Berberine and Itraconazole Are not Synergistic in Vitro against Aspergillus fumigatus Isolated from Clinical Patients

The incidence of Aspergillus fumigatus infections has become more frequent as a consequence of widespread immunosuppression. At present, the number of available antifungal agents in the clinic is limited, and most of them, such as itraconazole (ICZ), are toxic and show resistance. Berberine (BER) is a plant alkaloid used in the clinic mainly for alimentary infections. We have used BER and ICZ to measure in vitro resistance in A. fumigatus isolated from clinical patients. The minimum inhibitory concentration ranges of BER and ICZ were 4–256 and 0.031–0.250 μg/mL, respectively. In addition, against A. fumigatus IFM 40808 strain, the MIC50 values of BER and ICZ were 8 and 0.125 μg/mL. Using this strain, we compared the giant colonies with or without BER, and concluded that BER could restrain A. fumigatus mycelial growth and conidial pigment production. Combinations of the two drugs were also tested by the checkerboard assay to identify any functional interactions between them. Thirty-two out of 42 isolates had FICI values > 4.0, indicating that two drugs were mutually antagonistic. In conclusion, it is not advised that BER and ICZ be used in the clinic at the same time. Our results indicated that BER may inhibit A. fumigatus through the ergosterol biosynthesis pathway, like ICZ.


Introduction
Aspergillus fumigatus is an environmentally ubiquitous, spore-forming mold saprophyte. This airborne filamentous fungal pathogen is known to be a major cause of lethal lung infections in immunocompromised hosts and allergic asthma in atopic individuals [1]. Fungal infections including candidiasis, aspergillosis, cryptococcosis, and pneumocystosis often require hospitalization, with aspergillosis being the second most common cause [2]. Aspergillosis has high mortality rates, and its incidence has been increasing gradually [3]. Notwithstanding the increasing need for effective therapy, the range of antifungal agents available is limited, and some of the most effective agents are also toxic [4]. Treatment of A. fumigatus infections mainly involves azole derivatives, a class that includes itraconazole (C 35 H 38 Cl 2 N 8 O 4 , Figure 1), which has historically been the front-line drug in these treatments [5]. However, the pharmacological profile of azole drugs is determined and restricted by their liver toxicity, metabolic elimination, and pharmacokinetic drug-drug interactions involving CYP3A4 metabolic inhibition [6]. Berberine ([C 20 H 17 ClNO 5 ] + , Figure 2) is an isoquinoline plant alkaloid with a bright yellow color that is usually found in the stem bark, rhizomes and roots of the herb Berberis [7]. The alkaloid has multiple therapeutic actions and the use of berberine has been described for almost all disorders of the body. These plants are used medicinally in all traditional medical systems, and have a history of usage in Chinese and Korean medicine dating back at least 3,000 years. Berberine extract as a crude drug has been demonstrated to have significant antimicrobial activity against bacteria, viruses, protozoans, fungi, yeast [7,8], chlamydia and helminths (worms) [9]. The drug has been used in Chinese and Indian medicines for the treatment of bacterial diarrhea, intestinal parasitic infections, and ocular trachoma infections [10]. Testing of the effects of crude drugs on isolated fungi has been performed in China for over a thousand years as a means of identifying putative efficacious preparations. According to previous reports, berberine has both antifungal [4,[11][12][13][14] and antibacterial effects [15][16][17][18] under in vitro conditions. We show here that berberine has antifungal effects on the growth of A. fumigatus, as determined by the minimum inhibitory concentration (MIC) method. We have also investigated whether berberine has a synergic effect with itraconazole in vitro when tested against A. fumigatus and whether the alkaloid berberine is a valid therapeutic agent against A. fumigatus. We have reached the conclusion that berberine and itraconazole inhibit A. fumigatus by similar mechanisms of action and are not synergistic. After 5 days the bioassays showed that with increasing concentrations of BER, the growth of A. fumigates could be significantly inhibited. When the concentration was more than 256 μg/mL, there was no colonies visible on plates (see Figure 3), and therefore we concluded that the MIC 50 was 256 μg/mL on the agar plates.

Results and Discussion
We compared the giant colonies of A. fumigatus treated with DMSO alone and treated with BER (at the 1/2 MIC 50 values of 128 μg/mL) to investigate the effect of BER on colony size (Table 2), mycelial growth and conidial pigment at 3, 5, 7 and 10 day. Colonies changed from granular to cottony, velvety, or powdery. At 3 day, the colonies were green, darkening to blue-green at 5 day, with a white apron at the colony margin. A. fumigatus on PDA at 7 days had a typical green-gray surface pigment with a suede-like surface texture consisting of a dense felt of conidiophores. A. fumigatus treated with BER exhibited smaller colony size, slower mycelial growth, and reduced conidia. These cultures also lost conidial pigment such that the conidial surface observed was white rather than green-gray ( Figure 4). Figure 5 shows the results of morphological analysis of A. fumigatus in slide culture, with notable changes occurring in various morphological features ( Figures 5B-H). These results demonstrate that BER can restrain A. fumigatus growth, development and conidial pigmentation. was treated with DMSO only (the final concentrations of DMSO less than 1%), control (−) was only a PDA without fungus. All plates were incubated at 35 °C. After 7 days, bioassays showed, with increasing concentrations of BER, it could significantly inhibit the growth of A. fumigates. The MIC was taken as the lowest concentration of the drug that showed not any fungal colonies growth on the agar plates. When the concentration was more than 256 μg/mL, there were no colonies visible on plates.   . Some mycelia were wreathed and some phialides arose circumferentially and were biseriate or uniseriate, with circumferential conidia obscuring vesicles (E, F).

Fractional Inhibitory Concentration Index
Calculation of FICI values for drug combinations is a useful indicator of the nature of functional drug interactions. FICI values between 0.5 and 4.0 indicate no interaction, whereas values below 0.5 and above 4.0 signify synergistic and antagonistic interactions, respectively. Table 1 shows FICI values for the BER-ICZ combination in each of the 42 A. fumigatus strains used in this study. All calculated values were above 0.5, demonstrating that the drug interaction was not synergistic in any strain. Furthermore, 32 of the 42 strains gave a FICI value greater than 4.0, suggesting that the interaction between BER and ICZ in these strains was mutually antagonistic (i.e., acting via identical pathways).

Assessment of Ergosterol Content
The results indicated that the fungal dry weight was 254.40 ± 0.12 mg for the control group, 150.60 ± 0.14 mg for the BER group, and 97.49 ± 0.13 mg for the ICZ group. The HPLC results suggested that the retention time of ergosterol was about 10 min whatever the extraction process used and the chromatograms in this zone were very similar ( Figure 6). The standard curve was linear over the range of ergosterol concentrations from 0.0001 to 0.01 μg/mL, r 2 = 0.99983 with a slope of 25,924.25 and passing through the origin. The assay showed the variation coefficient less than 10%. The lower limit of the detector was established at detection 0.0001 μg/mL. Under our experimental conditions, ergosterol content in the control group was 0.067 mg/mL, in the BER group it was 0.025 mg/mL, and in the ICZ group it was 0.018 mg/mL. The results show ergosterol contents of BER and ICZ group were decreased (P < 0.01). Figure 6. HPLC analysis of ergosterol in A. fumigates. Red was Control, green was BER, blue was ICZ.

Real-Time PCR
Many genes encoding proteins in the ergosterol biosynthesis pathway show differential expression levels following exposure to BER and ICZ. Seventeen representative target genes and a control gene (GAPDH) were chosen for validation with real-time PCR, and results are shown in Figure 7. Gene expression profiles with BER were similar to those with ICZ, reflecting that the actions of these two agents are largely similar. The Erg5 (Cytochrome P450 sterol C-22 desaturase), Cyp51A (14-alpha sterol demethylase Cyp51A), Cyp51B (14-alpha sterol demethylase Cyp51B) and IMP (Integral membrane protein) genes have been shown to be downregulated by medicines, and expression of these genes in A. fumigatus treated with BER was more significant than in A. fumigatus treated with ICZ (Fold-change value, Erg5 was 0.347 ± 0.018 vs. 0.509 ± 0.073, P < 0.05; Cyp51A was 0.540 ± 0.017 vs. 0.974 ± 0.042, P < 0.05; Cyp51B was 0.645 ± 0.029 vs. 0.819 ± 0.070, P < 0.05; and IMP was 0.439 ± 0.036 vs. 0.927 ± 0.022, P < 0.05). However, most other genes except Erg27 (3-keto steroid reductase) and MnSOD (Mn superoxide dismutase) that were regulated by the BER and ICZ to similar degrees or were more strongly downregulated by ICZ (P < 0.05 or P < 0.01). and ▲▲ (P BER < 0.01), ★★ (P ICZ < 0.01). The fold expression in A. fumigatus treated with BER or ICZ as compared with that in untreated control. The calibrator was non-treatment, for which the fold expression was 1.0.

Discussion
BER is an alkaloid isolated from Chinese and Korean medicinal plants that notably inhibits the growth of a wide range of Candida species [19][20][21]. BER is clinically used in diseases such as urinary tract infections and conjunctivitis caused by bacteria like Escherichia coli, Staphylococcus aureus and Shigella dysenteriae. In recent years, research has shown that BER can also be used to treat fungal and viral infections and other diseases such as diabetes, hypertension, hyperlipidemia and arrhythmia [22][23][24][25][26][27][28]. Recently, investigators have used BER combined with amphotericin B to treat disseminated candidiasis in a mouse model, and others have investigated synergistic interactions of BER with fluconazole against fluconazole-resistant clinical isolates of Candida albicans [4,22,29,30].
Up to now, there has been no research on BER inhibition of A. fumigatus, or on the potential benefits of the combined effects of BER and ICZ. Our current study is the first to apply BER to clinically isolated A. fumigatus strains, and the first to use BER and ICZ in a combination treatment. Our results show that BER can inhibit the production of conidial pigment in A. fumigatus and consequently cause aberrations in A. fumigatus morphology and a reduction in its sporulation rate.
It is well known that drugs respond very differently in different background media. So we gave details as to how the MIC was obtained for BER on potato dextrose agar. We also show that BER and ICZ are mutually antagonistic in terms of their ability to inhibit A. fumigatus isolated from clinical patients. Furthermore, we have investigated the biomechanisms by which BER and ICZ inhibit the A. fumigatus ergosterol biosynthesis pathway. Ergosterol, one of the most basic components in fungal membranes, is involved in a multitude of biological functions, for instance, membrane fluidity regulation, distribution and activity of integral proteins and control of the cell cycle [31]. Indeed, controlling ergosterol and its biosynthetic pathway can impact fungal growth. Knowledge of the effects on the ergosterol biosynthesis in A. fumigatus has been key in the development of many antifungal drugs in clinical use and can hopefully aid the design of novel drugs [32]. Previous studies have shown that the majority of triazole drugs such as ICZ inhibit A. fumigatus isolates by targeting the ergosterol biosynthesis pathway [33]. In the present experiments, we found BER can inhibit ergosterol like ICZ. The results from our gene expression experiments also indicate that BER significantly inhibits gene expression in the A. fumigatus ergosterol biosynthesis pathway and that BER is significantly more effective than ICZ at inhibiting expression of the Erg5, Cyp51A, Cyp51B and IMP genes, which are related to pigment production in A. fumigatus conidia. It could be that inhibition of these genes causes albino characteristics in A. fumigatus conidia. The IMP gene is closely related to cell wall biosynthesis [34] and, by inhibiting its expression, BER may thus inhibit biosynthesis of fungal cell walls and cause growth and developmental aberrations in A. fumigates [35].

Strains, Media and Conditions
A. fumigatus strain IFM 40808 was isolated from the lung of a 54-year-old female Japanese patient with invasive aspergillosis, and provided from the Medical Mycology Research Center, Chiba University, Japan. The other A. fumigatus strains used in this study (41 strains Strains were cultured on potato dextrose agar medium (PDA; Becton Dickinson Co., Sparks, MD, USA) in C-shaped streaks at 35 °C for 3-4 days. Conidial suspensions were maintained in 0.9% NaCl-Tween 80 (0.01%) in sterile water at room temperature.

Antifungal Drug Susceptibility Testing
The minimum inhibitory concentration (MIC) of itraconazole (ICZ) (Sigma, Sigma-Aldrich Co., Louis, MO, USA) and berberine (BER) (Aldrich, Sigma-Aldrich Co., Louis, MO, USA) were tested following the Clinical and Laboratory Standards Institute document M38-A2 [37], with end points measured at 48 h (All drugs dissolved in dimethylsulfoxide (DMSO) (Sigma) and an initial ICZ concentration of 1,600 μg/mL，an initial BER concentration of 20,480 μg/mL). ICZ and BER were used over concentration ranges of 0.03-16 μg/mL and 1-512 μg/mL. A. fumigatus spores were harvested from the stock cultures and their concentration adjusted to 1.0 × 10 6 colony-forming units (CFU)/mL by 0.9% NaCl-Tween80 sterile water. Then spores were spotted onto the centre of PDA plates containing different concentrations of BER (512, 256, 128, 64, 32, 16, 8 and 4 μg/mL，BER was dissolved in DMSO), and control group drug was used DMSO only (the final concentrations of DMSO less than 1%). The plates were incubated at 35 °C, and growth of filamentous fungi on plates was monitored after 7 days. The MIC 50 was taken as the lowest concentration of the drug that showed not any fungal colonies growth on the agar plates [38].

Strain Culture
Giant colonies of A. fumigatus IFM 40808 treated without and with BER (at the 1/2 MIC 50 of plate) were compared for size of colony, mycelial growth and conidial pigment at 3, 5, 7, 10 day. We also examined the intact structures of normal and BER strains at 3 day using an Axiovert 200 MAT microscope (Zeiss, Freiberg, Germany) using slide culture techniques described previously [39].

