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 antifungals in combination with essential oils against the oomycete Pythium insidiosum.

AIMS: The aim of this study was to investigate the in vitro susceptibility of Pythium insidiosum to combinations of the antifungal drugs terbinafine or itraconazole with Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils (EOs). METHODS AND RESULTS: In vitro combinations of antifungal drugs with EOs were evaluated by checkerboard microdilution method against 20 Brazilian isolates of P. insidiosum. The tests were performed according to protocol M38-A2, and the interpretation of each combination result was based on the values of the fractional inhibitory concentration index. The combinations of itraconazole with EOs presented prominent synergistic effects against P. insidiosum isolates, and no antagonism was observed with these combinations. However, the combinations of terbinafine with EOs resulted in indifferent or antagonistic effects. CONCLUSIONS: The combination of plant-derived bioactive compounds with antifungal drugs may be an alternative therapy for the control of infections caused by P. insidiosum. Studies of new therapeutic protocols involving these proposed combinations are needed. SIGNIFICANCE AND IMPACT OF THE STUDY: The antimicrobial combinations using EOs with terbinafine or itraconazole can be an attractive therapeutic option for controlling P. insidiosum infections.

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.

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.

Different risk factors for candidemia occur for Candida species belonging to the C. parapsilosis complex.

Previous studies have demonstrated reduced virulence in the species that comprise the Candida parapsilosis complex. We investigated a cohort of 93 patients with candidemia caused by this complex. Most infections were caused by C. parapsilosis (80.6%), followed by C. orthopsilosis (18.3%) and C. metapsilosis (1.1%). Renal failure (P < 0.001) and chronic liver diseases (P = 0.019) were more frequently encountered with infections caused by the C. orthopsilosis group, suggesting an association with patients who had a greater state of immune suppression in comparison with infections caused by C. parapsilosis sensu stricto.

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.

Antifungal activity of nanocapsule suspensions containing tea tree oil on the growth of Trichophyton rubrum.

The aim of this study was to evaluate, for the first time, the antifungal efficacy of nanocapsules and nanoemulsions containing Melaleuca alternifolia essential oil (tea tree oil) in an onychomycosis model. The antifungal activity of nanostructured formulations was evaluated against Trichophyton rubrum in two different in vitro models of dermatophyte nail infection. First, nail powder was infected with T. rubrum in a 96-well plate and then treated with the formulations. After 7 and 14 days, cell viability was verified. The plate counts for the samples were 2.37, 1.45 and 1.0 log CFU mL(-1) (emulsion, nanoemulsion containing tea tree oil and nanocapsules containing tea tree oil, respectively). A second model employed nails fragments which were infected with the microorganism and treated with the formulations. The diameter of fungal colony was measured. The areas obtained were 2.88 ± 2.08 mm(2), 14.59 ± 2.01 mm(2), 40.98 ± 2.76 mm(2) and 38.72 ± 1.22 mm(2) for the nanocapsules containing tea tree oil, nanoemulsion containing tea tree oil, emulsion and untreated nail, respectively. Nail infection models demonstrated the ability of the formulations to reduce T. rubrum growth, with the inclusion of oil in nanocapsules being most efficient.

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.

Composition and evaluation of the antimicrobial activity of the essential oil of Senecio selloi Spreng DC / Composição e avaliação da atividade antimicrobiana do óleo essencial de Senecio selloi Spreng DC

The essential oil of the aerial parts of Senecio selloi Spreng. DC. was extracted by hydrodistillation and analyzed by GC/MS. Nineteen compounds were identified, representing 99.9% of the total. The main compounds were found to be sesquiterpene hydrocarbons (71.3%), most of them with a bisabolane skeleton (59.4%). The major constituent was α-zingiberene (54%), followed by monoterpene α-isolimonene (16%). The essential oil was also tested against two Gram-positive and two Gram-negative bacterial species, three yeasts, and an algae. From the strains assayed, only Bacillus subtilis ATCC 6633 showed susceptibility (MIC and MBC = 4400 µg/mL) to the essential oil.

O óleo essencial das partes aéreas de Senecio selloi Spreng DC. foi extraído por hidrodestilação e analisado por CG/EM. Dezenove constituintes foram identificados, representando 99,9% do total. Os principais compostos fornecidos foram sesquiterpenos hidrocarbonetos (71,3%), a maioria destes com esqueleto bisabolano (59,4%). O constituinte majoritário foi a-zingibereno (54%), seguido do monoterpeno a-isolimoneno (16%). O óleo essencial foi testado contra duas cepas Gram-positivas e duas Gram-negativas, três fungos e uma alga. De todas as linhagens testadas somente Bacillus subtilis ATCC 6633 mostrou suscetibilidade (CIM e CBM = 4400 µg/mL) para o óleo essencial.

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.

