Pythium insidiosum: insights into biofilm formation and antibiofilm activity of antifungal drugs.

In this study, we investigate the ability of Pythium insidiosum to form biofilms across various substrates and the antibiofilm efficacy of 8-hydroxyquinoline derivatives (8-HQs). Biofilms of P. insidiosum were cultured on polystyrene plates, contact lenses, and horsehair. We provide the first evidence of P. insidiosum's biofilm-forming capability, thus considerably expanding our understanding of its transmission and pathogenesis. Our results demonstrate that 8-HQs effectively inhibit biofilm formation and eradicate pre-existing biofilms, underscoring their potential as a novel treatment strategy for pythiosis, a disease currently lacking a gold-standard treatment. This finding has particular relevance for ocular pythiosis associated with contact lens usage and potential infection sources in animals. Our results contribute to the scientific knowledge base and directly impact innovative therapeutic interventions' development.

Mefenoxam and pyraclostrobin: toxicity and in vitro inhibitory activity against Pythium insidiosum.

The objective of this study is to verify in vitro susceptibility of Pythium insidiosum against the agricultural fungicides mefenoxam and pyraclostrobin and evaluate the toxicity of both compounds. Twenty-one P. insidiosum isolates were tested against mefenoxam and pyraclostrobin using the broth microdilution method. Minimum inhibitory and oomicidal concentrations for both compounds were established. Additionally, scanning electron microscopy was performed on P. insidiosum hyphae treated with the sublethal concentration of each fungicide. The toxicity of the compounds was evaluated in vivo Caenorhabditis elegans model. The concentration to inhibit 100% of P. insidiosum growth ranged from 0·625 to 10 µg ml-1 for mefenoxam and from 0·019 to 5 µg ml-1 for pyraclostrobin. The SEM analysis revealed changes on the surface of the hyphae treated with the fungicides, suggesting possible damage caused by these compounds. There was no evidence of toxicity in vivo models. Mefenoxam and pyraclostrobin did not show toxicity at the doses evaluated and have inhibitory effects on the pathogenic oomycete P. insidiosum. However, further evaluations of their pharmacokinetics and toxicity in different animal species and possible pharmacological interactions are necessary to infer a possible use in the clinical management of pythiosis.

Clioquinol and 8-hydroxyquinoline-5-sulfonamide derivatives damage the cell wall of Pythium insidiosum.

AIMS: To evaluate the antimicrobial activity and to determine the pharmacodynamic characteristics of three 8-hydroxyquinoline derivatives (8-HQs) against Pythium insidiosum, the causative agent of pythiosis. METHODS AND RESULTS: Antimicrobial activity was tested by broth microdilution and MTT assays. The antimicrobial mode of action was investigated using sorbitol protection assay, ergosterol binding assay, and scanning electron microscopy. Clioquinol, PH151, and PH153 were active against all isolates, with MIC values ranging from 0.25 to 2 µg ml-1. They also showed a time- and dose-dependent antimicrobial effect, damaging the P. insidiosum cell wall. CONCLUSIONS: Together, these results reinforce the potential of 8-HQs for developing new drugs to treat pythiosis.

Short communication: Algicide activity of antimicrobial peptides compounds against Prototheca bovis.

This study evaluated the in vitro activity of antimicrobial peptides pexiganan (MSI-78), h-Lf1-11, LL-37, cecropin B, magainin-2, and fengycin B against the veterinary mastitis agent Prototheca bovis. The results showed that pexiganan, h-Lf1-11, LL-37, and cecropin B were able to inhibit the growth and had effect on algicide P. bovis isolates (n = 32). The minimum inhibitory concentration ranged from 5 to 10 µg/mL for pexiganan, and algicide effect was detected from 5 to 20 µg/mL. The minimum inhibitory concentration ranged from 10 to 80 µg/mL for h-Lf1-11, 20 to 80 µg/mL for LL-37, and 40 to 160 µg/mL for cecropin B. These findings present a promising and novel alternative for P. bovis treatment and growth control.

Efficacy of miltefosine therapy against subcutaneous experimental pythiosis in rabbits.

