Clade related antifungal resistance among South African candida albicans isolates

Abstract

Background: Azoles and polyenes are antifungal agents used for treatment and/or prophylaxis of C. albicans infections, and a high increase in antifungal resistance in clinical isolates of C. albicans in HIV/AIDS patients has been reported. Five genetic clades were described among C. albicans isolates using DNA fingerprinting methods (clades I, II, III, SA and NG). Although these clades have been described, little is known about their phenotypic characteristics, and not much is known about antifungal resistance with regard to each of these clades. The widespread use of fluconazole has led to its increased resistance reported world-wide. Resistance to fluconazole can be caused by point mutations in the ERG11 gene or overexpression of this gene, however, not much is known about the contribution of these mutations and over-expression to fluconazole resistance among different clades of C. albicans, and whether mutations or over-expression are clade-related. There is evidence to suggest that phenotypic switching may play a significant role in the ability of Candida strains to survive under adverse conditions and perhaps cause more severe forms of disease in the immunocompromised host (Vargas et al., 2004). Only limited studies on the relationship between phenotypic switching and fluconazole resistance of C. albicans have been done, and not much is known about this relationship among different clades of C. albicans. Objectives: This study undertook to investigate: (1) the induction of antifungal resistance among South African C. albicans isolates belonging to different clades, (2) the contributions of mutations in the ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, (3) the contributions of over-expression of ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, (4) and the relationship between fluconazole resistance and phenotypic switching among C. albicans isolates belonging to different clades. Study populations and Methods: To investigate the induction of antifungal resistance among South African C. albicans isolates belonging to different clades, a total of 100 C. albicans isolates (20 from each of clades I, II, III, SA and NG) were used. These yeast isolates were obtained from surveillance cultures on patients attending HIV/AIDS clinics in the Pretoria region. Resistance to fluconazole, miconazole, amphotericin B and nystatin was induced in all 100 isolates according to the modified National Committee of Clinical vi Laboratory Standards (NCCLS) broth microdilution method. Survival and retention of resistance among fluconazole resistant (n=100), miconazole resistant (n=100), amphotericin B resistant (n=100) and nystatin resistant (n==100) isolates after two years of storage at -80oC was determined in the presence of highest concentrations of each antifungal. To investigate the contributions of mutations in the ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, 30 isolates were used. These consisted of 3 isolates with induced fluconazole resistance and their 3 matching fluconazole susceptible isolates from each of clades I, II, III, SA, and NG. DNA was extracted, PCR performed with a high-fidelity Pwo DNA polymerase), and PCR products sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit on the GeneAmp® PCR System 9700. Obtained sequences were compared with the published ERG11 sequence from a wild-type, fluconazole-susceptible C. albicans strain (Lai and Kirsch, 1989). To investigate the contributions of over-expression of the ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, 30 isolates were used. These consisted of 3 isolates with induced fluconazole resistance and their 3 matching fluconazole susceptible isolates from each of clades I, II, III, SA, and NG. RNA was extracted, cDNA synthesized and Real time PCR performed on a Rotor-Gene 6000 instrument. Relative gene expression of ERG11 gene among resistant isolates, relative to susceptible isolates was quantified after normalization with the 18SrRNA house-keeping gene. To investigate the relationship between fluconazole resistance and phenotypic switching among C. albicans isolates belonging to different clades, 30 isolates were used. These consisted of 3 isolates with induced fluconazole resistance and their 3 matching fluconazole susceptible isolates from each of clades I, II, III, SA, and NG. Primary and secondary cultures were prepared on Lee’s medium agar supplemented with arginine and zinc, and containing phloxine B. The switched colonies were counted and colony morphologies viewed and photographed. Phenotypic switching behavior and different colony morphologies obtained between the resistant and susceptible isolates from different clades were compared. Switch phenotypes among fluconazole resistant isolates in different clades were compared. Switch phenotypes and MIC levels among fluconazole resistant isolates from different clades were compared. Results: Resistance to nystatin, AmB, fluconazole and miconazole was successfully induced in all of 20 (100%) C. albicans isolates from each of clades I, II, III, SA and NG. When survival and retention of resistance were determined, all 20 (100%) fluconazole resistant vii isolates from