Pythium insidiosum infections have been widely studied in an attempt to develop an effective therapeutic protocol for the treatment of human and animal pythiosis. Several antifungal agents are still prescribed against this oomycete, although they present contradictory results. To evaluate the susceptibility profile and to verify the morphological alterations in P. insidiosum isolates treated with amorolfine hydrochloride and azithromycin, alone or in combination. Susceptibility tests for P. insidiosum isolates (n = 20) against amorolfine hydrochloride (AMR) and azithromycin (AZM) were performed according to Clinical and Laboratory Standards Institutes (CLSI) protocol M38-A2. Combinations of both drugs were evaluated using the checkerboard microdilution method. Additionally, transmission and scanning electron microscopy were performed in order to verify the morphological alterations in P. insidiosum isolates in response to these drugs. All P. insidiosum isolates had a minimum inhibitory concentration (MIC) ranging from 16 to 64 mg/l and 8 to 64 mg/l for amorolfine hydrochloride and azithromycin, respectively. Synergistic interactions between the drugs were not observed, with antagonism in 59.8% of isolates, and indifferent interactions in 36.2%. Electron microscopy showed changes in the surface of P. insidiosum hyphae, disorganization of intracellular organelles, and changes in the plasma membrane and cell wall of oomycetes treated with the drugs. This is the first study to demonstrate in vitro anti-P. insidiosum effect of amorolfine hydrochloride. These results indicate the therapeutic potential of this drug against cutaneous and subcutaneous forms of pythiosis, but further studies are necessary to confirm this potential.
The purpose of this review was to address the applicability of nanotechnology in veterinary medicine, with an emphasis on research in Brazil from 2013 to 2020. Firstly, we introduced to the general aspects of applicability of nanotechnology in veterinary medicine, and lately we pointed the research involving nanoscience performed in Brazil, in the studied period. Nanotechnology is the field of science that has the capacity to organize matter in nanoscale structures (1 to 100 nm), enabling innovations in different areas including biotechnology, agriculture, disease diagnosis, food and clothing industry, electronics, and pharmacological therapies. In veterinary medicine, several studies are being carried out in the world, mainly in the areas that involve search of new treatment options and the development of immunotherapy, as well as in the diagnosis of diseases. In Brazil, it is clear that the use of nanotechnology in veterinary medicine is still incipient, but it can be considered a growing area. In addition, several points have to be reflected and researched, including some adverse effects and implications to validate the safe use of nanotechnology in veterinary medicine. Therefore, this review highlighted the nanotechnology as a promise alternative in the current context of Brazilian technological innovation involving animal health, as well as a possible diagnostic tool and highlighting its potential therapeutic use in disease control in veterinary medicine. Regarding future perspectives, we believed that greater investment in science and technology could contribute to the advancement and strengthening of nanotechnology in Brazil.
We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates.
Summary Background The evolution of pathogenic mechanisms is a major challenge, which requires a thorough comprehension of the phylogenetic relationships of pathogens. Peronosporaleans encompasses a heterogeneous group of oomycetes that includes some animal/human pathogens, like Pythium insidiosum. Objective We analysed here the phylogenetic positioning and other evolutionary aspects related to this species and other peronosporaleans, using a multi‐locus approach with one mitochondrial and three nuclear genes. Methodology Phylogenetic patterns of 55 oomycetes were inferred by maximum likelihood and Bayesian analysis, and a relaxed molecular clock method was applied to infer the divergence time of some peronosporaleans branches. Results Pythium insidiosum was monophyletic with a major and polytomous clade of American isolates; however, Pythium spp. was found to be paraphyletic with Phytopythium sp. and Phytophthora spp. In general, peronosporaleans subdivided into four lineages, one of which evidenced a close relationship of P insidiosum, P aphanidermatum and P arrhenomanes. This lineage diverged about 63 million years ago (Mya), whereas P insidiosum diversified at approximately 24 Mya. The divergence of American and Thai isolates seems to have occurred at approximately 17 Mya, with further American diversification at 2.4 Mya. Conclusion Overall, this study clarifies the phylogenetic relationships of P insidiosum regarding other peronosporaleans in a multi‐locus perspective, despite previous claims that phylogenomic analyses are needed to accurately infer the patterns and processes related to the evolution of different lineages in this group. Additionally, this is the first time that a molecular clock was applied to study the evolution of P insidiosum.
This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 μg mL−1, and the MIC90 and MIC50 were 16 μg mL−1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti-P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models.
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