We evaluated the in vitro activity of miltefosine against 29 Pythium spp. and the in vivo therapeutic response of 2mg/kg/day of miltefosine given orally to rabbit with pythiosis induced experimentally. The MICs (in µg/mL) of miltefosine was medium-dependent and ranged from 0.5 to 2 and 32-64 on RPMI 1640 and Mueller Hinton broth, respectively. The treatment with miltefosine demonstrated significantly lower subcutaneous lesion areas compared to the control group but was not sufficient for the complete remission of the lesions. This study indicates that miltefosine has limited efficacy against pythiosis and furthers in vitro and in vivo studies are necessary to determine the possible potential of this drug in the treatment of pythiosis.

In vitro activity of carvacrol, cinnamaldehyde and thymol combined with antifungals against Malassezia pachydermatis.

Malassezia pachydermatis is an important opportunistic agent of dermatitis and otitis in dogs. M. pachydermatis is generally treated with topical therapies using combinations of antifungal, antimicrobial and anti-inflammatory agents. We investigated the in vitro activities of carvacrol (CRV), cinnamaldehyde (CIN) and thymol (THY) alone and in combination with antifungal agents (fluconazole, itraconazole, ketoconazole, clotrimazole, miconazole, terbinafine and nystatin) against M. pachydermatis. The assays were performed according to the Clinical and Laboratory Standards Institute (CLSI), using Sabouraud dextrose broth and checkerboard microdilution. The mean fractional inhibitory concentration index (FICI) showed primary synergies for the combinations carvacrol+nystatin, thymol+nystatin, and carvacrol+miconazole (80%). In conclusion, the results obtained indicate that the phytochemicals tested showed relevant in vitro anti-M. pachydermatis activity. Future in vivo experiments are needed to elucidate the safety and therapeutic potential of these combinations.

Susceptibility profile of Candida rugosa (Diutina rugosa) against antifungals and compounds of essential oils.

Candida rugosa (recently reclassified Diutina rugosa) is an emerging pathogen affecting humans and animals. Candida resistance to existing drugs is an important factor to be monitored, as well as the need of researching alternatives to conventional antifungals. Here, we evaluated the in vitro effects of some antifungals and major components of essential oils by the broth microdilution method (CLSI M27-A3) against fifteen C. rugosa strains from animals isolated and molecular identificated. The results showed MIC90 of: 0.125µg/mL to ketoconazole and voriconazole, 0.25µg/mL to micafungin, 0.5µg/mL to anidulafungin, 1µg/mL to caspofungin, 2µg/mL to amphotericin B, itraconazole and flucytosin, 8µg/mL to fluconazole, 16µg/mL to nystatin and >128µg/mL to terbinafine. The compounds carvacrol (MIC90 320µg/mL), thimol (MIC90 320µg/mL) and cinnamaldehyde (MIC90 160µg/mL) demonstrated antifungal activity against the samples tested.

In vitro activity of essential oils extracted from condiments against fluconazole-resistant and -sensitive Candida glabrata.

In the present study, the antifungal activity of essential oils obtained from Origanum vulgare (oregano), Cinnamomum zeylanicum (cinnamon), Lippia graveolens (Mexican oregano), Thymus vulgaris (thyme), Salvia officinalis (sage), Rosmarinus officinalis (rosemary), Ocimum basilicum (basil) and Zingiber officinale (ginger) were assessed against Candida glabrata isolates. One group contained 30 fluconazole-susceptible C. glabrata isolates, and the second group contained fluconazole-resistant isolates derived from the first group after the in vitro induction of fluconazole-resistance, for a total of 60 tested isolates. The broth microdilution methodology was used. Concentrations of 50µg/mL, 100µg/mL, 200µg/mL, 400µg/mL, 800µg/mL, 1600µg/mL and 3200µg/mL of the essential oils were used, and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined. Thyme, sage, rosemary, basil and ginger essential oils showed no antifungal activity at the tested concentrations. Antimicrobial activity less than or equal to 3200µg/mL was observed for oregano, Mexican oregano and cinnamon essential oils. Both the oregano and Mexican oregano essential oils showed high levels of antifungal activity against the fluconazole-susceptible C. glabrata group, whereas the cinnamon essential oil showed the best antifungal activity against the fluconazole-resistant C. glabrata isolates.

In vitro activity of carvacrol and thymol combined with antifungals or antibacterials against Pythium insidiosum.

