tetracycline combinations in Galleria mellonella with resistant Candida albicans infection

Accepted Manuscript Title: In vivo activity of fluconazole/tetracycline combinations in Galleria mellonella with resistant Candida albicans infection Authors: Wenrui Gu, Qiong Yu, Cuixiang Yu, Shujuan Sun PII: DOI: Reference:

S2213-7165(17)30222-9 https://doi.org/10.1016/j.jgar.2017.11.011 JGAR 545

To appear in: Received date: Revised date: Accepted date:

21-3-2017 18-9-2017 20-11-2017

Please cite this article as: Wenrui Gu, Qiong Yu, Cuixiang Yu, Shujuan Sun, In vivo activity of fluconazole/tetracycline combinations in Galleria mellonella with resistant Candida albicans infection (2010), https://doi.org/10.1016/j.jgar.2017.11.011 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.

In vivo activity of fluconazole/tetracycline combinations in Galleria

Wenrui Gua,b, Qiong Yub, Cuixiang Yuc, Shujuan Sund#

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mellonella with resistant Candida albicans infection

School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province, P. R. Chinaa

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Department of Pharmacy, Southwest Hospital, Third Military Medical University,

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Chongqing, P. R. Chinab;

Shandong Province, P. R. China c;

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Respiration Medicine, Qianfoshan Hospital Affiliated to Shandong University, Jinan,

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Department of Pharmacy, Qianfoshan Hospital Affiliated to Shandong University, Jinan,

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Shandong Province, P. R. Chinad.

Corresponding author, Tel: 86-531-89268365, Fax: 86-531-82961267

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E-mail: [email protected]

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Highlights 

The antifungal effect of fluconazole combined with tetracycline is confirmed using Galleria mellonella. The combination increased the survival rate and decreased the tissue damage

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caused by C. albicans. 

The mechanism may be attributed to attenuating fungal virulence factors.

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Abstract

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Objective: The treatment of azoles-resistant Candida albicans infections continues to

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pose significant challenges. With limited options of licensed agents, the drug’s combination turns out to be a practical way. In our previous studies,

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minocycline/fluconazole (MINO/FLC) and doxycycline/fluconazole (DOXY/FLC)

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combinations shown synergistic effect in vitro. It is necessary to explore appropriate

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dosage, potential toxicity and in vivo efficacy. Methods: The Galleria mellonella infection model was employed to study the in vivo

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efficacy of MINO/FLC and DOXY/FLC by survival analysis, quantification of C.

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albicans CFU/mL, and histological analysis. Results: The survival rates of G. mellonella larvae infected with lethal doses of C. albicans increased significantly when drug combination was given compared to fluconazole treatment alone. The fungal burden reduced by almost 4-fold and

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histopathology study showed that fewer infected areas in larvae were observed and the destructive degree was slighter when larvae were exposed to combined drugs. Conclusions: The findings suggest that the combination of tetracycline and fluconazole

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has antifungal activity against azoles-resistant Candida albicans in vivo. This is in agreement with several previous in vitro studies and provides preliminary in vivo evidence that such a combination might be useful therapeutically.

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Keywords: Candida albicans; minocycline; doxycycline; Galleria mellonella;

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Invertebrate model

1. Introduction

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Treatment of infections caused by fungi is challenging owing to appearance of drug

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resistance as well as lack of novel agents[1] . The most commonly used agents versus

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Candida albicans are azoles. With increasing resistance to these agents, the selection of effective therapy for Candida albicans infections is particularly challenging. As a result

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of the dearth of therapeutic options, there is now considerable interest in the potential

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combination therapies. Minocycline (MINO) and doxycycline (DOXY), as tetracycline derivatives, are

antimicrobial drugs with a large spectrum of antibiotic activity[2]. It has been reported to have an antifungal effect when used alone or combined with other antimicrobial drugs in vitro[3–6]. On the basis of predecessors’ study, we have carried on a series of studies and 3

found that both minocycline and doxycycline showed synergistic effect in vitro when combined with fluconazole (FLC) against azoles-resistant C. albicans. In brief, with the combination of fluconazole and minocycline, the MIC80 of fluconazole for the resistant

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strain CA10 decreased from 512mg/L to 2mg/L. And the fractional inhibiroty

concentration index (FICI) was 0.035[7]. Similarly, with the combination of fluconazole and doxycycline, the MIC80 of CA10 decreased to 4mg/L, and the FICI was 0.023[8].

