Antimicrobial Agents and Chemotherapy, July 1999, p. 1621-1630, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Multiple Resistant Phenotypes of Candida albicans
Coexist during Episodes of Oropharyngeal Candidiasis in Human
Immunodeficiency Virus-Infected Patients
Jose L.
Lopez-Ribot,1,*
Robert K.
McAtee,1
Sofia
Perea,1
William R.
Kirkpatrick,1
Michael G.
Rinaldi,2,3 and
Thomas F.
Patterson1
Departments of
Medicine1 and
Pathology,2 The University of Texas
Health Science Center at San Antonio, and South Texas
Veterans Health Care System, Audie L. Murphy
Division,3 San Antonio, Texas 78284
Received 8 February 1999/Returned for modification 29 March
1999/Accepted 7 May 1999
 |
ABSTRACT |
Mechanisms of resistance to azoles in Candida albicans,
the main etiologic agent of oropharyngeal candidiasis (OPC), include alterations in the target enzyme (lanosterol demethylase) and increased
efflux of drug. Previous studies on mechanisms of resistance have been
limited by the fact that only a single isolate from each OPC episode
was available for study. Multiple isolates from each OPC episode were
evaluated with oral samples plated in CHROMagar Candida with and
without fluconazole to maximize detection of resistant yeasts. A total
of 101 isolates from each of three serial episodes of OPC from four
different patients were evaluated. Decreasing geometric means of
fluconazole MICs with serial episodes of infection were detected in the
four patients. However, 8-fold or larger (up to 32-fold) differences in
fluconazole MICs were detected within isolates recovered at the same
time point in 7 of 12 episodes. Strain identity was analyzed by DNA
typing techniques and indicated that isolates from each patient
represented mainly isogenic strains, but differed among patients. A
Northern blot technique was used to monitor expression of
ERG11 (encoding lanosterol demethylase) and genes coding
for efflux pumps. This analysis revealed that clinical isolates
obtained from the same patient and episode were phenotypically
heterogeneous in their patterns of expression of these genes involved
in fluconazole resistance. These results demonstrate the complexity of
the distribution of the molecular mechanisms of antifungal drug
resistance and indicate that different subpopulations of yeasts may
coexist at a given time in the same patient and may develop resistance
through different mechanisms.
| |
INTRODUCTION |
Oropharyngeal candidiasis (OPC)
occurs in as many as 90% of patients with human immunodeficiency virus
(HIV) or AIDS (6, 12). OPC can be an early indicator of HIV
infection, but oral candidiasis is associated with worsening immune
function and may predict progressive immunodeficiency independently of
CD4 lymphocyte counts (2, 17). Azole antifungal drugs,
particularly fluconazole, have proven effective in treating mucosal
candidiasis even in individuals with advanced immunodeficiency
(20, 21). However, development of resistance, especially in
patients with extensive prior azole use, is common (7, 18,
23). Recently the National Committee for Clinical Laboratory
Standards (NCCLS) has approved standardized methods for susceptibility
testing (13). Also, extensive clinical data have been used
to establish the correlation between high in vitro MICs (mycological
resistance) and clinical outcome (22). Mycological
resistance may not always be predictive of a poor outcome, but
increased failure rates occur against resistant yeasts (fluconazole
MICs, >64 µg/ml). Strains for which fluconazole MICs were 16 to 32 µg/ml demonstrate dose-dependent susceptibility and may respond to
higher doses of drug (20, 22).
At the cellular level, development of fluconazole resistance may emerge
as a result of replacement of a susceptible strain by another,
intrinsically resistant strain or species (reviewed in reference
37). At the molecular level, two major mechanisms appear to be responsible for development of fluconazole resistance in
strains of Candida albicans. The first mechanism involves an altered target site, the cytochrome P-450 lanosterol 14
-demethylase, either by overproduction of the enzyme or due to point mutations in its
encoding gene (ERG11) leading to amino acid substitutions resulting in decreased affinity of the enzyme for azole derivatives (9, 25, 32, 33, 36). A second major mechanism is through increased efflux of drug, mediated by two types of multidrug efflux pumps, the major facilitators and the ABC transporters (1, 10, 28,
34, 35). The MDR1 gene encodes a major facilitator implicated in resistance (3), and its overexpression leads to fluconazole resistance exclusively among azole drugs (10, 26,
28). The genes coding for several ABC transporters in C. albicans have been identified, including several CDR
genes (19, 26). CDR1 and CDR2 were the
first two members of this family identified in C. albicans,
and both CDR1 and CDR2 have been described as
playing a role in fluconazole resistance (10, 27, 28). Other
azole drugs are also substrates for ABC transporters, and, thus,
overexpression of CDR genes results in cross-resistance to
other azole derivatives (10, 26, 28).
In general, description of these molecular mechanisms of resistance has
been performed by analyzing their role in serial isolates with
increasing resistance to the drug recovered from the same patient, as
detected by antifungal susceptibility testing (1, 4, 10, 28,
35). However, most studies evaluating resistance have been
limited due to the fact that only single isolates from each time point
were available for study. Methods that increase detection of
subpopulations of yeasts at the time of initial isolation, such as our
novel agar dilution screening technique (14, 15), may be
very useful to provide a more comprehensive assessment of the
mechanisms of resistance.
