Exposure of Culex quinquefasciatus to the oomycete Pythium insidiosum: A protocol for in vitro studies.

Pythium insidiosum causes pythiosis, an infection that affects different species of mammals, including humans, and inhabits marshy ecosystems of tropical, subtropical, and temperate regions worldwide. Therefore, this study proposes a protocol to expose Culex quinquefasciatus to P. insidiosum zoospores. Cx. quinquefasciatus immatures (eggs, larvae, and pupae) were exposed to zoospores (8x103 zoospores/mL) of the oomycete for 24 h. The exposure of Cx. quinquefasciatus to the zoospores from L1 to the emergence of adults was evaluated, and P. insidiosum detection was performed by microbiological culture, polymerase chain reaction, and histopathological analysis of stage 4 larvae. The protocol used to produce Cx. quinquefasciatus colonies and adapted for this study proved viable for research on the interaction between P. insidiosum and this Culicidae species. Moreover, P. insidiosum presence was evident in all larval stages of the mosquito, although the presence of the oomycete was not detected in the eggs, pupae, and adults. This study is a pioneer in the development of a protocol to evaluate Cx. quinquefasciatus exposure to P. insidiosum zoospores, and under experimental conditions, P. insidiosum can establish itself in Cx. quinquefasciatus larval stages. The developed protocol is expected to serve as a basis for developing studies to evaluate the interactions of P. insidiosum with these mosquitoes and shed more light on the participation of culicids in expanding the ecological niche of P. insidiosum.

Oomicidal activity of polypyrrole nanoparticles against Pythium 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.

Anti-Pythium insidiosum activity of MSI-78, LL-37, and magainin-2 antimicrobial peptides.

We investigated the anti-Pythium insidiosum activity of the antimicrobial peptides (AMPs) MSI-78, LL-37, and magainin-2. To detect the minimum inhibitory concentration (MIC), fourteen clinical strains were incubated with the AMPs following the CLSI M38-A2 protocol. All three AMPs showed antimicrobial activity with an MIC range of 20-80 mg/L against all strains. We concluded that the evaluated AMPs have great potential as anti-Pythium insidiosum agents, and their activity deserves to be more explored in further research. Antimicrobial peptides were tested against Pythium insidiosum, a microorganism that causes a difficult-to-treat disease in animals and humans. These peptides have been shown to be able to kill P. insidiosum and may be candidates for use in the treatment of this infection.

In vitro activity of the antimicrobial peptides h-Lf1-11, MSI-78, LL-37, fengycin 2B, and magainin-2 against clinically important bacteria.

We investigated the antibacterial activity of the antimicrobial peptides h-Lf1-11, MSI-78, LL-37, fengycin 2B, and magainin-2. The minimum inhibitory concentration (MIC) was determined by microdilution technique according to CLSI (M07-A9, 2012) against Escherichia coli, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, carbapenem-resistant Klebsiella pneumoniae, and Acinetobacter baumannii. The MSI-78 showed potent bactericidal activity with MIC range of 1.25-40 mg/L against all bacterial strains. The h-Lf1-11, magainin-2, and LL-37 exhibited moderate activity (MIC range of 40-160, 80-160, and 40-160 mg/L, respectively) while the fengycin 2B did not show significant activity against all bacterial strains tested. These results revealed that MSI-78, h-Lf1-11, magainin-2, and LL-37 have great potential as antibacterial agents and their activity deserves to be more explored in further studies for the treatment of antibiotic-resistant bacteria.

Activity of MSI-78, h-Lf1-11 and cecropin B antimicrobial peptides alone and in combination with voriconazole and amphotericin B against clinical isolates of Fusarium solani.

Fusarium infections have been associated with high mortality rates due to the lack of definition of an ideal treatment strategy. Antimicrobial peptides (AMPs) have potential antifungal activity. Therefore, investigating the in vitro activity of these molecules alone and in association with conventional antifungals against clinical isolates of Fusarium was the aim of this study. Fusarium solani (n=10) strains were tested against the AMPs, MSI-78, h-Lf1-11 and cecropin B in accordance with CLSI protocol. Further, a checkerboard assay for its combination with amphotericin B or voriconazole, was carried out. MSI-78, h-Lf1-11 and cecropin B exhibited antifungal activity against F. solani strains tested with MICs ranging from 20 to 320mg/L. Satisfactory percentage of synergism was demonstrated for all evaluated combinations, ranging from 70 to 100%. The use of AMPs combined with amphotericin B and voriconazole antifungals has great synergistic potential and deserve to be evaluated in murine models of fusariosis.

In vitro anti-Pythium insidiosum activity of amorolfine hydrochloride and azithromycin, alone and in combination.

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.

New insights on evolutionary aspects of Pythium insidiosum and other peronosporaleans.

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.

Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR / Genotipagem de isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único baseado em PCR multiplex

ABSTRACT: 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.

RESUMO: O objetivo deste estudo foi genotipar isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único (SNP) de sequências de rDNA. Anteriormente, um multiplex-PCR baseado em SNP foi capaz de distinguir P. insidiosum em três diferentes clados. Dessa forma, utilizamos este método para avaliar isolados clínicos de P. insidiosum da América do Sul (n=32), cepas padrão da Costa Rica (n=4), Tailândia (n=3), Japão (n=1) e Índia (n=1), uma cepa padrão de Pythium aphanidermatum e isolados ambientais brasileiros de Pythium torulosum; Pythium rhizo-oryzae e Pythium pachycaule voucher (n=3). Os isolados analisados foram alocados aos clados: I (americanos), II (isolados da Índia, Japão e Tailândia), e III (um isolado tailandês). P. aphanidermatum, P.torulosum, P.rhizo-oryzae e P.pachycaule voucher não foram amplificados. Pela primeira vez, um isolado de P. insidiosum do Uruguai foi incluído em análises moleculares. Através da multiplex-PCR baseada em SNP, foi possível realizar a identificação e genotipagem dos isolados sul-americanos de P. insidiosum, demonstrando características genéticas semelhantes entre esses isolados.