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Gentamicin synergises with azoles against drug-resistant Candida albicans
Accepted Manuscript Title: Gentamicin synergizes with azoles against resistant candida albicans Author: Mengjiao Lu, Cuixiang Yu, Xueyan Cui, Jinyi Shi, Lei Yuan, Shujuan Sun PII: DOI: Reference:
S0924-8579(17)30352-7 https://doi.org/doi:10.1016/j.ijantimicag.2017.09.012 ANTAGE 5269
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
23-5-2017 14-9-2017
Please cite this article as: Mengjiao Lu, Cuixiang Yu, Xueyan Cui, Jinyi Shi, Lei Yuan, Shujuan Sun, Gentamicin synergizes with azoles against resistant candida albicans, International Journal of Antimicrobial Agents (2017), https://doi.org/doi:10.1016/j.ijantimicag.2017.09.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Gentamicin synergizes with azoles against resistant Candida albicans
2 Mengjiao Lua, Cuixiang Yub, Xueyan Cuid, Jinyi Shid, Lei Yuanc, Shujuan Sund*
3 4 5 6
a
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Province, P.R. China;
8
b
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Ji’nan, Shandong Province, People’s Republic of China;
School of Pharmaceutical Sciences, Shandong University, Ji’nan, 250012, Shandong
Respiration Medicine, Qianfoshan Hospital Affiliated to Shandong University,
10
c
11
China;
12
d
13
Ji’nan, 250014, Shandong Province, P.R. China.
Department of Pharmacy, Baodi People’s Hospital, Baodi, 301800, Tianjin, P.R.
Department of Pharmacy, Qianfoshan Hospital Affiliated to Shandong University,
14 15 16
Correspondence to
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Shujuan Sun, Department of Pharmacy,
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Qianfoshan Hospital Affiliated to Shandong University,
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Ji’nan, 250014, P.R. China.
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Tel: 86-531-89268365
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Fax: 86-531-82961267
22
E-mail: [email protected]
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24 25 26 27 28 29
Highlight Gentamicin synergizes with azoles against planktonic cells of resistant C. albicans. Gentamicin synergizes with fluconazole against preformed biofilms of C. albicans. Gentamicin enhanced in vivo efficacy of fluconazole against resistant C. albicans.
30
Gentamicin suppressed efflux pump of resistant C. albicans.
31
Gentamicin plus fluconazole reduces phospholipase activity of
32
resistant C. albicans.
33
ABSTRACT
34
Candida species are the primary opportunistic pathogens of nosocomial fungal
35
infections, causing both superficial and life-threatening systemic infections.
36
Combination therapy for fungal infections has attracted considerable attention,
37
especially for those caused by drug-resistant fungi. Gentamicin, an aminoglycoside
38
antibiotic, has a weak antifungal activity against Fusarium. The aim of this study was
39
to investigate the interactions of gentamicin with azoles against the Candida species
40
and the underlying mechanism. In the checkerboard assay, gentamicin was found to
41
not only worked synergistically with azoles against planktonic cells of drug-resistant
42
C. albicans with a fractional inhibitory concentration index (FICI) of 0.13 to 0.14, but
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also synergized with fluconazole against C. albcians biofilms preformed in less than
44
12h. The synergism of gentamicin with fluconazole was also confirmed in vivo by a
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Galleria mellonella infection model. Additionally, mechanism studies showed that
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gentamicin not only suppressed the efflux pump of resistant C. albicans in a
47
dose-dependent manner but also inhibited extracellular phospholipase activities of
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resistant C. albicans when combined with fluconazole. These findings suggested that
49
gentamicin enhances the efficacy of azoles against resistant C. albicans via efflux
50
inhibition and extracellular phospholipase activities decrease.
51 52 53
Keywords: Gentamicin; Azoles; Synergy; Drug-resistant C. albians; G. mellonella model;
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1. Introduction
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Candida, which includes approximately 200 yeast species, remains the most most
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common cause of invasive fungal infections [1]. Among these species, C. albicans is
59
still the predominant cause of candidal infections, while the incidence of infections
60
due to non-albicans Candida species is increasing [2, 3]. To treat with candidiasis,
61
azoles, such as fluconazole (FLC), itraconazole (ITZ) and voriconazole (VRC), have
62
been extensively used in clinical practice in virtue of their great efficacy and low
63
toxicity. However, owing to the long-term or extensive application of azoles, drug
64
resistance of Candida species has frequently emerged, especially FLC resistance
65
[4-6]. Moreover, biofilms formed on medical devices act as a barrier to the diffusion
66
of antifungal agents, thereby enhancing the resistance of Candida spp. to antifungals
67
[7, 8]. Therefore, it is of great importance to search for new antifungal approaches to
68
eliminating the phenomenon of resistance in Candida species. Since the development
69
of new antifungals is beset with difficulties, the combination of non-antifungals with
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antifungals maybe a feasible policy to solve the problem [9]. Research on
71
combination therapies to enhance the susceptibility of Candida species to antifungals
72
has attracted considerable attention.
73 74
Aminoglycoside antibiotics (AmAns) are a class of glycoside antibiotics, which are
75
formed by the connection of amino sugars and amino alcohols via oxygen bridges.
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AmAns are effective against Gram negative infections and are always used in the
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clinic because of their vast antibacterial spectrum, fine curative effect and other
78
advantages. In past decades, extensive efforts have been dedicated to finding new
79
antimicrobial activities of AmAns, such as antifungal effects. For example, synthetic
80
AmAns analogues derived from kanamycin [10-12] and tobramycin [13, 14] are
81
reported to possess antifungal activity or synergize with antifungals. In addition,
82
gentamicin (GM), a conventional aminoglycoside antibiotic, is reported to exert a
83
weak antifungal effect against Fusarium species [15]. However, there are no reports
84
focusing on the anti-Candida activity of GM or its combined effects with azoles
85
against the Candida species.
86 87
In this study, we evaluated the in vitro efficacy of GM alone or in concomitant use
88
with azoles against Candida species via a checkerboard assay. We also investigated
89
the combined effects of GM with FLC against preformed biofilms of C. albcians. In
90
addition, a Galleria mellonella infection model was established to determine the
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combined efficacy of GM and FLC in vivo. Notably, we further investigated the
92
underlying mechanisms of the synergism between GM and FLC by assessing the
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impact of GM on the efflux pump and the extracellular phospholipases activity.
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2. Materials and Methods
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2.1. Strains and media
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The strains used in this study included 4 Candida albicans, 2 Candida glabrata, 3
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Candida krusei and a quality-control strain, Candida albicans ATCC10231. The
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quality-control strain was kindly provided by the Institute of Pharmacology, School of
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Pharmacy, Shandong University, Ji'nan, Shandong Province, China, and the others
101
were isolated from a clinical laboratory at the Shandong Provincial Qianfoshan
102
Hospital, Jinan, China. Frozen stocks of the isolates were stored at -80°C. Before each
103
experiment, the isolates were activated on yeast-peptone-dextrose (YPD) solid
104
medium (2% agar, 2% peptone, 1% yeast extract and 2% glucose) for 24 h at 35˚C at
105
least twice. RPMI-1640 medium (PH 7.0) buffered with morpholinepropanesulfonic
106
acid was used to dilute the drugs and yeast cells.
107 108
2.2. Antimicrobial agents
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FLC was purchased from Shandong Chengchuang Pharmaceutical Co., Ltd. China,
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and the others (VRC, ITZ, and gentamicin sulfate) were purchased from Dalian
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Meilun Biotech Co., Ltd. China. Stock solutions of FLC and gentamicin sulfate were
112
prepared in sterile distilled water at 2,560 μg/mL. VRC and ITZ were dissolved to a
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concentration of 2,560 μg/mL with dimethylsulfoxide (DMSO). Stock solutions were
114
sterilized using 0.22 micron filters, aliquoted and stored at 4°C.
115 116
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2.3. Antifungal susceptibility testing
118
The minimum inhibitory concentrations (MICs) of GM and the azoles against
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Candida spp. were determined by broth microdilution as described by the Clinical and
120
Laboratory Standards Institute guidelines (CLSI, document M27-A3). C. albicans
121
ATCC 10231 was used to ensure quality control. The yeast, at the final concentration
122
of 2.5×103 colony forming units (CFU)/mL, was inoculated in 96-well microtiter
123
plates. The drug-free well was set as the growth control and the wells containing only
124
RPMI-1640 medium act as negative controls. After an incubation for 24 h at 35°C,
125
the MICs were determined by both visual reading and the optical density values
126
measured at 492 nm with a microplate reader. The background optical density values
127
were subtracted during the subsequent analysis and quantification. The MIC was
128
defined as the lowest concentration of the drug that inhibited fungal growth by 80%
129
(MIC80) compared with that of the growth control.