In the present study, a total of 101 isolates from three serial OPC
episodes from four different patients were included for analysis of
resistance to azoles. Evidence is provided for (i) the heterogeneity of
the susceptibility to fluconazole between isolates recovered during the
same episode of OPC, (ii) the complexity of expression of genes
implicated in development of fluconazole resistance, and (iii) the
presence at the same time point of different subpopulations of yeast
exhibiting different resistance phenotypes.
| |
MATERIALS AND METHODS |
Clinical samples and isolates.
Yeast isolates were obtained
by direct swab or by oral saline rinses from four HIV-infected patients
with recurrent OPC enrolled in a longitudinal study to assess
significance of fluconazole resistance. At the time of initial
isolation, oral samples were plated on CHROMagar Candida (CHROMagar,
Paris, France) with and without fluconazole to maximize detection of
resistant yeasts as previously described by our group (14,
15). Briefly, dilutions of oral samples were added to plates
containing solid medium with and without fluconazole from which
representative colonies were recovered. Patients were treated initially
with fluconazole at 100 mg/day, and doses were increased to up to 800 mg/day if necessary for clinical resolution in an effort to achieve
therapeutic response after development of clinical resistance
(20). In all four patients, therapeutic response was
achieved by increasing the dose of fluconazole to a range of 200 to 800 mg/day. The identity of these clinical isolates as C. albicans was confirmed by standard biochemical and microbiological
procedures, including carbohydrate assimilation patterns (API 20C;
Analytab Products, BioMerieux, France), germ tube formation in
serum-containing medium, and color of colonies in chromogenic medium
(CHROMagar Candida). Only patient A had Candida species
other than albicans at the time of the second and third
episodes, but the predominant isolates were C. albicans. All
other patients had C. albicans only. Isolates were stored at
room temperature as suspensions in sterile deionized water.
Strain identification.
Strain identity was investigated by
karyotyping, restriction fragment length polymorphism (RFLP), and DNA
fingerprinting with the moderately repetitive probe Ca3 (provided as a
gift from D. Soll, University of Iowa) (29). Briefly,
chromosomes from the different isolates were prepared in agarose plugs,
separated by pulsed-field gel electrophoresis (Bio-Rad, Hercules,
Calif.), stained with ethidium bromide, and photographed under UV
light. RFLP patterns were generated by digestion of genomic DNA with SfiI (Boehringer Mannheim, Indianapolis, Ind.). After
separation by pulsed-field gel electrophoresis, gels were stained with
ethidium bromide and photographed. Following documentation, the
materials present in the RFLP gels were transferred to nylon membranes
(Nytran; Schleicher & Schuell, Keene, N.H.) and hybridized with a Ca3
probe radioactively labeled by random priming (Random Primers DNA
Labeling System; GibcoBRL, Gaithersburg, Md.). The membranes were then washed and exposed to autoradiography film (Du Pont, Wilmington, Del.).
Pictures of the gels or films were scanned with the Adobe Photo Shop
program (Adobe Systems, Inc., Mountain View, Calif.). For preparation
of figures, digital images were processed by using the Adobe Photo Shop program.
Drug susceptibility testing and MIC determinations.
Antifungal susceptibilities to fluconazole were determined by NCCLS
method M-27A with broth macrodilution techniques and reading of the
endpoints at 48 h (13). Isolates for which fluconazole MICs were 
8 µg/ml are considered susceptible. MICs of 16 to 32 µg/ml indicate susceptible but dose-dependent isolates. Isolates for
which MICs were 
64 µg/ml are considered resistant to the drug
(22). Additional susceptibility testing of selected isolates with itraconazole (Janssen Pharmaceutica, Beerse, Belgium),
ketoconazole (Janssen Pharmaceutica), voriconazole (Pfizer Inc.,
Sandwich, United Kingdom), SCH 56592 (Schering Plough, Kenilworth,
N.J.), amphotericin B (Bristol-Myers Squibb, Princeton, N.J.), and
terbinafine (Novartis, Vienna, Austria) was determined according to
NCCLS method M-27A by using a broth microdilution procedure and reading of the endpoints at 48 h (13).
Northern (RNA) blot analysis.
Isolates from the stocks in
water were subcultured onto plates containing Sabouraud dextrose agar
48 h prior to propagation in YEPD medium (2% yeast extract, 1%
peptone, 2% glucose). Total RNA from the different isolates grown to
mid-logarithmic phase in YEPD medium was obtained with the RNAeasy mini
kit (Qiagen, Inc., Santa Clarita, Calif.) according to the
manufacturer's instructions. Equal amounts (approximately 5 µg) of
RNA as determined by A260 measurements were
separated by electrophoresis (24). The gels were
photographed and subsequently transferred to nylon membranes (Nytran;
Schleicher & Schuell) by using the Turboblotter apparatus (Schleicher & Schuell). Probes for ERG11, MDR1, and
CDR genes were prepared as described before (10).
The resulting CDR probe is based on the whole sequence of
CDR1 and has been shown to cross-hybridize with other
members of this gene family (10, 27, 28, 34). Probes
specific for the CDR1 and CDR2 genes were
prepared by PCR amplification as described before (10, 27).
All probes were labelled by random priming (Random Primers DNA Labeling
System; GibcoBRL), and hybridizations were performed with Rapid-hyb
buffer (Amersham Life Science, Inc., Arlington Heights, Ill.) according to the manufacturer's instructions. After hybridization, blots were
washed and exposed to autoradiography film (Du Pont). Nylon membranes
were probed sequentially with the different probes following stripping
of the previously bound probe (10). Autoradiograms were
scanned by using the Adobe Photo Shop program (Adobe Systems, Inc.).
