Journalof Medical& VeterinaryMycology1997,35, 285-287
Accepted I April 1997
Short communication
In vitro growth-inhibitory activity of pneumocandins
L-733,560 and L-743,872 against putatively amphotericin
B- and fluconazole-resistant Candida isolates: influence of
assay conditions
P. W . N E L S O N ,
M. L O Z A N O - C H I U
& J. H. R E X *
Division of Infectious Diseases, Department of Internal Medicine, Center for the Study of Emerging and ReemergingPathogens,
University of Texas Medical School, Houston, Texas 77030, USA
L-733,560 and L-743,872 are water-soluble pneumocandins with potent antifungal
activity. By beginning with the NCCLS M27-T method and varying test format
(microdilution vs macrodilution), time of reading (24 h vs 48 h), and test medium
(RPMI 1640 vs Antibiotic Medium 3), we have demonstrated that the MICs for these
compounds can be altered significantly: the microdilution format, reading after 24 h
and use of Antibiotic Medium 3 all reduced the measured MIC. No cross-resistance
was demonstrated with either fluconazole or amphotericin B.
Keywords
candin
amphotericin B, antifungal susceptibility testing, fluconazole, pneumo-
Introduction
The pneumocandins are acyl-substituted cyclic hexapeptides (lipopeptides) that are related structurally to
the echinocandins and exert their antifungal effect by
inhibiting the synthesis of 1,3-fl-D-glucan [1-3]. Lipopeptides L-733,560 and L-743,872 are water-soluble
pneumocandins that exhibit potent activity against
Candida spp., Aspergillus fumigatus and Pneumocystis
carinii [1-3]. In this study, we have investigated the
activity of these two agents against a collection of
fluconazole- and amphotericin B-susceptible and
-resistant Candida isolates using the M27-T reference
method developed by the National Committee for
Clinical Laboratory Standards [4]. We have also
examined the effect varying the M27-T method by
changing test format (macrodilution vs microdilution),
time of reading (24 h vs 48 h) and medium (RPMI 1640 vs
Antibiotic Medium 3). As each of these test modifications
can alter the measured MIC [5] and have an effect on the
degree of correlation between results in vitro and outcome
Correspondence: Dr J. H. Rex, 6431 Fannin, 1.728 JFB, Houston,
TX 77030, USA. Tel: (713) 500-6738: Fax: (713) 500-5495: Internet:
[email protected].
© 1997 ISHAM
in vivo [6,7], an understanding of the effects of these
factors is important.
Materials and methods
Antifungal agents
L-733,560 and L-743,872 were from Merck Research
Laboratories (Rahway, N J), amphotericin B was from
E. R. Squibb (Princeton, N J) and fluconazole was from
Pfizer Pharmaceuticals (Groton, CT). Stock solutions
of L-733,560 and L-743,872 at 640pg m1-1 in sterile
water were stored at - 7 0 °C for a maximum of 72 h
before use. Amphotericin B and fluconazole stock
solutions were prepared as recommended in the M27-T
document [4].
Isolates
A collection of 30 Candida isolates were used (Table 1).
The amphotericin B-resistant isolates have been previously described [6,7], while the other isolates were
bloodstream isolates from a recent candidaemia therapy
trial [8]. The isolates were grouped by relative fluconazole
susceptibility following the recently proposed NCCLS
Nelson et aL
Table 1 MICs for fluconazole and amphotericin B obtained by
M27-T and modified M27-T conditions, respectively, against
Candida isolates used in this study
Group*
N
Fluconazole
M27-T M I C t
A M B resistant
A M B and F L U resistant
F L U resistant
Susceptible
7
1
7
15
0-06254
> 64
64- > 64
0.25 32
Modified
Amphotericin B
M27-T MIC:~
1- > 64
> 64
0.0625 0.25
0.0625 0-5
*AMB, amphotericin B; F L U , fluconazole.
?All MICs are reported in/~g ml ~.
~M27-T was modified by use of Antibiotic Medium 3 broth, a
microdilution format, and reading after 24 h of incubation. These
modifications are known to enhance the ability of M27-T to detect
resistance to amphotericin B [6,7]. The isolates included in the
amphotericin B resistant group were C. albicans (2), C. lusitaniae
(4) and C. parapsilosis (t). The one isolate which exhibited
resistance to both amphotericin B and fluconazole was a C.
tropicalis. The isolates in the fluconazole resistant group were
C. glahrata (4), C. krusei (1), C. lipolytica (1) and C2 tropicalis (1).
The susceptible group included (2 albicans (4), C glabrata (3),
C. guilliermondii (1), C. hesitaniae (1), C. parupsilosis (3) and
C. tropicalis (3).
interpretive guidelines [9] and for amphotericin B
susceptibility using recent data from our laboratory [10].
Susceptibility testing
Susceptibility testing was performed at final drug concentrations of 0.0625-64/~g ml-1 for all drugs and followed
both the macrodilution and microdilution procedures
of the M27-T document [4] with the modifications
described below. In some assays, Antibiotic Medium 3
(lot JD4ZSG from Becton Dickinson Microbiology
Systems, Cockeysville, MD) replaced the RPM! 1640
medium specified by M27-T. After reconstitution according to the manufacturer's directions, Antibiotic Medium 3
was filter sterilized, supplemented with glucose to achieve
the final concentration of 2% (2 g 100 ml 1), and buffered
with 10 mM sodium phosphate to pH 7-0. Endpoints
were determined after 24 and 48 h incubation, and the
amphotericin B endpoint was the least drug concentration
that allowed no visible growth of turbidity. The endpoint
for the other drugs was the drug concentration which
reduced visual turbidity to an optically clear or prominent
decrease in turbidity (also known as the MIC-0 and
MIC-2 endpoints, respectively) [4] when compared with
the drug-free growth control well.
