In vitro antifungal susceptibility of Candida species isolated from diabetic patients

Introduction: This study aims to evaluate the antifungal susceptibility of different species of Candida isolated from diabetic patients against eight antifungal agents. Methods: Susceptibility testing of 111 clinical isolates of Candida species was performed against 8 antifungals using the M27-A3 protocol of the Clinical and Laboratory Standards Institute (CLSI). Results: Voriconazole, lanoconazole, and caspofungin showed the highest in vitro activity against all the isolates of C. albicans. Resistance against the tested antifungals was only observed in the C. albicans isolates. Conclusions: Our finding revealed that resistance against amphotericin B, itraconazole, ketoconazole, posaconazole, and fluconazole can be observed in C. albicans.

Diabetes mellitus (DM) is the most prevalent type of diabetes and leads to harmful effects on multiple organs.Diabetes is prevalent among all age groups and has been predicted to show an increase from 171 million cases in 2000 to 366 million cases in 2030 1 .DM patients are known to be susceptible to infections.Candida species, especially Candida albicans, are a part of the normal flora of the oral cavity, intestinal tract, vagina, and skin in healthy individuals 2 .DM can be the underlying disorder for environmental changes of the oral cavity and provide favorable conditions for candidal colonization and cause an infection.This can result in a wide variety of clinical manifestations from superficial to systemic infections caused by different species of Candida 2 .Oral colonization and a high density of Candida species is more common among diabetic patients than nondiabetics 3 .Although C. albicans is considered the most common cause of candidal infections, the prevalence of non-albicans species has recently increased 4 .On the other hand, reports on the trends in the rates of resistance to azoles by Candida species isolated from patients with diabetes are increasing.This has been seen particularly in C. albicans that are typically azolesusceptible 5 .This epidemiologic shift is greatly impacted by pre-exposure to broad-spectrum azoles in patients who receive these agents either as antifungal therapy or prophylactic agents 4 .Accordingly, in this study, we aimed to evaluate the antifungal susceptibility of different isolated species of Candida from diabetic patients 6 against eight antifungal agents.
From February 2014 to June 2014, 300 patients with DM from Mazandaran, a Northern Province of Iran, were included in the study.The patients with any pre-existing fungal infections were excluded.The patients gave informed consent to participate in the research, and the study design was approved by the ethics committee of the Mazandaran University of Medical Sciences.All the isolates were cultured on Sabouraud's Dextrose Agar (Difco Laboratories Detroit, MI, USA) supplemented with chloramphenicol (0.5mg/mL) (SC).The plates were incubated at 27 -30 0 C for up to 7 days.The grown yeast-like colonies were identified to the species level by restriction fragment length polymorphism (RFLP) analysis, as described previously 7 .There was a modification in the procedure after the addition of the first restriction enzyme, MspI (Roche Molecular, Mannheim, Germany).To supplement the digestion of the polymerase chain reaction (PCR) products, a second restriction enzyme, Bln1 (Fermentas, Germany), was added, after which the same procedure was followed.
Genomic deoxyribonucleic acid (DNA) was extracted as per the phenol-chloroform protocol after the disruption of the yeast cells by glass beads, as described previously 6 .
Antifungal susceptibility testing was performed using broth microdilution based on the M27-A3 protocol of the Clinical and Laboratory Standards Institute (CLSI) 7 .Candida krusei (ATCC 6258) and Candida parapsilosis (ATCC 22019) were used as the quality control species in all the experiments.All the isolates of the Candida species were examined against 8 antifungal agents including itraconazole (ITR), ketoconazole (KET), voriconazole (VOR), lanoconazole (LAN), fluconazole (FLU), amphotericin B (AMB) (Sigma-Aldrich, St. Louis, MO, USA), posaconazole (POS) (Schering-Plough B.V., Boxmeer, the Netherlands), and caspofungin (CAS) (Pfizer, Capelle aan den Ijssel, the Netherlands).AMB, ITR, VOR, POS, KET, and LAN were dissolved in dimethyl sulphoxide (DMSO) while FLU and CAS were dissolved in deionized water.Serial twofold dilutions of the drugs were carried out to obtain a final concentration between 64 to 0.13μg/mL for FLU and between 16 to 0.03μg/ mL for the rest of the tested drugs.The antifungal agents were diluted in standard Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) buffered to pH 7.0 with 0.165mol L -1 morpholine propane sulfonic acid buffer with L-glutamine without bicarbonate (MOPS, Sigma-Aldrich, St. Louis, MO, USA).According to the CLSI protocol 7 , the minimum inhibitory concentration (MIC) of each antifungal drug was evaluated after 24h at 35°C.
