Occidiofungin's chemical stability and in vitro potency against Candida species.
ABSTRACT Occidiofungin is a cyclic glyco-lipopeptide produced by Burkholderia contaminans. MICs against Candida species were between 0.5 and 2.0 μg/ml. Occidiofungin retains its in vitro potency in the presence of 5% and 50% human serum with a minimal lethal concentration (MLC) of 2 and 4 μg/ml, respectively. Time-kill and postantifungal effect (PAFE) experiments of occidiofungin against Candida albicans were performed. The results demonstrate that occidiofungin is fungicidal. Occidiofungin was also found to be a very stable molecule. It is resistant to extreme temperatures and pH and maintains its activity following exposure to gastric proteases.
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ABSTRACT: Burkholderia contaminans strain MS14 produces the antifungal compound occidiofungin, which is responsible for significant antifungal activities against a broad range of plant and animal fungal pathogens. Occidiofungin is a cyclic glycolipopeptide made up of eight amino acids and one xylose. A 56-kb ocf gene cluster was determined to be essential for occidiofungin production. In this study the ocfC gene, which is located downstream of ocfD and upstream of ocfB gene in the ocf gene cluster, was examined. Antifungal activity of the ocfC gene mutant MS14KC1 was reduced against the indicator fungus Geotrichum candidum compared with the wild-type strain. Furthermore, the analysis of the protein sequence suggests that the ocfC gene encodes a glycosyltransferase. Biochemical analyses using NMR and Mass spectroscopy revealed that the ocfC mutant produced the occidiofungin without the xylose. Purified ocfC mutant MS14KC1 product had similar level of bioactivity as compared to the wild-type product. The revertant MS14KC1-R of the ocfC mutant produced the same antifungal activity level on plate assays and the same antifungal compound based on HPLC and mass spectroscopy analysis as the wild type strain MS14. Collectively, the study demonstrates the ocfC gene encodes a glycosyltransferase responsible to add a xylose to the occidiofungin molecule and that the presence of the xylose is not important for antifungal activity against Candida species. The finding provides a novel variant for future studies aimed at evaluating its use for inhibiting clinical and agricultural fungi and the finding could also simplify the chemical synthesis of occidiofungin variants.Applied and environmental microbiology 02/2013; · 3.69 Impact Factor
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ABSTRACT: Members of the Burkholderia cepacia complex (Bcc) have recently gained notoriety as significant bacterial pathogens due to their extreme levels of antibiotic resistance, their transmissibility in clinics, their persistence in bacteriostatic solutions, and their intracellular survival capabilities. As pathogens, the Bcc are known to elaborate a number of virulence factors including proteases, lipases and other exoproducts, as well as a number of secretion system associated effectors. Through random and directed mutagenesis studies, we have identified a Bcc gene cluster capable of expressing a toxin that is both hemolytic and required for full Bcc virulence. The Bcc toxin is synthesized via a non-ribosomal peptide synthetase mechanism, and appears to be related to the previously identified antifungal compound burkholdine or occidiofungin. Further testing shows mutations to this gene cluster cause a significant reduction in both hemolysis and Galleria mellonella mortality. Mutation to a glycosyltransferase gene putatively responsible for a structural-functional toxin variant causes only partial reduction in hemolysis. Molecular screening identifies the Bcc species containing this gene cluster, of which several strains produce hemolytic activity.Virulence 05/2012; 3(3):286-98. · 2.79 Impact Factor
Occidiofungin’s Chemical Stability and In Vitro Potency against
Dayna Ellis,aJiten Gosai,aCharles Emrick,aRachel Heintz,aLanette Romans,aDonna Gordon,aShi-En Lu,bFrank Austin,c
and Leif Smithd
Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi, USAa; Department of Biochemistry, Molecular Biology, Entomology and Plant
Pathology, Mississippi State University, Mississippi State, Mississippi, USAb; College of Veterinary Medicine, Department of Pathobiology and Population Medicine,
Mississippi State, Mississippi, USAc; and Department of Biological Sciences, Texas A&M University, College Station, Texas, USAd
Occidiofungin is a cyclic glyco-lipopeptide produced by Burkholderia contaminans. MICs against Candida species were between
0.5 and 2.0 ?g/ml. Occidiofungin retains its in vitro potency in the presence of 5% and 50% human serum with a minimal lethal
against Candida albicans were performed. The results demonstrate that occidiofungin is fungicidal. Occidiofungin was also
of currently available antifungal agents regarding their spectra of
activity and toxicities. Furthermore, there is a demand for new
antifungals, given the prevalence of azole-resistant fungal patho-
gens (1, 8). There are four major therapeutic groups of antifungal
three groups primarily target ergosterol production or bind to
ergosterol, disrupting the fungal membrane (10, 18, 24). Ergos-
terol, much like cholesterol found in mammalian cells, is impor-
tant for maintaining proper cell permeability and fluidity. The
echinocandins, the fourth group, are synthetically modified lipo-
peptides that originate from the natural compounds produced by
fungi (15, 16). The antifungal activity of echinocandins is attrib-
uted to selective inhibition of 1,3-?-glucan synthesis by their
function as noncompetitive inhibitors of 1,3-?-glucan synthase
possible development of new therapeutics.
Occidiofungin is an antifungal peptide produced by the bacte-
rium Burkholderia contaminans MS14. This bacterial strain was
isolated from soil that suppressed brown patch disease of lawn
the genetic locus and regulatory elements of the antifungal com-
pound (11–13). Structural characterization determined that oc-
cidiofungin is a cyclic glyco-lipopeptide (11, 25). Four structural
variants of the antifungal peptide, named occidiofungin A to D,
have been identified. They have masses of 1,200.39 Da, 1216.41
Da, 1234.17 Da, and 1250.41 Da, which correspond to the addi-
tion of oxygen and/or chlorine to the first compound. The target
and the mechanism of action of occidiofungin are still unknown.
The antifungal has been shown to inhibit a wide array of fungi
(25), such as Alternaria alternata, Aspergillus fumigatus, Aspergil-
lus niger, Fusarium oxysporum, Geotrichum candidum, Macropho-
mina phaseolina, Microsporum gypseum, Penicillium sp., Rhizocto-
nia solani MSCOT-1, and Trichophyton mentagrophytes. In
addition, two Pythium species were sensitive at nanomolar con-
centrations: Pythium spinosum and Pythium ultimum. Toxicolog-
ical evaluation of occidiofungin has been performed (W. Tan, et
al., submitted for publication). B6C3F1 mice were given a single
dose of occidiofungin up to 20 mg/kg of body weight or a daily
dose for 5 days at 2 mg/kg of body weight. Key effects were a
this study, we tested the sensitivity of Candida species to occidio-
fungin with and without the presence of human serum. We also
performed simultaneous time-kill curves and postantifungal ef-
fect (PAFE) experiments on Candida albicans (ATCC 66027).
Lastly, we tested occidiofungin’s chemical stability against ex-
treme pH and temperature, as well as its stability against gastric
MATERIALS AND METHODS
In vitro susceptibility testing. Occidiofungin was purified as previously
described (12). Microdilution broth susceptibility testing was performed
medium(buffered to a pH of 7.0 with morpholinepropanesulfonic acid
[MOPS]). Stock solutions (100?) of occidiofungin were prepared in di-
methyl sulfoxide (DMSO). MIC endpoints for occidiofungin were deter-
growth (an optically clear well) after 24 h of incubation. Susceptibility
used as a negative control.
Serum MICs. Susceptibility testing, as described above, was per-
formed in duplicate according to the CLSI M27-A3 method in RPMI
medium or YPD (yeast extract-peptone-dextrose) growth medium in the
presence of 5% and 50% (vol/vol) human serum (Sigma-Aldrich, St.
Louis, MO) (29). Given the turbidity of 50% serum, visual MIC determi-
the microtiter well was plated to determine the minimum lethal concen-
tration (MLC). MLC is defined in terms of a well that has no viable cells
(colonies) in the 50 ?l spread on a fresh agar plate. This is in accordance
Received 5 July 2011 Returned for modification 22 July 2011
Accepted 10 November 2011
Published ahead of print 21 November 2011
Address correspondence to Leif Smith, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0066-4804/12/$12.00Antimicrobial Agents and Chemotherapyp. 765–769aac.asm.org
with the fungicidal activity being defined as a 99.9% reduction in the
number of CFU compared to the starting inoculum, which is approxi-
mately 1,000 CFU per ml (19).
