Pythium Insidiosum Biological Characteristics and Treatment: A Strain Cured by Repurposing of Existing Drugs in China

doi:10.21203/rs.3.rs-4992114/v1

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Pythium Insidiosum Biological Characteristics and Treatment: A Strain Cured by Repurposing of Existing Drugs in China

https://doi.org/10.21203/rs.3.rs-4992114/v1

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Background

Pythiosis is an infectious disease caused by P. insidiosum that threatens humans and animals. The number of people being diagnosed with this disease has been increasing worldwide. Pythiosis has a high rate of morbidity and mortality. Early diagnosis and prompt treatment are crucial in determining the prognosis of patients. The character of P. insidiosum has geographical variants, and a comprehensive investigation of it is essential in China.

Methods

In this study, a strain of P. insidiosum was visually and genetically identified, and isolated from a patient at a hospital in Guangzhou. A novel sporulation technique was used to produce zoospores. Microscopic observation was employed to understand the biological properties of P. insidiosum. Drug susceptibility studies on the isolates were conducted in vitro and in vivo.

Results

Our results provide comprehensive evidence that this strain was P. insidiosum, supported by molecular biology, morphology, and biological processes. Drug susceptibility studies showed P. insidiosum was more sensitive to antibiotics than antifungals, with tetracyclines and macrolides being the most sensitive in vitro. In vivo, doxycycline and azithromycin were administered to immunodeficient mice infected with P. insidiosum subcutaneously. The treatment significantly increased the survival rate of infected mice (p < 0.05) and alleviated the histopathology while decreasing the fungal burden in infected mice.

Conclusion

Our study provides theoretical and technical support for effectively treating pythiosis in humans and animals in China.

Oomycetes

Pythiosis

Pythium insidiosum

Identification

antimicrobial treatment

P. insidiosum is a fungus-like pathogenic aquatic oomycete disease that can infect both humans and a wide range of animals, primarily horses, dogs, and cattle. P. insidiosum is the only pathogen in the Pythium that can cause Pythiosis in mammals [1, 2]. P. insidiosum is widespread in temperate, tropical, and subtropical areas and is swamps' major ecological species [3–5]. P. insidiosum has long been mistaken for an organism from the fungi kingdom because, throughout the culture phase, it produces sparse septal hyphae similar to fungi. P. insidiosum 's life cycle comprises the development of sporangia, the release of zoospores, their germination, and infection. The infective unit of P. insidiosum, the zoospore, enters the host tissue as hyphae that are forming to start the infection [6].

Pythiosis is a non-infectious condition brought on by P. insidiosum infection that is underdiagnosed and difficult to treat. Interestingly, 79.2% of affected animals were in America and Brazil, while 94.3% of human cases were in India and Thailand [7]. However, the illness can exist in any country, not limited to the endemic areas. Pythiosis is significantly linked to high morbidity and mortality, but the latter is based on the clinical features and species of the animal. For instance, lung, circulatory, and disseminated infections frequently result in mortality regardless of the host species, whereas eye infections frequently result in losing the eye and do not cause death. Pythiosis is commonly treated with surgical resection, immunotherapy, and antibacterial drugs [8, 9]. The majority of treatment for Pythiosis is surgical surgery, although this course of action can considerably raise the economic burden, postoperative complications, and uncontrolled infections. It has been demonstrated that the use of P. insidiosum antigens (PIA) as a therapy for both human and animal pythiosis is promising [10]. However, The PIA therapeutic property needs improvement for a better prognosis for pythiosis patients [9]. Yolanda et al. [11] reported that cyazofamid is a promising candidate for treating pythiosis because it consistently and potently inhibits genetically diverse isolates of P. insidiosum while exhibiting minimal toxicities against various cell types. However, there is still a long way to go before it can be used clinically. Antimicrobial medication therapy achieves therapeutic efficacy by eliminating invasive microorganisms while minimizing its costs and adverse effects. The repurposing of existing drugs is a safe and efficient strategy for anti-P. insidiosum drug discovery [12]. However, the results of antimicrobials against P. insidiosum are inconsistent in vitro and in vivo. Therefore, there is an urgent need for effective medications to treat pythiosis in both humans and animals. On the other hand, P. insidiosum has geographical variants [1]. Thus, a comprehensive investigation is necessary, particularly in regions where it occurs rarely as China.

