Chemical composition, antifungal activity, antibiofilm and citotoxicity of the essential oil of Citrus deliciosa tenore.

The leaves of Citrus deliciosa Tenore were collected in southern Brazil, dried, and subjected to the hydrodistillation process to obtain the essential oil. The extraction of essential oil yielded 0.97% ± 0.04. The chromatographic profile of this compound revealed 11 substances, being methyl N-methyl-anthranilate (75.1%), γ-terpinene (13.8%), and Limonene (7%) as major substances. The essential oil shows antifungal action against all tested yeasts, with promising action against Cryptococcus neoformans, Cryptococcus gattii, and Trichosporon asahii. This compound was also able to inhibit the biofilm production of Candida albicans, Candida glabrata, Candida parapsilosis, and T. asahii. The essential oil of tangerine showed weak antioxidant action. It did not show cytotoxicity in human mononuclear cells. It is hoped that these results may guide future studies for the production of formulations that can be used in the treatment of biofilms caused by yeasts, as well as in candidiasis, cryptococcosis, and/or trichosporonosis.

Antibacterial, antifungal, and anti-biofilm effects of sulfamethoxazole-complexes against pulmonary infection agents.

Antibiotic resistance associated with pulmonary infection agents has become a public health problem, being considered one of the main priorities for immediate resolution. Thus, to increase the therapeutic options in the fight against resistant microorganisms, the synthesis of molecules from pre-existing drugs has shown to be a promising alternative. In this sense, the present work reports the synthesis, characterization, and biological evaluation (against fungal and bacterial agents that cause lung infections) of potential metallodrugs based on sulfamethoxazole complexed with AuI, AgI, HgII, CdII, NiII, and CuII. The minimal inhibitory concentration (MIC) value was used to evaluate the antifungal and antibacterial properties of the compounds. In addition, it was also evaluated the antibiofilm capacity in Pseudomonas aeruginosa, through the quantification of its biomass and visualization using atomic force microscopy. For each case, molecular docking calculations were carried out to suggest the possible biological target of the assayed inorganic complexes. Our results indicated that the novel inorganic complexes are better antibacterial and antifungal than the commercial antibiotic sulfamethoxazole, highlighting the AgI-complex, which was able to inhibit the growth of microorganisms that cause lung diseases with concentrations in the 2-8 µg mL-1 range, probably at targeting dihydropteroate synthetase - a key enzyme involved in the folate synthesis. Furthermore, sulfamethoxazole complexes were able to inhibit the formation of bacterial biofilms at significantly lower concentrations than free sulfamethoxazole, probably mainly targeting the active site of LysR-type transcriptional regulator (PqsR). Overall, the present study reports preliminary results that demonstrate the derivatization of sulfamethoxazole with transition metal cations to obtain potential metallodrugs with applications as antimicrobial and antifungal against pulmonary infections, being an alternative for drug-resistant strains.

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.

Mucoadhesive gellan gum hydrogel containing diphenyl diselenide-loaded nanocapsules presents improved anti-candida action in a mouse model of vulvovaginal candidiasis.

The aim of this study was to evaluate the in vitro antifungal action of a diphenyl diselenide-loaded poly(ε-caprolactone) nanocapsules suspension (NC-1) and incorporate it into a gellan gum hydrogel formulation in order to assess its in vivo efficacy in an animal model of vulvovaginal candidiasis. Nanocapsules suspensions containing the compound (NC-1 ∼ 5 mg/mL) or not (NC-B) were prepared by the interfacial deposition of preformed polymer method. To estimate in vitro antifungal effect, the broth microdilution test was applied. The results showed that NC-1 had equal or lower MIC values when compared to free compound against fifteen Candida strains. Following, the hydrogel was prepared by direct thickening of the nanocapsules suspension by gellan gum addition. The animal model of vulvovaginal candidiasis was induced by infecting female Swiss mice with Candida albicans strains. The animals were topically treated with 20 µL of hydrogels (NC-1 and free compound - 0.1 mg of diphenyl diselenide/once a day for seven days) and then the total fungal burden was assessed after the euthanasia. The results showed that the hydrogels presented pH in the acidic range, compound content close to theoretical value, homogeneous particle distribution with nanometric size, high physicochemical and microbiological stability as well as great bioadhesive property. The nano-based presented superior pharmacological action in comparison to the hydrogel containing non-encapsulated diphenyl diselenide. The results demonstrated that the nanoencapsulation maintained the effective antifungal action of diphenyl diselenide. The nano-based hydrogel formulation may be considered a promising approach against vulvovaginal candidiasis.