The mechanism of action was suggested to be the enhancement of intracellular calcium

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release, the downregulation of fluconazole-inducible efflux pump gene overexpression,

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and the penetrating ability on biofilms[7–9].

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Since these combinations appear to be a promising treatment option based on the in vitro data, animal studies are necessary before it can be considered for clinical treatment.

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Besides, it can inform on appropriate dosage, potential toxicity and in vivo efficacy.

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Mammalian models are routinely employed since it is considered that the data generated

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are most relevant to human infections[10]. However, these experiments are complex, expensive and time consuming, as well as there being legitimate ethical concerns over the

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use of such animals. So it is reasonable that preliminary experiments of in vivo activity

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can be carried on through the use of invertebrate infection models. Galleria mellonella, the larva of the wax moth has been proposed as an inexpensive and easy alternative that is able to generate reliable and reproducible data which mirror almost exactly those obtained using higher animals. A number of other research has recently used Galleria mellonella to investigate the in vivo activity of antimicrobial agents against pathogenic 4

microorganism, including bacteria and fungi[11–16]. In the present study, we first established an infect model with azoles-resistant C. albicans, and then employed it to study the in vivo activity of MINO/FLC and

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DOXY/FLC combination in an attempt to gain further insights into whether it should be explored for the treatment of resistant C. albicans infections.

2. Materials and methods 2.1. Candida albicans cultivation

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The resistant isolates of Candida albicans (CA10) were used in this study. Their

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susceptibilities were determined according to Clinical and Laboratory Standards Institute

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M27-A3 document, with C. parapsilosis ATCC 22019 as the reference strain. The MIC80 of fluconazole, itraconazole and voriconazole are 256-512mg/L, 4 mg/L, and 4 mg/L,

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respectively[7,8,17]. Frozen stocks of isolates were maintained at -80 °C until testing.

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After thawing, the yeast cells were subcultured on the yeast–peptone–dextrose (YPD)

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solid medium (1% yeast extract, 2% peptone, 2% glucose and 2% agar) at least twice at 35°C before each experiment to ensure viability and purity. RPMI1640 was used as liquid

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medium to diluting drugs and strains.

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2.2. Drugs

All 3 drugs (FLC, MINO and DOXY) were purchased from Dalian Meilun Biotech

Co. Ltd., China. The stock solution was prepared following the manufacturer’s instructions: they were dissolved in sterile demineralized water respectively at room temperature to achieve a stock solution of 2,560 mg/L. All stock solutions were stored at 5

-20°C.

From the stock solutions, 2-fold serial dilutions were prepared.

2.3. Survival assay of G. mellonella model in different yeast concentrations Galleria mellonella larvae (0.25±0.03g) were placed in Petri dishes and incubated

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at 37°C in the dark the night before the experiments. Larvae with dark spots or apparent melanization were excluded. Yeasts were grown overnight in liquid Sabouraud, washed

with PBS, and suspended in the same buffer. For survival assays, larvae were inoculated

with 1×107, 5×106, 1×106 and 5×105 cells/ larva of C. albicans. The range of inoculation

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concentration modified by reference to Liliana et al’s study[16]. The inocula were

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prepared in PBS plus 20 mg/L of ampicillin to prevent bacterial contamination. Yeast

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suspensions were injected in the haemocele through the last left pro-leg of the larvae using a 10 μL microliter syringe (Gaoge, China). The infected larvae were incubated at

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37° C, and the death was daily monitored during 4 days. Larvae death was monitored by

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visual inspection of the color (brown-dark brown) and by lack of movement after

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touching them with forceps. A group of larvae inoculated with PBS-ampicillin were studied in parallel in every infection investigation as controls. For each condition, a total

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of 20 larvae were used, and each experiment was repeated at least three times.