Time-kill experiments and PAFE. Time-kill and PAFE experiments
provided important information about the activity of occidiofungin.
These studies determined whether occidiofungin is fungicidal or fungi-
static and its rate of activity. Fungicidal activity is defined as a 3-log re-
effect occidiofungin has on cells exposed for a short time period. This
provides information about the affinity occidiofungin has to the cellular
target and whether the antifungal activity can be washed away following
exposure. Time-kill assays are essentially a measurement of CFU count
following the addition of the antifungal to the yeast. Time-kill and PAFE
experiments were done according to a method reported by Clancy et al.
(2). Candida albicans (ATCC 66027) colonies on 24-h-old YPD plates
were suspended in 9 ml of sterile water. The density was adjusted to a 0.5
McFarland standard (2, 6, 7, 19, 20) and was diluted 10-fold with RPMI
1640 medium to a final volume of 10 ml containing a final concentration
35°C with agitation. For the time-kill experiment, 100-?l samples were
drawn, serially diluted, and plated on YPD medium for colony counts.
PAFE experiments were performed in a similar manner, except that fol-
ilarly, 100-?l samples at times 0, 2, 4, 8, 12, and 24 h were drawn and
plated for counting CFU. Each experiment was completed at least twice
for each sample.
Temperature stability. A temperature stability assay was performed
following a method similar to that reported by Wu et al. (30), except that
the procedure was optimized for determining the MIC using the CLSI
M27-A3 method. Occidiofungin was suspended in 0.5 ml of DMSO at a
Each tube was then placed in a water bath at 50, 60, 70, and 100°C for 30
min. Then, 200 ?l from each tube was added to the first well of each row
of a 48-well microtiter plate and serially diluted 2-fold in 100 ?l of RPMI
medium. Lastly, C. albicans (ATCC 66027) suspension was prepared us-
bator for 24 h at 35°C. The MIC was determined as described above.
pH stability. A pH stability assay was performed following a method
similar to that reported by Wu et al. (30), except that the procedure was
optimized for determining the MIC using the CLSI M27-A3 method.
Occidiofungin (160 ?g) was dried in eight separate 1.8-ml Eppendorf
1 ml of RPMI 1640 medium. Using stock solutions of 6 M HCl and 6 M
NaOH, the pHs of the samples were adjusted accordingly, and then sam-
ples were left at room temperature for 2 h. After a 2-h exposure, the pH
was readjusted to pH 7.0. The 160 ?g/ml solution was subsequently di-
luted 2-fold, resulting in concentrations of 160, 80, 40, 20, 10, 5, and 2.5
was prepared, and 900 ?l of the suspension was added to each well con-
taining the antifungal solution. MICs were determined as described
Protease stability. A protease stability assay was performed following
a method similar to that reported by Wu et al. (30), except that the pro-
cedure was optimized for determining the MIC using the CLSI M27-A3
method. Occidiofungin’s stability was tested against the digestive pro-
stock solution (5.19 mg of trypsin in 1 ml of a 1 mM HCl solution) were
used. For the chymotrypsin assay, a 2? stock solution of Tris buffer con-
in 1 ml of 1 mM HCl–2 mM calcium chloride) were used. For the pepsin
(0.8 mg of pepsin in 1 ml of sterile deionized water) were used. The
reaction mixtures having a final volume of 250 ?l were prepared and
consisted of 25 ?l of 10? protease stock solution, 125 ?l of 2? stock
reaction solution was added to two 1.8-ml Eppendorf tubes containing 8
was prepared, and 900 ?l of the suspension was added to each tube con-
of CFU was counted after 4 h of incubation by serial dilution and plating.