Here, a strain of P. insidiosum was isolated from a 56-year-old Chinese male who was cured with existing drugs [13]. We identified it visually and genetically. To comprehend the biological properties of P. insidiosum, we used microscopic observation of the transition of P. insidiosum from hyphae to spore form. To explore a better assessment of P. insidiosum. we used a novel sporulation technique to produce zoospores more quickly and plentifully. We conducted the drug-sensitive test not only in vitro but also in vivo on infected mice models. We speculated that this strain of P. insidiosum is sensitive to some antibacterial drugs. This will help repurpose existing drugs as a safe and efficient strategy for anti-P. insidiosum.

Isolation and Identification

The P. insidiosum (GZ2020) was isolated from a skin infection patient from The Third Affiliated Hospital of Sun Yat-sen University. We placed infected tissues on blood agar plates or 2% Sabouraud dextrose agar (SDA) and incubated them at 37°C for 2 d. After hyphae growth, the culture was purified by passaging using dot-planting. According to previous studies, hyphae were picked and stained with lactophenol cotton blue [14, 15].

Genomic DNA from mycelia was extracted and gDNA amplification was performed using primers for different genes [16]. The polymerase chain reaction (PCR) products were separated by 1% agarose gel electrophoresis and stained for observation. Internal transcribed spacer(ITS) amplification primers were ITS4 (5'-TCCTCCGCTTATTatGC-3') and ITS (5'-CCTTGTTACGACTTTTCC-3') [17]. Nuclear large subunit (LSU) amplification primers were LSULROR (5'-ACCCGCTGAACTTAAGC-3') and LSULR5 (5'-TCCTGaGGGAaACTTCG-3') [18]. Cytochrome oxidase (COI) amplification primers were FM52R (5'-TTAGaATGGAATTAGCACAAC-3') and FM55 (5'-GGCATA CCAGCTAAACCTAA-3') [18–20]. All extracted gDNA samples were recruited for species identification using the DNA barcode sequences (i.e., ITS and LSU) as the primary DNA barcode. PCR amplification was carried out in a 50 µL reaction containing 2 µL gDNA template, the universal fungal ITS primers (1 µL each of ITS1 and ITS4) or the universal fungal LSU primers (1 µL each of LSULROR and LSULR5), 21 µL ddH2O, and 25 µL of 2Taq PCR MasterMix. The reaction was carried out with the following thermal cycling conditions: the initial 95℃ denaturation for 2 min, 35 cycles of 94℃ denaturation for 40 s, 56℃ annealing for 40 s and 72℃ extension for 80 s, and the final 72℃extension for 10 min. The secondary DNA barcode sequences (i.e., COI were also amplified from gDNA samples of P insidiosum. PCR amplification was set up in a 25- µL reaction, containing 1 µL gDNA template, the oomycete-specific cox1 primers (1 µL each of FM52R and FM55), 10 µL ddH2O, and 12 µL Green enzyme (Green Taq Mix). The thermal cycle settings included the initial 95℃ denaturation for 3 min, 35 cycles of 94℃ denaturation for 40 s, 5 annealing for 50 s, and 72℃ extension for 70 s, and the final extension at 72℃ for 10 min.

We spliced the sequences into complete sequences using DNAMAN (Version 9.0, USA) and compared the amplified primer sequences with BLAST software on the NCBI website. Homology searches were performed in the GenBank database and compared with the identified P. insidiosum sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi). We utilized MAFTT (Version 7.0, JPN) to download sequences with high similarity and similar genera. Then we used MEGA (Version 6.0, USA) with the maximum likelihood method to construct a phylogenetic—tree to determine if they were in the same branch as P. insidiosum.

Zoospore Induction

This study used a new method of inducing sporulation (the supplementary material Fig. 1 shows the details) in P. insidiosum, which requires a multi-ionic solution combined with plant sap. multi-ionic solution: Liquid A includes K2HPO4 · 3H2O 11.4 g, KH2PO4 6.8 g, (NH4)2HPO4 6.6g, distilled water 50mL, Liquid B includes MgCI2 ·6H2O 2.54 g, CaCl2 ·2H20 1.84 g, distilled water 25 mL. Mix 0.5 mL of Liquid A and 0. 1 mL of Liquid B in 1000 mL of distilled water, adjusted pH range 7.0 ~ 7.1, stored at 4℃ [21]. Extraction of plant sap: Weigh 100g of grass leaves, add 500mL of distilled water, extract into sap, sterilize, and store at 4°C. Agar plugs with hyphae on the edge of fresh blood agar plates were inserted into 2% SDA plates, and incubated at 37°C for one week. The agar plugs with mycelium were placed in centrifuge tubes with 2% SDB culture solution and set at 37°C, 180 rpm for 4 h. The broth was removed from the centrifuge tubes, and an equal amount of distilled water was added and incubated at 37°C, 180 rpm for 1 h. The centrifuge tubes were incubated with induction solution and plant sap at a volume ratio of 1:1 at 37°C, 180 rpm for 10–24 h. Observation of the zoospores produced and the release of the zoospores from the sporangia under a light microscope. A Neubauer chamber is used to count the zoospores after zoosporogenesis [22, 23].

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

Oomycete preparation was used to remove P. insidiosum from the induction solution. We soaked samples in 1 mL of 2.5% glutaraldehyde fixative, rinsed them with 0. 1 mL phosphate buffer, washed them in 0. 1 mL phosphate buffer, and fixed them in 1% osmium solution. After drying with ethanol and dehydrating the samples, the Pythium insidious developmental cycle was observed by SEM.

To evaluate the ultrastructural features of the hyphae, we applied the oomycete preparation to remove the hyphae from the induction solution. Samples were placed in 1 mL of a fixative solution containing 2.5% glutaraldehyde and 2% paraformaldehyde (2.5% ga + 2% pfa). The samples were then rinsed with 0.1 mL of phosphate buffer and fixed with a solution of osmium (1% osmium + 1% potassium ferricyanide, prepared at 0.2 MPB). Subsequently, the samples were stained with uranyl acetate, dehydrated with ethanol, washed with acetone, and infiltrated with a mixture of acetone and embedding agent (resin). Finally, the samples were polymerized for 24 hours at 70°C [24]. Sections of 70–100 nm in thickness were obtained in an ultrathin sectioning machine. 70–100 nm thick sections were obtained using an ultrathin sectioning machine. The sections were then placed on a nickel grid and stained with uranyl acetate solution and lead citrate solution. The internal morphology of the hyphae was examined using TEM on the nickel grid.

Drug Sensitivity in Vitro

The susceptibility of P. insidiosum was tested using the broth microdilution method, following the modified Clinical and Laboratory Standards Institute (CLSI) M38-A2 protocol by Pereira et al [25]. In our study, zoospores were used for the drug susceptibility assay. The zoospores were resuspended in RPMI-1640 broth with a pH of 6.9–7.1, supplemented with 0.164 mL of 3-[N-morpholino] propane sulfonic acid (MOPS), to achieve a final concentration of 2–3×10 cells/mL [26]. Microdilution 96-well plates were utilized to test the zoospore suspension (inoculum) against either RPMI-1640 alone (as a no-drug control) or various drug concentrations. After 24 hours of incubation at 37°C, the minimum inhibitory concentration (MIC) was determined according to the other studies [27]. We conducted three biological replicates to assess the sensitivity of P. insidiosum to several antibiotics.

Animal Model and Drug Sensitivity in Vivo

This study utilized seven-week-old female BALB/c mice (18-22g) that were pathogen-free and obtained from the Guangdong Medical Lab Animal Center in Guangzhou, China. The animal experimental procedures were conducted following the guidelines of the South China Agricultural University (SCAU) Institutional Laboratory Animal Care and Use and were approved by the SCAU Institutional Ethics Committee (2020C407). To induce neutropenia, two intraperitoneal injections of cyclophosphamide were administered, with doses of 150 mg/kg on 4 days before infection and 100 mg/kg on 1 day before infection. On day 0, the mice were anesthetized using an intraperitoneal injection of ketamine-xylazine (Syntec Brasil Ltd., Brazil) at doses of 50 and 10 mg/kg, respectively. Subsequently, subcutaneous injections infected the mice with 2 × 10^4 zoospores/mouse [28]. The infected mice were randomly divided into four groups, each consisting of 10 animals. From day 0 (3 hours after zoospore inoculation) to day 14 after infection, the mice received one of the following treatments: (i) Saline (PI group), (ii) Azithromycin (50 mg/kg of body weight every 12 hours; AZM group), (iii) Doxycycline (40 mg/kg of body weight every 12 hours; DOX group), or (iv) control (negative control without any treatment). The experimental process is shown in Fig. 5a. Throughout the study, the animals were monitored 3 to 4 times daily for clinical signs such as changes in breathing, weight, mobility disorders, etc. Criteria for euthanizing the animals during the study included physical and mental alertness, chronic diarrhea, and bleeding. At the end of the experiment, all surviving animals were euthanized by deepening the anesthesia using thiopental (60 mg/kg). Samples were taken from the kidney, liver, lung, and spleen to be cultured in Sabouraud dextrose agar. A nested PCR (nPCR) assay was conducted on the samples to detect P. insidiosum DNA [29]. Histopathological analysis was also performed. The quantity of fungal presence in the tissue slices was measured using a GMS stain, which enabled the observation of P. insidiosum in the organs. Additionally, a hematoxylin-eosin (H&E) stain was utilized to identify inflammatory responses.