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.

Trends in Azole Resistance in Aspergillus fumigatus, the Netherlands, 1994-2016.

We investigated azole resistance in Aspergillus fumigatus in a tertiary reference hospital in the Netherlands during 1994-2016. The 5-year patient-adjusted proportion of resistance increased from 0.79% for 1996-2001 to 4.25% for 2002-2006, 7.17% for 2007-2011, and 7.04% for 2012-2016. However, we observed substantial variation between years.

In vitro interactions of azoles and echinocandins against clinical strains of Aspergillus flavus.

Combinations of an azole (itraconazole, voriconazole, or posaconazole) with an echinocandin (caspofungin, micafungin, or anidulafungin) were tested against 20 clinical isolates of Aspergillus flavus according to EUCAST guidelines. The interactions were determined using two endpoints-minimal effective concentration (MEC) and minimal inhibitory concentration (MIC)-via calculation of the fractional inhibitory concentration (FIC) index. A higher prevalence of synergistic interactions was observed for MIC, whereas indifference was the most frequent outcome according to MEC among the 20 strains. Combined treatment of A. flavus with these two important classes of antifungals should be explored further in in vivo studies.

In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil

ABSTRACT The in vitro susceptibility of 105 clinical and environmental strains of Aspergillus fumigatus and Aspergillus flavus to antifungal drugs, such as amphotericin B, azoles, and echinocandins was evaluated by the broth microdilution method proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Following the EUCAST-proposed breakpoints, 20% and 25% of the clinical and environmental isolates of A. fumigatus, respectively, were found to be resistant to itraconazole (Minimal Inhibitory Concentration, MIC > 2.0 mg/L). Voriconazole showed good activity against A. fumigatus and A. flavus strains, except for one clinical strain of A. fumigatus whose MIC was 4.0 mg/L. Posaconazole (≤0.25 mg/L) also showed appreciable activity against both species of Aspergillus, except for six A. fumigatus strains with relatively higher MICs (0.5 mg/L). The MICs for Amphotericin B ranged from 0.06 to 1.0 mg/L for A. fumigatus, but were much higher (0.5-8.0 mg/L) for A. flavus. Among the echinocandins, caspofungin showed a geometric mean of 0.078 and 0.113 against the clinical and environmental strains of A. flavus, respectively, but had elevated minimal effective concentrations (MECs) for seven of the A. fumigatus strains. Anidulafungin and micafungin exhibited considerable activity against both A. fumigatus and A. flavus isolates, except for one environmental isolate of A. fumigatus that showed an MEC of 1 mg/L to micafungin. Our study proposes that a detailed investigation of the antifungal susceptibility of the genus Aspergillus from different regions of Brazil is necessary for establishing a response profile against the different classes of antifungal agents used in the treatment of aspergillosis.

Comparison Between Etest and Broth Microdilution Methods for Testing Itraconazole-Resistant Aspergillus fumigatus Susceptibility to Antifungal Combinations.

The checkerboard broth microdilution assay (BMD) is the most frequently used method for the in vitro evaluation of drug combinations. However, its use to evaluate the effect of antifungal drugs on filamentous fungi is sometimes associated with endpoint-reading difficulties, and different degrees of interaction are assigned to the same drug combination. We evaluated combinations of the azoles, itraconazole, posaconazole, and voriconazole, with the echinocandins, anidulafungin, caspofungin, and micafungin, against 15 itraconazole-resistant Aspergillus fumigatus clinical strains via the checkerboard BMD and Etest assay. Readings after 24 and 48 h, considering the two reading endpoints, the minimum inhibitory concentration (MIC) and minimum effective concentration (MEC), were performed for both methods. Our results showed that the correlation coefficients between the BMD and Etest methods were quite diverse to the drug combinations tested. The highest correlation coefficients of the Etest with the BMD assays (MEC and MIC reading) were the Etest-MIC reading at 24 h and the Etest-MEC reading at 48 h. Improvements in experimental conditions may increase the correlation between the two methods and ensure that Etest assay can be safely used in the evaluation of antifungal combinations against Aspergillus species.

