Evaluation of antifungal effect and toxicity of xanthyletin and two bacterial metabolites against Thai isolates of Pythium insidiosum

Pythiosis is a harmful disease caused by Pythium insidiosum, an aquatic oomycete. Therapeutic protocols based on antifungal drugs are often ineffective because the cytoplasmic membrane of P. insidiosum does not contain ergosterol. Therefore, the treatment of pythiosis is still challenging, particularly making use of natural products and secondary metabolites from bacteria. In this study, xanthyletin and substances obtained from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 exhibited anti-P. insidiosum activity and, moreover, xanthyletin was non-toxic against human cell lines. The hyphae of P. insidiosum treated with these three substances exhibited lysis holes on a rough surface and release of anamorphic material. Therefore, xanthyletin could be considered a promising alternative agent for treating cutaneous pythiosis in the near future.


Results
Crude extracts and fractions from P. stutzeri ST1302 and K. pneumoniae ST2501 and their anti-P. insidiosum activity. Crude extracts from P. stutzeri ST1302 (brown liquid) and K. pneumoniae ST2501 (dark brown liquid) showed anti-P. insidiosum activity as screened by the disc diffusion method. After that, the crude extracts were fractionated by activity-guided separation liquid column chromatography. The crude extracts from K. pneumoniae ST2501 were divided into 5 fractions; fraction number 1 was brown semisolid, fraction number 2 was brown oil, fraction number 3 was brown solid, fraction number 4 was black oil and fraction number 5 was black semisolid. The crude extracts from P. stutzeri ST1302 were divided into 14 fractions; fraction no. 1 was yellow oil, fraction no. 2 was orange oil, fractions no. 3, 4, 5, 7 and 9 were deep yellow solid, fraction no. 6 was yellow solid, fractions no. 8, 12, 13 and 14 were brown oil, and fractions no. 10 and 11 were white solid. Fractions no. 1 to 4 of the crude extracts from K. pneumoniae ST2501 and fractions no. 3, 4, 6, 9 and 12 of the crude extracts from P. stutzeri ST1302 exhibited anti-P. insidiosum activity ( Table 1). The strongest anti-P. insidiosum activity (the largest inhibition zone) was exhibited by fraction no. 6 of the crude extract from P. stutzeri ( Fig. 1) eluted with 20% methanol in dichloromethane and fraction no. 1 of the crude extract from K. pneumoniae eluted with 100% dichloromethane. For further testing, only two fractions mentioned above with the strongest anti-P. insidiosum activity together with xanthyletin were chosen.

Minimum fungicidal concentrations (MFCs) determined by broth dilution susceptibility testing.
The MFCs of xanthyletin, fraction no. 1 of the crude extract from K. pneumoniae ST2501, and fraction no. 6 of the crude extract from P. stutzeri ST1302 against 11 strains of P. insidiosum isolated from human pythiosis in Thailand were 0.003, 1.563-3.125, and 3.125 mg/mL, respectively (Table 1).
Hyphal morphology evaluated by scanning electron microscopy. Figure 2 shows morphological hyphae of P. insidiosum (negative control; P. insidiosum survival) and hyphae changes when P. insidiosum was treated with thimerosal (positive control; P. insidiosum death) and treated with three anti-P. insidiosum substances. The negative control showed cylindrical hyphae with a smooth surface ( Fig. 2A). Hyphae from the positive control were with a rough surface and high amount of anamorphic material released from the organism (Fig. 2B). Morphology of hyphae treated with three anti-P. insidiosum substances including xanthyletin (Fig. 2C), fraction no. 6 of the crude extract from P. stutzeri ST1302 (Fig. 2D) and fraction no. 1 of the crude extract from K. pneumoniae ST2501 (Fig. 2E) The crude extract from Pseudomonas stutzeri ST1302  www.nature.com/scientificreports www.nature.com/scientificreports/ Cytotoxicity assay. The toxicity testing of xanthyletin and extracts from P. stutzeri ST1302 and K. pneumoniae ST2501 covered the whole ranges of dilutions tested for MFC determination by the broth dilution method. The effect of anti-P. insidiosum substances on NHDF cells was dose-dependent. The IC50 of xanthyletin, fraction no. 6 of the crude extract from P. stutzeri ST1302 and fraction no. 1 of the crude extract from K. pneumoniae ST2501 were 0.11, 4.69, and 7.81 mg/mL, respectively (Fig. 3). Further, at the MFC of xanthyletin, NHDF cells showed more than 90% viability, compared to only 55% and 70% viability in case of the MFCs of the tested fractions from P. stutzeri and K. pneumoniae, respectively.

