Clioquinol and 8-hydroxyquinoline-5-sulfonamide derivatives damage the cell wall of Pythium insidiosum.

AIMS: To evaluate the antimicrobial activity and to determine the pharmacodynamic characteristics of three 8-hydroxyquinoline derivatives (8-HQs) against Pythium insidiosum, the causative agent of pythiosis. METHODS AND RESULTS: Antimicrobial activity was tested by broth microdilution and MTT assays. The antimicrobial mode of action was investigated using sorbitol protection assay, ergosterol binding assay, and scanning electron microscopy. Clioquinol, PH151, and PH153 were active against all isolates, with MIC values ranging from 0.25 to 2 µg ml-1. They also showed a time- and dose-dependent antimicrobial effect, damaging the P. insidiosum cell wall. CONCLUSIONS: Together, these results reinforce the potential of 8-HQs for developing new drugs to treat pythiosis.

Immunotherapy based on Pythium insidiosum mycelia drives a Th1/Th17 response in mice.

Pythium insidiosum is an oomycete that affects mammals, especially humans and horses, causing a difficult-to-treat disease. Typically, surgical interventions associated with antimicrobial therapy, immunotherapy, or both are the preferred treatment choices. PitiumVac® is a therapeutic vaccine prepared from the mycelial mass of P. insidiosum and is used to treat Brazilian equine pythiosis. To better understand how PitiumVac® works, we analyzed the composition of PitiumVac® and the immune response triggered by this immunotherapy in mice. We performed an enzymatic quantification that showed a total glucan content of 21.05% ± 0.94 (α-glucan, 6.37% ± 0.77 and (1,3)(1,6)-ß-glucan, 14.68% ± 0.60) and mannose content of 1.39% ± 0.26; the protein content was 0.52 mg ml-1 ± 0.07 mg ml-1. Healthy Swiss mice (n = 3) were subcutaneously preimmunized with one, two, or three shots of PitiumVac®, and immunization promoted a relevant Th1 and Th17 responses compared to nonimmunization of mice. The highest cytokine levels were observed after the third immunization, principally for IFN-γ, IL-17A, IL-6, and IL-10 levels. Results of infected untreated (Pythiosis) and infected treated (Pythiosis + PVAC) mice (n = 3) showed that PitiumVac® reinforces the Th1/Th17 response displayed by untreated mice. The (1,3)(1,6)-ß-glucan content can be, at least in part, related to this Th1/Th17 response.

Efficacy of Azithromycin and Miltefosine in Experimental Systemic Pythiosis in Immunosuppressed Mice.

We evaluated the efficacy of azithromycin (50 mg/kg, every 12 h [q12h] orally) and miltefosine (25 mg/kg, q24h orally) treatments in an experimental model of vascular/disseminated pythiosis in immunosuppressed mice. Azithromycin was the only treatment able to reduce mortality. The histopathological findings showed acute vascular inflammation, pathogen dissemination, necrotizing myositis, neuritis, and arteritis. The results suggest that azithromycin, but not miltefosine, may have clinical relevance in the treatment of vascular/disseminated pythiosis.

Screening of antibacterial drugs for antimicrobial activity against Pythium insidiosum.

We tested 25 isolates of Pythium insidiosum to investigate their susceptibility to antibacterial drugs that act through inhibition of protein synthesis or other mechanisms of action. We observed that tetracycline, erythromycin, linezolid, nitrofurantoin, Synercid (quinupristin and dalfopristin), chloramphenicol, clindamycin, cetrimide, and crystal violet had inhibitory activity against P. insidiosum. Those in vitro results suggest that antibacterials that inhibit protein synthesis should be the primary antimicrobials investigated for the treatment of pythiosis in animals and humans.

In vitro assessment of antifungal, antibacterial, and antiprotozoal drugs against clinical isolates of Conidiobolus lamprauges.

We have determined the in vitro activity of antifungal, antibacterial, and antiprotozoal drugs alone and in combination against seven Conidiobolus lamprauges clinical isolates. The assays were based on the M38-A2 protocol and the checkerboard microdilution method. The lowest inhibitory concentrations were observed for amphotericin B, miconazole (MCZ), terbinafine, and miltefosine (MTF) (MIC range 0.25-1; 2-8; 0.25-2; 2-16 µg/ml, respectively). The main synergism observed was through the combination of azithromycin (AZI)+MTF and dapsone (DAP)+MTF (100%), AZI+DAP (85.7%), AZI+MCZ (57.1%) as well as MCZ plus CTX and DAP (42.9%). The in vitro activities suggest that the combination of MTF and AZI or DAP are promising candidate therapies for conidiobolomycosis.

In vitro combination of antifungal agents against Malassezia pachydermatis.