Statistical Analysis

Results were presented as means ± standard deviation (SD). The statistical analysis was performed with GraphPad Prism (Version 8, USA). Unless otherwise specified, we used the unpaired T-test or one-way ANOVA to assess the statistical significance of comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Isolation and Identification

Based on their microscopic features (Fig.1), the isolate was identified as a Pythium species. At 37°C, P. insidiosum was characterized by white, sunflower-shaped radiating colonies on brain heart infusion agar (Fig.1a); white, short, fluffy colonies on Columbia blood agar as our published paper [13]; and submerged, white to colorless colonies with an irregular radiate pattern on Sabouraud agar (Fig. 1b). The hyphae of P. insidiosum had lateral branches with diameters ranging from 4-10μm. Older hyphens usually showed transverse septa, with flowing protoplasmic nutrients inside the hyphae (Fi.1c). P. insidiosum from plate cultures exhibited sparse septate hyphae in lactophenol blue as our published paper [13]. Zoospores were only developed in water cultures. And they were stimulated by the presence of ions such as K+, Ca2+, and Mg2+, and chemically attracted by plant material, animal hairs, or pieces of animal tissue. After swimming in the external water for some time, the zoospores stopped, started to encapsulate, and their flagella fell off, forming a spherical shape. This resulted in the formation of resting spores. When the external conditions were suitable, the resting spores would germinate again to form hyphae (Fig.1d). According to the BLAST sequencing results from the NCBI database, the LSU gene amplification primer sequence shares a 100.00% sequence identity with P. insidiosum, while the COI gene amplification primers have a 99.00% sequence identity with P. insidiosum. The sequences of P. insidiosum were obtained through homology searches in the GenBank database. A phylogenetic tree was created using MEGA 6.0 and the greatest likelihood approach, and the comparison results are displayed in Fig.1e and Fig.1f, indicating that the isolated strain is Pythium Insidiosum.

Reproduction of Zoospores and Scanning Electron Microscopy (SEM)

When exposed to small amounts of various ions, such as Mg2+, P. insidiosum reproduces zoosporangia upon contact with grass leaf fragments. Zoospore release can be observed through microscopy (Fig.2, 3). The medium first drives the vegetative hyphae to become sporangia. At the early stage, sporangia can't be distinguished from vegetative hyphae (Fig. 3 a, b). As they mature, they form a globose, hyaline cyst with a diameter of 20–60μm. The sporangial protoplasm was poured into a discharge tube (Fig. 3c). Through progressive cleavage, biflagellate zoospores were formed inside the vesicle. The undifferentiated vesicle undergoes an internal developmental process, and the zoospores are released (the supplementary material Video.1 shows the details). This process takes about 35 minutes. After emergence, the zoospores mechanically break the vesicle's wall and swim for approximately 20 minutes. These zoospores had two flagella that were uneven in length and reniform, belonging to the secondary dinoflagellate type. The shorter preflagellate was of the tinsel type and had mastigonemas, while the whiplash-type posterior flagellum had none. Eventually, the flagellum of the zoospore drops off when it encysts and becomes a resting spore (Fig.3 d-f). When external conditions are suitable, the zoospores re-sprout to form hyphae (Fig. 3 g-i). We also saw the flow of protoplasm within the hypha (the supplementary material Video.1 shows the details).