Samples of 18S rRNA in the gels were used as a control for loading and
subsequent normalization of signals in the autoradiograms (10,
24). For preparation of figures, digital images were processed
with the Adobe Photo Shop program.
| |
RESULTS |
Analysis of isolates from patient A.
Table
1 shows results of fluconazole
susceptibility testing for isolates recovered from patient A. Testing
of 10 C. albicans isolates recovered during the first
episode of OPC indicated the presence of highly susceptible isolates
only (fluconazole MICs, 0.5 to 1 µg/ml; geometric mean, 0.6 µg/ml).
Decreased susceptibility was observed for all five isolates recovered
during the second episode (geometric mean of fluconazole MICs, 12.1),
including the presence of a highly resistant isolate (isolate A.2.1;
fluconazole MIC, 128 µg/ml), together with isolates with elevated in
vitro susceptibilities but still in the susceptible range (fluconazole MICs, 4 to 8 µg/ml). A 32-fold difference in fluconazole MICs for
isolates recovered at the same time point suggested the presence in the
oral cavity of a heterogeneous yeast population. Increasing resistance
was detected in representative isolates recovered during the third
episode, with all three isolates demonstrating resistance (isolate
A.3.1, fluconazole MIC, 128 µg/ml) or dose-dependent susceptibility
(isolates A.3.2 and A.3.3, fluconazole MICs, 32 and 16 µg/ml,
respectively) and an overall geometric mean of fluconazole MICs of 40.3 µg/ml.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Fluconazole MICs for multiple C. albicans
isolates recovered from three serial episodes of OPC from patient A
|
|
DNA typing techniques indicated development of resistance in a
persistent strain (Fig. 1). Minor
differences in the karyotyping patterns across the different isolates
indicated a certain degree of instability of their chromosomal
organization. Also, isolate A.2.1 showed markedly different karyotyping
and RFLP patterns, but the same Ca3 fingerprinting pattern, suggesting
this isolate may constitute a different substrain or variant of the
same persistent strain rather than an unrelated strain. Interestingly,
this isolate was the only resistant isolate (among other susceptible
isolates) detected during the second episode of OPC, but analysis of
representative isolates of the third OPC episode revealed that this
substrain did not persist, but rather development of resistance
occurred in the same strain, representing the majority of yeasts
present in the oral cavity during the first two episodes.
View larger version (66K):
[in this window]
[in a new window]
|
FIG. 1.
Karyotype (A), RFLP analysis generated by digestion with
SfiI of genomic DNA (B), and fingerprinting analysis with
the moderately repetitive probe Ca3 (C) of representative C. albicans clinical isolates recovered from three OPC episodes from
patient A. Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Further demonstration of phenotypic heterogeneity within isolates
recovered at the same time point was evidenced by the analysis of
expression of genes implicated in the development of fluconazole resistance, as shown in Fig. 2. Three
isolates from the first OPC episode and for which the fluconazole MIC
was the same (0.5 µg/ml) exhibited different patterns of gene
expression. While isolates A.1.4 and A.1.7 showed expression of
MDR1 and low constitutive levels of expression of
CDR1, isolate A.1.1 exhibited increased levels of
CDR1 but negligible levels of MDR1. Analysis of
gene expression for isolate A.2.1 revealed moderate overexpression of
MDR1 and strong expression of CDR1 and
CDR2 genes, which were correlated with its high in vitro
resistance to the antifungal drug (MIC, 128 µg/ml). Two other
isolates recovered at the same time (isolates A.2.2. and A.2.3) showed
distinct patterns of gene expression despite displaying the same in
vitro fluconazole MIC (8 µg/ml). Negligible levels of MDR1
but increased message for CDR genes (especially
CDR2) was detected in isolate A.2.2. On the other hand,
constitutive MDR1 expression accompanied by moderate expression of CDR genes (mainly CDR1) was
detected for isolate A.2.3. Strong expression of CDR genes
together with a more moderate overexpression of ERG11 was
detected in isolates A.3.1 and A.3.2 (fluconazole MICs, 128 and 32 µg/ml, respectively) recovered during the third OPC episode, but not
in isolate A.3.3 (fluconazole MIC, 16 µg/ml) isolated at the same
time point, which showed moderate expression of MDR1 only.
Overall, development of resistance in this patient correlated with
increased message for CDR genes (both CDR1 and
CDR2), but overexpression of either MDR1 or
ERG11 was detected in some isolates and was usually detected
in conjunction with overexpression of CDR.
View larger version (52K):
[in this window]
[in a new window]
|
FIG. 2.
Northern-blot analysis of total RNA obtained from
multiple C. albicans clinical isolates recovered from each
of three serial episodes of OPC in patient A. The membranes were probed
with ERG11, MDR1, a nonspecific probe for
CDR genes, and probes specific for CDR1 and
CDR2. The bottom panel shows amounts of 18S rRNA used to
standardize signal levels according to lane loading parameters.
Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Analysis of isolates from patient B.