The yeast Malassezia pachydermatis is a common commensal and occasional opportunistic pathogen of theskin microbiota of animals and humans. In this study, the susceptibility of M. pachydermatis isolates to fluconazole (FLC), itraconazole (ITZ), ketoconazole (KTZ), clotrimazole (CLZ), and miconazole (MCZ) alone and in combination with terbinafine (TRB), nystatin (NYS), and caspofungin (CSP) was evaluated in vitro based on the M27-A3 technique and the checkerboard microdilution method using Sabouraud dextrose broth with 1% tween 80 (SDB). Based on the mean FICI values, the main synergies observed were combinations of ITZ+CSP and CLZ+CSP (55.17%). The most significant combinations deserve in vivo evaluations because might provide effective alternative treatments against M. pachydermatis due to their synergistic interactions.

In vitro combination between antifungals and diphenyl diselenide against Cryptococcus species.

Cryptococcus species are an encapsulated fungal pathogen that cause cryptococcal meningitis. There are limited therapeutic options for this infection. The management includes the use of different antifungals such as amphotericin B, flucytosine, or fluconazole, either alone or in combination. However, numerous therapeutic failures, as well as the limited effectiveness of such therapeutics, have been described. Diphenyl diselenide is a chemically synthesised molecule with was found to have antimicrobial activity. In this study, we evaluated the antifungal activities of fluconazole, amphotericin B and flucytosine, in combination with diphenyl diselenide against 30 clinical isolates of Cryptococcus spp. using CLSI M27-A3 method and the checkerboard microdilution technique. Our results show that the combination of flucytosine and diphenyl diselenide displayed 100% of synergism. However, when we analysed (PhSe)2 plus AMB or FLZ we observed around 70% of indifference. Our results suggest that the combination of diphenyl diselenide with other antifungal agents deserves attention as a new option for the development of alternative therapies for cryptococcosis.

In Vitro Activities of Miltefosine and Antibacterial Agents from the Macrolide, Oxazolidinone, and Pleuromutilin Classes against Pythium insidiosum and Pythium aphanidermatum.

We tested 29 isolates of Pythium insidiosum and one isolate of Pythium aphanidermatum to investigate their susceptibility to miltefosine and antibacterial drugs from the macrolide, oxazolidinone, and pleuromutilin classes. We found that miltefosine, azithromycin, clarithromycin, josamycin, linezolid, sutezolid, retapamulin, tiamulin, and valnemulin had inhibitory and cidal activity against the pathogens at concentrations ranging from 0.25 to 64 µg/ml. Our results suggest that these antimicrobials are promising candidates for future studies on pythiosis in animals and humans.

An experimental murine model of otitis and dermatitis caused by Malassezia pachydermatis.

We report a malasseziosis model in immunocompromised Swiss mice. For this model, the mice were immunosuppressed with a combination of cyclophosphamide at 150 mg/kg and hydrocortisone acetate at 250 mg/kg. Two groups were formed according to the site of inoculation. Dermatitis group received an intradermal injection of 5 × 106 cell/mouse at a shaved dorsal region, while the otitis group received the same inoculum in the middle ear. Five animals/group were euthanised at different times, and the skin and ear were histopathologically analysed. During the first euthanasia, which occurred after inoculation, microscopic examination showed that all mice presented budding yeast-like in a tissue sample. The presence of yeasts decreased over time being undetected on the 17th day (dermatitis group) and the 21st day (otitis group) after inoculation. This is the first murine model for malasseziosis that can be useful for evaluating new treatment approaches.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection.

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.

In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum.

We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC90 between 2 and 32 µg∕ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum.

In Vitro and In Vivo Antimicrobial Activities of Minocycline in Combination with Azithromycin, Clarithromycin, or Tigecycline against Pythium insidiosum.

The present study investigated the in vitro and the in vivo interactions among azithromycin, clarithromycin, minocycline, and tigecycline against Pythium insidiosum. In vitro antimicrobial activities were determined by the broth microdilution method in accordance with CLSI document M38-A2, and the antibiotic interactions were assayed using the checkerboard MIC format. In vivo efficacy was determined using a rabbit infection model. The geometric mean MICs of azithromycin, clarithromycin, minocycline, and tigecycline against P. insidiosum were, respectively, 1.91, 1.38, 0.91, and 0.79 µg/ml. By checkerboard testing, all combinations resulted in in vitro synergistic interactions (>60%). Antagonism was not observed. The in vivo studies showed that azithromycin (20 mg/kg/day twice daily) alone or in combination with minocycline (10 mg/kg/day twice daily) significantly decreased the fungal burden. This study demonstrates that azithromycin possesses potent curative efficacy against subcutaneous pythiosis in the rabbit model.

In vitro interaction of antifungal and antibacterial drugs against Cryptococcus neoformans var. grubii before and after capsular induction.

This study evaluated the synergistic interactions between amphotericin B (AMB) and azithromycin (AZM), daptomycin (DAP), linezolid (LNZ), minocycline (MINO), fluconazole (FLZ), flucytosine (5FC), linezolid (LZD), or tigecycline (TIG) against clinical isolates of Cryptococcus neoformans var. grubii before and after capsule induction. High synergism (>75%) was observed for the combinations, AMB+5FC, AMB+TIG, AMB+AZM, AMB+LZD and AMB+MINO but only in the strains after capsule induction. The results show that the presence of the capsule may lower the minimum inhibitory concentrations (MICs) of antifungal agents, but antimicrobial activity can be improved by combining antifungal and antibacterial agents.