The plates were incubated at 35°C for 24 h, and CFU counts were deter-
mined. The assays were repeated for 60- and 120-min exposures to the
protease, no occidiofungin, and protease inhibitors for trypsin (phenyl-
methylsulfonyl fluoride [PMSF]), chymotrypsin, and pepsin (0.5 ?M
E-64 [Sigma], 0.1 ?M leupeptin, 0.7 ?M pepstatin A, 0.6 ?M bestatin,
37.5 ?M [4-(2-aminoethyl)benzenesulfonyl fluoride] AEBSF, 1 mM
PMSF, and 3 U of aprotinin [Trasylol]). Each experiment was completed
Protease activity of trypsin, chymotrypsin, and pepsin was confirmed
using bovine serum albumin (BSA) as the substrate in place of occidio-
4.4 ?g/?l. After 0, 30, 60, and 120 min at 35°C, 20-?l aliquots were re-
moved, and the reaction was stopped by addition of 80 ?l of SDS loading
SDS-PAGE gel, and proteins were visualized by Coomassie blue staining.
Susceptibility testing. The occidiofungin MIC for C. albicans, C.
glabrata, C. tropicalis, and C. parapsilosis was determined to be
within a range of 2.0 to 0.5 ?g/ml (Table 1). A caspofungin- and
fluconazole-resistant strain was tested. The MIC for the
the MIC for the fluconazole-resistant strain, C. albicans 2677, was
1.0 ?g/ml. The most direct approach to determine the effect of
protein binding on the antifungal activity of occidiofungin is to
test the compound in the presence of serum. This provides infor-
TABLE 1 Occidiofungin MICs
C. albicans TE
C. albicans LL
C. albicans 66027
C. albicans 24067
C. albicans 11034a
C. albicans 2677b
C. glabrata 66032
C. glabrata 200989
C. glabrata 2001
C. tropicalis 66029
C. tropicalis 13803
C. parapsilosis 90018
C. parapsilosis 34136
C. parapsilosis 90875
aCaspofungin-resistant strain (MIC ? 8 ?g/ml).
bFluconazole-resistant strain (MIC ? 64 ?g/ml).
Ellis et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
present in blood. This information is also important for future
were determined using 5% and 50% (vol/vol) human serum in
glabrata (ATCC 66032) would not grow in the presence of 50%
values following the standard CLSI protocol against these two
strains using YPD medium and RPMI 1640 medium. Given the
the microtiter plate was plated to determine the minimum lethal
concentration (MLC). Activity of occidiofungin was moderately
inhibited by 5% and 50% human serum. The MIC at 5% human
MLC at 50% human serum was 4 ?g/ml. Presumably, the 2- to
4-fold reduction in activity in the presence of serum is attributed
of serum is not uncommon for lipopeptides (3, 29).
Time-kill and PAFE experiments. The occidiofungin MICs
for the time-kill experiments were 2 ?g/ml at 24 h (Fig. 1A). The
in cell density compared to the cell density in the CLSI protocol.
Time-kill experiments revealed that occidiofungin is fungicidal
(20). There was a log decrease in cell density after a 2-h exposure
with 1? MIC and 2? MIC and a 2-log decrease in cell density
following a 2-h exposure to occidiofungin with 8? MIC. There
was greater than a 3-log reduction by 8 h and 24 h with 8? MIC
and 2? MIC, respectively. A 2-log decrease in cell density per-
be only a moderate effect on the rate of growth at 0.5? MIC in
these experiments but no reduction from the starting inoculum.
cidal activity and that the fungal target needs to be saturated for
effect. The rapid drop in cell density after a 2-h exposure suggests
that the target for occidiofungin is critical for survival, while the
slow gradual decrease in cell density after a 2-h exposure may be
attributed to either the chance that these cells have not been satu-
rated with occidiofungin or possibly that they were in a different
cellular growth phase that is less susceptible to occidiofungin’s
gin at 2? MIC was washed away (Fig. 1B). However, the effect of
exposure was still visible 3 h and 7 h after occidiofungin was re-
resulted in a greater than 3-log reduction in the cell count at 12 h.