Fluconazole MICs for
C. albicans isolates recovered from three serial episodes of
OPC from patient B are shown in Table 2. Susceptibility testing of seven representative isolates recovered during the first OPC episode demonstrated fluconazole susceptibility (geometric mean of fluconazole MICs, 0.8 µg/ml), although
susceptibility results were distributed among a wide range of MICs
(0.25 to 4 µg/ml). Decreased susceptibility was detected for all six
isolates recovered during the second OPC episode (geometric mean of
fluconazole MICs, 5.0 µg/ml; range, 4 to 8 µg/ml) and the seven
isolates evaluated from the third OPC episode (geometric mean of
fluconazole MICs, 5.9 µg/ml; range, 4 to 16 µg/ml).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Fluconazole MICs for multiple C. albicans
isolates recovered from three serial episodes of OPC from patient B
|
|
Investigation of strain identity by using DNA typing techniques
revealed the presence of three different strains during the first OPC
episode, as detected by differences in karyotyping, RFLP, and Ca3
fingerprinting patterns (Fig. 3).
However, isolates recovered during the second and third episodes showed
a single pattern for each technique used, which was the same as the one displayed by three of five isolates in the first episode. Again, these
results suggest the development of decreasing susceptibility to the
azole agent in the same persistent strain, which was already present
during the first OPC episode.
View larger version (89K):
[in this window]
[in a new window]
|
FIG. 3.
Karyotype (A), RFLP analysis generated by digestion with
SfiI of genomic DNA (B), and fingerprinting analysis with
the moderately repetitive probe Ca3 (C) of representative C. albicans clinical isolates recovered from three OPC episodes from
patient B. Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Northern blot analysis in this series of isolates (Fig.
4) revealed a correlation between
overexpression of CDR genes (both CDR1 and
CDR2) and isolates with decreased fluconazole susceptibility in all three episodes. Levels of ERG11 remained constant
throughout the series. MDR1 levels were below the detection
limit for all isolates from this patient.
View larger version (55K):
[in this window]
[in a new window]
|
FIG. 4.
Northern blot analysis of total RNA from obtained from
multiple C. albicans clinical isolates recovered from each
of three serial episodes of OPC in patient B. The membranes were probed
with ERG11, MDR1, a nonspecific probe for
CDR genes, and probes specific for CDR1 and
CDR2. The bottom panel shows amounts of 18S rRNA used to
standardize signal levels according to lane loading parameters.
Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Analysis of isolates from patient C.
Table
3 shows results of fluconazole
susceptibility testing for isolates recovered from patient C during
three serial OPC episodes. Fifteen isolates recovered from each of the
first two episodes showed a decreased fluconazole susceptibility
(geometric mean of fluconazole MICs, 9.2 and 8.8 µg/ml, respectively,
for isolates recovered from the first and second episodes), including the presence of isolates exhibiting dose-dependent susceptibility (fluconazole MIC, 16 µg/ml). Isolates recovered from the third episode showed a further decrease in fluconazole susceptibility (geometric mean of fluconazole MICs, 27.4 µg/ml) and included mostly
resistant (isolate C.3.4; fluconazole MIC, 64 µg/ml) and dose-dependent susceptible (MICs, 16 to 32 µg/ml) isolates. However, a susceptible isolate was also present (isolate C.3.1; fluconazole MIC,
4 µg/ml).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Fluconazole MICs for multiple C. albicans
isolates recovered from three serial episodes of OPC from patient C
|
|
Investigation of strain identity by DNA typing techniques demonstrated
that all isolates were highly related, as determined by similar
karyotype (although slight differences in mobility were detected in
several isolates in this series), RFLP, and Ca3 fingerprinting patterns
(Fig. 5). Thus, development of resistance in this series of isolates also occurred in a persistent strain.
View larger version (74K):
[in this window]
[in a new window]
|
FIG. 5.
Karyotype (A), RFLP analysis generated by digestion with
SfiI of genomic DNA (B), and fingerprinting analysis with
the moderately repetitive probe Ca3 (C) of representative C. albicans clinical isolates recovered from three OPC episodes from
patient C. Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Analysis of expression of genes implicated in the development of
fluconazole resistance is shown in Fig.
6. Dose-dependent susceptible isolates
C.1.5 and C.1.10 (fluconazole MICs, 16 µg/ml) showed increased
expression of CDR genes (especially CDR2)
compared to that of the susceptible isolate C.1.3 (fluconazole MIC, 4 µg/ml) recovered at the same time during the first OPC episode. In
the case of isolate C.1.5, but not C.1.10, this overexpression was accompanied by a strong expression of MDR1. In vitro MIC
data for isolates recovered during the second episode closely mirrored expression of CDR genes, particularly CDR2.
However, while levels of ERG11 expression remained quite
constant along the series, isolate C.2.8 showed elevated levels of this
gene along with overexpression of CDR. A great degree of
heterogeneity was detected for isolates from the third OPC episode. As
expected, the susceptible isolate C.3.1 (fluconazole MIC, 4 µg/ml)
showed no elevated message for any of these genes. Isolate C.3.2
(fluconazole MIC, 32 µg/ml) showed overexpression of CDR2.
However, the resistant isolate C.3.4 (fluconazole MIC, 64 µg/ml)
showed no apparent overexpression of any of these genes, suggesting
that an alternate mechanism or mechanisms may be responsible for its
increased fluconazole resistance.
View larger version (61K):
[in this window]
[in a new window]
|
FIG. 6.
Northern blot analysis of total RNA from obtained from
multiple C. albicans clinical isolates recovered from each
of three serial episodes of OPC in patient C. The membranes were probed
with ERG11, MDR1, a nonspecific probe for
CDR genes, and probes specific for CDR1 and
CDR2. The bottom panel shows amounts of 18S rRNA used to
standardize signal levels according to lane loading parameters.
Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Analysis of isolates from patient D.
Eightfold or larger
differences in fluconazole MIC values were detected within C. albicans isolates recovered at the same time in each of three
episodes of OPC in patient D (Table 4). These results indicate coexistence of subpopulations of yeast with
different sensitivities to azole derivatives. Overall, susceptibility tests showed decreasing fluconazole susceptibility with repetitive episodes, as revealed by the increase in the geometric mean of fluconazole MICs for isolates recovered from serial episodes (0.9 µg/ml for the initial episode in which all six isolates were
susceptible to the drug versus 2.1 and 5.7 µg/ml for 12 and 6 isolates recovered during the second and third episodes, respectively).
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Fluconazole MICs for multiple C. albicans
isolates recovered from three serial episodes of OPC from patient D
|
|
Once more, DNA strain typing techniques confirmed the high degree of
relatedness among all isolates recovered from this patient (Fig.
7) and development of decreased
susceptibility to fluconazole in a persistent strain. Minor differences
in electrophoretic mobility were detected between different isolates.
Also, differences were detected by RFLP and Ca3 probe fingerprinting
for isolate D.1.6 that may represent a substrain or variant of the same
strain.
View larger version (93K):
[in this window]
[in a new window]
|
FIG. 7.
Karyotype (A), RFLP analysis generated by digestion with
SfiI of genomic DNA (B), and fingerprinting analysis with
the moderately repetitive probe Ca3 (C) of representative C. albicans clinical isolates recovered from three OPC episodes from
patient D. Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Northern blot analysis of isolates from this patient (Fig.
8) revealed strong baseline expression of
MDR1 and, to a lesser extent, CDR1 for isolate
D.1.2, susceptible to fluconazole (MIC, 0.5 µg/ml). Isolate D.1.4
(fluconazole MIC, 2 µg/ml) showed a low level of expression of
MDR1 and levels of CDR genes below the detection
limits. In contrast, increased message for CDR1 only was
detected in isolate D.1.6 (fluconazole MIC, 0.25 µg/ml). All isolates
recovered during the second OPC episode (D.2.1, D.2.2, and D.2.3;
fluconazole MICs, 16, 1, and 8 µg/ml, respectively) expressed
MDR1, with increased expression correlating with increasing MICs. For these three isolates, levels of expression of CDR
genes were below the detection limit. On the other hand, both
dose-dependent susceptible isolates recovered during the third episodes
(isolates D.3.3 and D.3.4; fluconazole MICs, 16 µg/ml) showed similar
levels of overexpression of CDR genes, including
CDR1 and CDR2, but negligible levels of
MDR1. Analysis of isolate D.3.1 (fluconazole MIC, 2 µg/ml)
revealed moderate levels of expression of MDR1 but
negligible message for CDR genes. Levels of expression of
ERG11 varied slightly throughout the series. Of notice,
ERG11 mRNA was almost undetectable for isolate D.1.6
representing a strain variant. Overall, isolates from this patient
showed dominance of MDR1 overexpression during initial
episodes followed by higher levels of expression of CDR genes in the final episode.
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 8.
Northern-blot analysis of total RNA from obtained from
multiple C. albicans clinical isolates recovered from each
of three serial episodes of OPC in patient D. The membranes were probed
with ERG11, MDR1, a nonspecific probe for
CDR genes, and probes specific for CDR1 and
CDR2. The bottom panel shows amounts of 18S rRNA used to
standardize signal levels according to lane loading parameters.
Fluconazole (FLU) MICs are shown at the top (micrograms per
milliliter).
|
|
Antifungal susceptibility testing against a panel of antifungal
drugs.
Antifungal susceptibility testing against amphotericin B,
itraconazole, ketoconazole, voriconazole, SCH 56592, and terbinafine was performed according to NCCLS method M-27A by using a broth microdilution technique and reading of endpoints at 48 h. Table 5 shows MICs of the different antifungal
agents for the 36 isolates (3 isolates per episode from the four
different patients) included in the study of gene expression. Two
laboratory strains were used as controls. Isolates were all susceptible
to amphotericin B (48-h MIC range, 0.125 to 0.25 µg/ml). Levels of
susceptibility to other azole derivatives varied among the different
isolates and often paralleled increases in MICs of fluconazole,
especially in those isolates demonstrating overexpression of
CDR genes. Although several isolates displayed somewhat
decreased susceptibilities to a given azole derivative, the MICs
obtained were not high enough to be considered resistant according to
currently accepted interpretative breakpoints (13, 22).
Interpretative criteria have not been defined for the investigational
azole derivatives voriconazole and SCH 56592. The ranges of MICs of
terbinafine were 0.5 to >2 µg/ml.
