bWe 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. P ythiosis is a life-threatening and chronic pyogranulomatous disease caused by the fungus-like pathogen Pythium insidiosum, the main oomycete species capable of infecting humans and other animals (1, 2). Although there have been a few reports of clinical cures of cases of pythiosis with antifungal therapy (3, 4), the data from the literature on the clinical management of pythiosis patients with treatment by antifungals indicate that such therapy has been largely ineffective (2,5,6). The genus Pythium is unable to synthesize ergosterol, the active target of most antifungals, which partly explains why this class of drugs has been ineffective (2).Studies on the in vitro susceptibility of the clinical isolates of P. insidiosum to antifungal drugs have shown divergent results, and there are no international protocols approved for evaluating the in vitro susceptibility of P. insidiosum. Interestingly, previous studies have shown that P. insidiosum is quite sensitive to antibacterials belonging to the classes of macrolides, tetracyclines, and glycylcyclines (7,8) and that its combination with antifungal drugs can result in synergistic interactions in vitro (9). However, the effects of antibacterial agents that inhibit protein synthesis on P. insidiosum have not been studied extensively. Thus, the objective of this study was to compare the in vitro susceptibilities of P. insidiosum to a number of antibacterial and antifungal drugs using the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth microdilution, CLSI M51-A disk diffusion, and Etest methods.We evaluated the susceptibility of 25 Brazilian P. insidiosum strains isolated from equine pythiosis lesions. All of the isolates were identified using a PCR-based assay according to Botton et al. (10). The reference strains included P. insidiosum CBS 101555 from an equine pythiosis case and P. insidiosum CBS 119452 and Pythium aphanidermatum CBS 128995 from human pythiosis cases.The broth microdilution (BMD) reference assay was carried out following the CLSI M38-A2 guidelines (11), as previously described (12, 13). The final concentrations of the antimicrobial agents tested in the wells ranged from 1,024 to 0.5 g/ml for mupirocin and tobramycin and from 256 to 0.125 g/ml for azithromycin, clarithromycin, clindamycin, chloramphenicol, doxycycline, erythromycin, florfenicol, fluconazole, fusidic acid, lincomycin, linezolid, minocycline, roxithromycin, terbinafine, tetracycline, tigecycline, and tilmicosin. The final conce...
eThe 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 g/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.
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%). Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum, which can cause infections in humans and in animals, such as horses, bovines, cats, dogs, and sheep. Clinically, the disease may manifest in cutaneous, gastrointestinal, vascular, and systemic forms and has been described in tropical and subtropical areas (1). The hyphae of P. insidiosum are morphologically similar to those of certain mucoraceous molds, but P. insidiosum is not a true fungus because it does not synthesize ergosterol, which is the target of most antifungal drugs. Despite this challenge, two cases of pythiosis in humans, one case of ocular pythiosis and one case of pleuropericarditis (2, 3), have been successfully treated using combination antifungal therapy. However, combination antifungal therapy has been ineffective in cases of vascular and disseminated human pythiosis (4).Previous studies have shown that the growth of P. insidiosum is inhibited in vitro by the glycylcycline, macrolide, and tetracycline classes of antibacterial drugs (5, 6). However, studies evaluating the antimicrobial combination of antibacterial and antifungal agents against P. insidiosum have not been performed. In this context, this study evaluated the in vitro combination of the antibacterial drugs azithromycin, clarithromycin, minocycline, or tigecycline with the antifungal drugs amphotericin B, itraconazole, voriconazole, terbinafine, anidulafungin, caspofungin, or micafungin against P. insidiosum.Twenty-eight P. insidiosum isolates obtained from Brazilian cases of equine pythiosis and the reference strains ATCC 58.637 and CBS 101555 were evaluated in this study. The identities of the clinical isolates were confirmed using PCR-based assays (7). The antibacterial drugs azithromycin (Pharma Nostra, Rio de Janeiro, Brazil), clarithromycin (Genix, Anápolis, Brazil), minocycline (Pharma Nostra), and tigecycline (Pfizer, New York, NY) and the antifungal drugs amphotericin B (Sigma-Aldrich, St. Louis, MO), itraconazole (Frangon do Brasil Farmacêutica Ltda., São Paulo, Brazil), voriconazole (Pfizer), terbinafine (Pharma Nostra), anidulafungin (Pfizer), caspofungin (Merck, Darmstadt, Germany), and micafungin (Astellas, Chuo, Japan) were obtained commercially and diluted in dimethyl sulfoxide or distilled water, as recommended, to generate stock solutions. The concentrations of the antimicrobial agents tested were 0.03 to 16 g/ml and 1 to 512 g/ml for the antibacterial and antifungal dr...
bThis study evaluated the in vitro activity of aminoglycoside antibiotics and tigecycline against Pythium insidiosum. The susceptibility tests were carried out using the broth microdilution method in accordance with the CLSI document M38-A2. MIC values for gentamicin, neomycin, paromomycin, and streptomycin ranged from 32 to 64 mg/liter, and the minimal fungicidal concentration (MFC) ranged from 32 to 128 mg/liter, which are incompatible with safe concentrations of these drugs in plasma in vivo. Tigecycline showed the lowest MIC (0.25 to 2 mg/liter) and MFC (1 to 8 mg/liter) range values. The in vitro susceptibility observed to tigecycline makes this drug a good option in future tests in vitro and in vivo for the management of pythiosis. Pythiosis is a chronic disease that affects humans, mammals, and birds and is caused by the aquatic oomycete Pythium insidiosum. It occurs in tropical, subtropical, and temperate regions, and there have been no reports of direct transmission among animals or between animals and humans. Cutaneous, vascular, ocular, and systemic forms (humans) and subcutaneous and gastrointestinal forms (horses, dogs, cattle, cats, and sheep) are the clinical presentations most commonly observed (3).Pythiosis progresses rapidly and can become life threatening if it is not treated in the early stages. The absence of ergosterol in the cell wall of this oomycete restrains the treatment of pythiosis with antifungal therapy due to the fact that most antifungal drugs act by inhibiting the synthesis of ergosterol. Radical surgery and immunotherapy are among the best therapeutic options, but the improved cure rates are directly associated with early diagnosis of pythiosis (14).Members of the genus Pythium are known to be susceptible to some antibiotics of the tetracycline, macrolide, aminoglycoside, and amphenicol classes (5,6,8,12). McMeekin and Mendoza (9) reported that streptomycin may have a stimulatory or inhibitory effect on P. insidiosum depending on the culture conditions, and Loreto et al. (7) described an in vitro inhibitory activity of minocycline against P. insidiosum. In this context, this study aims to evaluate the in vitro susceptibility of the aminoglycosides gentamicin, neomycin, paromomycin, and streptomycin and the minocycline-derived tigecycline against isolates of P. insidiosum.Twenty-three clinical Brazilian isolates of P. insidiosum from equine pythiosis cases and the ATCC 58.637 reference strain were tested in this study. The identities of the isolates were confirmed by a PCR-based assay (4).Gentamicin, neomycin, paromomycin, and streptomycin were obtained from Sigma Chemical Co. (St. Louis, MO). Tigecycline (Tygacil; Pfizer) was purchased commercially. The drugs were dissolved in dimethyl sulfoxide to obtain stock solutions (12,800 mg/ liter) and stored at Ϫ70°C.At present, there is no standardized susceptibility testing method for oomycetes. Because P. insidiosum produces hyphae and based on the results of previous in vitro and in vivo studies (7,11), the susceptibility test wa...
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