In vitro and ex vivo anti-Pythium insidiosum potential of ozonated sunflower oil

Abstract

This study sought to evaluate the in vitro and ex vivo susceptibility of Pythium insidiosum to ozonized sunflower oil (OSO) and verify the morphological alterations of OSO-exposed hyphae. Susceptibility assays were performed according to the broth microdilution protocol M38-A2/CLSI, and the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Non-ozonated sunflower oil (SO) was used as the oil control. Additionally, kunkers from equine pythiosis were exposed to OSO. Damages caused by OSO and SO on P. insidiosum hyphae ultrastructure were verified using scanning electron microscopy. The MIC range for OSO was 7000 to 437.5 mg/mL, and the values for SO were higher, ranging from 56000 to 14000 mg/mL. The MOC was equal to MIC for both oil formulations. The OSO fully inhibited the oomycete growth from kunkers, although there was P. insidiosum growth in the kunker control in 24 h of incubation. The SEM analyses showed that both OSO and SO caused morphological alterations in P. insidiosum hyphae, highlighting the presence of cavitation along the hyphae with loss of continuity of the cell wall, which was more evident in the OSO-treated hyphae. The OSO had the best oomicidal activity, leading us to believe that our findings may support future research containing this formulation to be applied in integrative medicine protocols to control pythiosis in animals and humans.

Introduction

Pythium insidiosum is an oomycete that inhabits aquatic environments in tropical, subtropical, and temperate climates. It is the main agent responsible for pythiosis (a rapidly evolving and life-threatening disease) in mammals, with horses, humans, and dogs being the most affected species [1, 2]. P. insidiosum is a fungal-like organism that shares some characteristics with true fungi, including somatic structures formed by hyphae and asexual spore production (zoospores). However, it differs from these eukaryotes by having an incomplete sterol biosynthesis pathway and lacking ergosterol in its cytoplasmic membrane [3, 4]. Various difficulties in treating infections caused by P. insidiosum have been reported in the affected species when treated with antifungal drugs that inhibit ergosterol synthesis, such as azoles and amphotericin B [1–3]. This concerning issue has led researchers in recent decades to seek compounds with anti-P. insidiosum activity, including repositioned drugs [5–7], natural bioactive compounds [8–15], and nanostructured compounds [15–17]. Although some of these evaluated compounds have demonstrated in vitro and in vivo activity against this oomycete pathogen, there is still no standardized therapeutic protocol to treat the disease in different susceptible hosts, thereby justifying the continuity of research for new antimicrobial compounds for potential use in integrative pythiosis treatment.

In this sense, ozone (O₃), a highly reactive oxidizing molecule formed by triatomic oxygen atoms, has been investigated as a potential therapeutic agent. The medical use of the gaseous mixture of O₃/O₂ (95–99.95% oxygen and 0.05–5% ozone), called ozone therapy, is widely employed as a complementary treatment in various pathological processes [18]. Ozone can be administered systemically and locally; in the latter, it can be used as ozonated water, an O₂-O₃ gas mixture, or ozonated oil [19].

The presence of fatty acids in the composition of vegetable oils, including sunflower, canola, olive, and coconut oil, allows the ozone molecule to be incorporated into the oil, generating compounds such as aldehydes, peroxides, and carboxylic acids [20–22]. These compounds are responsible for the antimicrobial action of ozonized oils against bacteria [23], fungi [24], viruses [25], and oomycetes [26].

Therefore, this study sought to evaluate the in vitro and ex vivo susceptibility of P. insidiosum to ozonated sunflower oil (OSO) and verify morphological changes in OSO-exposed hyphae.

Material and methods

Pythium insidiosum isolates

Forty-eight P. insidiosum isolates, including nine standard strains (CBS101555, CBS57585, CBS57685, and CBS70283 from equines; CBS119453, CBS119454, CBS119455, and CBS673.83 from humans, and CBS77784 from mosquito larvae) and clinical isolates (SISGEN A139392) from equines (n = 37) and canine (n = 2) were used. The isolates belonged to the Mycology Laboratory of the Universidade Federal de Pelotas (UFPel), Brazil. All clinical isolates were morphologically and molecularly characterized according to Azevedo et al. [27] and Weiblen et al. [28].

Inoculum preparation

The inoculum was prepared from a mycelial culture of P. insidiosum according to Fonseca et al. [29]. The inoculum suspension was obtained from a P. insidiosum culture on 0.1% yeast agar at 37 ºC/96 h. The culture was then hydrated with 10 mL of sterile distilled water, and the mycelium was scraped with a sterilized scalpel blade. The solution was transferred to a tube, and the inoculum was adjusted to a transmittance of 80–85% at 530 nm. Subsequently, the inoculum was diluted at 1:10 in RPMI 1640 medium and buffered at pH 7.0 with 0.165 M MOPS for carrying out susceptibility tests. To test the viability of the inoculum, an aliquot of 100 μL was added to 900 μL of Sabouraud broth and incubated at 37ºC for 48 h.

Susceptibility assays

In vitro susceptibility assay

The susceptibility assays were performed according to the broth microdilution protocol (M38-A2) [30] adapted by Pereira et al. [31]. The sunflower oil (SO) (peroxide index 0.6 mEq/kg) and ozonized sunflower oil (OSO) (peroxide index ~ 600 mEq/kg) were obtained commercially (Ferquima Ltd., Brazil and Ozone&Life, Brazil, respectively). The chemical composition of the vegetable oils was previously determined by the manufacturer.

The susceptibility tests were carried out by diluting the oils in RPMI 1640 buffered medium at pH 7.0 with 0.165 M MOPS, and added 50 μL of Tween 80. Both oils concentrations ranged from 56000 to 109.37 µg/mL. Aliquots (100 μL) of each dilution were dispensed into each microplate well and mixed with an equal inoculum volume. A positive control (inoculum + RPMI) and a negative control (oil + RPMI) were used for each test. All assays were performed in quadruplicate. The minimum inhibitory concentration (MIC) was determined after 48 h of incubation in an orbital shaker under constant stirring (60 rpm) at 37 °C and consisted of visual observation of 100% mycelium growth inhibition. The concentrations capable of inhibiting 50 and 90% of the isolates were named MIC₅₀ and MIC₉₀, respectively. Concentrations above the MIC were used to determine the minimum oomycidal concentration (MOC). After the MIC determinations, 100 μL aliquots were taken from each well without growth and added to tubes containing 900 μL of Sabouraud broth. The tubes were incubated at 37 °C/48 h. The lowest concentration that did not show growth was considered the MOC.

Ex vivo susceptibility assay

Four samples of kunkers were obtained from equines (n = 4) with cutaneous/subcutaneous pythiosis in Rio Grande do Sul State, Brazil. These kunkers were removed from the clinical lesions with the help of surgical forceps and immediately packed in flasks containing sterile distilled water with ampicillin (100 µg/mL). Thereafter, the kunkers were processed according to Fonseca et al. [32]. In summary, the kunkers were fragmented with a sterile scalp and washed for 20 min in an antibiotic solution containing benzathine penicillin G (10,000,000 IU), procaine penicillin G (10,000,000 IU), and dihydrostreptomycin (20 g). Afterward, the fragments were washed in sterile distilled water, and ten pieces were transferred to Petri dishes containing 0.1% yeast agar. The surface of each kunker fragment was covered with OSO (50 μL). For the control, ten fragments of kunkers were placed on the surface of yeast agar 0.1% without OSO. The assays were performed in duplicate at 37 °C/96 h.

Scanning electron microscopy (SEM)

The SEM analysis was performed according to Valente et al. [33]. P. insidiosum (CBS 101555) hyphae were treated with OSO and SO at sublethal doses of 1750 and 28000 mg/mL, respectively, and the mycelial growth control (without treatment) was fixed in 1 mL of glutaraldehyde (2.5%) at 4 °C/48 h. The mycelium was washed in sterile distilled water (dH₂O) and subjected to several baths (20 μL/20 s each) containing increasing concentrations of ethanol (30, 50, 70, 95, and 100%). The hyphae were then placed on microscope coverslips and dried in an oven at 37 °C/24 h. Lastly, the samples were transferred to stubs, covered with gold–palladium (60 s, 1.8 mM, 2.4 kv), and visualized at 15 kv at 500–1500 × magnitude.

Statistical analyses

The MIC values were submitted to a normality test, and the response variable did not show normality, so data were subjected to Tukey's test. Analyses were performed using the SAS statistical software (version 9.4) assuming a 5% probability.

Results

The in vitro susceptibility results showed that the anti-P. insidiosum activity of the OSO (MIC range: 7000–437.5 mg/mL) was superior (p < 0.05) to the SO (MIC range: 56000–14000 mg/mL). The MIC₅₀ and MIC₉₀ values of the OSO were 1750 and 7000 mg/mL, respectively; for the SO, these values were 28000 mg/mL (MIC₅₀) and 56000 mg/mL (MIC₉₀). The MOC results were identical to the MICs for both compounds (Table 1).