Fractional Inhibitory Concentration Index
Drug interactions were tested using the checkerboard microdilution method [40]. The checkerboard tests were performed by a broth microdilution reference procedure at a final inoculum of 1.0 × 10 6 -5.0 × 10 6 CFU/mL, using RPMI 1640 medium (Roswell Park Memorial Institute, Sigma, Sigma-Aldrich Co., Louis, MO, USA) buffered with 0.165 M MOPS (3-(N-morpholino)propanesulfonic acid). Final concentrations ranged from 0.036 to 0.5 μg/mL for ICZ, and 1 to 128 μg/mL for BER. Ninety-six shadow masks were incubated at 35 °C for 48 h prior to analysis. The fractional inhibitory concentration index (FICI) is the sum of the MIC of each drug in combination divided by the MIC of the drug used alone. A FICI value ≤0.5 is classed as 'synergy', whereas a FICI value >4.0 denotes 'antagonism'. Values >0.5 but ≤4.0 indicate that there is 'no interaction' between the drugs [41].

Total Ergosterol Extraction
The well-developed A. fumigatus sample was cultured on a slant which was rinsed with 10 mL sterile water (0.9% NaCl-Tween80). Conidial suspension (10 6 CFU/mL) was mixed with RPMI 1640 medium at 1:10. The media joined the MIC 50 of BER and ICZ respectively (All drugs were dissolved in DMSO), and compared with control group (receiving DMSO only). The fungi were incubated at 35 °C, at 110 rpm shaking. A. fumigatus were grown separately in 150 mL flasks containing adequate medium. After 3 days, mycelia were collected by filtration and washed with sterile phosphate-buffered saline (PBS, pH = 7.4), and then freeze-dried (LABCONCO FreeZone Triad Freeze Dry Systems, Kansas City, MO, USA) and weighted (fungal dry weight).
To a weight of dried mycelia (50 mg) was added 25% alcoholic KOH (potassium hydroxide solution) (3:2 methanol-ethanol, 25 mL) and then the mixture was homogenised by vortexing for 2 min. The mixed culture was incubated in a 90 °C water bath for 2.5 h. After cooling to room temperature, the saponified mixture was extracted in a fume hood with ether (20 mL), and vigorously vortexed for 15 min at room temperature. After 2.5 h the upper ether layer was transferred to a clean glass tube and evaporated with a centrifugal evaporator (EYELA, CVE-2000, Tokyo, Japan) at 20 °C. These dry residues were re-dissolved again in 1 mL of methanol and used for high performance liquid chromatography (HPLC) analysis.

Real-Time PCR
Using A. fumigatus IFM 40808, we compared the ergosterol biosynthesis pathway of A. fumigatus treated with either BER or ICZ (each at the MIC 50 i.e., the MIC at which 50% of isolates are inhibited). Conidial suspensions were maintained at 1.0 × 10 6 CFU/mL and inoculated in RPMI 1640 medium with DMSO, 1640 medium with BER (at the MIC 50 ), 1640 medium with ICZ (at the MIC 50 ). Cultures were incubated aerobically at 35 °C/110 rpm for 96 h, and examined daily before being collected and freeze-dried. Total RNA (messenger RNA) was prepared using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) and VERSA Mini Nucleic Acid Extraction Workstation (Aurora Biomed, Vancouver, BC, Canada). The RNA was then reverse transcribed using the Quantitect Reverse Transcription Kit (Qiagen). The resultant cDNAs (complementary DNA) were subsequently analyzed by quantitative PCR (polymerase chain reaction) using a SYBR green master mix in an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA, USA) following the manufacturer's recommended protocols. Table 3 shows the primers used in the real-time PCR experiments. Amplifications were performed with the following parameters: an initial preheat at 95 °C for 10 s, followed by 45 cycles at 95 °C for 5 s, 60 °C for 32 s, to detect and quantify the fluorescence at a temperature above the denaturation of primer-dimers. Once amplifications were completed, melting curves were obtained to identify PCR products. For each sample, PCR amplifications to quantify the expression of the constitutively-expressed GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene were performed as a reference using the primer pair F-GAPDH-F + F-GAPDH-R (Table 3) [43]. The experiment was repeated five times. The expression of each tested gene in the BER-treated or ICZ-treated sample relative to that of untreated sample was calculated using the 2 −ΔΔCt method [44][45][46].

Statistical Analysis
Statistical analysis was performed with SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). For all results analyses, Student's t test was used. P < 0.05 was considered statistically significant.

Conclusions
In conclusion, our study shows that while BER is effective in restraining the growth of several different A. fumigatus strains, it is not synergistic with ICZ against A. fumigatus isolated from clinical patients. Therefore, it is not advised that BER and ICZ be used at the same time in the clinic. This observation is the first report showing evidence of mutual antagonism between BER and ICZ. BER significantly inhibits gene expression in the ergosterol biosynthesis pathway of A. fumigatus like ICZ.