We describe the in vitro activities of the combinations of carvacrol and thymol with antibiotics (azithromycin, clarithromycin, minocycline and tigecycline) and antifungal agents (amphotericin B, caspofungin, itraconazole and terbinafine) against 23 isolates of the oomycete Pythium insidiosum. The assays were based on the M38-A2 technique and checkerboard microdilution. Based on the mean FICI values, the main synergies observed were combinations of carvacrol+itraconazole and thymol+itraconazole (96%), thymol+clarithromycin (92%), carvacrol+clarithromycin (88%), thymol+minocycline (84%), carvacrol+minocycline (80%), carvacrol+azithromycin (76%), thymol+azithromycin (68%), carvacrol+tigecycline (64%) and thymol+tigecycline (60%). In conclusion, we found that combinations of carvacrol or thymol with these antimicrobial agents might provide effective alternative treatments for cutaneous pythiosis due to their synergistic interactions. Future in vivo experiments are needed to elucidate the safety and therapeutic potential of these combinations.

Cytoprotective and genoprotective effects of ß-glucans against aflatoxin B1-induced DNA damage in broiler chicken lymphocytes.

The polysaccharide ß-glucan presents beneficial effects on the immune system, although the mechanisms of the immunomodulatory effect remain poorly understood. The potential cytoprotective and genoprotective effects of ß-glucans were evaluated in broiler chicken lymphocytes exposed to increasing concentrations of aflatoxin B1 (AFB1) and/or ß-glucans. AFB1 significantly decreased cell viability at the concentrations of 10 and 20 µg/ml at 72 h of incubation (p<0.01 and p<0.001, respectively). Moreover, the AFB1 concentrations of 1, 10 and 20 µg/ml increased DNA fragmentation levels at 24 h (p<0.001). Conversely, lymphocyte death was prevented by ß-glucans at the concentrations of 1% and 10%, indicating a cytoprotective effect. Reactive oxygen species levels were increased in the cells treated with 20 µg/ml AFB1 at 24 h (p<0.05) and 10% ß-glucans with or without AFB1 at 24, 48 and 72 h of incubation (p<0.001). DNA damage increased by more than 100% in AFB1-treated lymphocytes when compared to control group. ß-glucans at 1% was able to fully revert the AFB1-induced lymphocyte DNA damage, indicating a genoprotective effect and maintaining DNA integrity. In conclusion, ß-glucans showed in vitro dose-dependent cytoprotective and genoprotective effects in broiler chicken lymphocytes exposed to AFB1.

Micafungin alone and in combination therapy with deferasirox against Pythium insidiosum.

This study evaluated the in vitro and in vivo activity of micafungin alone and in combination with the iron chelator deferasirox against Pythium insidiosum. Micafungin showed a poor in vitro activity when it was used alone, but synergistic interactions were observed for 88.2% of the strains when the drug was combined with deferasirox. Smaller lesions were observed in infected rabbits receiving the combination therapy, although it favored disease dissemination to the lungs. The present results show that micafungin alone is ineffective against P. insidiosum, and the combination micafungin-deferasirox might have deleterious effects for the host.

Efficacy of a Brazilian calcium montmorillonite against toxic effects of dietary aflatoxins on broilers reared to market weight.

1. The protective effect of a natural Brazilian calcium montmorillonite (CaMont) against aflatoxins was studied in broiler chickens. 2. A total of 1056-d-old Cobb male broilers were housed in experimental pens (22 chickens per pen) for 42 d. Three levels of CaMont (0, 2.5 and 5 g/kg) and two levels of aflatoxins (0 and 3 mg/kg) were assayed. Each treatment had 8 replicate pens of 22 broiler chickens each. 3. Of all the chickens tested in the experiment, the ones treated with aflatoxins were the most adversely affected. CaMont treatment at concentrations of 2.5 and 5 g/kg improved body weight of chickens at 42 d of age by 13.3% and 22.7%, increased daily feed intake by 9.7% and 24.7%, and improved the productive efficiency index of chickens by 53% and 66.5%, respectively. 4. Dietary CaMont positively affected parameters such as weight of liver, heart and gizzard; however, serum potassium concentration decreased by 15.3% compared with that of chickens given only the aflatoxin-contaminated diet. 5. CaMont did not cause adverse effects in chickens that did not receive aflatoxins. 6. CaMont at pH 8.5 partially reduced the toxic effects of aflatoxins in broilers when included at levels of 2.5 and 5 g/kg in the diet.

Iron chelation therapy as a treatment for Pythium insidiosum in an animal model.

OBJECTIVES: Iron plays an important role in the pathogenesis of Pythium insidiosum. Human pythiosis frequently occurs in iron-overloaded thalassaemic patients and experimentally infected animals develop iron deficiency anaemia. Therefore, we sought to determine the in vitro and in vivo activities of the iron chelator deferasirox against P. insidiosum. METHODS: In vitro, the MIC and minimum fungicidal concentration (MFC) values of deferasirox for 17 strains of P. insidiosum were determined in accordance with CLSI document M38-A2. In vivo studies were carried out in 20 inoculated rabbits divided into four groups: placebo, immunotherapy obtained from vortexed P. insidiosum cultures (14 day intervals), deferasirox (15 mg/kg/day) and a combination of immunotherapy and deferasirox. Five non-infected animals were used as controls. RESULTS: The MIC and MFC values of deferasirox for P. insidiosum ranged from 12.5 to 50 mg/L and from 50 to 100 mg/L, respectively. Treatment with deferasirox alone ameliorated anaemia and normalized the serum iron levels and hepatic iron concentration in the animals. However, the mean lesion size, although decreased, did not differ significantly from that in the placebo group. The results of immunotherapy plus iron chelation therapy were worse than those of immunotherapy alone. Moreover, the disease spread to the lung tissue in 5 out of 10 deferasirox-treated animals. CONCLUSIONS: Despite its limited in vitro and in vivo activity, deferasirox improved iron deficiency anaemia in P. insidiosum-infected rabbits. Further studies are needed to investigate the immunomodulatory properties observed in this study and the benefits and drawbacks of using iron-chelating drugs as an adjuvant therapy in pythiosis.

Insights into the pathophysiology of iron metabolism in Pythium insidiosum infections.

Pythium insidiosum causes life-threatening disease in mammals. Animals with pythiosis usually develop anemia, and most human patients are reported to have thalassemia and the major consequence of thalassemia, iron overload. Therefore, this study evaluated the iron metabolism in rabbits experimentally infected with P. insidiosum. Ten infected rabbits were divided into two groups: one groups received a placebo, and the other was treated with immunotherapy. Five rabbits were used as negative controls. The hematological and biochemical parameters, including the iron profile, were evaluated. Microcytic hypochromic anemia was observed in the infected animals, and this condition was more accentuated in the untreated group. The serum iron level was decreased, whereas the transferrin level was increased, resulting in low saturation. The level of stainable iron in hepatocytes was markedly decreased in the untreated group. A high correlation was observed between the total iron binding capacity and the lesion size, and this correlation likely confirms the affinity of P. insidiosum for iron. The data from this study corroborate the previous implications of iron in the pathogenesis of pythiosis in humans and animals.

Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences.

Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum.

Phylogenetic analysis of Pneumocystis from pig lungs obtained from slaughterhouses in southern and midwestern regions of Brazil / Análise filogenética de Pneumocystis obtidos de pulmões de suínos provenientes de abatedouros da região sul e centro oeste do Brasil

The Pneumocystis genus is comprised of pathogens dwelling in the lungs of terrestrial, aerial, and aquatic mammals. Occasionally they induce severe pneumonitis, particularly in hosts with severe impairment of the immune system and progressively may fill pulmonary alveolar cavities causing respiratory failure. Molecular genetic studies revealed that Pneumocystis gene sequences present a marked divergence with the host species concerned. In the present study, the genetic diversity of Pneumocystis obtained from lungs of swines was examined by analyzing mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences. The samples were obtained from two slaughterhouses located in two Brazilian states. Phylogenetic analysis demonstrated that genetic groupings within Pneumocystis organisms were in accordance with those of the corresponding hosts and that two clusters were formed. In conclusion, these data show that there are genetically distinct porcine Pneumocystis genotypes with at least two separate clusters in Brazil.