Table 1.

In vitro activity of ozonized sunflower oil (OSO) and sunflower oil (SO) against Pythium insidiosum isolates (n = 48)

Number of isolates (%) with the following MIC1/MOC2									MIC Range	MIC503	MIC904	GM5
(mg/mL)
	56,000	28,000	14,000	7000	3500	1750	875	437.5
OSO	-	-	-	7(14.9)	16(33.3)	15(31.3)	6(12.5)	4(8.3)	7000–437.5	1750	7000	2204.8
SO	12(25)	28(58.3)	8(16.7)	+	+	+	+	+	56,000–14000	28,000	56,000	29,664.9

¹Minimum inhibitory concentration, ²Minimum oomicidal concentration, ³Minimal concentration to inhibit the growth of 50% of isolates, ⁴Minimal concentration to inhibit the growth of 90% of isolates, ⁵Geometric mean. ( +) All isolates (n = 48) grew at this concentration. (-) All isolates (n = 48) had their growth inhibited at this concentration

In the ex vivo susceptibility tests, evaluation of the P. insidiosum mycelial growth from the OSO-covered kunkers revealed that the ozonated oil completely inhibited the growth of the oomycete. Nevertheless, the fragments of kunkers without OSO (control) showed P. insidiosum mycelial growth within 24 h of incubation (Fig. 1).

Fig. 1.

Ex vivo susceptibility assay. A Control (kunkers fragments not treated with ozonized sunflower oil (OSO): abundant mycelial growth of P. insidiosum is observed from the kunkers fragments. B (kunkers fragments treated with 50 μL of OSO): is evidenced the inhibition of mycelial growth of P. insidiosum from kunkers. Cultures in 0.1% yeast agar and incubated at 37ºC by 48 h

Figure 2 shows the images of P. insidiosum (CBS 101555) hyphae untreated and treated with SO and OSO obtained by SEM. The untreated hyphae (control) did not show changes, with a turgid appearance and homogeneous surface (Fig. 2A and B). The hyphae of the SO-treated oomycete presented a dehydrated appearance, showing surface roughness and areas with increased electron density (Fig. 2C); some hyphae showed cavitation and loss of cell wall continuity (Fig. 2D). Moreover, we observed that the OSO-treated hyphae showed intense dehydration, with withered, brittle, and twisted aspects and cavitation along the cell wall (Fig. 2E and F).

Fig. 2.

Ultrastructure of Pythium insidiosum (CBS 101555) visualized in scanning electron microscopy (SEM). A and B Control (untreated P. insidiosum): intact hyphae with turgid appearance and homogeneous surface. C and D P. insidiosum: hyphae treated with 28000 mg/L sunflower oil. C. Dehydrated hyphae showing surface roughness and areas with increased electrodensity; D. Dehydrated hyphae exhibiting cavitation (arrow) and loss of cell wall continuity. E and F P. insidiosum: hyphae treated with 1750 mg/L ozonated sunflower oil. E. Dehydrated and twisted hyphae showing multiple electrodensity points due to cavitation along the cell wall; F. Hyphae showing cavitation in the cell wall (arrow). Hyphae with distended and thinned cell membrane, leaving only remnants of the integral wall (arrowhead). Dehydrated and withered hyphae (white arrow)

Discussion

Plant products have seen widespread use and have been vastly studied in the microbiological field. In this regard, the interest in vegetable oils has increased given their potential antimicrobial role, enabling one to increase their properties by adding ozone. In the last decades, ozonated vegetable oils have been evaluated for their antibacterial and antifungal activity, especially in microorganisms resistant to conventional antimicrobial agents. Additionally, ozonated vegetable oils have demonstrated efficacy in healing cutaneous wounds [22].