Discussion
It is well known that the therapy of pythiosis has posed a challenge in recent decades as the current treatment options are highly variable and often lead to failure 14 . Therefore, many studies have searched for antimicrobial agents, natural compounds or secondary metabolites from microorganisms against P. insidiosum and results of in vitro testing are quite promising 6,7,[15][16][17][18][19][20][21] . The present study evaluated antimicrobial effects against P. insidiosum of xanthyletin and two fractions of the crude extracts from P. stutzeri ST1302 and K. pneumoniae ST2501. The MFC results of fraction no. 1 from the crude extract from K. pneumoniae against P. insidiosum strains SIMI-6666, SIMI-2989-42, SIMI-7873 and SIMI-7874 at higher concentrations than the other strains correlated with the previous study 21 . Xanthyletin showed the best anti-P. insidiosum activity and the MFC of 0.003 mg/mL was well correlated with compound 5 from Scaevola taccada fruits exhibiting good anti-P. insidiosum activity (the minimum inhibitory concentration 0.005 mg/mL) 6 . SEM analysis of P. insidiosum treated with anti-P. insidiosum substances revealed lysis holes on a rough surface and anamorphic material released from hyphae, suggesting effects on protein composition in the cell wall or cell membrane of this oomycete. Our previous study using Fourier transform infrared spectroscopy found anti-P. insidiosum activity of xanthyletin and secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501, characterized by clearly changed proteins in P. insidiosum 21 . Thimerosal is a well-known compound used as a preservative in various cosmetics, vaccines and drug products because of its bactericidal and antifungal properties including anti-P. insidiosum activity. However, it is toxic to humans, causing mortality in the fetus, birth defects and neurodevelopmental disorders as evidenced by a case of a pregnant woman who received a vaccine preserved with thimerosal. Besides, cellular apoptosis and abnormal functions of T-and B-cells that affected cytokine production, cell growth and proliferation following chronic exposure to low levels of methyl mercury have also been reported 22,23 . Recently, we used 0.02% (w/v) thimerosal as a positive control because it can kill P. insidiosum although this amount is not suitable for use in humans and animals. The mechanism of its action is not fully understood yet and we suspected it to be related to interactions with some proteins in P. insidiosum leading to the death of the microorganism 21 . Xanthyletin is a pyranocoumarin that has been shown to be cytotoxic in vitro against tumor cells such as Caco-2, HCT-8 and HEp-2 cell lines using an MTT assay 24 . Moreover, xanthyletin showed cytotoxic activity against human cancer HeLa cells without toxicity to normal cells 9 . The NHDF cell culture was selected for cytotoxicity assay as these cells are a common part of the human dermis and toxic effects of drugs on them are undesirable. Moreover, one of the important clinical signs of human pythiosis is skin damage. In the present study, xanthyletin at 0.003 mg/mL showed the best anti-P. insidiosum activity and exhibited low toxicity for NHDF cells. However, this study evaluated anti-P. insidiosum activity against only 11 strains of P. insidiosum isolated from pythiosis patients in Thailand. Therefore, further studies www.nature.com/scientificreports www.nature.com/scientificreports/ on geographically and genetically diverse P. insidiosum strains and anti-P. insidiosum activity against pythiosis in animal models should be conducted to confirm the properties of these anti-P. insidiosum substances.

Conclusion
As seen from the evaluation of anti-P. insidiosum effects and toxicity of xanthyletin and secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501, xanthyletin showed excellent anti-P. insidiosum activity as well as the lowest toxic effects in vitro on NHDF cells. Therefore, it could be considered a promising alternative agent for treating cutaneous pythiosis in the near future.

Material and Methods
Microorganisms. P. insidiosum isolates (n = 11) from human pythiosis were identified by molecular analysis in a study by Chaiprasert et al. 25 . The cultures were maintained on Sabouraud Dextrose Agar (SDA; Himedia) at room temperature and subcultured once a month. According to a study by Thongsri et al., they were rechecked by their morphology on the SDA at 25 °C, microscopically by detection of perpendicular sparsely septate hyphae and induced zoospore formation and by PCR identification 7 .

