Koymans L, Bille J. Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother . 1998;42(2):241-53.,4444. Flowers SA, Colón B, Whaley SG, Schuler MA, Rogers PD. Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob Agents Chemother . 2015;59(1):450-60., in agreement with the MIC profiles. Although these mutations are synonymous, it is possible that they could influence the efficiency of translation, resulting in alterations in protein production, as has been suggested previously4545. Jacobson GN, Clark PL. Quality over quantity: optimizing co-translational protein folding with non-'optimal' synonymous codons. Curr Opin Struct Biol. 2016;38:102-10.. For example, the F105L mutation was previously correlated with fluconazole resistance4040. Marichal P, Koymans L, Willemsens S, Bellens D, Verhasselt P, Luyten W, et al. Contribution of mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans. Microbiology. 1999;145(Pt 10):2701-13., where as another study suggested that this mutation alone does not result in an alteration in the active site of 14-α-demethylase4646. Park HG, Lee IS, Chun YJ, Yun CH, Johnston JB, Montellano PR, et al. Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans. Arch Biochem Biophys. 2011;509(1):9-15..

Several studies have recently investigated the fluconazole susceptibility of Candida isolates from HIV, cancer, and immunocompromised patients in Brazil4747. Pinto PM, Weikert RC, Lyon JP, Cury VF, Arantes RR, Koga-Ito CY, et al. In vitro antifungal susceptibility of clinical isolates of Candida spp. obtained from patients with different predisposing factors to candidiasis. Microbiol Res. 2008;163(5):579-85.,4848. Terças AL, Marques SG, Moffa EB, Alves MB, Azevedo CMPS, Siqueira WL, et al. Antifungal drug susceptibility of Candida species isolated from HIV-Positive patients recruited at a public hospital in São Luís, Maranhão, Brazil. Front Microbiol . 2017;8:298.. However, no other studies have investigated the fluconazole susceptibility of Candida species associated with diabetic and kidney transplant patients. Although Chaves et al.4949. Chaves GM, Diniz MG, Da Silva-Rocha WP, de Souza LB, Gondim LA, Ferreira MA, et al. Species distribution and virulence factors of Candida spp. isolated from the oral cavity of kidney transplant recipients in Brazil. Mycopathologia. 2013(3-4);175:255-63.evaluated the diversity of Candida isolates from kidney transplant recipients in Brazil, the authors did not investigate the fluconazole susceptibility profiles

We therefore provide the first comparison of fluconazole susceptibility profiles of Candida species isolated from diabetic and kidney transplant patients in Brazil. Although the therapeutic and prophylactic use of fluconazole is widespread, fluconazole resistant Candida isolates were not observed in this study. However, fluconazole MIC values were higher for isolates from diabetic and kidney transplant patients compared with the control group. Further studies should therefore be performed to verify whether continued exposure to fluconazole may result in the identification of resistant Candida strains in these patients.

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Publication Dates

  • Publication in this collection
    07 Mar 2019
  • Date of issue
    2019

History

  • Received
    09 Nov 2018
  • Accepted
    25 Jan 2019
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