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Antifungal susceptibilities of multiple C. albicans isolates from each of three episodes of OPC from four
HIV-infected patientsa
|
|
| |
DISCUSSION |
Recent studies have demonstrated the multifactorial nature of
resistance. Antifungal resistance may result from replacement of a
susceptible strain by a more resistant isolate or by the development of
resistance in the original strain mediated by multiple mechanisms at
the molecular level. The use of a novel agar dilution screening
technique developed by our group (14, 15) increases detection of subpopulations of yeasts at the time of initial isolation based on the different susceptibilities of individual colonies to the
antifungal agent, thus allowing a comprehensive assessment of the
epidemiology of resistance. By using this technique for initial
sampling of oral rinses and swabs, it was obvious that different
colonies present in the plates differed in their susceptibilities to
the antifungal agent, confirming previous results by our group and
others (14, 15, 30, 31). These differences were further confirmed by susceptibility testing by the NCCLS standard methodology (Tables 1 to 4). Susceptibility tests revealed 8-fold or larger (up to
32-fold) differences in fluconazole MICs for isolates recovered at the
same time point in 7 of 12 OPC episodes in four patients. In general,
discrepancies up to two tube dilutions are considered "acceptable"
when performing standardized antifungal susceptibility testing
according to NCCLS methodologies (16). Thus, eightfold or
larger differences should be considered highly significant.
Investigation of strain identity by a combination of DNA typing
techniques revealed that in all four patients studied, development of
resistance occurred in a single, persistent strain. Of note, in patient
A, a highly resistant isolate recovered during the second episode
(isolate A.2.1) showed karyotyping and RFLP patterns different from
those of all other isolates from the same patient (Fig. 1A and B).
However, the fact that this isolate displayed exactly the same Ca3
fingerprinting pattern as those of all other isolates (Fig. 1C)
suggests that it may represent a substrain or a strain variant rather
than a completely different strain of C. albicans. One would
expect that this resistant substrain should be selected over the more
prevalent but less resistant population of yeasts, as has been
described previously (38). However, representatives of this
substrain were not further recovered, but rather development of high
levels of resistance occurred in the more prevalent (but more
susceptible) strain. This could be an indication that development of
resistance may not always confer an ecological advantage, as suggested
by the fact that some highly resistant isolates demonstrate decreased
virulence in vivo (5).
We have previously demonstrated a high degree of complexity in the
molecular mechanisms responsible for the development of fluconazole
resistance with five distinct patterns of gene expression associated
with the development of fluconazole resistance in serial C. albicans isolates from five different HIV-infected patients with
OPC (10). The present study provides evidence for an
additional level of complexity in the molecular mechanisms of
fluconazole resistance, as demonstrated by differences in expression of
genes implicated in the development of fluconazole resistance
(ERG11, MDR1, CDR1, and
CDR2) within isolates recovered at the same time point from
the same OPC episode and from the same patient. This analysis revealed
that isolates obtained from the same patient and episode were
heterogeneous in their patterns of expression of these genes. As shown
in Table 5, in general, high levels of mRNA for CDR genes
correlated with decreased susceptibilities to other azole derivatives
(i.e., isolates A.3.1 and A.3.2 from patient A). Isolates D.2.1 and
D.2.3 showed overexpression of MDR1 only, and although their
susceptibilities to itraconazole and SCH 56592 were unchanged (as
expected, since fluconazole is the only substrate for Mdr1p), the MICs
of ketoconazole (both isolates) and voriconazole (isolate D.2.1 only)
for them were slightly elevated. Thus, since the present study is
limited to the study of expression of these set of genes implicated in
the development of fluconazole resistance, and since levels of gene expression did not always parallel the MICs, it should be noted that
other mechanisms (changes in the ergosterol biosynthesis pathway, point
mutations in the gene coding for the target enzyme for azole
derivatives, and other yet uncharacterized resistance mechanisms) may
be operational in these series of isolates, which may contribute to the
overall decrease in susceptibility.
It is not clear how this high degree of phenotypic heterogeneity
originates in an apparently homogeneous population of yeasts (as
indicated by typing techniques). In the case of genotypic microheterogeneities described by some authors, the most likely explanation was that these microheterogeneities arise due to physical or functional separation of two populations (8). In this
regard, although the oral cavity is many times considered a uniform
environment, in truth, its great anatomical diversity results in the
presence of several habitats, each of them characterized by different
physicochemical properties (11). Thus, subpopulations of
yeast in each of these microniches may evolve differently not only
genotypically, but also phenotypically in trying to adapt to a
particular microhabitat. Also unknown are the factors (related to the
organism, the host, and the environment) affecting patterns of gene
expression associated with resistance. For example, levels of
antifungal drug attained in each of these diverse microniches in the
oral cavity may be different and may result in different degrees of
antifungal pressure and ultimately lead to microheterogeneity in
patterns of expression of genes associated with development of
resistance, as observed in this study. Other factors that could
influence gene expression include host defense mechanisms and the
microbiota occupying the same ecological niche (11).
Overall, this report shows that C. albicans isolates
obtained from the same patient and episode were phenotypically
heterogeneous in their susceptibilities to fluconazole and in their
patterns of expression of certain genes involved in resistance to this antifungal agent. These results further demonstrate the complexity of
the distribution of the molecular mechanisms of antifungal drug
resistance and indicate that different subpopulations of yeasts may
coexist at a given time in the oral cavity of the same patient and may
develop resistance through different mechanisms.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from Pfizer, Inc., and by
Public Health Service grants 1 R29 AI42401 (to J.L.L.-R.), 1 R01
DE11381 (to T.F.P.), and M01-RR-01346 for the Frederic C. Bartter
General Clinical Research Center.
Chromogenic medium was provided by CHROMagar Company (Paris, France).