Transmission Electron Microscopy (TEM)

The P. insidiosum has a distinct cell wall consisting of several inner layers and typical internal organelles. The cell wall has smooth plasmalemma (cytoplasmic membrane) associated with interlaced and meshed inner layers that form a coarse and thick inner layer. (Fig.4). Cytoplasmic vesicles are transported from the cytosolic compartment to the plasmalemma and integrated into the inner layers. Once integrated, the cytoplasmic bilayer membrane maintains its integrity. Large vacuoles containing black electron-dense bodies (EDBs) were observed within the cytosolic compartment. Some of the samples had nuclei, each surrounded by a bilayered membrane and containing granular chromatin (Fig.4 a, d). Golgi structures with numerous Golgi vesicles pinching off from the Golgi apparatus were also detected in the hyphae. The Golgi vesicles were found integrating into the plasmalemma surrounding the cell wall. There were relatively large microtubules dispersed throughout the hyphae. Endoplasmic reticula (ER) were sometimes detected near the mitochondria with multiple ribosomes around the ER. Numerous small vacuoles with fine granular content, ranging in size and shape, were dispersed throughout the hyphae. Some stained samples had numerous mitochondria with tubular cristae.

Drug Sensitivity in Vitro

The results of the in vitro susceptibility tests for P. insidiosum are presented in Table 1. Various common antifungal and antibiotic drugs were evaluated for their effectiveness against P. insidiosum. P. insidiosum was more sensitive to antibiotics than antifungals. The minimum inhibitory concentration (MIC) values of antibiotics against P. insidiosum were significantly lower compared to those of antifungals. Notably, DOX and azithromycin AZM exhibited effective results against P. insidiosum.

Table 1 In vitro susceptibility results for antimicrobial drugs against P. insidiosum

Antimicrobial Class

Drug

MIC(μg/ml）

Antifungal drugs

Fluorocytosine

Amphotericin B

Itraconazole

Fluconazole

doxycycline（DOX）

Tigecycline（TIG）

Tetracycline(TET)

Chloramphenicol(CHL)

Linezolid(LZD)

256

128

>256

256

Antibiotic

Erythromycin（ERY）

Azithromycin（AZM） Gentamicin(GEN)

Florfenicol(FFC)

SMZ/TMP

>256

375

Animal Model and Drug Sensitivity in Vivo

Ulcerated lesions were observed in mice infected with P. insidiosum at the inoculation site. Fig.5b visualizes the recovery of infected wounds among different treatment groups. The study revealed that AZM and DOX treatments were effective in promoting wound healing compared to the PI group. Fig.5c shows the survival curves of mice in different treatment groups. The untreated control and PI groups exhibited survival rates of 100% and 20%, respectively. In contrast, the AZM and DOX treatment groups had 80% and 90% survival rates, respectively. AZM and DOX treatment significantly reduced mortality compared to the saline group (p = 0.0304, p= 0.0109). Microbiological organ cultures revealed that 80% of infected mice in the PI-treated group had established P. insidiosum infections in the liver tissues. The nPCR analysis of organs aligned with the culture results. Fig.5 (d-e) depicts the primary histological lesions and the tissue loading capacity of pythiosis in the kidneys, spleen, liver, and lungs of BALB/c mice (AZM, DOX, PI groups) that were subcutaneously infected with P. insidiosum.

In the PI group, Histological H&E staining revealed prominent neutrophilic periarteritis with thrombosis and perivascular inflammatory infiltration in the liver, lung, spleen, and kidney of the mice (Fig.5d). Kidney pathology showed thrombosis, increased interstitial space, renal vesicle atrophy, partial vesicle damage, infiltration of blood cells, and significant inflammatory cell infiltration around renal tubules. Liver pathology exhibited thrombosis, blurred liver lobule borders, small hepatocyte nuclei, congestion in the portal area, central venous area, hepatic blood sinusoids, and a substantial infiltration of inflammatory cells. In the lungs, multifocal bleeding accompanied by fibrin and mild edema was observed, along with significant congestion in the septal capillaries (Fig.5d). Spleen histology showed atrophy of the splenic white marrow, unclear boundary between splenic cortex and medulla, spleen thrombosis, and infiltration of inflammatory cells (Fig.5d). In contrast, mice in the AZM and DOX treatment groups exhibited improved histological features compared to the PI group, indicating the therapeutic effect of AZM and DOX on pythiosis. Gomori methenamine silver (GMS)staining revealed fewer positively stained P. insidiosum in the tissues of mice in the AZM and DOX treatment groups. GMS staining of the PI group demonstrated a higher presence of black-dyed hyphae in the liver, while no hyphae were observed in other organs (Fig.5e). In contrast, GMS staining of the tissues from mice treated with AZM and DOX showed no black-dyed hyphae, indicating a significant reduction in fungal burden. Thus, treatment with the antibiotics AZM and DOX resulted in marked improvement in histological lesions and a substantial decrease in fungal load in vivo.