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2.4. Efficacy of MINO/FLC and DOXY/FLC in G. mellonella infected with C. albicans Larvae killing assays were carried out at 37 ° C as previously described above, using a dose of 4×106 yeast cells/larva. Infected larvae were treated with fluconazole (4, 8, 16 or 32mg/ kg, Meilun, Dalian, China), minocycline (25.6mg/kg, Meilun, Dalian, China) or 6

doxycycline (25.6mg/kg, Meilun, Dalian, China). Combinations of MINO/FLC and DOXY/FLC were also used. Parallel groups of uninfected larvae treated with the same concentrations of drugs were also included to eliminate possible drug toxicity and other

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effects of the drugs as factors contributing to the observed results. Antifungals were

administered 2h post-infection. Survival was monitored every 24 h and lasted for 4 days. Each experiment used groups containing 20 larvae and experiments were repeated twice using larvae from different batches.

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2.5. Fungal burden determination

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Fungal burden was determined by CFU counts at different times after inoculation.

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For this purpose, four groups of 80 larvae were selected, all of which received 2.5×106 cells/ larva of C. albicans strain. While one group treated with PBS as blank control, the

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others were treated with FLC (1.6μg/larvae), MINO (5.12μg/larvae) and the combination

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of MINO/FLC. Similarly, another 80 larvae were divided into groups and treated with

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PBS, FLC (3.2μg/larvae), DOXY (5.12μg/larvae) and the combination of DOXY/FLC. Every 24h, 5 larvae were taken from each group, washed with ethanol and cut into small

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pieces with a scalpel. No discrimination was made between live or dead larvae. The

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material was suspended in 1 ml of PBS-ampicillin and homogenized gently with a vortex of a few seconds. The homogenate was 10- fold diluted with same buffer. 5 μl of the resulting dilutions were inoculated onto Sabouraud agar plates. The plates were incubated during 24 h at 37°C, and CFUs enumerated, with results expressed as the average and standard deviation. 7

2.6. Histological study of larvae tissue Histological experiment was carried on to evaluate the presence of C. albicans inside tissue of G. mellonella and observe the damage to the surrounding normal tissue.

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Larvae from different groups (uninfected, infected, treated with FLC and treated with

drug combination) were collected on the third day after infection. Larvae were fixed for 24 h in 4% buffered formalin and dehydrated with increasing concentrations of ethanol (70%, 80%, 90%, 96% and 100%). The samples were then treated with xylene and

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paraffin embedded. Tissue sections of 8 microns were stained with periodic acid Schiff

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(PAS) and observed under an OLYMPUS FSX100 fluorescence microscope with 4.2×

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and 10× objectives. Samples from non-infected larvae were included as control. 2.7. Statistics

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Graphs and Statistics analyzes were performed with Graph Pad Prisma 5 (La Jolla

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CA, USA). Survival curves were analyzed by Kaplan-Meier method and fungal burden

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were analyzed using t-Test.

3. Results

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3.1. Survival assay of G. mellonella model in different yeast concentrations

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First, the most suitable concentration of C. albicans causing larvae infection was

investigated. The range of inoculation concentration was 5×105 to 1×107 refering to Liliana et al’s study[16]. Moreover, to confirm that the death was not a consequence of a shock due to large amounts of liquid injected in the larvae, a group of larvae were inoculated with PBS as control. As expected, larval survival was significantly dependent 8

on the number of yeast cells infected (Figure 1). Most reproducible results were found when larvae were infected with 5106 cells/larva. For further experiments, the inoculum of 5106 cells/larva were used.

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3.2. Activities of FLC, MINO and DOXY in the G. mellonella infection model

To determine whether the combination of MINO/FLC and DOXY/FLC have

synergistic effect in vivo, G. mellonella were infected with resistant C. albicans (CA10) and treated with different drugs (FLC, MINO, DOXY and their combinations). The

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results showed that treatment with FLC could increase the survival rate at the

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concentration of 320 mg/L. However, the difference of survival at 160 mg/L and 320

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mg/L were not statistically significant (P> 0.05) using Kaplan Meier method. At higher concentrations (640mg/L), there was a decrease in the survival, which might be explained

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by the toxicity of the antifungal at this high concentration (Figure 2A). When treated with

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the combination of FLC and MINO, the highest survival rate was observed in the group

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at the concentration of 160mg/L (Figures 2B). So the survival rate of the FLC group and the combination group was compared in the same concentration (160mg/L). As for the

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combination of FLC and DOXY, the highest survival rate was also observed in the group

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at the concentration of 320 mg/L (Figure 2C). So the comparison was applied at the concentration of 320 mg/L. As shown in Figure 2D and 2E, drug combination groups showed significant antifungal activity. 3.3. Fungal burden determination and histopathology The fungal burden was determined by recovering the yeast cells from the larvae 9

infected with C. albicans and treated with FLC, MINO, DOXY or MINO/FLC and DOXY/FLC. Referred to the result mentioned above, the concentration of FLC and MINO are 1.6μg/larvae and 5.12μg/larvae and the concentration of FLC and DOXY are

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3.2μg/larvae and 5.12μg/larvae respectively. Treatment of larvae with FLC decreased the number of CFUs by nearly 2-fold compared to control group. The combination of FLC and MINO reduced the fungal burden by almost 4-fold. The DOXY/FLC combination

showed similar result and the greatest distinction was found in the second and the third

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day. The larval burden of combination group presented an up-trend with the time, which

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may suggest a limited interaction time and the antifungal activity is strong within 48

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hours after treatment. (Figure 3).

Histopathology studies of larvae infected with C. albicans were performed at days 3

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post-infection. Yeast cells and filaments were observed in the tissue, both in the treated

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and untreated larvae, primarily in clusters. However, in larvae of untreated and FLC

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groups, there were a higher number of infected areas compared to the larvae with the combination treatment. Moreover, the fungi were mainly found in defined structures

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surrounded by G. mellonella cells (Figure 4).

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4. Discussion A number of studies have found that the antifungal activity of fluconazole can be

enhanced when combined with other drugs, such as calcium channel blockers, anticancer drugs, immunosuppressants and antimicrobials[18–23]. In our previous studies, the strong synergistic activity of DOXY/FLC and MINO/FLC have been observed in vitro 10

against resistant C. albicans isolates. And to the best of our knowledge, there is a lack of study which carrying on the antifungal activity of fluconazole/ tetracycline in vivo at the present moment. So in this study, we employed G. mellonella infection system to find that

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DOXY/FLC and MINO/FLC combinations showed antifungal activity against azoles-resistant C. albicans.

Mammalian infection models are commonly used to investigate unconventional antimicrobial therapies in order to clarify in vivo efficacy. However, the use of

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invertebrate hosts has recently facilitated the study of fungal pathogenesis and antifungal

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activity. Galleria mellonella is a lepidopteran that has important advantages as host

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model. The larvae can be incubated in a range of temperature between 25°C to 37°C, which is suitable to simulate the natural fungal habitat and the infection conditions in

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mammals[24]. In addition, G. mellonella allows the use of precise doses of both the

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pathogen and antimicrobial agents by infection. Moreover, some aspects of the innate

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reponse are conserved between G. mellonella and mammals[25]. The massive melanization induced by G. mellonella in response to infection make it easy to observe

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the viability of larvae[25–27]. Ethical issues, cost and reproducibility are other benefits of

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this model[26–28]. In this work, we used G. mellonella to investigate the antifungal efficacy of

DOXY/FLC and MINO/FLC combinations.