We thank the Fungus Testing Laboratory at The University of Texas
Health Science Center at San Antonio for performing antifungal susceptibility testing.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, Division of Infectious Diseases, The University of Texas
Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7881. Phone: (210) 567-1981. Fax: (210) 567-3303. E-mail: ribot{at}uthscsa.edu.
| |
REFERENCES |
| 1.
|
Albertson, G. D.,
M. Niimi,
R. D. Cannon, and H. F. Jenkinson.
1996.
Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance.
Antimicrob. Agents Chemother.
40:2835-2841[Abstract].
|
| 2.
|
Challacombe, S. J.
1994.
Immunological aspects of candidiasis.
Med. Oral Pathol.
78:202-210.
|
| 3.
|
Fling, M. E.,
J. Kopf,
A. Tamrkin,
J. A. Gorman,
H. A. Smith, and Y. Koltin.
1991.
Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate.
Mol. Gen. Genet.
227:318-329[Medline].
|
| 4.
|
Franz, R.,
S. L. Kelly,
D. C. Lamb,
D. E. Kelly,
M. Ruhnke, and J. Morschhäuser.
1998.
Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains.
Antimicrob. Agents Chemother.
42:3065-3072[Abstract/Free Full Text].
|
| 5.
|
Graybill, J. R.,
E. Montalbo,
W. R. Kirkpatrick,
M. F. Luther,
S. G. Revankar, and T. F. Patterson.
1998.
Fluconazole versus Candida albicans: a complex relationship.
Antimicrob. Agents Chemother.
42:2938-2942[Abstract/Free Full Text].
|
| 6.
|
Klein, R. S.,
A. H. Carol,
C. B. Small,
B. Moll,
M. Lesser, and G. H. Friedland.
1984.
Oral candidiasis in high-risk patients as the initial manifestation of the acquired immunodeficiency syndrome.
N. Engl. J. Med.
311:354-3588[Abstract].
|
| 7.
|
Law, D.,
C. B. Moore,
H. M. Wardle,
L. A. Ganguli,
M. G. L. Keaney, and D. W. Denning.
1994.
High prevalence of antifungal resistance in Candida spp. from patients with AIDS.
J. Antimicrob. Chemother.
34:659-668[Abstract/Free Full Text].
|
| 8.
|
Lockhart, S. R.,
J. J. Fritch,
A. S. Meier,
K. Schröppel,
T. Srikantha,
R. Galask, and D. R. Soll.
1995.
Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing.
J. Clin. Microbiol.
33:1501-1509[Abstract].
|
| 9.
|
Löffler, J.,
S. L. Kelly,
H. Hebart,
U. Schumacher,
C. Lass-Flörl, and H. Einsele.
1997.
Molecular analysis of cyp51 from fluconazole-resistant Candida albicans strains.
FEMS Microbiol. Lett.
151:263-268[Medline].
|
| 10.
|
López-Ribot, J. L.,
R. K. McAtee,
L. N. Lee,
W. R. Kirkpatrick,
T. C. White,
D. Sanglard, and T. F. Patterson.
1998.
Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.
Antimicrob. Agents Chemother.
42:2932-2937[Abstract/Free Full Text].
|
| 11.
|
Marcotte, H., and M. C. Lavoie.
1998.
Oral microbial ecology and the role of salivary immunoglobulin A.
Microbiol. Mol. Biol. Rev.
62:71-109[Abstract/Free Full Text].
|
| 12.
|
Meunier, F.
1989.
Candidiasis.
Eur. J. Clin. Microbiol. Infect. Dis.
8:438-447[Medline].
|
| 13.
|
National Committee for Clinical Laboratory Standards.
1997.
Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard. NCCLS document M27-A.
National Committee for Clinical Laboratory Standards, Wayne, Pa.
|
| 14.
|
Patterson, T. F.,
W. R. Kirkpatrick,
S. G. Revankar,
R. K. McAtee,
A. W. Fothergill,
D. I. McCarthy, and M. G. Rinaldi.
1996.
Comparative evaluation of macrodilution and chromogenic agar screening for determining fluconazole susceptibility of Candida albicans.
J. Clin. Microbiol.
34:3237-3239[Abstract].
|
| 15.
|
Patterson, T. F.,
S. G. Revankar,
W. R. Kirkpatrick,
O. Dib,
A. W. Fothergill,
S. W. Redding,
D. A. Sutton, and M. G. Rinaldi.
1996.
Simple method for detecting fluconazole-resistant yeasts with chromogenic agar.
J. Clin. Microbiol.
34:1794-1797[Abstract].
|
| 16.
|
Pfaller, M. A.,
J. H. Rex, and M. G. Rinaldi.
1997.
Antifungal susceptibility testing: technical advances and potential clinical implications.
Clin. Infect. Dis.
24:776-784[Medline].
|
| 17.
|
Plettenberg, A.,
E. Reisinger,
U. Lenzner,
H. Listemann,
M. Ernst,
P. Kern,
M. Dietrich, and W. Meigel.
1990.
Oral candidosis in HIV-infected patients. Prognostic value and correlation with immunological parameters.
Mycoses
33:421-425[Medline].
|
| 18.
|
Powderly, W. G.
1994.
Resistant candidiasis.
AIDS Res. Hum. Retroviruses
10:925-929[Medline].
|
| 19.
|
Prasad, R.,
W. P. De Wergifosse,
A. Goffeau, and E. Balzi.
1995.
Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals.
Curr. Genet.
27:320-329[Medline].
|
| 20.
|
Revankar, S. G.,
W. R. Kirkpatrick,
R. K. McAtee,
O. P. Dib,
A. W. Fothergill,
S. W. Redding,
D. A. McGough,
M. G. Rinaldi, and T. F. Patterson.
1996.
Detection and significance of fluconazole resistance in oropharyngeal candidiasis in HIV-infected patients.
J. Infect. Dis.
174:821-827[Medline].
|
| 21.
|
Revankar, S. G.,
W. R. Kirkpatrick,
R. K. McAtee,
O. P. Dib,
A. W. Fothergill,
S. W. Redding,
M. G. Rinaldi, and T. F. Patterson.
1998.
A randomized trial of continuous or intermittent therapy with fluconazole for oropharyngeal candidiasis in human immunodeficiency virus-infected patients: clinical outcomes and development of fluconazole resistance.
Am. J. Med.
105:7-10[Medline].
|
| 22.
|
Rex, J. H.,
M. A. Pfaller,
J. N. Galgiani,
M. S. Bartlett,
A. Espinel-Ingroff,
M. A. Ghannoum,
M. Lancaster,
F. C. Odds,
M. G. Rinaldi,
T. J. Walsh, and A. L. Berry.
1997.
Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections.
Clin. Infect. Dis.
24:235-247[Medline].
|
| 23.
|
Rex, J. H.,
M. G. Rinaldi, and M. A. Pfaller.
1995.
Resistance of Candida species to fluconazole.
Antimicrob. Agents Chemother.
39:1-8[Medline].
|
| 24.
|
Sambrook, J. F.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 25.
|
Sanglard, D.,
F. Ischer,
L. Koymans, and J. Bille.
1998.
Amino acid substitutions in the cytochrome P-450 lanosterol 14-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents.
Antimicrob. Agents Chemother.
42:241-253[Abstract/Free Full Text].
|
| 26.
|
Sanglard, D.,
F. Ischer,
M. Monod, and J. Bille.
1996.
Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors.
Antimicrob. Agents Chemother.
40:2300-2305[Abstract].
|
| 27.
|
Sanglard, D.,
F. Ischer,
M. Monod, and J. Bille.
1997.
Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene.
Microbiology
143:405-416[Abstract].
|
| 28.
|
Sanglard, D.,
K. Kuchler,
F. Ischer,
J.-L. Pagani,
M. Monod, and J. Bille.
1995.
Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters.
Antimicrob. Agents Chemother.
39:2378-2386[Abstract].
|
| 29.
|
Schmid, J.,
E. Voss, and D. R. Soll.
1990.
Computer-assisted methods for assessing strain relatedness in Candida albicans by fingerprinting with the moderately repetitive sequence Ca3.
J. Clin. Microbiol.
28:1236-1243[Abstract/Free Full Text].
|
| 30.
|
Schoofs, A.,
F. C. Odds,
R. Colebunders,
M. Ieven,
L. Wouters, and H. Goossens.
1997.
Isolation of Candida species on media with and without added fluconazole reveals high variability in relative growth susceptibility phenotypes.
Antimicrob. Agents Chemother.
41:1625-1635[Abstract].
|
| 31.
|
Takasuka, T.,
G. G. Baily,
M. Birch,
M. J. Anderson,
D. Law, and D. W. Denning.
1998.
Variation in morphotype, karyotype and DNA type of fluconazole resistant Candida albicans from an AIDS patient.
J. Infect.
36:57-62[Medline].
|
| 32.
|
Vanden Bossche, H.,
P. Marichal, and F. C. Odds.
1994.
Molecular mechanisms of drug resistance in fungi.
Trends Microbiol.
2:393-400[Medline].
|
| 33.
|
Vanden Bossche, H.,
P. Marichal,
J. Gorrens,
D. Bellens,
H. Moereels, and P. A. J. Janssen.
1990.
Mutation in cytocrome P450-dependent 14 demethylase results in decreased affinity for azole antifungals.
Biochem. Soc. Trans.
18:56-59[Medline].
|
| 34.
|
Walsh, T. J.,
M. Kasai,
A. Francesconi,
D. Landsman, and J. Chanock.
1997.
New evidence that Candida albicans possesses additional ATP-binding cassette MDR-like genes: implications for antifungal azole resistance.
J. Med. Vet. Mycol.
35:133-137[Medline].
|
| 35.
|
White, T. C.
1997.
Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus.
Antimicrob. Agents Chemother.
41:1482-1487[Abstract].
|
| 36.
|
White, T. C.
1997.
The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14 demethylase in Candida albicans.
Antimicrob. Agents Chemother.
41:1488-1494[Abstract].
|
| 37.
|
White, T. C.,
K. A. Marr, and R. A. Bowden.
1998.
Clinical, cellular, and molecular factors that contribute to antifungal drug resistance.
Clin. Microbiol. Rev.
11:382-402[Abstract/Free Full Text].
|
| 38.
|
White, T. C.,
M. A. Pfaller,
R. G. Rinaldi,
J. Smith, and S. W. Redding.
1997.
Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient.
Oral Dis.
3:S102-S109.
|
Antimicrobial Agents and Chemotherapy, July 1999, p. 1621-1630, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