New insights into the in vitro susceptibility of Pythium insidiosum.

We have determined the in vitro activity of several antibacterial and antifungal drugs against Pythium insidiosum using broth microdilution (BMD), disk diffusion, and Etest methods. The largest zones of inhibition (disk diffusion) and the lowest BMD and Etest MICs were observed for azithromycin, clarithromycin, linezolid, mupirocin, doxycycline, minocycline, and tigecycline. The in vitro activities observed suggest that antibacterials, which act by inhibiting protein synthesis, are promising candidate therapies for the treatment of pythiosis.

In vitro synergism observed with azithromycin, clarithromycin, minocycline, or tigecycline in association with antifungal agents against Pythium insidiosum.

We describe here the in vitro activities of azithromycin, clarithromycin, minocycline, or tigecycline alone and in combination with amphotericin B, itraconazole, terbinafine, voriconazole, anidulafungin, caspofungin, or micafungin against 30 isolates of the oomycete Pythium insidiosum. The assays were based on the CLSI M38-A2 technique and the checkerboard microdilution method. The main synergisms observed were through the combination of minocycline with amphotericin B (73.33%), itraconazole (70%), and micafungin (70%) and of clarithromycin with micafungin (73.33%).

Treating Pythiosis with Antibacterial Drugs Targeting Protein Synthesis: An Overview.

This review article explores the effectiveness of antibacterial drugs that inhibit protein synthesis in treating pythiosis, a difficult-to-treat infection caused by Pythium insidiosum. The article highlights the susceptibility of P. insidiosum to antibacterial drugs, such as macrolides, oxazolidinones, and tetracyclines. We examine various studies, including in vitro tests, experimental infection models, and clinical case reports. Based on our synthesis of these findings, we highlight the potential of these drugs in managing pythiosis, primarily when combined with surgical interventions. The review emphasizes the need for personalized treatment strategies and further research to establish standardized testing protocols and optimize therapeutic approaches.

Epidemiology of horse pythiosis in the Pantanal of Mato Grosso: Exploring the host-parasite-vector relationship.

Horse pythiosis is considered an endemic disease in the Brazilian Pantanal region, causing devastating health and economic losses. This study aimed to enhance the understanding of pythiosis epidemiology, map the distribution of horse body lesions, and investigate the correlation between these lesions and warm body surface areas, potentially implicating hematophagous vectors in the disease's transmission. A prospective study was conducted on equids in the Pantanal Mato-grossense and adjacent areas from 2012 to 2022, with 112 horses and three mules diagnosed with pythiosis. Clinical and epidemiological data, lesions' photographic records, and healthy equids' thermal imaging were collected. Most pythiosis cases occurred between January and March, correlating with regional flood cycles. Most lesions were found on limbs and the ventral abdomen, with dark-colored horses exhibiting a higher frequency of lesions. Interestingly, the thermal mapping revealed that warm areas on a healthy horse's body overlapped significantly with lesion distribution - blood-sucking insects also prefer these areas. The results suggest that pythiosis lesions in horses correlate with warmer areas of the animal body, reinforcing the hypothesis of vector involvement in disease transmission. This study underscores the need for further observational research to fully understand the complex epidemiological dynamics of pythiosis in horses.

Toll-deficient Drosophila is susceptible to Pythium insidiosum infection.

There is a paucity of animal models of pythiosis, a life-threatening disease of humans and animals, the immunopathogenesis of which is poorly understood. A pythiosis model was developed by injecting Toll (Tl)-deficient Drosophila melanogaster flies with Pythium insidiosum zoospores. The infected Tl mutant flies had significantly lower survival rates (73.7%) than did control flies. This study reveals the important role of Tl pathway activation in fly immune response to pythiosis.

In vivo effect of minocycline alone and in combination with immunotherapy against pythium insidiosum.

This study examined the effect of minocycline alone and in combination with immunotherapy against pythiosis. Twenty rabbits, aged three months old and subcutaneously inoculated with Pythium insidiosum zoospores were divided into four groups (n = 5): treated with minocycline (10 mg/kg/day twice daily), treated with immunotherapy (34 mg subcutaneously every 14 days), treated with minocycline plus immunotherapy, and untreated (control group). The treatments were started 30 days after inoculation and continued for 70 days. The subcutaneous nodular injury areas in infected groups were measured every seven days after the beginning of treatment. Only the rabbits that developed lesions were selected for this study. When compared with the control group over 70 days, the minocycline and minocycline plus immunotherapy groups of rabbits with pythiosis showed significantly reduced injuries. The histopathology showed the presence of inflammation, macrophages and eosinophils. Grocott's staining revealed irregular hyphae-like structures that were ramified and occasionally septate. Our results suggest that minocycline has fungistatic activity and that the combination of minocycline and immunotherapy is more effective than the individual therapies tested.