In vitro anti-Pythium insidiosum activity of biogenic silver nanoparticles.

Pythium insidiosum belongs to the phylum Oomycota. It is capable of infecting mammals causing a serious condition called pythiosis, which affects mainly horses in Brazil and humans in Thailand. The objective of the present study was to verify the in vitro anti-P. insidiosum activity of a biogenic silver nanoparticle (bio-AgNP) formulation. The in vitro assays were evaluated on P. insidiosum isolates (n = 38) following the M38-A2 protocol. Damage to the P. insidiosum hyphae ultrastructure was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bio-AgNP inhibition concentrations on P. insidiosum isolates ranged from 0.06 to 0.47 µg/ml. It was observed through SEM that P. insidiosum hyphae treated showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. The TEM of treated hyphae did not differentiate organelle structures; also, the cellular wall was rarefied, showing wrinkled and partly ruptured borders. The bio-AgNP evaluated has excellent in vitro anti-P. insidiosum activity. However, further studies on its in vivo action are necessary as so to determine the possibility of its use in the treatment of the disease in affected hosts.

In Vitro Activity of Melaleuca alternifolia (Tea Tree) in Its Free Oil and Nanoemulsion Formulations Against Pythium insidiosum.

Pythium insidiosum is an important aquatic oomycete which can cause pythiosis in both animals and humans. This microorganism shows low susceptibility to antifungal drugs available. This study analyzed the in vitro antimicrobial activity of Melaleuca alternifolia in its free oil (FO) and nanoemulsion (NE) formulations against Brazilian P. insidiosum isolates. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 document adapted to phytopharmaceuticals. Twenty-six P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100 % growth inhibition. Melaleuca alternifolia essential oil or FO was obtained commercially. The NE containing 1 % M. alternifolia essential oil was prepared by the spontaneous emulsification method. All P. insidiosum isolates evaluated showed minimum inhibitory concentrations (MIC) ranging from 531.5 to 2125 µg/mL for the FO formulation; MIC50 and MIC90 showed values between 1062.5 and 2125 µg/mL, respectively. When the NE formulation was evaluated, MIC values ranged from 132.7 to 2125 µg/mL and both MIC50 and MIC90 corresponded to 1062.5 µg/mL. FO and NE formulations of M. alternifolia showed antimicrobial activity against P. insidiosum. This study demonstrated that M. alternifolia oil can be an additional therapy in pythiosis treatment; however, further studies are needed to evaluate the applicability of the plant essential oils in the treatment of clinical pythiosis.

In Vitro Susceptibility of Pythium insidiosum to Melaleuca alternifolia, Mentha piperita and Origanum vulgare Essential Oils Combinations.

Pythium insidiosum is the etiologic agent of pythiosis, a severe and emerging disease that affects mammals. Failure of conventional antifungal therapies is partially justified by the absence of ergosterol in the plasma membrane of this oomycete. Despite research advancement, the treatment of pythiosis has not been not fully established. The present study investigated the in vitro susceptibility profile of Brazilian isolates of P. insidiosum (n = 20) against Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils, and their combinations. Susceptibility tests were performed according to CLSI M38-A2 protocol, and combinations were evaluated by the microdilution cherkerboard method. All tested essential oils showed antimicrobial activity against P. insidiosum, and the greatest activity of O. vulgare was highlighted. Synergistic and/or indifferent effect was observed for all combinations evaluated, especially the M. piperita and O. vulgare combination, which showed 65 % synergism. This is the first study to report in vitro combinations of essential oils against P. insidiosum indicating the susceptibility of this oomycete to M. alternifolia, M. piperita and O. vulgare essential oils, as well as their combinations.