In our study, the OSO showed superior anti-P. insidiosum activity, with MIC ranging from 7000 to 437.5 mg/mL, while the SO presented MIC ranging from 56000 to 14000 mg/mL. Ferreira et al. [26] evaluated the antimicrobial activity of an OSO with a low peroxide index (< 500 mEq/kg⁻¹) and a high peroxide index (> 600 mEq/kg⁻¹) and a SO against four P. insidiosum isolates; they reported that only OSO with high peroxide index could completely inhibit the radial mycelial growth of P. insidiosum. In the present study, the unozonized SO inhibited the growth of the oomycete in microdilution assays, differing from the findings of Ferreira et al. [26]. This difference is likely due to the different methods employed and the number of isolates evaluated in both studies. Furthermore, despite many authors correlating the antimicrobial activity of ozonated oils to the peroxide index and believing that activity is greater when the peroxide index is higher [22, 26], some investigations have demonstrated that an increase in peroxide index does not affect oil activity [22, 34]. This is probably because the antimicrobial mechanism of ozonated vegetable oils is related to oxidizing species [35, 36]. In addition, the presence of fatty components in the composition of vegetable oils is responsible for incorporating the ozone molecule into the oil, where ozone reacts with carbon chains, producing new molecules and the formation of new compounds, such as aldehydes, peroxides, and carboxylic acids, which are responsible for the antimicrobial action of these oils [20–22].

Our study demonstrated the superior in vitro oomicidal activity of the OSO and that this formulation could completely inhibit P. insidiosum growth from kunkers extracted from equine pythiosis cutaneous lesions. This finding is exciting because kunkers are formed inside the large tumor masses that characterize equine pythiosis lesions and are constituted by viable clusters of P. insidiosum hyphae covered by inflammatory cells [37]. Thus, the high proportion of unsaturated fatty acids in sunflower oil facilitates absorption and penetration into the skin [26], and associated with its anti-P. insidiosum activity, allowing us to infer that the integrative topical use of OSO in cutaneous wounds of pythiosis may be an excellent tool in wound recovery. Furthermore, Di Filippo et al. [38] demonstrated that topical application of ozonated sunflower seed oil accelerated acute cutaneous wound repair in horses, preventing tissue hypergranulation and infections. In this sense, our research group has routinely integrated topical OSO into equine cutaneous pythiosis therapeutic protocols, especially those involving surgery, immunotherapy, and systemic and local ozone therapy, and the data obtained have been encouraging (unpublished data). These results leverage future research with OSO-containing formulations and ozone therapy that can be applied to treat other clinical forms of pythiosis in animals and humans.

Notably, SEM analysis demonstrated that both OSO and SO caused relevant morphological changes in P. insidiosum hyphae, especially cavitation along the hyphae, with loss of cell wall continuity, which was more evident in the OSO-treated hyphae; these changes are likely caused by the antimicrobial properties of the OSO. Pietrocola et al. [39] and Xiang et al. [40] suggested that in bacteria, compounds generated by the ozonation of oils interact with the cell wall and plasma membrane, altering cell permeability and causing irreversible cytoplasmic damage, preventing protein synthesis and causing the death of microorganisms. In fungi, ozone can enter the fungal cytoplasm, disrupt critical cellular functions, increase nutrient leakage, and inhibit fungal enzyme production [18]. However, the mechanisms of action on P. insidiosum requires further investigation in future studies.

Ozonated sunflower oil has activity against bacteria, including those causing mastitis in cattle such as Staphylococcus aureus, Escherichia coli, and Streptococcus uberis [34, 41], as well as action on yeast fungi, including Candida species [22], filamentous fungi such as Aspergillus fumigatus, and dermatophytes (Epidermophyton floccosum, Microsporum canis, and Trichophyton rubrum) [22, 24, 42, 43], and dimorphic fungi such as Sporothrix schenckii [44]. In fact, Ferreira et al. [26] recently verified the action of OSO against the oomycete P. insidiosum.

The results of this study affirm the anti-P. insidiosum potential of sunflower oil in both ozonated and non-ozone forms. Nevertheless, it is noteworthy that in the ozonated form, the in vitro results were significantly superior (p < 0.05). Thus, we believe this formulation can be proposed for the integrative treatment of chronic local infections, such as pythiosis.

Conclusions

This study demonstrated the in vitro activity of OSO and SO against P. insidiosum isolates and highlighted the improved oomicidal activity of ozonated oil. The results presented herein serve to leverage future research with OSO-containing formulations that can be applied in integrative medicine protocols for controlling pythiosis in animals and humans.

Author contributions

All authors contributed to the study conception and design; Braga CQ, Zambrano CG, Bermann CS, Milech A and Ianiski LB conducted experiments. Braga, CQ, Pereira DIB and Botton SA analysed the data and wrote the first draft of the manuscript; Soares MP performed analysis of electron microscopy images; Pötter L performed the statistical analysis. all authors read and approved the manuscript.

Funding

This research was supported by Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES—Finance Code 001) and National Council for Scientific and Technological Development – CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for student and researcher scholarships.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.