Suscetibilidade in vitro e in vivo de Pythium insidiosum frente a antibacterianos (macrolídeos, oxazolidinonas e pleuromutilinas) e miltefosina

Abstract

Pythiosis is an infection caused by the oomycete Pythium insidiosum that affects humans and other wild and domestic mammals, presenting difficulties in both diagnosis and treatment. Pythiosis cases do not respond satisfactorily to antifungal drugs due to peculiarities of the cell wall and cell membrane of P. insidiosum, such as the absence of ergosterol, which is the primary target of these drugs. Due to the difficulties in the treatment, surgical excision is an often-necessary approach but presents a relapse of approximately 40%. Also, the use of immunotherapies from the microorganism cultures shows good therapeutic activity in equines but has a varied activity in other animals and humans. Therefore, there is no standard pharmacological approach entirely adequate for the treatment of pythiosis. In this context, the present study aims to: (i) carry out an in vitro screening of the anti-P. insidiosum activity of several classes of antibacterials; (ii) to evaluate the in vitro antimicrobial activity of antibacterials of the of macrolides, oxazolidinones and pleuromutilins classes and the antiparasitic miltefosine against P. insidiosum; (iii) standardize a new experimental pythiosis model using Swiss mice immunosuppressed with cyclophosphamide; (iv) to evaluate the in vivo efficacy, in a pythiosis model in mice, of the antibacterial drugs with the lowest Minimum Inhibitory Concentrations (MICs) observed in the in vitro tests; (v) to evaluate the efficacy of miltefosine in the treatment of pythiosis in an experimental pythiosis model in rabbits. The results obtained in this study are: (i) the screening of the anti-P. insidiosum activity showed that, except for the aminoglycoside drugs, all antibacterial inhibitors of protein synthesis showed anti-P. insidiosum activity and that antimicrobial drugs with a mechanism other than inhibition of protein synthesis did not demonstrate antimicrobial activity against P. insidiosum, except for cetrimide, crystal violet, and nitrofurantoin; (ii) in the evaluation of in vitro susceptibility of 29 strains of P. insidiosum and 1 strain of P. aphanidermatum, the lowest MICs / Minimum Oomicidal Concentrations (COMs) (in μg/mL) were observed for azithromycin (1-32 / 4 -32), clarithromycin (0.5-64 / 1-64), linezolid (1-64 / 8-64), sutezolid (4-64 / 4-64), retapamulin (0.25-32 / 0.5-64), valnemulin (0.25-16 / 0.25-32) and miltefosine (0.5-4 / 0.5-4); (iii) the evaluation of the cyclophosphamide-induced immunosuppression protocol (2 doses of 100 mg/kg, 4 and 1 days before infection) in Swiss mice was shown to be a useful model for the experimental induction of systemic/vascular pythiosis with index mortality rate of 70%; (iv) the evaluation of the efficacy of treatment of the experimental pythiosis in mice by survival analysis showed that azithromycin (50 mg/kg /12/12h) was effective in reducing mortality from 70% (untreated group) to 20%; treatment with miltefosine did not significantly influence mortality compared to disease control (60% and 70%, respectively); (v) miltefosine (2 mg/kg/day) showed limited activity in the treatment of rabbits experimentally infected with P. insidiosum, reducing the progression of subcutaneous lesions when compared to the control, but without achieving the clinical cure of the animals. From these results, we can conclude and suggest that the antibacterial drugs that act through the inhibition of protein synthesis are compounds that have an antimicrobial potential for use in the treatment of animal and human pythiosis. Future studies, with the evaluation of the pharmacokinetic and pharmacodynamic parameters of treatments with the proposed drugs in experimental models of pythiosis and the assessment of the combination of these treatments with the anti-P. insidiosum immunotherapy, are necessary and will guide the choice of the best classes of drugs for the treatment of pythiosis.