clades I, II, SA, NG, and 19 (95%) from clade III survived and retained their resistance. Of miconazole resistant isolates, all 20 (100%) isolates from clade I, II, and SA, and 19 (95%) from clade III and NG survived and retained their resistance. Of AmB resistant isolates, 12 (60%) from Clade NG survived and retained their resistance; 9 (45%) from Clade I; 8 (40%) from Clade III; 7 (35%) from Clade II and 6 (30%) from Clade SA survived and retained their resistance. Of the isolates resistant to nystatin, 12 (60%) from clade I survived and retained their resistance, 8 (40%) from clade II, 10 (50%) from clade III, 11 (55%) from clade SA, and 15 (75%) from clade NG survived and retained their resistance. No mutations associated with fluconazole resistance were observed in all isolates from clades I and II. Mutations associated with fluconazole resistance were observed in 33.3% of isolates from each of clades III, SA and NG , and some of the mutations observed in resistant isolates from clades III and NG were novel. A total of 50 novel mutations that have not been described previously were observed in both fluconazole resistant and susceptible isolates from this study. Previously described mutations, which were associated with fluconazole resistance, namely, D116E, K128T, V437I and E266D were also observed in this study. When relative ERG11 gene expression was quantified among fluconazole resistant and susceptible isolates from various clades, over-expression of ERG11 gene was observed in 66.6% of isolates from each of clades I, II and SA, and in 33.3% of isolates from each of clades III and NG. When the relationship between fluconazole resistance and phenotypic switching was investigated, phenotypic switching was related to resistance in 66.6% of the resistant isolates tested from each of clades I, II and III, and in 33.3% of the resistant isolates tested from each of clades SA and NG. When the switch phenotypes and MIC levels of resistant isolates from different clades were compared, stipple was the most common switch phenotype observed in all clades, and it was associated with the highest fluconazole MIC levels among isolates from all clades. Conclusions: The results of this study showed that resistance to polyenes and azoles could readily be induced in C. albicans isolates from all clades, and that induction was not claderelated. The ease with which azole and polyene resistance could be induced in this study can hold serious implications, especially in HIV/AIDS patients who are already immunocompromised, and in whom azoles/polyenes are mostly used for C. albicans infections. viii The study also showed that mutations contributed to fluconazole resistance in isolates from clades III, SA and NG, but not clades I and II, showing clade-relatedness. Novel mutations were observed, and their contribution to fluconazole resistance is at this stage not known. Genetic analysis of these mutations needs to be studied further to determine their significance to azole resistance, especially in C. albicans isolates from HIV/AIDS patients in South Africa. The results of the study showed that over-expression of ERG11 gene contributed to fluconazole resistance in isolates from all clades. However, over-expression was observed in more isolates from clades I, II and SA, and in less isolates from clades III and NG, showing clade-relatedness of ERG11 over-expression. The occurrence of over-expression of ERG11 gene in these clades is a cause for concern, especially in HIV/AIDS patients with OPC, as the increased expression of ERG11 allows for the cells to persist within the host, which in turn leads to the subsequent development of other more stable resistant isolates. In this study, phenotypic switching was found to be related to fluconazole resistance in isolates from all clades, with a high number of switch phenotypes occurring more in isolates from clade II as compared to others. This suggests that isolates belonging to this clade may survive better under adverse conditions than isolates from other clades. These results suggest that further study of differences between different C. albicans clades may be warranted, and that isolates from this clade need to be studied further. The stipple phenotype was found to be the most dominant in isolates from all clades, and was found to be associated with the highest fluconazole MICs levels. These findings suggest that the evaluation of colony phenotypes and their antifungal susceptibilities in C. albicans isolates may be useful in therapy. Recommendations: A continued analysis of clade-specific phenotypic characteristics of C. albicans isolates is recommended. Pathogens that can potentially infect HIV-infected individuals need to be studied to subspecies level in order to improve treatment of these patients. Continued antifungal surveillance is needed to predict the evolution of resistance in a particular population and to take timely measures. Evaluation of colony phenotypes and their antifungal susceptibilities in C. albicans isolates is recommended as this may be useful in therapy. Genetic analysis of the novel mutations observed is recommended to determine their significance to azole resistance, especially in C. albicans isolates from HIV/AIDS patients in South Africa.