The fungicidal effect of a 1-h exposure with 8? MIC was perma-
iments. Presumably, occidiofungin has a strong interaction with
the fungal cells, given that three washes were not enough to elim-
inate its fungicidal activity at 8? MIC. Another possibility is that
occidiofungin may have an intracellular target. Irreversible cellu-
lar damage following a 1-h exposure is not probable, given that
viable cell counts were present 7 h after exposure in the PAFE
Chemical stability experiments. Occidiofungin was exposed
to 50, 60, 70, and 100°C for 30 min. In this temperature stability
experiment, the MICs of occidiofungin against C. albicans 66027
were 1 ?g/ml for all temperatures. This suggests that occidiofun-
gin is stable following exposure to extreme temperatures. Occid-
study. The MICs for all pH samples were 1 ?g/ml. Lastly, occid-
iofungin’s stability against the gastric proteases trypsin, chymo-
120 min was measured. The potency of occidiofungin did not
change following exposure to these proteases under the optimal
conditions for their proteolytic activity. Presumably, the cyclic
nature of occidiofungin provides protection against proteolytic
In vitro potency of occidiofungin against Candida species is in the
submicromolar range and is not drastically inhibited by the pres-
ence of serum. The reduction in activity in the presence of serum
is similar to the reduction observed for anidulafungin (29). In
addition, occidiofungin is fungicidal against C. albicans. The ini-
tial 1- to 2-log drop in cell density following exposure to occidio-
TABLE 2 Occidiofungin serum MICs and MLCsa
Isolate% Human serumMIC (?g/ml) MLC (?g/ml)
C. albicans 66027
C. albicans 66027
C. glabrata 66032
C. glabrata 66032
aOnly the MLC is reported for 50% serum because of its turbidity.
?, 2? MIC (4 ?g/ml); ‘, 1? MIC (2 ?g/ml); ?, 0.5? MIC (1 ?g/ml); and ?, control (0 ?g/ml).
Stability and In Vitro Potency of Occidiofungin
February 2012 Volume 56 Number 2 aac.asm.org 767
and PAFE experiments (2). The differences in activities in the
time-kill and PAFE experiments suggest that occidiofungin has a
separate target from the lipopeptide caspofungin and, presum-
ably, from other echinocandins. These experiments support the
need for efficacy studies aimed at understanding occidiofungin’s
ability to treat systemic Candida infections.
Azoles are the only oral bioavailable drug for the treatment of
fungal infections. Lipophilic drugs generally have good colonic
gin is attributed to drug permeability limitations to absorption
(22). However, in the same study caspofungin was reported to
have some instability at an acidic pH. A detailed analysis of the
chemical stability of echinocandins, caspofungin, anidulafungin,
or micafungin is not available. Occidiofungin is stable under ex-
treme temperature and pH. Furthermore, the compound is not
inactivated by the gastric proteases trypsin, chymotrypsin, and
pepsin. Given occidiofungin’s chemical stability, studies aimed at
understanding occidiofungin’s oral bioavailability are warranted.
burkholdines (28). Occidiofungin is the first antifungal from this
group to demonstrate its physical stability and in vitro activity
against a human fungal pathogen. These data suggest that further
this small group of antifungals are necessary. These compounds
may provide a new line of treatment for life-threatening fungal
We are grateful to Thomas D. Edlind, Drexel University College of Med-
icine, for providing many of the Candida isolates used in the study. We
also thank Mahmoud Ghannoum, University Hospitals Case Medical
Center, for the fluconazole- and caspofungin-resistant isolates tested in
This work was supported in part by P20RR016476 (National Center
of Southern Mississippi to D.G.
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FIG 2 (A) Bioactivity assay assessing proteolytic stability of occidiofungin.
Activity is reflective of the CFU count/ml. Occidiofungin (8 ?g/ml) was ex-
posed to trypsin (black), chymotrypsin (gray), and pepsin (white) for 30, 60,
and 120 min. Control 1, control 2, and control 3 are the reaction buffer with
protease, the reaction buffer alone, and the reaction buffer with protease and
protease inhibitors, respectively. (B) Confirmation of protease activity using
followed by separation through a 12% SDS-PAGE gel. The first and last lanes
are controls and contain BSA in reaction buffer without added protease for 0
and 120 min, respectively.
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Stability and In Vitro Potency of Occidiofungin
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