This study describes in detail the morphological characterization and biological features of a strain of P. insidiosum that was isolated from hospital patients in Guangzhou, China. Culture remains the gold standard and provides advantages for additional susceptibility testing in P. insidiosum [30]. The study showed that the P. insidiosum presented different colonial morphology in different mediums. The traditional method of cultivating and staining the isolated P. insidiosum is compatible with the earlier diagnostic findings [31]. This may be because the different nutrients in different mediums affected the growths of P. insidiosum and could provide some evidence for morphological identification. This may infer that P. insidiosum can be cultured on different mediums without affecting the identification of its colonies. Since P. insidiosum primarily takes on hyphae form in a lab setting, further research into its life cycle necessitates the induction of P. insidiosum zoospores. In this study, a new sporulation method was developed based on the improvements of earlier studies, and it has the advantage of being more effective and convenient than earlier sporulation techniques, not only increasing yield but also streamlining the procedures and cutting down on time. This will be helpful for further study on P. insidiosum.

Using a microscope, the complete life cycle of P. insidiosum was continually observed, and it was possible to see the dynamic process by which hyphae gradually changed into zoospores throughout a cycle. We found that P. insidiosum can produce zoospores when particular conditions are met and that zoospores constitute the main element of the organism's infection of the host. When P. insidiosum produces sporangia and releases zoospores, it may expand its ecological niche by colonizing other plants, similar to how other fungi that produce zoospores do. Previously, researchers proposed that P. insidiosum utilizes aquatic plant substrates to perpetuate the asexual stage of its life cycle. This hypothesis was supported by laboratory experiments in which P. insidiosum could infect a range of herbaceous plants [21, 32, 33]. We observed a similar phenomenon.

It was difficult and insufficient to identify P. insidiosum just based on morphology alone due to the restricted morphological differentiation in culture [1]. Direct sample multiplex-PCR, which is a potential tool for rapid diagnosis of equine pythiosis, overcomes limitations of morphological identification and provides a definitive diagnosis [34]. Molecular phylogenetic analysis is a trustworthy method for identifying P. insidiosum using ITS, COX, and LSU [18, 35]. Our results suggest that the isolate is more closely related to P. insidiosum than to any other Pythium species, indicating that it might be a single species. This improves the accuracy and dependability of our subsequent findings. To gain a better understanding of the internal morphological and structural characteristics of P. insidiosum, we used electron microscopy to observe the transformation of P. insidiosum from hyphae to zoospores and to examine its internal cellular structure. This laid the foundation for further in-depth studies.

We conducted experiments to investigate the effects of various popular classes of antibiotics and antifungals on the in vitro isolate of P. insidiosum, to discover an effective treatment for Pythiosis. The results showed that P. insidiosum was more susceptible to antibiotics than to antifungal medications. This conclusion is similar to the previous study, which found that antibiotics inhibit P. insidiosum 100 times more than anti-fungal agents [36]. Tetracyclines and macrolides may be potential candidates for infection control. This result is consistent with these studies too [36, 37]. However, as we know, there is no standard drug sensitivity method. We measured MIC values using the broth method and pathogenic spores. This may be a reliable and effective drug sensitivity test method. In vitro drug sensitivity tests, sulfamethoxazole had not shown effective bacteriostatic effects. This is inconsistent with the clinical outcome of this patient. The reason is unknown, which may include as follows: firstly, in vitro, experiments often fail to take into account metabolic processes in the body, such as the drug may be metabolized into different metabolites in the body, affecting the efficacy of the drug [38]; secondly, cell interactions and signaling in vivo can influence experimental results, and in vitro experiments often fail to fully simulate such complex cell interactions [39].

Studies conducted in vivo have indicated that DOX and AZM are the main antibiotics used for treating Pythiosis. Rabbits are often employed as experimental infection models, but they are not natural hosts of the disease and have limited immunological and biomolecular reagents. As a result, in this study, a mouse model was used instead. Certain immunocompetent mice infected with Pythiosis can recover through the typical Th1/Th17 response. The mouse immunodeficiency model was adopted for this research, and systemic infection with Pythiosis was made possible through subcutaneous injection of P. insidiosum [22]. The infected mice showed systemic illness, which successfully imitated vascular/spread Pythiosis in humans with animal infections. According to in vivo investigations, AZM and DOX alone demonstrated positive therapeutic effects in treating Pythiosis in mice, correlating with the in vitro findings. Both medications considerably improved the histological changes in mice, dramatically improved the survival rates of infected mice(p<0.05), and significantly decreased the fungal load in the liver. These results were similar to those of some previous studies [40–42], although this study relied on only one P. insidiosum strain. The mechanisms of action of AZM and DOX against P. insidiosum remain unclear. Possible mechanisms include inhibiting protein synthesis, cell wall synthesis, and/or amino acid transport [41]. In China, where infection rates are very low, it is essential to thoroughly analyze data on this strain to develop effective treatments for the disease.

Limitations of this study include the limited number of isolates tested and have not yet explored the specific dosages and durations required for conducting drug combination experiments. We also have not conducted a further study of its mechanism of therapeutical effect. Thus, further research, including a larger number of isolates, especially from genetically diverse P. insidiosum strains, different drug regimen treatments, and associations with other antimicrobials, immunotherapies, and surgery, will provide a better understanding of the therapeutic potential of AZM and DOX in pythiosis treatment, as well as the pathogenesis of P. insidiosum infection in humans and animals. Our efforts also should include the possible mechanisms of sulfanilamide being effective in our patients.

Early detection and appropriate therapy are essential for the effective management of human and animal pythiosis. Practitioners and pathologists must be knowledgeable about the typical clinicopathological characteristics of P. insidiosum as well as the available confirmatory tests and treatment options to successfully identify and treat patients infected with pythiosis in new geographic areas. In this study, we identified and developed a novel spore-producing method for a strain of P. insidiosum isolated from patients in a hospital in Guangzhou, China, to gain insight into its biological characteristics. Finally, through in vitro and in vivo drug susceptibility testing, we successfully found effective drugs for the treatment of Pythiosis, which will provide theoretical and technical support for the rapid diagnosis and treatment of outbreaks of such diseases in inexperienced areas with few epidemics. In summary, we recommend that AZM and DOX be considered in future in vivo experimental studies that evaluate the treatment of pythiosis.

PIA, P. insidiosum antigens; SDA, Sabouraud dextrose agar; PCR, polymerase chain reaction; ITS, internal transcribed spacer; LSU, nuclear large subunit; COI, Cytochrome oxidase; SEM, Scanning Electron Microscopy; TEM, Transmission Electron Microscopy; CLSI, Clinical and Laboratory Standards Institute; MOPS, 3-[N-morpholino] propane sulfonic acid; MIC, minimum inhibitory concentration; SCAU, South China Agricultural University; AZM, azithromycin; DOX, Doxycycline; nPCR, nested PCR; EDBs, electron-dense bodies; ER, Endoplasmic reticula; GMS, Gomori methenamine silver.

Ethics approval and consent to participate

The animal experimental procedures were approved by the SCAU Institutional Ethics Committee (2020C407). The clinical trial number was not applicable since the study was not a Clinical trial.

Clinical trial number

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

All authors manifest no financial interest or non-financial interest in the subject matter or materials discussed in this manuscript to disclose. The authors also have no competing interests relevant to the content of this article to declare.

Funding

This work was supported by the Guangdong Major Project of Basic and Applied Basic Research (grant 2020B0301030007).

Authors' contributions

Xiaoyun Liu: Methodology, Original draft preparation, and Review and editing. Qiuyue Diao: Methodology, Formal analysis and investigation, Original draft preparation, and Review and editing. MingLiang Li, Zehua Cui, Tijiang Shan, Jiaobao Huang, Jiayin Liang, and Xiaoping Liao: Methodology.

Yuting Yang, Hao Ren, Haiyan Zhang, and Huiling He: Formal analysis and investigation. Fengli Zhou: Review and editing. Jian Sun: Conceptualization and Funding acquisition. Kouxing Zhang: Conceptualization, Review and editing.

Acknowledgments

Thanks to the authors of this article for their collaboration.

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No competing interests reported.

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supplementary material Fig. 1 Schematic diagram of a new method for inducing sporulation.

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Pythium Insidiosum Biological Characteristics and Treatment: A Strain Cured by Repurposing of Existing Drugs in China

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Isolation and Identification

Zoospore Induction

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

Drug Sensitivity in Vitro

Animal Model and Drug Sensitivity in Vivo

Statistical Analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1