130 131
2.4. Planktonic checkerboard assay
132
The interaction of GM with the azoles against Candida planktonic cells was assessed
133
by a broth microdilution in 96-well microtiter plate according to the CLSI guidelines
134
(document M27-A3). For the checkerboard method [16, 17], drugs at the final
135
concentrations of 0.25-128 μg/mL for FLC, 0.03-16 μg/mL for ITZ/VRC, and 4-256
136
μg/mL for GM, were added to the wells. Meanwhile, the cell suspensions (2.5×103
137
CFU/mL) were added to each well. After an incubation for 24 h at 35°C, the MICs
138
were determined as the susceptibility testing. The obtained data were analyzed using
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two different models as follows: the FICI model and the ΔE model. The FICI is
140
expressed with the equation: FICI = FICA + FICB = MICAcomb/MICAalone +
141
MICBcomb/MICBalone and is interpreted as antagonistic when FICI ≥ 4, as indifferent
142
when FIC < 0.5-4 and as synergistic when FICI ≤ 0.5 [18]. The ΔE model was
143
described as the following equation: ΔE = EA×EB-Eobserved. EA and EB are the
144
experimental percentages of fungal growth when each drug acts alone, and the
145
Eobserved is the observed percentage of growth in combination of drugs A and B [19].
146
When the ΔE and its 95% confidence interval (CI) were positive, synergy was
147
defined. When the ΔE and its 95% CI were negative, antagonism was defined. In
148
other cases, the conclusion was independence.
149 150
2.5. Biofilms checkerboard assay
151
The interaction between GM and FLC against C. albicans (CA8, CA10 and CA16)
152
biofilms was assessed with preformed biofilms at different stages as previous
153
description with some modification [20]. Briefly, the biofilm was preformed by
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adding 200-µl 2.5×103 CFU/ml of the suspension into a 96-well plate and
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anaerobically incubating the plates over four time intervals (4, 8, 12, and 24 h) at
156
35°C. Subsequently, the biofilm was washed with sterile phosphate-buffered saline
157
(PBS) three times to remove the planktonic and nonadherent cells. The drugs were
158
then added at the final concentrations of 16-1024 μg/mL for GM or 2-1024 μg/mL for
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FLC. The drug-free well was set as the control growth. After further incubation for 24
160
h at 35°C, the biofilm was washed with sterile PBS for three times, and its metabolic
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activity was examined by the
162
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)
163
reduction assay [21]. Colorimetric change was measured in a microtiter plate reader at
164
492 nm. The sessile minimum inhibitory concentration (sMIC) was defined as the
165
lowest concentration of drug that reduced the biofilm metabolic activity by 80%
166
(sMIC80) compared to that of the control growth.
167 168
2.6. G. mellonella infection model
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To determine the in vivo combined effects of GM and FLC, a G. mellonella infection
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model was established [22, 23], and two resistant C. albcians isolates CA10 and
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CA16 were used. In the experiment, larvae in the final instar were chosen to be absent
172
of gray markings and similar in size (approximately 0.25 g). A survival assay was
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conducted with four groups of 20 randomly chosen larvae. The larvae were injected
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with 10-μL of C. albicans (5×108 CFU/mL) via the last left pro-leg. Before the
175
injection, the area was sterilized by an alcohol swab. Two hours after the infection,
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four groups of the larvae were injected via the last right pro-leg with 10-μL of sterile
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PBS, 160 μg/mL of FLC, 160 μg/mL of GM, 160 μg/mL of GM plus 160 μg/mL of
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FLC, respectively. Then, the larvae were incubated at 35˚C in the dark, and we
179
monitored the death daily for survival over four days. The larvae were considered
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dead if they give no response to touch. For the fungal burden analysis, another four
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groups of larvae were injected with yeast and drugs as described above. During the
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4-days incubation, three larvae from each group were taken randomly daily, washed
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with ethanol and homogenized in sterile PBS-ampicillin. Then, the homogenates were
184
diluted, and a 10-μL inoculum was added onto YPD agar. After an incubation for 24 h
185
at 35°C, colony counts were performed to determine the CFU/larva. Histological
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studies were performed on the four groups of larvae injected with yeast and drugs as
187
described above and one group of larvae untreated with yeast and drugs. After a
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two-day incubation, two larvae from every group were taken, washed, and cut into
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histological sections (15 μm). Then, the sections were stained with Periodic acid
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Schiff (PAS) and observed under fluorescence microscope with the 4.2× objective.
191 192
2.7. Rhodamine 6G efflux assay
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The rhodamine 6G (Rh6G) efflux assay was applied to determine whether GM affects
194
the efflux pump activity of a drug-resistant C. albicans isolate (CA10) [24]. Yeast
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cells (1×105 CFU/mL) were incubated in YPD liquid medium overnight at 35°C.
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Then, the cells were collected, washed with glucose-free PBS and the concertration
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were adjusted to 1×107 CFU/ml. Subsequently, the Rh6G solution was added into cell
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suspension at the final concentration of 10 μM for a 50 minute incubation at 35°C,
199
and then, the suspension was exposed to an ice-water bath for 10 minutes. The cells
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were then collected, washed with glucose-free PBS and resuspended in glucose-PBS
201
(5%). Meanwhile, GM was added at different final concentrations of 64, 128, and 256
202
μg/mL, and the Rh6G-alone group was served as the control group. Then, the
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fluorescence intensity of the intracellular Rh6G was recorded every 30 minutes using
204
a flow cytometer with excitation at 488 nm and emission at 530 nm.
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2.8. Extracellular phospholipase activity assays
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Extracellular phospholipase activity was assayed according to Gu et al.[23] and Price
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et al.[25]. The yeast suspension (106 CFU/ml) was incubated with FLC (1 μg/mL),
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GM (64 μg/mL), GM (64 μg/mL) plus FLC (1 μg/mL), and no drugs, respectively.
210
After a 24 h incubation at 35°C, 10 μl of the cell suspensions were inoculated onto the
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egg yolk agar medium plates (0.01 M NaCl, 0.025 M CaCl2, 1% peptone, 10% egg
212
yolk, 3% glucose, and 2% agar), and then, the plates were then incubated for 72h at
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35°C. The phospholipase activity (Pz) values were expressed by the following
214
equation: Pz = Colony diameter/(Colony diameter + precipitation zone diameter). As
215
previously described, phospholipase activity was classified as very high (Pz ≤ 0.69),
216
high (Pz = 0.70 to 0.79), low (Pz = 0.80 to 0.89), very low (Pz = 0.90 to 0.99), and
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negative (Pz = 1).
218 219
2.9. Statistical analysis
220
Each experiment was performed three times. The graphs and statistical analyses were
221
performed with Graph Pad Prisma 5 software and SPSS Statistics V17.0. The survival
222
curve was analyzed by the Kaplan-Meier method and the log-rank test. The fungal
223
burden and Rh6G effluxes data were analyzed using a Student’s t-test and a one-way
224
ANOVA. P < 0.05 was considered statistically significant.
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3. Results
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3.1. GM Synergizes with Azoles against resistant C. albicans but not susceptive C.
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albicans or resistant non-albicans Candida strains.
230
The MICs of GM and the azoles against Candida species are shown in Table 1. The
231
MICs of the azoles indicated that CA4 and CA8 were susceptive strains, while the
232
others were all resistant strains. The MICs of GM against all the tested strains were
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proved to be >512 μg/mL, indicating a very limited intrinsic antifungal activity.
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However, when GM was used in combination with azoles against drug-resistant C.
235
albicans (CA10, CA16), synergistic effects were observed with FICIs of 0.13-0.14,
236
demonstrating that GM significantly increased the susceptibility of resistant C.
237
albcians to azoles. The synergism of GM with azoles against resistant C. albicans was
238
also demonstrated by the ΔE method (Fig. 1), with high percentages of strong
239
synergistic interactions (∑SYN) from 1302% to 1890% for CA10 and a ∑SYN from
240
1487% to 2757% for CA16.
241 242
For the susceptive C. albicans isolates and the resistant non-albicans Candida (Table
243
1), indifference was observed, with FICI ranging from 0.51 to 2 when GM was used
244
combined with azoles. The very low ∑SYN shown by the ΔE method also indicated
245
that there were no interactions when GM combined with the azoles against susceptive
246
C. albicans isolates and resistant non-albicans Candida (data not shown). Moreover,
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no antagonism was observed in the combination of GM and FLC against all the tested
248
strains.
249 250 251
3.2. GM Enhances the Efficacy of FLC against C. albicans biofilms at different stages.
252
The interactions of GM with FLC against preformed biofilm were tested against CA8,
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CA10 and CA16, and the results are shown in Table 2. The data demonstrated that
254
GM synergized with FLC against susceptive C. albicans biofilms preformed over 4,
255
8, and 12h (FICI 0.06-0.25). For resistant C. albicans, synergism was only observed
256
against biofilms preformed over 4 and 8 h with a FICI < 0.5. As the biofilm matured,
257
the synergism weakened and was scarcely observed on the biofilm that preformed
258
over 24 h. These data indicated that GM obviously enhanced the efficacy of FLC
259
against the biofilm that formed at early stage but not more mature biofilm.
260 261
3.3. GM combined with FLC Prolonged the Survival rate of G. mellonella.
262
Survival rate. In vivo studies on the combined effects of GM and FLC were
263
performed using G. mellonella larvae infected with two resistant C. albicans isolates
264
(CA10, CA16). Over a 4-day period, the GM groups showed a low survival rate, with
265
20% for the larvae infected with CA10 and 35% for the larvae infected with CA16,
266
which was slightly higher than the control groups (15% and 20%) (Fig. 2). Notably,
267
the survival rate of the larvae treated with GM plus FLC was 75% for the larvae
268
infected with CA10 and 80% for the larvae infected with CA16, which was
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significantly higher than that of the larvae treated with FLC (35% and 40%) (P <
270
0.01). These results indicated that GM combined with FLC might be more effective
271
than a FLC monotherapy to treat with candidiasis in the clinic.
272 273
Fungal burden analysis. To detect the combined effect of GM with FLC on the
274
fungal burden of the infected larvae, a fungal burden analysis was performed. The
275
data shown in Fig. 3 demonstrates that there was a gradual increased in the fungal
276
burden in all the groups, and the fungal burden of the drug monotherapy groups was
277
similar to the control group. Of note, compared with the control group and the drug
278
monotherapy group, a significant lower fungal burden was observed in t of GM plus
279
FLC group (P < 0.01), indicating that GM combined with FLC decreased the fungal
280
burden significantly.
281 282
Histological study. Histological study was carried out to describe infected tissues of
283
G. mellonella larvae. The results were completely in accordance with that of the
284
fungal burden analysis. As shown in Fig. 4, the infected tissues of larvae were
285
presented as melanized nodules after the PAS staining. The melanized nodules in the
286
GM plus FLC groups were barely detectable, while there were large numbers of
287
melanized nodules in the other three groups. In addition, the areas of melanized
288
nodules in these three groups were much larger than those of the combination groups.
289
These findings demonstrated that compared with an FLC monotherapy, GM
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combined with FLC significantly reduced the tissue damage of resistant C. albicans to
291
larvae.
292 293
3.4. GM inhibites the efflux pumps of resistant C. albicans in a dose-dependent
294
manner.
295
The efflux pump activity of CA10 was investigated by the Rh6G assay to test whether
296
GM affects drug efflux. As time went on, the fluorescent intensity demonstrated a
297
gradual decrease both in the control group and the GM group (Fig. 5A). However, the
298
fluorescent intensity in the GM group was significantly higher than that of control
299
group all the time (P < 0.05), indicating that GM inhibited the energy-dependent
300
efflux pump activity of CA10. Interestingly, the inhibition was
301
concentration-dependent, as the higher concentrations of GM yielded the higher
302
intracellular levels of Rh6G at the endpoint of the test time (Fig. 5B).
303 304 305
3.5. GM combined with FLC Reduces the Extracellular Phospholipase activity of C. albicans.
306
The extracellular phospholipase activities of C. albicans treated with different drugs
307
are presented in Table 3. The PZ values of the control group and the drug
308
monotherapy groups are both < 0.7, demonstrating a very high phospholipase activity.
309
Furthermore, the PZ value of the GM plus FLC group was 0.86±0.01, which was much
310
higher than that of FLC group (0.63±0.01), indicating that GM plus FLC significantly
311
decreased the extracellular phospholipase activity of a drug-resistant C. albicans
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strain. The result suggested that the inhibition of extracellular phospholipase activity
313
was involved in the mechanisms of the increased efficacy of FLC to resistant C.
314
albicans induced by GM.
315
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316 317
4. Discussion
318
Candida is one of the most common causes of fungal disease in humans and its
319
resistance to antifungal agents has emerged frequently in the last several decades [26].
320
Combination therapies of antifungal drugs with non-antifungal agents, such as
321
amiodarone [27], minocycline [28], and cyclosporine A [29], might be an ideal
322
approach to treat candidiasis caused by drug-resistant Candida spp. . In this paper, we
323
proved that the combination of GM with azoles showed a synergism against the
324
resistant C. albicans, and indifference was observed against susceptible C. albicans
325
and resistant non-albicans Candida in vitro. The MICs of the azoles against resistant
326
C. albcians decreased from 512 μg/mL to 1 μg/mL for FLC, from 16 μg/mL to 0.25
327
μg/mL for ITZ, and from 16 μg/mL to 0.03 μg/mL for VRC in the presence of GM.
328
These observations indicated that GM might be a candidate to combine with azoles
329
against resistant C. albicans.
330 331
Numerous studies reveal that C. albicans biofilm possess an intrinsic resistance to
332
most of the current antifungal drugs in the clinic [7, 30]. Biofilm-induced drug
333
resistance complicates the use of FLC as a single-drug treatment option and is starting
334
to emerge as a growing clinical problem. Here, we found that although the synergistic
335
inhibition was not observed against mature biofilm of both the susceptive and
336
resistant C. albicans, the biofilm that performed for less than 12h was synergistically
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inhibited by a combination of GM with FLC, indicating a potential use of this
338
combination in prevention or early treatment of bioflim-related diseases.
339 340
In recent years, G. mellonella has been an attractive host to study pathogens and
341
antimicrobial agents, given its significant ethical, logistical and economic advantages
342
[31, 32]. In this study, we chose this model to evaluate the in vivo combined effects of
343
GM and FLC. The exposure of infected larvae to GM plus FLC significantly
344
enhanced the survival rate compared with drug monotherapy (Fig. 2) (P<0.05).
345
Furthermore, the fungal burden analysis and histopathology study also proved that the
346
efficacy of FLC in vivo could be enhanced by GM. The fungal burden of the infected
347
larvae showed that the GM plus FLC therapy was more efficacious than the FLC
348
monotherapy in clearing C. albcians from the larvae, as the fungal burden was always
349
less than that of the FLC monotherapy over a 4-day period (Fig. 3). To complement
350
the in vivo study, a histopathology analysis was also performed. The GM plus FLC
351
therapy resulted in a significant decrease in melanized nodules, while the drug
352
monotherapy scarcely affected the histopathology of the infected larvae (Fig. 4).
353
Therefore, the in vivo data were in accordance with the antifungal effect demonstrated
354
in vitro and indicated the potential use of this combination in vivo against resistant C.
355
albcians.
356 357
It is well known that a decrease in the drug concentration in the fungal cells could be
358
induced by the over-expression of efflux pumps [33, 34]. Numerous studies suggest
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359
that inhibiting the function of the efflux pump might be an important synergistic
360
mechanism of drug interactions against resistant isolates [35, 36]. In this study, we
361
found a significant inhibitory effect of GM on the efflux pump of C. albicans (Fig.
362
5A). Interestingly, the inhibitory effect occurred in a dose-dependent manner (Fig.
363
5B). These observations indicated that the synergism between GM and FLC might be
364
mediated by suppressing the efflux pump of C. albcians.
365 366
Phospholipases is one of the most important extracellular hydrolytic enzymes and
367
plays an important role in the pathogenicity of the Candida species [37, 38]. In
368
addition, phospholipases B1 protein (Plb1p) is detected at the tips of hyphae during
369
tissue invasion [39, 40]. In this report, we found that GM combined with FLC
370
decreased the extracellular phospholipase activity of resistant C. albicans with a Pz
371
value > 0.80 (Table 3). The results indicated that the inhibition of extracellular
372
phospholipase activities might be a synergistic mechanism of GM combined with
373
FLC against resistant C. albicans.
374 375
AmAns are antimicrobial drugs with a broad spectrum of antibiotic activity, and some
376
AmAns analogues obtained from structure modification are reported to have
377
antifungal activities. For example, K20 and FG08 derived from kanamycin A and B,
378
displayed antifungal activity, while K20 also synergizes with azoles against Candida
379
species and Cryptococcus neoformans [10-12]. Additionally, C12 and C14 derived
380
from tobramycin also displayed antifungal activities against various fungi and work
Page 19 of 42
381
synergistically with azoles against Candida albicans [13]. In our study, GM, a
382
conventional aminoglycoside antibiotic, showed synergistic inhibitory activities with
383
azoles against resistant C. albicans, although a very weak antifungal activity of GM
384
against Candida spp. was observed. Different from the studies of the AmAns
385
analogues, we further evaluated the combined effects of GM with FLC against the
386
preformed biofilm of C. albicans, and the synergism was observed for the biofilm that
387
was performed for less than 12 h. Furthermore, we used a G. mellonella infection
388
model to determine the in vivo combined effects of GM with FLC, and we found that
389
GM significantly enhanced the efficacy of FLC in vivo, with a high survival rate
390
based on a survival assay, a decreased fungal burden via a fungal burden analysis, and
391
showed fewer melanized nodules in a histological study. We further investigated the
392
synergistic mechanism by assaying Rh6G efflux and extracellular phospholipase
393
activity, and GM suppressed the efflux pump in a dose-dependent manner and
394
significantly reduced the extracellular phospholipase activity of resistant C. albicans
395
when combined with FLC. Taken together, these findings suggest the possibility of
396
combining AmAns or their analogues with azoles to treat fungal infections with a
397
higher efficiency or at lower administration doses.
398 399
In past decades, we have been devoting our efforts towards the discovery of
400
fluconazole sensitizer, and indeed we have evaluated the sensitization of a series of
401
different compounds on fluconazole. Inspired from Shrestha's studies on the
402
antifungal activity of aminoglycosides derivatives, we systematically evaluated the
Page 20 of 42
403
sensitization effect of gentamycin to three azole antifungal agents (FLC, ITZ, and
404
VRC) in this paper. This paper provides an advance over recent studies of ours and in
405
the field by first finding that the conventional aminoglycoside antibiotic GM not only
406
synergized with the azoles against drug-resistant C. albicans in vitro, but also
407
enhanced the efficacy of FLC in a G. mellonella larvae infection model.
408 409
In conclusion, GM synergized with azoles against resistant C. albicans in vitro, and
410
enhanced the efficacy of FLC against preformed biofilm of both susceptive and
411
resistant C. albicans. Moreover, GM plus FLC prolonged the survival rate of G.
412
mellonella larvae infected with resistant C. albicans, decreased the fungal burden, as
413
well as reduced damage to tissues. Mechanism studies elucidated that the synergism is
414
related to suppressing the efflux pump and inhibiting extracellular phospholipases
415
activity. These findings together with studies on AmAns analogues indicated that
416
AmAns and their analogues might be developed as potential antifungal agents or
417
sensitizers of antifungals for therapeutic applications.
418 419
Declarations
420
Funding: Financial support was received from the Department of Science and
421
Technology of Shandong Province, Shandong Provincial Natural Science Foundation,
422
China [2016GSF201187, 2015GSF118022].
423
Competing Interests: None declared
424
Ethical Approval: Not required
Page 21 of 42
425
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Cavalcanti BC, et al. Synergistic effects of amiodarone and fluconazole on Candida
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fluconazole and minocycline against mixed cultures of Candida albicans and
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Staphylococcus aureus. J Microbiol Immunol 2015;48:655-61.
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a synergistically enhances the susceptibility of Candida albicans biofilms to
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fluconazole by multiple mechanisms. BMC Microbiol 2016;16:113.
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mechanisms. Curr Genet 2013;59:251-64.
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Investigation of multidrug efflux pumps in relation to fluconazole resistance in
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Candida albicans biofilms. J Antimicrob Chemother 2002;49:973-80.
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[34] Rocha MF, Bandeira SP, de Alencar LP, Melo LM, Sales JA, Paiva MA, et al.
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Azole resistance in Candida albicans from animals: Highlights on efflux pump
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activity and gene overexpression. Mycoses 2017.
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[35] Holmes AR, Keniya MV, Ivnitski-Steele I, Monk BC, Lamping E, Sklar LA, et
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al. The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of
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fungal ABC and MFS transporter efflux pump activities which reverses the azole
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resistance of Candida albicans and Candida glabrata clinical isolates. Antimicrob
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Agents Chemother 2012;56:1508-15.
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[36] De Cremer K, Staes I, Delattin N, Cammue BP, Thevissen K, De Brucker K.
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Combinatorial drug approaches to tackle Candida albicans biofilms. Expert Rev Anti
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infect Ther 2015;13:973-84.
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Candida species: current epidemiology, pathogenicity, biofilm formation, natural
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[39] Niewerth M, Korting HC. Phospholipases of Candida albicans. Mycoses
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[40] Djordjevic JT. Role of phospholipases in fungal fitness, pathogenicity, and drug
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development - lessons from cryptococcus neoformans. Front Microbiol 2010;1:125.
535
Page 27 of 42
536
Fig 1. Three-dimensional plots of GM combined with azoles against CA10 and
537
CA16. The ΔE values were depicted on the z-axis to construct a three-dimensional
538
plot, and peaks above and below the 0 plane indicate synergistic and antagonistic
539
combinations, respectively. The color-coding on the right indicates that the closer to
540
the top of the bar, the more effective the drug combination.
541 542
Fig 2. Survival rate of the G. mellonella larvae infected with CA10 and CA16
543
over a 4-day period. G. mellonella larvae were injected with 10 μL of C. albicans
544
(5×108 CFU/mL) and were treated with PBS, FLC (160 μg/mL), GM (160 μg/mL),
545
and GM (160 μg/mL) plus FLC (160 μg/mL), respectively. The data came from the
546
means of three independent experiments and the log-rank test was performed.
547
0.01 when compared with the FLC-treated group.
＊＊
P<
548 549
Fig 3. Fungal burden of the G. mellonella larvae infected with CA10 and CA16
550
over a 4-day period. G. mellonella larvae were injected with 10 μL of C. albicans
551
(5×108 CFU/mL) and were treated with PBS, FLC (160 μg/mL), GM (160 μg/mL),
552
and GM (160μg/mL) plus FLC (160 μg/mL), respectively. For clarity, the fungal
553
burden of the GM groups is not shown because the data were similar to that of the
554
control groups. The data are means ± standard deviations from three independent
555
experiments, and the statistical significances were determined by a Student’s t-test.
556
＊
557
Fig 4. Histopathology of the G. mellonella larvae infected with CA10 and CA16 at
＊
P < 0.01 when compared with the FLC-treated group.
Page 28 of 42
558
2 day-post-infection. G. mellonella larvae were injected with 10 μL of C. albicans
559
(5×108 CFU/mL) and were treated with PBS, FLC (160 μg/mL), GM (160 μg/mL),
560
and GM (160 μg/mL) plus FLC (160 μg/mL), respectively. The larvae of the blank
561
control groups were not treated with yeast and drugs. The melanized nodules in the
562
tissue sections are the stained yeast clusters and hyphae.
563 564
Fig 5. Inhibitory effect of GM on the efflux of Rh6G in resistant C. albicans
565
(CA10). (A) The fluorescent intensity was detected after the treatment with GM (256
566
μg/mL) over 150 min. The data are the means ± standard deviations from three
567
independent experiments. The statistical significances were determined by Student’s
568
t-test. (B) The fluorescent intensities were detected after 150 minutes of treatment
569
with different concentrations of GM (64, 128, and 256 μg/mL). The data are the
570
means ± standard deviations from three independent experiments. The statistical
571
significances were determined by One-way ANOVA. n.s., P > 0.05;
572
< 0.01.
＊
P < 0.05,
＊＊
P
573 574
575
Page 29 of 42
576
Table 1. In vitro interactions of GM with the azoles against Candida spp. MIC80 (μg/mL) Strains Alone Combined FICI Interpretation Azoles GM Azoles GM FLC CA4 1 >512 1 >512 2 NI CA8 2 >512 1 32 0.56 NI CA10 >512 >512 1 64 0.13 SYN CA16 >512 >512 1 64 0.13 SYN CG2 128 >512 128 >512 2 NI CG3 64 >512 64 >512 2 NI CK8 64 >512 64 128 1.25 NI CK9 64 >512 64 128 1.25 NI CK10 128 >512 128 >512 2 NI ITZ CA4 0.25 >512 0.13 256 1 NI CA8 0.25 >512 0.13 64 0.63 NI CA10 >16 >512 0.25 64 0.14 SYN CA16 >16 >512 0.25 64 0.14 SYN CG2 16 >512 16 >512 2 NI CG3 8 >512 8 64 0.63 NI CK8 4 >512 4 16 1.03 NI CK9 16 >512 16 >512 2 NI CK10 >16 >512 16 256 1.5 NI VRC CA4 0.06 >512 0.03 8 0.51 NI CA8 0.13 >512 0.06 32 0.56 NI CA10 >16 >512 0.03 64 0.13 SYN CA16 >16 >512 0.03 64 0.13 SYN CG2 4 >512 16 >512 2 NI CG3 2 >512 2 >512 2 NI CK8 2 >512 2 32 1.06 NI CK9 2 >512 2 8 1.02 NI CK10 4 >512 4 >512 2 NI
577
The MICs and FICI values are shown as the median of three independent experiments.
578
CA, Candida albicans; CG, Candida glabrata; CK, Candida krusei; NI, no interaction;
579
SYN, synergism.
580
Page 30 of 42
581 582
Table 2. In vitro interactions of GM with FLC against preformed biofilm of C. albicans. sMIC80 of drugs (µg/ml) Time Isolates Alone Combined FICI Interpretation (h) FLC GM FLC GM 4 >1024 >1024 2 64 0.06 SYN 8 >1024 >1024 2 128 0.12 SYN CA8 12 >1024 >1024 4 256 0.25 SYN 24 >1024 >1024 >128 >1024 1.1 NI
CA10
4 8 12 24
>1024 >1024 >1024 >1024
>1024 >1024 >1024 >1024
2 4 8 >1024
64 128 512 >1024
0.06 0.13 0.51 2
SYN SYN NI NI
CA16
4 8 12 24
>1024 >1024 >1024 >1024
>1024 >1024 >1024 >1024
2 2 8 >1024
64 128 512 >1024
0.06 0.13 0.51 2
SYN SYN NI NI
583
The time indicated the incubation period of biofilm formation.
584
The sMICs and FICI values are shown as the median of three independent experiments.
585
NI, no interaction; SYN, synergism.
586 587
Page 31 of 42
588 589
Table 3. Extracellular phospholipase activity of resistant C. albicans (CA10) treated with drugs Drugs Pz value ± SD Phospholipase activity No drug 0.62±0.01 Very High FLC 0.63±0.01 Very High GM 0.61±0.01 Very High GM+FLC 0.86±0.03 low
590
Control, C. albicans (CA10) without drugs; FLC, C. albicans (CA10) treated FLC (1 μg/mL)
591
alone; GM, C. albicans (CA10) treated GM (64 μg/mL) alone; FLC+GM, C. albicans (CA10)
592
treated FLC (1 μg/mL) with GM (64 μg/mL); SD, standard deviation.
593
All data are the averages of triplicate experiments.
594
595
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S0924-8579(17)30352-7 https://doi.org/doi:10.1016/j.ijantimicag.2017.09.012 ANTAGE 5269
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
23-5-2017 14-9-2017
Please cite this article as: Mengjiao Lu, Cuixiang Yu, Xueyan Cui, Jinyi Shi, Lei Yuan, Shujuan Sun, Gentamicin synergizes with azoles against resistant candida albicans, International Journal of Antimicrobial Agents (2017), https://doi.org/doi:10.1016/j.ijantimicag.2017.09.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Gentamicin synergizes with azoles against resistant Candida albicans
2 Mengjiao Lua, Cuixiang Yub, Xueyan Cuid, Jinyi Shid, Lei Yuanc, Shujuan Sund*
3 4 5 6
a
7
Province, P.R. China;
8
b
9
Ji’nan, Shandong Province, People’s Republic of China;
School of Pharmaceutical Sciences, Shandong University, Ji’nan, 250012, Shandong
Respiration Medicine, Qianfoshan Hospital Affiliated to Shandong University,
10
c
11
China;
12
d
13
Ji’nan, 250014, Shandong Province, P.R. China.
Department of Pharmacy, Baodi People’s Hospital, Baodi, 301800, Tianjin, P.R.
Department of Pharmacy, Qianfoshan Hospital Affiliated to Shandong University,
14 15 16
Correspondence to
17
Shujuan Sun, Department of Pharmacy,
18
Qianfoshan Hospital Affiliated to Shandong University,
19
Ji’nan, 250014, P.R. China.
20
Tel: 86-531-89268365
21
Fax: 86-531-82961267
22
E-mail: [email protected]
Page 1 of 42
23
24 25 26 27 28 29
Highlight Gentamicin synergizes with azoles against planktonic cells of resistant C. albicans. Gentamicin synergizes with fluconazole against preformed biofilms of C. albicans. Gentamicin enhanced in vivo efficacy of fluconazole against resistant C. albicans.
30
Gentamicin suppressed efflux pump of resistant C. albicans.
31
Gentamicin plus fluconazole reduces phospholipase activity of
32
resistant C. albicans.
33
ABSTRACT
34
Candida species are the primary opportunistic pathogens of nosocomial fungal
35
infections, causing both superficial and life-threatening systemic infections.
36
Combination therapy for fungal infections has attracted considerable attention,
37
especially for those caused by drug-resistant fungi. Gentamicin, an aminoglycoside
38
antibiotic, has a weak antifungal activity against Fusarium. The aim of this study was
39
to investigate the interactions of gentamicin with azoles against the Candida species
40
and the underlying mechanism. In the checkerboard assay, gentamicin was found to
41
not only worked synergistically with azoles against planktonic cells of drug-resistant
42
C. albicans with a fractional inhibitory concentration index (FICI) of 0.13 to 0.14, but
43
also synergized with fluconazole against C. albcians biofilms preformed in less than
44
12h. The synergism of gentamicin with fluconazole was also confirmed in vivo by a
Page 2 of 42
45
Galleria mellonella infection model. Additionally, mechanism studies showed that
46
gentamicin not only suppressed the efflux pump of resistant C. albicans in a
47
dose-dependent manner but also inhibited extracellular phospholipase activities of
48
resistant C. albicans when combined with fluconazole. These findings suggested that
49
gentamicin enhances the efficacy of azoles against resistant C. albicans via efflux
50
inhibition and extracellular phospholipase activities decrease.
51 52 53
Keywords: Gentamicin; Azoles; Synergy; Drug-resistant C. albians; G. mellonella model;
54
Page 3 of 42
55 56
1. Introduction
57
Candida, which includes approximately 200 yeast species, remains the most most
58
common cause of invasive fungal infections [1]. Among these species, C. albicans is
59
still the predominant cause of candidal infections, while the incidence of infections
60
due to non-albicans Candida species is increasing [2, 3]. To treat with candidiasis,
61
azoles, such as fluconazole (FLC), itraconazole (ITZ) and voriconazole (VRC), have
62
been extensively used in clinical practice in virtue of their great efficacy and low
63
toxicity. However, owing to the long-term or extensive application of azoles, drug
64
resistance of Candida species has frequently emerged, especially FLC resistance
65
[4-6]. Moreover, biofilms formed on medical devices act as a barrier to the diffusion
66
of antifungal agents, thereby enhancing the resistance of Candida spp. to antifungals
67
[7, 8]. Therefore, it is of great importance to search for new antifungal approaches to
68
eliminating the phenomenon of resistance in Candida species. Since the development
69
of new antifungals is beset with difficulties, the combination of non-antifungals with
70
antifungals maybe a feasible policy to solve the problem [9]. Research on
71
combination therapies to enhance the susceptibility of Candida species to antifungals
72
has attracted considerable attention.
73 74
Aminoglycoside antibiotics (AmAns) are a class of glycoside antibiotics, which are
75
formed by the connection of amino sugars and amino alcohols via oxygen bridges.
76
AmAns are effective against Gram negative infections and are always used in the
Page 4 of 42
77
clinic because of their vast antibacterial spectrum, fine curative effect and other
78
advantages. In past decades, extensive efforts have been dedicated to finding new
79
antimicrobial activities of AmAns, such as antifungal effects. For example, synthetic
80
AmAns analogues derived from kanamycin [10-12] and tobramycin [13, 14] are
81
reported to possess antifungal activity or synergize with antifungals. In addition,
82
gentamicin (GM), a conventional aminoglycoside antibiotic, is reported to exert a
83
weak antifungal effect against Fusarium species [15]. However, there are no reports
84
focusing on the anti-Candida activity of GM or its combined effects with azoles
85
against the Candida species.
86 87
In this study, we evaluated the in vitro efficacy of GM alone or in concomitant use
88
with azoles against Candida species via a checkerboard assay. We also investigated
89
the combined effects of GM with FLC against preformed biofilms of C. albcians. In
90
addition, a Galleria mellonella infection model was established to determine the
91
combined efficacy of GM and FLC in vivo. Notably, we further investigated the
92
underlying mechanisms of the synergism between GM and FLC by assessing the
93
impact of GM on the efflux pump and the extracellular phospholipases activity.
94
Page 5 of 42
95
2. Materials and Methods
96
2.1. Strains and media
97
The strains used in this study included 4 Candida albicans, 2 Candida glabrata, 3
98
Candida krusei and a quality-control strain, Candida albicans ATCC10231. The
99
quality-control strain was kindly provided by the Institute of Pharmacology, School of
100
Pharmacy, Shandong University, Ji'nan, Shandong Province, China, and the others
101
were isolated from a clinical laboratory at the Shandong Provincial Qianfoshan
102
Hospital, Jinan, China. Frozen stocks of the isolates were stored at -80°C. Before each
103
experiment, the isolates were activated on yeast-peptone-dextrose (YPD) solid
104
medium (2% agar, 2% peptone, 1% yeast extract and 2% glucose) for 24 h at 35˚C at
105
least twice. RPMI-1640 medium (PH 7.0) buffered with morpholinepropanesulfonic
106
acid was used to dilute the drugs and yeast cells.
107 108
2.2. Antimicrobial agents
109
FLC was purchased from Shandong Chengchuang Pharmaceutical Co., Ltd. China,
110
and the others (VRC, ITZ, and gentamicin sulfate) were purchased from Dalian
111
Meilun Biotech Co., Ltd. China. Stock solutions of FLC and gentamicin sulfate were
112
prepared in sterile distilled water at 2,560 μg/mL. VRC and ITZ were dissolved to a
113
concentration of 2,560 μg/mL with dimethylsulfoxide (DMSO). Stock solutions were
114
sterilized using 0.22 micron filters, aliquoted and stored at 4°C.
115 116
Page 6 of 42
117
2.3. Antifungal susceptibility testing
118
The minimum inhibitory concentrations (MICs) of GM and the azoles against
119
Candida spp. were determined by broth microdilution as described by the Clinical and
120
Laboratory Standards Institute guidelines (CLSI, document M27-A3). C. albicans
121
ATCC 10231 was used to ensure quality control. The yeast, at the final concentration
122
of 2.5×103 colony forming units (CFU)/mL, was inoculated in 96-well microtiter
123
plates. The drug-free well was set as the growth control and the wells containing only
124
RPMI-1640 medium act as negative controls. After an incubation for 24 h at 35°C,
125
the MICs were determined by both visual reading and the optical density values
126
measured at 492 nm with a microplate reader. The background optical density values
127
were subtracted during the subsequent analysis and quantification. The MIC was
128
defined as the lowest concentration of the drug that inhibited fungal growth by 80%
129
(MIC80) compared with that of the growth control.
130 131
2.4. Planktonic checkerboard assay
132
The interaction of GM with the azoles against Candida planktonic cells was assessed
133
by a broth microdilution in 96-well microtiter plate according to the CLSI guidelines
134
(document M27-A3). For the checkerboard method [16, 17], drugs at the final
135
concentrations of 0.25-128 μg/mL for FLC, 0.03-16 μg/mL for ITZ/VRC, and 4-256
136
μg/mL for GM, were added to the wells. Meanwhile, the cell suspensions (2.5×103
137
CFU/mL) were added to each well. After an incubation for 24 h at 35°C, the MICs
138
were determined as the susceptibility testing. The obtained data were analyzed using
Page 7 of 42
139
two different models as follows: the FICI model and the ΔE model. The FICI is
140
expressed with the equation: FICI = FICA + FICB = MICAcomb/MICAalone +
141
MICBcomb/MICBalone and is interpreted as antagonistic when FICI ≥ 4, as indifferent
142
when FIC < 0.5-4 and as synergistic when FICI ≤ 0.5 [18]. The ΔE model was
143
described as the following equation: ΔE = EA×EB-Eobserved. EA and EB are the
144
experimental percentages of fungal growth when each drug acts alone, and the
145
Eobserved is the observed percentage of growth in combination of drugs A and B [19].
146
When the ΔE and its 95% confidence interval (CI) were positive, synergy was
147
defined. When the ΔE and its 95% CI were negative, antagonism was defined. In
148
other cases, the conclusion was independence.
149 150
2.5. Biofilms checkerboard assay
151
The interaction between GM and FLC against C. albicans (CA8, CA10 and CA16)
152
biofilms was assessed with preformed biofilms at different stages as previous
153
description with some modification [20]. Briefly, the biofilm was preformed by
154
adding 200-µl 2.5×103 CFU/ml of the suspension into a 96-well plate and
155
anaerobically incubating the plates over four time intervals (4, 8, 12, and 24 h) at
156
35°C. Subsequently, the biofilm was washed with sterile phosphate-buffered saline
157
(PBS) three times to remove the planktonic and nonadherent cells. The drugs were
158
then added at the final concentrations of 16-1024 μg/mL for GM or 2-1024 μg/mL for
159
FLC. The drug-free well was set as the control growth. After further incubation for 24
160
h at 35°C, the biofilm was washed with sterile PBS for three times, and its metabolic
Page 8 of 42
161
activity was examined by the
162
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)
163
reduction assay [21]. Colorimetric change was measured in a microtiter plate reader at
164
492 nm. The sessile minimum inhibitory concentration (sMIC) was defined as the
165
lowest concentration of drug that reduced the biofilm metabolic activity by 80%
166
(sMIC80) compared to that of the control growth.
167 168
2.6. G. mellonella infection model
169
To determine the in vivo combined effects of GM and FLC, a G. mellonella infection
170
model was established [22, 23], and two resistant C. albcians isolates CA10 and
171
CA16 were used. In the experiment, larvae in the final instar were chosen to be absent
172
of gray markings and similar in size (approximately 0.25 g). A survival assay was
173
conducted with four groups of 20 randomly chosen larvae. The larvae were injected
174
with 10-μL of C. albicans (5×108 CFU/mL) via the last left pro-leg. Before the
175
injection, the area was sterilized by an alcohol swab. Two hours after the infection,
176
four groups of the larvae were injected via the last right pro-leg with 10-μL of sterile
177
PBS, 160 μg/mL of FLC, 160 μg/mL of GM, 160 μg/mL of GM plus 160 μg/mL of
178
FLC, respectively. Then, the larvae were incubated at 35˚C in the dark, and we
179
monitored the death daily for survival over four days. The larvae were considered
180
dead if they give no response to touch. For the fungal burden analysis, another four
181
groups of larvae were injected with yeast and drugs as described above. During the
182
4-days incubation, three larvae from each group were taken randomly daily, washed
Page 9 of 42
183
with ethanol and homogenized in sterile PBS-ampicillin. Then, the homogenates were
184
diluted, and a 10-μL inoculum was added onto YPD agar. After an incubation for 24 h
185
at 35°C, colony counts were performed to determine the CFU/larva. Histological
186
studies were performed on the four groups of larvae injected with yeast and drugs as
187
described above and one group of larvae untreated with yeast and drugs. After a
188
two-day incubation, two larvae from every group were taken, washed, and cut into
189
histological sections (15 μm). Then, the sections were stained with Periodic acid
190
Schiff (PAS) and observed under fluorescence microscope with the 4.2× objective.
191 192
2.7. Rhodamine 6G efflux assay
193
The rhodamine 6G (Rh6G) efflux assay was applied to determine whether GM affects
194
the efflux pump activity of a drug-resistant C. albicans isolate (CA10) [24]. Yeast
195
cells (1×105 CFU/mL) were incubated in YPD liquid medium overnight at 35°C.
196
Then, the cells were collected, washed with glucose-free PBS and the concertration
197
were adjusted to 1×107 CFU/ml. Subsequently, the Rh6G solution was added into cell
198
suspension at the final concentration of 10 μM for a 50 minute incubation at 35°C,
199
and then, the suspension was exposed to an ice-water bath for 10 minutes. The cells
200
were then collected, washed with glucose-free PBS and resuspended in glucose-PBS
201
(5%). Meanwhile, GM was added at different final concentrations of 64, 128, and 256
202
μg/mL, and the Rh6G-alone group was served as the control group. Then, the
203
fluorescence intensity of the intracellular Rh6G was recorded every 30 minutes using
204
a flow cytometer with excitation at 488 nm and emission at 530 nm.
Page 10 of 42
205 206
2.8. Extracellular phospholipase activity assays
207
Extracellular phospholipase activity was assayed according to Gu et al.[23] and Price
208
et al.[25]. The yeast suspension (106 CFU/ml) was incubated with FLC (1 μg/mL),
209
GM (64 μg/mL), GM (64 μg/mL) plus FLC (1 μg/mL), and no drugs, respectively.
210
After a 24 h incubation at 35°C, 10 μl of the cell suspensions were inoculated onto the
211
egg yolk agar medium plates (0.01 M NaCl, 0.025 M CaCl2, 1% peptone, 10% egg
212
yolk, 3% glucose, and 2% agar), and then, the plates were then incubated for 72h at
213
35°C. The phospholipase activity (Pz) values were expressed by the following
214
equation: Pz = Colony diameter/(Colony diameter + precipitation zone diameter). As
215
previously described, phospholipase activity was classified as very high (Pz ≤ 0.69),
216
high (Pz = 0.70 to 0.79), low (Pz = 0.80 to 0.89), very low (Pz = 0.90 to 0.99), and
217
negative (Pz = 1).
218 219
2.9. Statistical analysis
220
Each experiment was performed three times. The graphs and statistical analyses were
221
performed with Graph Pad Prisma 5 software and SPSS Statistics V17.0. The survival
222
curve was analyzed by the Kaplan-Meier method and the log-rank test. The fungal
223
burden and Rh6G effluxes data were analyzed using a Student’s t-test and a one-way
224
ANOVA. P < 0.05 was considered statistically significant.
225
Page 11 of 42
226 227
3. Results
228
3.1. GM Synergizes with Azoles against resistant C. albicans but not susceptive C.
229
albicans or resistant non-albicans Candida strains.
230
The MICs of GM and the azoles against Candida species are shown in Table 1. The
231
MICs of the azoles indicated that CA4 and CA8 were susceptive strains, while the
232
others were all resistant strains. The MICs of GM against all the tested strains were
233
proved to be >512 μg/mL, indicating a very limited intrinsic antifungal activity.
234
However, when GM was used in combination with azoles against drug-resistant C.
235
albicans (CA10, CA16), synergistic effects were observed with FICIs of 0.13-0.14,
236
demonstrating that GM significantly increased the susceptibility of resistant C.
237
albcians to azoles. The synergism of GM with azoles against resistant C. albicans was
238
also demonstrated by the ΔE method (Fig. 1), with high percentages of strong
239
synergistic interactions (∑SYN) from 1302% to 1890% for CA10 and a ∑SYN from
240
1487% to 2757% for CA16.
241 242
For the susceptive C. albicans isolates and the resistant non-albicans Candida (Table
243
1), indifference was observed, with FICI ranging from 0.51 to 2 when GM was used
244
combined with azoles. The very low ∑SYN shown by the ΔE method also indicated
245
that there were no interactions when GM combined with the azoles against susceptive
246
C. albicans isolates and resistant non-albicans Candida (data not shown). Moreover,
Page 12 of 42
247
no antagonism was observed in the combination of GM and FLC against all the tested
248
strains.
249 250 251
3.2. GM Enhances the Efficacy of FLC against C. albicans biofilms at different stages.
252
The interactions of GM with FLC against preformed biofilm were tested against CA8,
253
CA10 and CA16, and the results are shown in Table 2. The data demonstrated that
254
GM synergized with FLC against susceptive C. albicans biofilms preformed over 4,
255
8, and 12h (FICI 0.06-0.25). For resistant C. albicans, synergism was only observed
256
against biofilms preformed over 4 and 8 h with a FICI < 0.5. As the biofilm matured,
257
the synergism weakened and was scarcely observed on the biofilm that preformed
258
over 24 h. These data indicated that GM obviously enhanced the efficacy of FLC
259
against the biofilm that formed at early stage but not more mature biofilm.
260 261
3.3. GM combined with FLC Prolonged the Survival rate of G. mellonella.
262
Survival rate. In vivo studies on the combined effects of GM and FLC were
263
performed using G. mellonella larvae infected with two resistant C. albicans isolates
264
(CA10, CA16). Over a 4-day period, the GM groups showed a low survival rate, with
265
20% for the larvae infected with CA10 and 35% for the larvae infected with CA16,
266
which was slightly higher than the control groups (15% and 20%) (Fig. 2). Notably,
267
the survival rate of the larvae treated with GM plus FLC was 75% for the larvae
268
infected with CA10 and 80% for the larvae infected with CA16, which was
Page 13 of 42
269
significantly higher than that of the larvae treated with FLC (35% and 40%) (P <
270
0.01). These results indicated that GM combined with FLC might be more effective
271
than a FLC monotherapy to treat with candidiasis in the clinic.
272 273
Fungal burden analysis. To detect the combined effect of GM with FLC on the
274
fungal burden of the infected larvae, a fungal burden analysis was performed. The
275
data shown in Fig. 3 demonstrates that there was a gradual increased in the fungal
276
burden in all the groups, and the fungal burden of the drug monotherapy groups was
277
similar to the control group. Of note, compared with the control group and the drug
278
monotherapy group, a significant lower fungal burden was observed in t of GM plus
279
FLC group (P < 0.01), indicating that GM combined with FLC decreased the fungal
280
burden significantly.
281 282
Histological study. Histological study was carried out to describe infected tissues of
283
G. mellonella larvae. The results were completely in accordance with that of the
284
fungal burden analysis. As shown in Fig. 4, the infected tissues of larvae were
285
presented as melanized nodules after the PAS staining. The melanized nodules in the
286
GM plus FLC groups were barely detectable, while there were large numbers of
287
melanized nodules in the other three groups. In addition, the areas of melanized
288
nodules in these three groups were much larger than those of the combination groups.
289
These findings demonstrated that compared with an FLC monotherapy, GM
Page 14 of 42
290
combined with FLC significantly reduced the tissue damage of resistant C. albicans to
291
larvae.
292 293
3.4. GM inhibites the efflux pumps of resistant C. albicans in a dose-dependent
294
manner.
295
The efflux pump activity of CA10 was investigated by the Rh6G assay to test whether
296
GM affects drug efflux. As time went on, the fluorescent intensity demonstrated a
297
gradual decrease both in the control group and the GM group (Fig. 5A). However, the
298
fluorescent intensity in the GM group was significantly higher than that of control
299
group all the time (P < 0.05), indicating that GM inhibited the energy-dependent
300
efflux pump activity of CA10. Interestingly, the inhibition was
301
concentration-dependent, as the higher concentrations of GM yielded the higher
302
intracellular levels of Rh6G at the endpoint of the test time (Fig. 5B).
303 304 305
3.5. GM combined with FLC Reduces the Extracellular Phospholipase activity of C. albicans.
306
The extracellular phospholipase activities of C. albicans treated with different drugs
307
are presented in Table 3. The PZ values of the control group and the drug
308
monotherapy groups are both < 0.7, demonstrating a very high phospholipase activity.
309
Furthermore, the PZ value of the GM plus FLC group was 0.86±0.01, which was much
310
higher than that of FLC group (0.63±0.01), indicating that GM plus FLC significantly
311
decreased the extracellular phospholipase activity of a drug-resistant C. albicans
Page 15 of 42
312
strain. The result suggested that the inhibition of extracellular phospholipase activity
313
was involved in the mechanisms of the increased efficacy of FLC to resistant C.
314
albicans induced by GM.
315
Page 16 of 42
316 317
4. Discussion
318
Candida is one of the most common causes of fungal disease in humans and its
319
resistance to antifungal agents has emerged frequently in the last several decades [26].
320
Combination therapies of antifungal drugs with non-antifungal agents, such as
321
amiodarone [27], minocycline [28], and cyclosporine A [29], might be an ideal
322
approach to treat candidiasis caused by drug-resistant Candida spp. . In this paper, we
323
proved that the combination of GM with azoles showed a synergism against the
324
resistant C. albicans, and indifference was observed against susceptible C. albicans
325
and resistant non-albicans Candida in vitro. The MICs of the azoles against resistant
326
C. albcians decreased from 512 μg/mL to 1 μg/mL for FLC, from 16 μg/mL to 0.25
327
μg/mL for ITZ, and from 16 μg/mL to 0.03 μg/mL for VRC in the presence of GM.
328
These observations indicated that GM might be a candidate to combine with azoles
329
against resistant C. albicans.
330 331
Numerous studies reveal that C. albicans biofilm possess an intrinsic resistance to
332
most of the current antifungal drugs in the clinic [7, 30]. Biofilm-induced drug
333
resistance complicates the use of FLC as a single-drug treatment option and is starting
334
to emerge as a growing clinical problem. Here, we found that although the synergistic
335
inhibition was not observed against mature biofilm of both the susceptive and
336
resistant C. albicans, the biofilm that performed for less than 12h was synergistically
Page 17 of 42
337
inhibited by a combination of GM with FLC, indicating a potential use of this
338
combination in prevention or early treatment of bioflim-related diseases.
339 340
In recent years, G. mellonella has been an attractive host to study pathogens and
341
antimicrobial agents, given its significant ethical, logistical and economic advantages
342
[31, 32]. In this study, we chose this model to evaluate the in vivo combined effects of
343
GM and FLC. The exposure of infected larvae to GM plus FLC significantly
344
enhanced the survival rate compared with drug monotherapy (Fig. 2) (P<0.05).
345
Furthermore, the fungal burden analysis and histopathology study also proved that the
346
efficacy of FLC in vivo could be enhanced by GM. The fungal burden of the infected
347
larvae showed that the GM plus FLC therapy was more efficacious than the FLC
348
monotherapy in clearing C. albcians from the larvae, as the fungal burden was always
349
less than that of the FLC monotherapy over a 4-day period (Fig. 3). To complement
350
the in vivo study, a histopathology analysis was also performed. The GM plus FLC
351
therapy resulted in a significant decrease in melanized nodules, while the drug
352
monotherapy scarcely affected the histopathology of the infected larvae (Fig. 4).
353
Therefore, the in vivo data were in accordance with the antifungal effect demonstrated
354
in vitro and indicated the potential use of this combination in vivo against resistant C.
355
albcians.
356 357
It is well known that a decrease in the drug concentration in the fungal cells could be
358
induced by the over-expression of efflux pumps [33, 34]. Numerous studies suggest
Page 18 of 42
359
that inhibiting the function of the efflux pump might be an important synergistic
360
mechanism of drug interactions against resistant isolates [35, 36]. In this study, we
361
found a significant inhibitory effect of GM on the efflux pump of C. albicans (Fig.
362
5A). Interestingly, the inhibitory effect occurred in a dose-dependent manner (Fig.
363
5B). These observations indicated that the synergism between GM and FLC might be
364
mediated by suppressing the efflux pump of C. albcians.
365 366
Phospholipases is one of the most important extracellular hydrolytic enzymes and
367
plays an important role in the pathogenicity of the Candida species [37, 38]. In
368
addition, phospholipases B1 protein (Plb1p) is detected at the tips of hyphae during
369
tissue invasion [39, 40]. In this report, we found that GM combined with FLC
370
decreased the extracellular phospholipase activity of resistant C. albicans with a Pz
371
value > 0.80 (Table 3). The results indicated that the inhibition of extracellular
372
phospholipase activities might be a synergistic mechanism of GM combined with
373
FLC against resistant C. albicans.
374 375
AmAns are antimicrobial drugs with a broad spectrum of antibiotic activity, and some
376
AmAns analogues obtained from structure modification are reported to have
377
antifungal activities. For example, K20 and FG08 derived from kanamycin A and B,
378
displayed antifungal activity, while K20 also synergizes with azoles against Candida
379
species and Cryptococcus neoformans [10-12]. Additionally, C12 and C14 derived
380
from tobramycin also displayed antifungal activities against various fungi and work
Page 19 of 42
381
synergistically with azoles against Candida albicans [13]. In our study, GM, a
382
conventional aminoglycoside antibiotic, showed synergistic inhibitory activities with
383
azoles against resistant C. albicans, although a very weak antifungal activity of GM
384
against Candida spp. was observed. Different from the studies of the AmAns
385
analogues, we further evaluated the combined effects of GM with FLC against the
386
preformed biofilm of C. albicans, and the synergism was observed for the biofilm that
387
was performed for less than 12 h. Furthermore, we used a G. mellonella infection
388
model to determine the in vivo combined effects of GM with FLC, and we found that
389
GM significantly enhanced the efficacy of FLC in vivo, with a high survival rate
390
based on a survival assay, a decreased fungal burden via a fungal burden analysis, and
391
showed fewer melanized nodules in a histological study. We further investigated the
392
synergistic mechanism by assaying Rh6G efflux and extracellular phospholipase
393
activity, and GM suppressed the efflux pump in a dose-dependent manner and
394
significantly reduced the extracellular phospholipase activity of resistant C. albicans
395
when combined with FLC. Taken together, these findings suggest the possibility of
396
combining AmAns or their analogues with azoles to treat fungal infections with a
397
higher efficiency or at lower administration doses.
398 399
In past decades, we have been devoting our efforts towards the discovery of
400
fluconazole sensitizer, and indeed we have evaluated the sensitization of a series of
401
different compounds on fluconazole. Inspired from Shrestha's studies on the
402
antifungal activity of aminoglycosides derivatives, we systematically evaluated the
Page 20 of 42
403
sensitization effect of gentamycin to three azole antifungal agents (FLC, ITZ, and
404
VRC) in this paper. This paper provides an advance over recent studies of ours and in
405
the field by first finding that the conventional aminoglycoside antibiotic GM not only
406
synergized with the azoles against drug-resistant C. albicans in vitro, but also
407
enhanced the efficacy of FLC in a G. mellonella larvae infection model.
408 409
In conclusion, GM synergized with azoles against resistant C. albicans in vitro, and
410
enhanced the efficacy of FLC against preformed biofilm of both susceptive and
411
resistant C. albicans. Moreover, GM plus FLC prolonged the survival rate of G.
412
mellonella larvae infected with resistant C. albicans, decreased the fungal burden, as
413
well as reduced damage to tissues. Mechanism studies elucidated that the synergism is
414
related to suppressing the efflux pump and inhibiting extracellular phospholipases
415
activity. These findings together with studies on AmAns analogues indicated that
416
AmAns and their analogues might be developed as potential antifungal agents or
417
sensitizers of antifungals for therapeutic applications.
418 419
Declarations
420
Funding: Financial support was received from the Department of Science and
421
Technology of Shandong Province, Shandong Provincial Natural Science Foundation,
422
China [2016GSF201187, 2015GSF118022].
423
Competing Interests: None declared
424
Ethical Approval: Not required
Page 21 of 42
425
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tropicalis resistant to fluconazole. Antimicrob Agents Chemother 2013;57:1691-700.
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[28] Li H, Zhang C, Liu P, Liu W, Gao Y, Sun S. In vitro interactions between
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Staphylococcus aureus. J Microbiol Immunol 2015;48:655-61.
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[29] Jia W, Zhang H, Li C, Li G, Liu X, Wei J. The calcineruin inhibitor cyclosporine
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a synergistically enhances the susceptibility of Candida albicans biofilms to
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fluconazole by multiple mechanisms. BMC Microbiol 2016;16:113.
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[30] Mathe L, Van Dijck P. Recent insights into Candida albicans biofilm resistance
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mechanisms. Curr Genet 2013;59:251-64.
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microbiological and toxin research. Virulence 2016;7:840-5.
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[32] Tsai CJ, Loh JM, Proft T. Galleria mellonella infection models for the study of
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bacterial diseases and for antimicrobial drug testing. Virulence 2016;7:214-29.
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Investigation of multidrug efflux pumps in relation to fluconazole resistance in
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Candida albicans biofilms. J Antimicrob Chemother 2002;49:973-80.
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Azole resistance in Candida albicans from animals: Highlights on efflux pump
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Fig 1. Three-dimensional plots of GM combined with azoles against CA10 and
537
CA16. The ΔE values were depicted on the z-axis to construct a three-dimensional
538
plot, and peaks above and below the 0 plane indicate synergistic and antagonistic
539
combinations, respectively. The color-coding on the right indicates that the closer to
540
the top of the bar, the more effective the drug combination.
541 542
Fig 2. Survival rate of the G. mellonella larvae infected with CA10 and CA16
543
over a 4-day period. G. mellonella larvae were injected with 10 μL of C. albicans
544
(5×108 CFU/mL) and were treated with PBS, FLC (160 μg/mL), GM (160 μg/mL),
545
and GM (160 μg/mL) plus FLC (160 μg/mL), respectively. The data came from the
546
means of three independent experiments and the log-rank test was performed.
547
0.01 when compared with the FLC-treated group.
＊＊
P<
548 549
Fig 3. Fungal burden of the G. mellonella larvae infected with CA10 and CA16
550
over a 4-day period. G. mellonella larvae were injected with 10 μL of C. albicans
551
(5×108 CFU/mL) and were treated with PBS, FLC (160 μg/mL), GM (160 μg/mL),
552
and GM (160μg/mL) plus FLC (160 μg/mL), respectively. For clarity, the fungal
553
burden of the GM groups is not shown because the data were similar to that of the
554
control groups. The data are means ± standard deviations from three independent
555
experiments, and the statistical significances were determined by a Student’s t-test.
556
＊
557
Fig 4. Histopathology of the G. mellonella larvae infected with CA10 and CA16 at
＊
P < 0.01 when compared with the FLC-treated group.
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558
2 day-post-infection. G. mellonella larvae were injected with 10 μL of C. albicans
559
(5×108 CFU/mL) and were treated with PBS, FLC (160 μg/mL), GM (160 μg/mL),
560
and GM (160 μg/mL) plus FLC (160 μg/mL), respectively. The larvae of the blank
561
control groups were not treated with yeast and drugs. The melanized nodules in the
562
tissue sections are the stained yeast clusters and hyphae.
563 564
Fig 5. Inhibitory effect of GM on the efflux of Rh6G in resistant C. albicans
565
(CA10). (A) The fluorescent intensity was detected after the treatment with GM (256
566
μg/mL) over 150 min. The data are the means ± standard deviations from three
567
independent experiments. The statistical significances were determined by Student’s
568
t-test. (B) The fluorescent intensities were detected after 150 minutes of treatment
569
with different concentrations of GM (64, 128, and 256 μg/mL). The data are the
570
means ± standard deviations from three independent experiments. The statistical
571
significances were determined by One-way ANOVA. n.s., P > 0.05;
572
< 0.01.
＊
P < 0.05,
＊＊
P
573 574
575
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576
Table 1. In vitro interactions of GM with the azoles against Candida spp. MIC80 (μg/mL) Strains Alone Combined FICI Interpretation Azoles GM Azoles GM FLC CA4 1 >512 1 >512 2 NI CA8 2 >512 1 32 0.56 NI CA10 >512 >512 1 64 0.13 SYN CA16 >512 >512 1 64 0.13 SYN CG2 128 >512 128 >512 2 NI CG3 64 >512 64 >512 2 NI CK8 64 >512 64 128 1.25 NI CK9 64 >512 64 128 1.25 NI CK10 128 >512 128 >512 2 NI ITZ CA4 0.25 >512 0.13 256 1 NI CA8 0.25 >512 0.13 64 0.63 NI CA10 >16 >512 0.25 64 0.14 SYN CA16 >16 >512 0.25 64 0.14 SYN CG2 16 >512 16 >512 2 NI CG3 8 >512 8 64 0.63 NI CK8 4 >512 4 16 1.03 NI CK9 16 >512 16 >512 2 NI CK10 >16 >512 16 256 1.5 NI VRC CA4 0.06 >512 0.03 8 0.51 NI CA8 0.13 >512 0.06 32 0.56 NI CA10 >16 >512 0.03 64 0.13 SYN CA16 >16 >512 0.03 64 0.13 SYN CG2 4 >512 16 >512 2 NI CG3 2 >512 2 >512 2 NI CK8 2 >512 2 32 1.06 NI CK9 2 >512 2 8 1.02 NI CK10 4 >512 4 >512 2 NI
577
The MICs and FICI values are shown as the median of three independent experiments.
578
CA, Candida albicans; CG, Candida glabrata; CK, Candida krusei; NI, no interaction;
579
SYN, synergism.
580
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581 582
Table 2. In vitro interactions of GM with FLC against preformed biofilm of C. albicans. sMIC80 of drugs (µg/ml) Time Isolates Alone Combined FICI Interpretation (h) FLC GM FLC GM 4 >1024 >1024 2 64 0.06 SYN 8 >1024 >1024 2 128 0.12 SYN CA8 12 >1024 >1024 4 256 0.25 SYN 24 >1024 >1024 >128 >1024 1.1 NI
CA10
4 8 12 24
>1024 >1024 >1024 >1024
>1024 >1024 >1024 >1024
2 4 8 >1024
64 128 512 >1024
0.06 0.13 0.51 2
SYN SYN NI NI
CA16
4 8 12 24
>1024 >1024 >1024 >1024
>1024 >1024 >1024 >1024
2 2 8 >1024
64 128 512 >1024
0.06 0.13 0.51 2
SYN SYN NI NI
583
The time indicated the incubation period of biofilm formation.
584
The sMICs and FICI values are shown as the median of three independent experiments.
585
NI, no interaction; SYN, synergism.
586 587
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588 589
Table 3. Extracellular phospholipase activity of resistant C. albicans (CA10) treated with drugs Drugs Pz value ± SD Phospholipase activity No drug 0.62±0.01 Very High FLC 0.63±0.01 Very High GM 0.61±0.01 Very High GM+FLC 0.86±0.03 low
590
Control, C. albicans (CA10) without drugs; FLC, C. albicans (CA10) treated FLC (1 μg/mL)
591
alone; GM, C. albicans (CA10) treated GM (64 μg/mL) alone; FLC+GM, C. albicans (CA10)
592
treated FLC (1 μg/mL) with GM (64 μg/mL); SD, standard deviation.
593
All data are the averages of triplicate experiments.
594
595
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