Activity of essential oils from spices against Staphylococcus spp. isolated from bovine mastitis / Atividade de óleos essenciais de plantas condimentares frente Staphylococcus spp. isolados de mastite bovina

Avaliou-se a atividade antimicrobiana dos óleos essenciais (OE) de Origanum vulgare (orégano), Thymus vulgaris (tomilho), Lippia graveolens (lipia), Zingiber officinale (gengibre), Salvia officinalis (sálvia), Rosmarinus officinalis (alecrim) e Ocimum basilicum (manjericão), e de suas frações majoritárias, carvacrol e timol, frente a 32 isolados de Staphylococcus spp, oriundos de rebanhos leiteiros bovinos. A concentração inibitória mínima (CIM) e a concentração bactericida mínima foram determinadas por meio da técnica de microdiluição em caldo. Orégano, tomilho e lípia (Orégano Mexicano) apresentaram atividade antimicrobiana similar, médias geométrica de CIM de 1600µg mL-1; 1564µg mL-1; 1562µg mL-1, respectivamente, no entanto menos ativos que carvacrol, 584µg mL-1 e thymol, 427µg mL-1. Isolados com diferentes perfis de susceptibil idade aos antimicrobianos usados no tratamento de mastite bovina, quando subagrupados, foram inibidos por concentrações semelhantes de OE . Estes resultados confirmam a atividade antimicrobiana de OE e algumas frações majoritárias.

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.

In vitro interactions between amphotericin B and other antifungal agents and rifampin against Fusarium spp.

Fusarium species are common hyaline soil saprophytes and plant pathogens that are opportunistic fungal pathogens of immunocompromised patients. The treatment for fusariosis remains uncertain with an unfavourable prognosis; new possibilities for treatment, such as various synergistic drug interactions, must be uncovered. In this study, we evaluated the in vitro interactions of amphotericin B with caspofungin, ketoconazole, 5-flucytosine, itraconazole, miconazole, rifampin, fluconazole, terbinafine and voriconazole against isolates of Fusarium spp. using the chequerboard method with interactions evaluated by fractional inhibitory concentration indices. The highest percentages of synergistic interactions were observed for the combinations of amphotericin B and caspofungin (68.7%), amphotericin B and rifampin (68.7%), amphotericin B plus 5-flucytosine (59.3%) and amphotericin B with voriconazole (37.5%). The pattern of susceptibility to antifungal agents among Fusarium species and their consequence on the effects of drug combinations are also discussed.

Comparison of antioxidant activities and total polyphenolic and methylxanthine contents between the unripe fruit and leaves of Ilex paraguariensis A. St. Hil.

Ilex paraguariensis is used in Brazil as a stimulating beverage called "mate". Leaves and immature fruit extracts of Ilex paraguariensis were evaluated for their radical scavenging capacity, total methylxanthine and polyphenol contents. Antimicrobial activity of two enriched saponin fractions obtained from the fruits were also evaluated. The radical scavenging activity of the fractioned extracts was determined spectrophotometrically using 1,1-diphenylpicrylhydrazyl free radical (DPPH). The IC50o of L-ascorbic acid, ethyl acetate and n-butanol fractions from the leaves and ethyl acetate fraction from the fruits were 6.48 microg/mL, 13.26 microg/mL, 27.22 microg/mL, and 285.78 microg/mL, respectively. Total methylxanthine content was 1.16 +/- 0.06 mg/g dry weight in the fruits and 8.78 +/- 0.01 mg/g in the leaves. Total polyphenol content varied from 86.82 +/- 3 x 10(-4) to 199.91 +/- 3 x 10(-3) mg/g in leaf fractions and from 54.25 +/- 1 x 10(-3) to 110.36 +/- 4 x 10(-4) mg/g in fruit fractions. Enriched saponin fractions from the fruits showed no antimicrobial activity. To our knowledge, this are the first data available on the antioxidant/antimicrobial activities and polyphenol/methylxanthine contents of Ilex paraguariensis fruits.

In vitro activities of new and conventional antimycotics against fluconazole-susceptible and non-susceptible Brazilian Candida spp. isolates.

The substantial increase in the rate of azole resistant Candida spp. yeast infections has become a serious treatment problem requiring new and more active antifungal agents. In this study, the in vitro activities of ravuconazole and albaconazole were compared with those of amphotericin B, flucytosine, itraconazole and fluconazole against 162 Brazilian isolates of Candida spp. from which 48 isolates had previously shown lower susceptibility or resistance to fluconazole. Ravuconazole susceptibility ranged from 84.6% (Candida albicans) to 100% for other species and albaconazole MIC(90) was < or =1.0 microg ml(-1) for all the species emphasising the potent activity of these triazoles. To our knowledge this is the first study evaluating the susceptibility of C. dubliniensis to albaconazole.