O gênero Pneumocystis compreende patógenos que residem em pulmões de animais terrestres, aéreos e aquáticos. Pode ocasionar uma grave pneumonia, particularmente em hospedeiros com o sistema imunológico seriamente comprometido, o que ocorre por meio de uma progressiva disseminação nas cavidades alveolares, causando insuficiência respiratória. Estudos genéticos, baseados em métodos moleculares, revelaram que as sequências dos genes de Pneumocystis apresentam marcante divergência de acordo com a espécie de hospedeiro. Neste estudo, a diversidade genética das amostras obtidas a partir de pulmões de suínos, provenientes de dois abatedouros localizados em dois estados brasileiros, foi examinada por análise das sequencias dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis. O resultado confirma a tendência registrada em pesquisas com amostras de outros animais e permite concluir que existem, pelo menos, dois grupos filogenéticos distintos de Pneumocystis de suínos no Brasil.

In vitro susceptibility of fluconazole-susceptible and -resistant isolates of Malassezia pachydermatis against azoles.

OBJECTIVES: The first aim of this study was to evaluate the in vitro efficacies of fluconazole, ketoconazole, itraconazole and voriconazole on M. pachydermatis growth inhibition. This study also evaluated M. pachydermatis azole cross-resistance, comparing wild clinical isolates and the same isolates with in vitro-induced fluconazole resistance. METHODS: Two techniques were used: (1) a broth microdilution method based on protocol M27-A3 from the Clinical and Laboratory Standards Institute to determine the minimum inhibitory concentration (MIC) and (2) the Fekete-Forgács method to induce fluconazole resistance in vitro. The isolates were divided into two groups: group 1 included fluconazole-susceptible clinical isolates (n=30) and group 2 contained the same isolates with in vitro-induced fluconazole resistance (n=30). RESULTS: The two groups exhibited differences in susceptibility (p<0.001). Group 1 isolates were susceptible to azoles: ketoconazole (MIC 0.01-1.0 µg/mL), itraconazole (MIC 0.01-1.0 µg/mL), voriconazole (MIC 0.01-4.0 µg/mL), and fluconazole (MIC 0.01-4.0 µg/mL). Group 2 isolates demonstrated a wider range of MICs to azoles: ITZ (MIC 0.06-64.0 µg/mL), KTZ (MIC 0.25-32.0 µg/mL), VRZ (MIC 2.0-128.0 µg/mL), and FLZ (MIC 64.0-128.0 µg/mL). CONCLUSIONS: It was shown that FLZ-resistant M. pachydermatis isolates exhibit cross-resistance to other azoles, reinforcing the importance of susceptibility tests as a guide for the therapeutic prescription of antifungals in medical and veterinary mycology.

A suitable model for the utilization of Duddingtonia flagrans fungus in small-flock-size sheep farms.

Effective alternatives to anthelmintic treatment of nematode parasite infections of sheep are required because of the high prevalence of drug resistance. Within this context, the nematode-trapping fungus Duddingtonia flagrans has become a valuable component of various integrated control strategies. Toward this objective, a small quantity of lyophilized D. flagrans chlamydospores (10(6) spores per animal) was administered to sheep in a one-year plot study. Animals grazing on native pasture were divided into two homogeneous groups and were kept in 1-ha paddocks in the southern region of Brazil. The oral administration of chlamydospores led to a significant reduction (p<0.05) in the number of nematode eggs per gram of feces and in the larval availability on herbage (difference of 37.6%) in comparison to the control group. Control animals needed to be dewormed three times during the experiment, whereas the fungus-treated animals maintained a low parasite load, independent of seasonal variation. Although D. flagrans cannot serve as a panacea for nematode parasite control of livestock, it represents a significant advance toward rationalizing the use of endoparasitic drugs in small animals.

DRBC agar: a new tool for Candida dubliniensis identification.

Candida dubliniensis pathogenic species, which shares many phenotypic features with C. albicans, may be misidentified in the microbiology laboratory. The growth on DRBC agar at 25 degrees C was shown to be a new tool for differentiation between C. dubliniensis and C. albicans. All 27 isolates of C. dubliniensis showed in this medium rough colonies (peripheral hyphal fringes) and abundant chlamydospore production, while all 103 isolates of C. albicans showed smooth colonies without fringes or chlamydospores. DRBC agar allowed the differentiation of C. albicans from C. dubliniensis with 100 % sensitivity and specificity.