The survival assay showed that, the

combinations were highly effective in protecting larvae from lethal infections with resistant C. albicans. At concentrations equivalent to therapeutic doses in human, the 11

combined treatment significantly prolonged the rate of survival of G. mellonella, compared with larvae treated with FLC alone. The efficacies of the antimicrobials on infected larvae closely correlate with the drug susceptibilities of resistant C. albicans

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strain in vitro.

The fungal clearance rate in infection model can directly reflect the antifungal effect of treatments. The determination of larval burden post-infection showed that combined antifungal therapy significantly reduced the number of C. albicans cells

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detected inside the larvae. Also, the larval burden of combination group presented an

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up-trend with the time, which may suggest a limited interaction time. Another point

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worth noting is that the CFU of control group also decreased compared to the inoculum injected. This can be explained by the action of hemocytes which play an important role

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in in the larva’s cellular defense against bacteria and fungi like phagocytic cells[29,30].

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Histological examination plays an important role in studying the virulence of

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infection and in this study, microscopic observation indicates the correlation of the virulence with the degree of damage on the histological tissue of G. mellonella. Resistant

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C. albicans produced pseudohyphae and severe tissue damage in the larvae with

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numerous areas of infection, clustered yeast cells were also observed in larvae. With the combined treatment, fewer clustered yeast cells and filaments were observed. In conclusion, our findings confirm that the combination of fluconazole and tetracycline has antifungal activity against resistant C. albicans in vivo. The combination might be a promising therapeutic option for treatment of infections with resistant C. 12

albicans. Further pharmaceutical studies using mammalian models are required on pharmacokinetic, pharmacodynamics.

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Declarations

Funding: Financial support was received from the Department of Science and

Technology of Shandong Province, Shandong Provincial Natural Science Foundation,

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China [2016GSF201187, 2015GSF118022].

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Competing Interests: None

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Ethical Approval: Not required

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Figure 1. Survival curve of G.mellonella infected with different concentration of resistant C. albicans (CA10). The most reproducible results were found when larvae were

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infected with 5×106 C. albicans cells/larva.

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Figure 2. Efficacy of FLC alone (A) or in combination with MINO (B) or DOXY (C) during G. mellonella infection with resistant C. albicans (CA10). The concentration of yeast cells was 5×106 cells/larva. For comparison purposes, the curves of PBS, FLC alone, MINO alone, and FLC+MINO were extracted. These four curves were put in the same coordinate system (D) to compare the survival rates. The comparison of FLC and DOXY is

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the same (E).

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Figure 3. Effect of treatment with FLC and the combinations of MINO/FLC (A) or DOX/FLC (B) on larval burden of resistant C. albicans (CA10). All larvae were inoculated with 2.5×106 cells/larva CA10, and treated with 10 μl of each indicidual drug or combinations at 2h post-infection. Treatments consisted of PBS, FLC (6.4mg/kg) alone, MINO (20.48mg/kg) alone or combinations of

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FLC with MINO. The other treatment group consisted of PBS, FLC (12.8mg/kg) alone, DOX (20.48mg/kg) alone and the combination of DOX/FLC. For clarity, data for treatment with MINO or

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DOX was not shown because the data obtained closely followed that shown for Control group.

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Figure 4. Histopathology of G. mellonella infected with C. albicans and treated with different agents. Galleria mellonella was infected with 5 × 106 cells/larva of resistant C. albicans CA10(B-E). After 72 hours of infection, larvae were processed for histopathology as described in Material and

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Methods. (A), uninfected controls; (B), untreated controls; (C), larvae treated with FLC (16mg/kg); (D), larvae treated with the combination of FLC (16mg/kg) and MINO (25.6mg/kg); (E), larvae

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treated with the combination of FLC (16mg/kg) and DOX (25.6mg/kg).

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