Substances. Xanthyletin
Preparation and fractionation of crude extracts from P. stutzeri and K. pneumonia. The inoculations from P. stutzeri ST1302 and K. pneumoniae ST2501 were prepared according to McFarland Standard No.1 (3×10 8 CFU/mL) in saline; 700 µL of this concentration were inoculated into 700 mL of Brain Heart Infusion broth (BHI; Himedia) and incubated in a rotary shaker (200 rpm, 37 °C) for 3 days. Each culture broth was then centrifuged and the supernatant was filtered through a membrane filter (Millipore). The cell-free filtrate was concentrated ten-fold in a rotary evaporator (Rotavapor R-210, Büchi) and mixed three times with a two-fold volume of dichloromethane (Thermo Fisher Scientific) in case of P. stutzeri or with ethyl acetate (Thermo Fisher Scientific) in case of K. pneumoniae. Both dichloromethane and ethyl acetate layers were dried with anhydrous Na 2 SO 4 (Merck) and concentrated in a rotary evaporator. Then the crude extracts were fractionated by activity-guided separation liquid column chromatography using a protocol described by Wittayapipath et al. 21 .
Screening for anti-P. insidiosum activity by the disc diffusion method. Xanthyletin and the crude extracts from P. stutzeri and K. pneumoniae were tested for anti-P. insidiosum activity using the disc diffusion method. Stock solutions of each testing solution were prepared as 500 mg/mL. Paper discs (6 mm in diameter; Gibthai, Thailand) were placed onto SDA plates with P. insidiosum aged 2 days grown on the plates. Twenty microliters of testing solutions were applied to the discs (dichloromethane and ethyl acetate were used as controls) and then the testing plates were stored at room temperature for 2 h in a laminar flow biosafety cabinet to test the solution diffusion. Subsequently, they were incubated at 37 °C for 3, 6 and 9 days and inhibition zones were measured when the growth of P. insidiosum reached the control discs 21 .
Broth dilution susceptibility testing. The method by Trolezi et al. was used with some modifications 20 .
Briefly, blocks measuring 5 mm in diameter were cut out from SDA plates with P. insidiosum cultures with a cork borer. These mycelia-containing blocks were transferred into 1.95 mL of Sabouraud Dextrose Broth (SDB; Himedia) and incubated at 37 °C for 3 days. Dilutions of xanthyletin were prepared from 0.100 to 0.001 mg/mL, dilutions of P. stutzeri ST1302 and K. pneumoniae ST2501 crude extracts from 12.5 to 0.1 mg/mL by double dilution. Each 50 μL of diluted testing solutions were added to the cultures and incubated at 37 °C for 24 h. A P. insidiosum block in 2 mL of SDB was incubated at 37 °C for 4 days as a growth control. After expiration of the incubation time, the P. insidiosum agar blocks with mycelium were placed on SDA plates and incubated at 37 °C for 3, 6 and 9 days (depending on the growth speed) to evaluate the hyphal growth and determine the minimum fungicidal concentrations (MFCs). The MFCs were assessed as the lowest concentration of the solution without any apparent hyphal growth. All tests were performed in triplicate.
Scanning electron microscopy of the hyphal morphology. The protocols described by Trolezi et al. 20 and Mendoza et al. 3 were followed with some modifications. For SEM analysis, hyphae in blocks from each testing solution evaluated as MFCs were selected. P. insidiosum hyphal fragments from the growth control in broth dilution susceptibility testing served as a negative control, that is the agent survived. By contrast, P. insidiosum hyphal fragments treated with 0.02% (w/v) thimerosal (Sigma-Aldrich) were used as a positive control. Thimerosal is well known as a preservative in various cosmetics, vaccines and drug products to prevent harmful contamination with microorganism. Therefore, thimerosal completely killing P. insidiosum was used as a positive control in this study. Hyphal fragments were collected and washed three times in 0.1 M phosphate buffer solution (PBS). Then, they were fixed in 2.5% glutaraldehyde (Merck) in 0.1 M PBS at 4 °C for 1 h and washed in the same buffer. Subsequently, the samples were dehydrated with a graded ethanol series. The Emitech K500X sputter coater (Quorum Technologies) was used for gold sputtering of SEM samples. Finally, the hyphal morphology was evaluated by the S-3000N scanning electron microscope (Hitachi). Cytotoxicity assay. Xanthyletin and two anti-P. insidiosum substances from P. stutzeri ST1302 and K. pneumoniae ST2501 were evaluated for cytotoxicity using NHDF cell lines (Promocell). A colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) was applied to investigate the cytotoxicity 26,27 . Briefly, NHDF cell lines were counted with the Neubauer hemocytometer using trypan blue solution (10 4 cells; 100 μL/well). Then, the cells were cultured in the wells of 96-well flat-bottom microplates (Thermo Fisher Scientific) containing 100 μL of Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% solution of antibiotics (penicillin and streptomycin; Gibco) at 37 °C with 5% CO 2 and 95% humidity for 24 h. Subsequently, 100 μL of each anti-P. insidiosum substance double diluted with fresh medium was added to each well with the final concentrations in the wells ranging from 0.125 to 0.004 mg/mL in case of xanthyletin and from 12.5 to 0.391 mg/mL in case of both secondary metabolites from P. stutzeri and (2020) 10:4495 | https://doi.org/10.1038/s41598-020-61271-0 www.nature.com/scientificreports www.nature.com/scientificreports/ K. pneumoniae. The plates were then incubated under the same conditions. Further, the solutions in the wells were carefully discarded and cells were washed with PBS. Then, the MTT solution (0.5 mg/mL in PBS, Invitrogen) was added at an amount of 100 μL each into the wells and the mixture was kept at 37 °C with 5% CO 2 and 95% humidity for 4 h. After removal of the supernatant, the formazan crystals were dissolved by adding 100 μL of dimethyl sulfoxide per well and the absorbance was determined at a wavelength of 570 nm. The experiment was performed in triplicate and repeated three times under the same conditions. The rate of cytotoxicity was determined using the following formula: