ACS Infectious Diseases
Article
2
H), 1.66−1.40 (m, 10H). 3C NMR (151 MHz, DMSO-d6,
1
Biofilm Formation Assay. The assay was modified with
37,38
TMS): δ 148.89, 144.02, 142.54, 141.27, 131.03, 128.58,
reference to the previously reported protocol.
Azole-
1
5
2
27.92 (s, 2C), 126.94 (s, 2C), 126.60, 123.40, 72.20, 69.38,
7.81, 49.04, 48.90, 37.48 (s, 2C), 37.29, 36.19, 35.66, 32.29,
8.31, 28.25, 22.82. HRMS (ESI) m/z: calcd for C H ClNO
2
resistant C. albicans cells were incubated to the exponential
growth stage in YEPD medium and diluted with RPMI 1640
6
medium to 1 × 10 CFU/mL. The fungal suspension was
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34
+
[
M + H] 428.2351, found 428.2380. HPLC purity: 99.5%.
added to 96-well plates with 100 μL per well. The azole-
resistant C. albicans were static cultured at 37 °C for 1.5 h.
Then, the cells were washed three times with 150 μL of
phosphate buffer saline (PBS) and the nonadherent cells were
removed. Drugs including FLC and compound B2 or both
were added to each well, and then, the azole-resistant C.
albicans cells were further static cultured at 37 °C for 24 h.
After that, the upper RPMI 1640 medium liquid was absorbed,
removed, and gently washed three times with 150 μL of PBS.
Then, 120 μL of XTT/menadione solution was added to the
Retention time: 7.286 min, eluted with MeOH/0.1%TFA·H O
2
(
v/v, 8/2).
In Vitro Antifungal Potency Test. In vitro antifungal
activity of the compounds was evaluated by determining the
MIC . The test was performed using the microliquid dilution
method by referring to CLSI (M27-A3). Fungal cells were
incubated to the exponential growth stage in YEPD medium
and then collected and diluted with RPMI 1640 medium to 1
×
compounds were obtained by 2-fold serial dilutions in 96-well
plates. The fungal cells were incubated at 35 °C for 48 h,
except that C. neoformans H99 was incubated at 35 °C for 72 h.
Then, the optical density at 630 nm (OD630) was measured
with a spectrophotometer to calculate the inhibition rate of
each well, which was used to evaluate the antifungal activity of
the compounds. Each compound was tested in triplicate.
In Vitro Synergistic Antifungal Activity Test. The test
was performed using the checkerboard microdilution method
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0
35
3
10 CFU/mL. The different concentration solutions of
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6-well plates, and the cells were further static cultured at 37
°
C for 3 h, avoiding light. Then, 80 μL of supernatant was
absorbed from each well and transferred to a 96-well blank
plate. The OD492 value of each well was measured by a
spectrophotometer. Finally, the XTT reduction assay was used
to calculate the semiquantitative measurement of biofilm
formation rate, and the changes of XTT staining were recorded
by photos at the same time.
In Vivo Antifungal Potency. The assay was performed
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according to the previously reported protocol. Female mice
breed, ICR; weight, 18−22 g; age, 4−6 weeks) were used to
by referring to CLSI (M27-A3). Fungal cells were incubated
to the exponential growth stage in YEPD medium, then
collected and diluted with RPMI 1640 medium to 1 × 10
(
3
build the in vivo experimental model, which were from the
Shanghai Experimental Animal Center, Chinese Academy of
Sciences. The drug solutions were prepared in advance and
divided into four groups: control group (normal saline, NS),
FLC group (0.8 mg/kg, in NS), compound B2 group (10 mg/
kg, suspended in NS with 1.5% glycerin and 0.5% Tween 80),
and the coadministration group of FLC and compound B2. All
mice were intraperitoneal injected with 0.2 mL of cyclo-
phosphamide (100 mg/kg, in NS) before the inoculation,
which was used as an immunosuppressive agent to destroy the
immune system of mice. After 24 h, each mouse was inoculated
CFU/mL. Fungal cells containing the tested compounds with
different concentrations were added to 24-well plates for the
preparation of fungal suspension and then transferred to 96-
well plates. Then, FLC was added into the 96-well plates and
serially double-diluted. The azole-resistant C. albicans cells
were static cultured at 35 °C for 48 h. Then, the OD630 with a
spectrophotometer was measured to calculate the inhibition
rate of each well, which was analyzed referring to Loewe
additivity theory and expressed by the FICI. FICI < 0.5
indicates synergistic effect, FICI > 4 indicates antagonistic
effect, and 0.5 ≤ FICI ≤ 4 indicates indifferent. Each
compound was tested in triplicate.
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6
with a 0.2 mL inoculum of azole-resistant C. albicans (1 × 10
35
CFU/mL) via a tail vein. The mice were administered with
drug solutions by intraperitoneal injection once a day at regular
times. After continuous administration for 5 days, all the mice
were sacrificed, and then, they were dissected and their kidneys
were removed and weighed. Following this, the left kidneys
were put into centrifuge tubes; to each tube was added 1 mL of
NS. The kidney tissue homogenate was obtained through a
high-speed tissue homogenizer and further diluted with NS.
The diluent kidney tissue homogenates were spread on
Sabouraud dextrose agar (SDA) medium plates with
chloramphenicol (100 μg/mL) by an inoculating loop and
static cultured at 35 °C for 48 h. Finally, the number of single
colonies on each SDA medium were counted, and the fungal
burden of the kidney tissue was further calculated. ANOVA
was used to analyze the differences among the groups.
Time-Growth Curve Assay. The assay was modified with
1
2,13
reference to the previously reported protocol.
resistant C. albicans cells were incubated to the exponential
growth stage in YEPD medium and diluted with RPMI 1640
medium to 1 × 10 CFU/mL. Then, FLC and compound B2
or both were added into 5 mL of fungal suspension as drug
groups, and the cells without any treatment were used as the
control group. The azole-resistant C. albicans cells were
shaking-cultured (200 rpm/min) at 30 °C, and the OD630
with a spectrophotometer was measured to calculate the
Azole-
6
numbers of azole-resistant C. albicans cells at different specified
times. This assay was performed in triplicate.
Filamentation Assay. The assay was modified with
37
reference to the previously reported protocol. Azole-resistant
C. albicans cells were incubated to the exponential growth stage
in YEPD medium and diluted with spider medium to 1 × 10
CFU/mL. The fungal suspension was transferred to a 24-well
plate with 2 mL per well. Then, FLC and compound B2 or
both were added to the 24-well plate as drug groups, and the
cells without any treatment were used as the control group.
The azole-resistant C. albicans cells were static cultured at 37
Microscopic Observation of Fungal Morphology.
Microscopic morphology of azole-resistant C. albicans cells
was observed by TEM, which was a reference to the reported
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12
protocol with some modifications. Azole-resistant C. albicans
cells were incubated to the exponential growth stage in YEPD
5
medium and diluted with RPMI 1640 medium to 5 × 10
CFU/mL. Then, the drugs were added including FLC,
compound B2, or both into 10 mL of fungal suspension, and
the control group was treated without drugs. The cells of each
group were incubated with constant shaking (200 rpm/min) at
°C for 3 h, and then, the fungal morphological differences were
recorded on a live cell imaging inverted microscope.
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ACS Infect. Dis. 2021, 7, 650−660
Yang, Wanzhen