Results and discussion
The MICs obtained for the two pneumocandins are
summarized in Table 2. Macrodilution and microdilution
MIC ranges were similar for both drugs, with the microdilution MICs tending to be one dilution higher. As with
other antifungal agents [5], 48 h MICs were slightly higher
than the 24 h MICs. The most striking effect was related
to choice of medium: for both drugs, MICs in Antibiotic
Medium 3 broth were consistently lower than in RPMI
1640. While Antibiotic Medium 3 is an undefined medium
Table 2 MICs for pneumocandins L-733,560 and L-743,872 obtained under different assay conditions
R P M I 1640
Time of reading
G r o u p (N)
L-733,560
A M B resistant (7)
A M B and F L U resistant (1)
F L U resistant (7)
Susceptible (15)
All isolates (30)
Macrodilution: all isolates (30)
L-743,872
A M B resistant (7)
A M B and F L U resistant (1)
F L U resistant (7)
Susceptible (l 5)
All isolates (30)
Macrodilution: all isolates (30)
24 h range
<
<
<
<
<
<
0.0625 0-5
0.0625
0-0625 0.25
0-0625 1
0.0625-1
0.0625-0.5
0.125-0.5
< 0-0625
0.125 0.25
< 0.0625-0.5
< 0-0625-0-5
< 0.0625 0.5
Antibiotic M e d i u m 3
48 h range
24 h range
48 h range
0.125-1
< 0.0625
0-25-1
< 0.0625 2
< 0-0625 2
< 0-0625 16
<
<
<
<
<
<
0.0625
0-0625
0.0625-0.125
0.0625-0.5
0-0625 0.5
0.0625
<
<
<
<
<
<
0-0625-0.25
0-0625
0.0625 0.125
0.0625-2
0.0625 2
0-0625 0-5
0.25 1
05
0.25 1
0.125 1
0-125 1
< 0.0625-0-5
<
<
<
<
<
<
0.0625
0-0625
0-0625-0-125
0.0625-0.25
0-0625 0-25
0.0625
<
<
<
<
<
<
0.0625-0-125
0-0625
0.0625 0.25
0.0625-0.5
0.0625 0.5
0.0625
AMB, amphotericin B: FLU, fluconazole. All MICs are reported in /~g ml -~ and, except for as marked, were determined in the
microdilution format. The pooled results obtained in the macrodilntion format are shown in the final row of the sections on L-733,560 and
L-743,872.
r© 1997 ISHAM, Journal of Medical & Veterinary Mycology 35,285-287
In vitro activity of pneumocandins
for which lot-to-lot variability is possible, that used in this
study is a current lot that h a d been shown to perform in
the same manner as other current lots of this medium [10].
These results are consistent with d a t a generated for
amphotericin B susceptibility in these two media [6,7]
where, especially for truly susceptible isolates, M I C s in
Antibiotic M e d i u m 3 are lower than those obtained in
R P M I 1640. The cause of this effect is not known, but
in the case of amphotericin B this lowering o f M I C s
is critical to the detection of amphotericin B-resistant
isolates.
F o r each o f the eight combinations of test conditions,
the range and median M I C did not a p p e a r to be related to
amphotericin B or fluconazole susceptibility. One isolate
did, however, appear to have relatively elevated M I C s for
both lipopeptides. This isolate is a C lusitaniae designated
in previous studies as CL524, and it is known to be
susceptible b o t h to fluconazole (M27-T M I C of 0 - 5 # g
m1-1) and to amphotericin B ( M I C o f 0.125/~g ml -~
using Antibiotic M e d i u m 3 in the test format best able to
detect amphotericin B resistance [6]). This isolate had
L-733,560 microdilution M I C s o f 1 # g m l - i (24 h, R P M I
1640 broth), 2 ¢tg m l - ~ (48 h, R P M I 1640 broth), 0" 125/lg
m l - 1 (24 h, Antibiotic M e d i u m 3) and 2 # g ml-~ (48 h,
Antibiotic M e d i u m 3). The L-743,872 M I C s were 0 . 5 # g
m1-1 (24h, R P M I 1640 broth), l # g ml -~ (48h, R P M I
1640 broth), ~<0.0625 # g m l - ~ (24 h, Antibiotic M e d i u m
3) and 0'5 p g ml-L (48 h, Antibiotic M e d i u m 3). This is
also the isolate responsible for the L-733,560 M I C o f
16/tg m l - ~ after 48 h in R P M I 1640 in the macrodilution
results in Table 2.
While the consequences o f these observations in vivo are
not known, these d a t a demonstrate the effects o f test
format (especially o f medium) on M I C for the lipopeptides. Antibiotic M e d i u m 3 was chosen as the alternative medium for this study due to its ability to enhance
detection of amphotericin B-resistant Candida isolates
[6,7]. In this study, use o f Antibiotic M e d i u m 3 did not
appear critical to detection o f the isolate that tended to
have the highest M I C for both drugs (CL524), although
the clinical response o f this isolate to these c o m p o u n d s
remains to be determined. The meaning o f the small
difference in M I C s obtained by the m i c r o b r o t h vs macrobroth method is likewise unknown. Finally, these data
© i997 ISHAM, Journal of Medical & Veterinary Mycology 35, 285-287
support the recent work o f others demonstrating a lack of
cross-resistance between these lipopeptides and either
fluconazole or amphotericin B [2,3].
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water-soluble pneumocandin analogs L-733560, L-705589 and
L-731373 with mouse models of disseminated aspergillosis,
candidiasis, and cryptococcosis. Antimierob Agents Chemother
1995; 39: 1077-81.
2 Bartizal K, Scott T, Abruzzo GK, et al. In vitro evaluation of the
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