The MIC for susceptible (S), susceptible-dose dependent (SDD), and resistance (R) was defined according to the CLSI protocol 7 and the M27-S3 supplement of the CLSI 8 .The data was analyzed using Statistical Package for the Social Sciences (SPSS) software (version 19).
The patients' data was presented in our previous published study 9 .In brief, out of 300 patients, 224 (74.7%) were female.The mean age of the patients was 56.83 (range: 30 -90) years.The 51 -60 year age group had the most frequency (35.3%).According to the glycosylated hemoglobin (HbA1c) results, 52 (17.3%)DM patients were classified as suffering from controlled diabetes and 248 (82.7%) had uncontrolled diabetes.Of these two groups, Candida species were identified in 25% and 39.5% of patients with controlled and uncontrolled diabetes (P=0.143),respectively.Out of 300 patients, 111 (37%) cases were positive for Candida species growth.The Candida species were isolated from the oral mucosa (104), axilla (2), vagina (2), and the skin surfaces of chest area (3) of patients with diabetes.According to the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach, C. albicans (93.7%) was the most commonly isolated species, followed by Candida parapsilosis (2.7%), Candida glabrata (1.8%), and Candida tropicalis (1.8%).The geometric mean (GM) MICs, MIC 50 , and MIC 90 of ITR, KET, POS, VOR, LAN, FLU, CAS, and AMB against Candida isolates are summarized in Table 1.According to the number of each identified isolate from the patients, we considered only the evaluation of MICs obtained for C. albicans isolates.As shown in Table 1, VOR, LAN, and CAS showed the highest MICs against all the isolates of C. albicans with MICs ranging from 0.016 -2μg/mL.Resistance against the tested antifungals was observed in the C. albicans isolates.The most resistant isolates of C. albicans were observed against AMB (6.7%).Resistant isolates were not observed among the non-albicans species of Candida.
Due to the limited number of C. parapsilosis, C. glabrata, and C. tropicalis isolates, the calculation of relevant MIC 50 , MIC 90 , and GM was not possible.
Accordingly, due to the lack of data on the clinical breakpoint of LAN, the determination of the S, SDD, and R isolates of Candida species against LAN could not be done.
In the present study, we evaluated 111 isolated species of Candida against eight antifungals.C. albicans was the only species which showed resistance against the tested antifungals as follows: FLU (1.0%), KET (2.9%), POS (2.9%), ITR (4.8 %), and AMB (6.7%).All the isolates of C. albicans were susceptible to VOR and CAS, however, the SDD was observed in 1.9% of C. albicans to VOR.Kowalewska et al 10 reported that susceptible strains to AMB and ITR were reported in 100% and 28% of C. albicans isolated from the fecal samples of children with type 1 diabetes mellitus, respectively.In a study carried out by de Aquino Lemos et al 11 , all isolates of C. albicans (MIC ≤ 1μg/ml) showed a high susceptibility to AMB and CAS while only two isolates (6.4%) were resistant to FLU.Pfaller et al 12 also reported a high activity of CAS against the clinical isolates of C. albicans.However, there are also a few reports on the resistance of Candida species against amphotericin B 13 .Our findings corroborate these previously reported results regarding the efficacy of CAS against Candida species.Our results have also confirmed that CAS is more active than FLU against the clinical isolates of C. albicans (Table 1).
A remarkable point in our finding was the low MICs of LAN against all the isolates of Candida species.The MICs range and MIC 90 of LAN against C. albicans were 0.016 -2 and 1µg/mL, respectively.However, due to the lack of data on the clinical breakpoint of LAN, the determination of the S, SDD, and R isolates of the Candida species against LAN was not possible.LAN is known as a topical antifungal agent with activity against superficial mycoses especially dermatomycosis and cutaneous candidiasis 14 .In this study, the GM MIC of LAN against clinical isolates of C. albicans was 0.14µg/mL.Our findings showed a slight difference in the in vitro inhibition potency of LAN in comparison with that reported by Tatsumi et al. 15 The latter reported the GM MIC range of LAN against clinical isolates of C. albicans and several non-albicans species of Candida as 0.0625 -1.59µg/mL.
Our findings have revealed that C. albicans isolated from diabetic patients exhibited resistance to some antifungals including AMB, ITR, KET, POS, and FLU, the main antifungal agents against superficial and systemic candidal infections.Therefore, we strongly recommend performing the antifungal susceptibility test for all the isolated species of Candida to optimize the treatment of candidal infections.
Clinical and Laboratory Standards Institute; MIC: minimum inhibitory concentration; GM: geometric mean; S: susceptible; SDD: susceptible-dose dependent; R: resistance; C.: Candida.*The MIC for susceptible, susceptible-dose dependent, and resistance was defined according to the CLSI protocol8 and the M27-S3 supplement of the CLSI9 .

TABLE 1 :
Minimum inhibitory concentrations for of antifungal agents for Candida species determined by the CLSI broth microdilution methods.