Juliana S. M. Tondolo

Anesthesia and transport of fat snook Centropomus parallelus with the essential oil of Nectandra megapotamica(Spreng.) Mez

This study analyzed the chemical composition and anesthetic potential of essential oil (EO) of Nectandra megapotamica in fat snook (Centropomus parallelus). For the extraction of EO by hydrodistillation, leaves were separated in young (EO-Y) or old (EO-O), and the chemical composition of the EOs was determined by CG-MS. The anesthetic potential was assessed by the evaluation of induction and recovery time of anesthesia and stress response from anesthesia and transport. Three experiments were carried out: i) four different concentrations of each EO were tested to evaluate anesthesia induction and recovery time; ii) two concentrations of EO-O were tested for the evaluation of its effects on stress parameters (glucose, lactate, and Na+ and K+ plasma levels) caused by anesthesia; and iii) fish were transported in plastic bags, supplied with two concentrations of EO-O for the evaluation of water quality and mortality. All experiments were performed on fish acclimated to 0 and 33 ppt salinity. The main constituents of the Y and O-EOs were bicyclogermacrene (46.5/34.6%), α-pinene (26.8/26.2%), β-pinene (7.9/12.3%), and germacrene D (9.6/9.1%). Mild sedation was achieved at 30 °L L-1(1.3-3.2 min) and deep anesthesia at 150 °L L-1(5.6-8.0 min) with both EOs. The recovery time ranged from 1-10 min. The EO-O was not able to avoid the stress of anesthesia evidenced by elevated glucose and lactate plasma levels observed in all groups. Plasma levels of Na+ and K+ were not significantly affected by treatments. During transport, the use of EO-O did not prevent deterioration in water quality and the post-transport mortality. In conclusion, the EO of N. megapotamica has anesthetic activity in fat snook, but it was not able to prevent the stress of anesthesia and transport.

In Vitro Synergism Observed with Azithromycin, Clarithromycin, Minocycline, or Tigecycline in Association with Antifungal Agents against Pythium insidiosum

ABSTRACT We describe here the in vitro activities of azithromycin, clarithromycin, minocycline, or tigecycline alone and in combination with amphotericin B, itraconazole, terbinafine, voriconazole, anidulafungin, caspofungin, or micafungin against 30 isolates of the oomycete Pythium insidiosum. The assays were based on the CLSI M38-A2 technique and the checkerboard microdilution method. The main synergisms observed were through the combination of minocycline with amphotericin B (73.33%), itraconazole (70%), and micafungin (70%) and of clarithromycin with micafungin (73.33%).

Seasonal influence on the essential oil production of Nectandra megapotamica (Spreng.) Mez

This study evaluated the seasonal influence on the yield and chemical composition of the essential oil (EO) of Nectandra megapotamica. Fresh young (YL) and old leaves (OL) obtained from three trees in each season (Nov/2010 to Sep/2011) collected in Santa Maria-RS were hydrodistilled in triplicate. The chemical composition was determined by the gas chromatography coupled to mass spectrometry (GC-MS) and the yield on dry basis was evaluated by two-way ANOVA (seasons, development stage). Spring (Sp) and summer (Su) showed higher average incomes (0.45 and 0.33%), which occurred when flowering, fruiting, and growth of YL and senescence of OL took place, while autumn (Au) presented the lowest yield (0.25%) during the rustification of OL. The highest yield was obtained for the YL in Sp (0.59%) and the lowest for the OL in Au (0.21%). The major constituents of the EO were independent from the season and were identified as α-pinene, bicyclogermacrene, β-pinene, germacrene D, and limonene. Seasonality and phenology influenced the production of EO probably due to morphological and metabolic alterations in the leaves as well as due to the needs of the tree, such as attraction and/or protection.

In vitro and in vivo antimicrobial activities of minocycline in combination with azithromycin, clarithromycin or tigecycline against Pythium insidiosum

ABSTRACT The present study investigated the in vitro and the in vivo interactions among azithromycin, clarithromycin, minocycline, and tigecycline against Pythium insidiosum. In vitro antimicrobial activities were determined by the broth microdilution method in accordance with CLSI document M38-A2, and the antibiotic interactions were assayed using the checkerboard MIC format. In vivo efficacy was determined using a rabbit infection model. The geometric mean MICs of azithromycin, clarithromycin, minocycline, and tigecycline against P. insidiosum were, respectively, 1.91, 1.38, 0.91, and 0.79 μg/ml. By checkerboard testing, all combinations resulted in in vitro synergistic interactions (>60%). Antagonism was not observed. The in vivo studies showed that azithromycin (20 mg/kg/day twice daily) alone or in combination with minocycline (10 mg/kg/day twice daily) significantly decreased the fungal burden. This study demonstrates that azithromycin possesses potent curative efficacy against subcutaneous pythiosis in the rabbit model.

New Insights into the In Vitro Susceptibility of Pythium insidiosum

ABSTRACT We have determined the in vitro activity of several antibacterial and antifungal drugs against Pythium insidiosum using broth microdilution (BMD), disk diffusion, and Etest methods. The largest zones of inhibition (disk diffusion) and the lowest BMD and Etest MICs were observed for azithromycin, clarithromycin, linezolid, mupirocin, doxycycline, minocycline, and tigecycline. The in vitro activities observed suggest that antibacterials, which act by inhibiting protein synthesis, are promising candidate therapies for the treatment of pythiosis.

In vitro susceptibility of Conidiobolus lamprauges recovered from sheep to antifungal agents.

Data regarding the susceptibility of Conidiobolus lamprauges is limited and there is no consensus about the optimal treatment for infections caused by Conidiobolus spp. In this context, the objective of this study was to evaluate the in vitro susceptibility of six C. lamprauges strains isolated from sheep conidiobolomycosis to amphotericin B, ketoconazole, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, micafungin, flucytosine, and terbinafine using the CLSI M38-A2 microdilution technique. Terbinafine was the most active (MIC range <0.06-0.5 μg/mL). Resistance or reduced susceptibility was observed for amphotericin B and azole and echinocandin antifungals. Additional studies are necessary to determine the therapeutic potential of terbinafine as monotherapy or in combination therapy with other antifungals.

A simple, rapid and inexpensive screening method for the identification of Pythium insidiosum☆

Growth of Pythium insidiosum mycelia around minocycline disks (30μg) did not occur within 7days of incubation at 35°C when the isolates were grown on Sabouraud, corn meal, Muller-Hinton or RPMI agar. This technique offers a simple and rapid method for the differentiation of P. insidiosum from true filamentous fungi.

Chemically induced disseminated pythiosis in BALB/c mice: A new experimental model for Pythium insidiosum infection

Pythiosis is a severe and life-threatening disease that affects humans and various animal species. We report a model of vascular/disseminated pythiosis occurring after subcutaneous inoculation of 2 x 104 Pythium insidiosum zoospores/mL in immunocompromised BALB/c mice. For this model, we carried out two rounds of experiments. First, we evaluated two protocols of immunosuppression before inoculation: cyclophosphamide at 150 mg/kg (CYP group) and cyclophosphamide 200 mg/kg plus hydrocortisone acetate at 250 mg/kg (CYP+HCA group). It was not possible to obtain mortality in the CYP group; however, the combination of CYP+HCA altered disease outcomes, with mortality rates reaching 60%. Second, we used the CYP+HCA immunosuppression protocol to analyze the histological and immunological statuses triggered by disease. When we inoculated immunocompetent mice with P. insidiosum zoospores, self-healing occurred via increased levels of IL-2, IFN-γ and IL-17A, which are characteristic of the Th1/Th17 cytokine response. For infected and immunosuppressed mice, the cytokine profiles showed high levels of IL-10, IL-6 and TNF-α. Increased IL-10 values are related to fungal infection susceptibility and led us to speculate that infection may be established through suppression of the host immune response. In addition, histopathological evaluation of the kidneys and liver demonstrated the presence of hyphae and the cellular findings suggested an acute vascular inflammation that mimics vascular/disseminated pythiosis in humans. This is the first murine model for pythiosis that is useful both for understanding the pathogenesis of this disease and for evaluating new treatment approaches.

Extraction, characterization and biological activity of a (1,3)(1,6)-β-d-glucan from the pathogenic oomycete Pythium insidiosum.

Pythiosis is a life-threatening infectious disease caused by the pathogenic oomycete Pythium insidiosum. This study is the first to evaluate the P. insidiosum glucan content and its biological activities. The enzymatic quantification of the glucans in P. insidiosum mycelia showed that the β-glucan content was 18.99%±3.59. The cell wall polysaccharide extract consisted of ∼81.7% carbohydrates (exclusively glucose) and ∼18.3% residual amino acids and peptides. The results from monosaccharide composition, methylation and 1D/2D NMR spectroscopy analyses indicated the presence of a highly branched (1,3)(1,6)-β-d-glucan, with (1,6)-β-d-glucopyranosil side-branching unit on average every 1-2 repeat units. In vitro, the β-d-glucan extract could significantly promote spleen lymphocyte proliferation in human, equine and mouse cell cultures. BALB/c mice that were subcutaneously pre-immunized with three doses of 0.5, 2.5 and 5.0mg of β-glucan/mouse, showed a significant increase in IL-2, IL-6, IL-10, TNF-α and IL-17A production compared to non-immunized mice. These results suggested that β-d-glucan extract induces significant and specific Th17 cellular immune response and provided the theoretical basis for further experiments.

In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum.

ABSTRACT We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC90 between 2 and 32 μg∕ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum.