Thesis (PhD (Microbiological Pathology) --University of Limpopo (Medunsa Campus), 2010 / 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
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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
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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.
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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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/265 |
| Date | 29 May 2010 |
| Creators | Molepo, Julitha |
| Contributors | Nchabeleng, M |
| Publisher | University of Limpopo (Medunsa Campus) |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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