Sporothrix brasiliensis produces the highest levels of oxidative stress in a murine model among the species of the Sporothrix schenckii complex.

INTRODUCTION:: We compared indicators of oxidative stress in the tissue of mice infected with strains from Sporothrix schenckii complex. METHODS:: Mice were inoculated with Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa, Sporothrix mexicana or Sporothrix albicans. The activity of catalase and glutathione were accessed in the liver and spleen. RESULTS:: Animals infected with S. brasiliensis exhibited splenomegaly and significant decrease in catalase activity, and protein and non-protein thiol content compared to animals infected with the other species. CONCLUSIONS:: Sporothrix brasiliensis exhibits higher pathogenicity compared to other species of the Sporothrix schenckii complex by increasing oxidative stress in animal tissue.

Sporothrix brasiliensis produces the highest levels of oxidative stress in a murine model among the species of the Sporothrix schenckii complex

Abstract INTRODUCTION: We compared indicators of oxidative stress in the tissue of mice infected with strains from Sporothrix schenckii complex. METHODS: Mice were inoculated with Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa, Sporothrix mexicana or Sporothrix albicans. The activity of catalase and glutathione were accessed in the liver and spleen. RESULTS: Animals infected with S. brasiliensis exhibited splenomegaly and significant decrease in catalase activity, and protein and non-protein thiol content compared to animals infected with the other species. CONCLUSIONS: Sporothrix brasiliensis exhibits higher pathogenicity compared to other species of the Sporothrix schenckii complex by increasing oxidative stress in animal tissue.

Activity of Combined Antifungal Agents Against Multidrug-Resistant Candida glabrata Strains.

In this study, we evaluated the in vitro activity of echinocandins, azoles, and amphotericin B alone and in combination against echinocandin/azole-sensitive and echinocandin/azole-resistant Candida glabrata isolates. Susceptibility tests were performed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute document M27-A3. The checkerboard method was used to evaluate the fractional inhibitory concentration index of the interactions. Cross-resistance was observed among echinocandins; 15% of the isolates resistant to caspofungin were also resistant to anidulafungin and micafungin. Synergistic activity was observed in 70% of resistant C. glabrata when anidulafungin was combined with voriconazole or posaconazole. Higher (85%) synergism was found in the combination of caspofungin and voriconazole. The combinations of caspofungin with fluconazole, posaconazole and amphotericin B, micafungin with fluconazole, posaconazole and voriconazole, and anidulafungin with amphotericin B showed indifferent activities for the majority of the isolates. Anidulafungin combined with fluconazole showed the same percentage of synergism and indifference (45%). Antagonism was detected in 50% of isolates when micafungin was combined with amphotericin B. Combinations of echinocandins and antifungal azoles have great potential for in vivo assays which are required to evaluate the efficacy of these combinations against multidrug-resistant C. glabrata strains.

Antifungal activities of tacrolimus in combination with antifungal agents against fluconazole-susceptible and fluconazole-resistant Trichosporon asahii isolates

ABSTRACT The antifungal activity of tacrolimus in combination with antifungal agents against different fungal species has been previously reported. Here we report the in vitro interactions between tacrolimus and amphotericin B, fluconazole, itraconazole, and caspofungin against 30 clinical isolates of both fluconazole-susceptible and fluconazole-resistant Trichosporon asahii. For these analyses, we used the broth microdilution method based on the M27-A3 technique and checkerboard microdilution method. Tacrolimus showed no activity against T. asahii strains (minimal inhibitory concentrations, MICs > 64.0 µg mL−1). However, a larger synergistic interaction was observed by the combinations tacrolimus + amphotericin B (96.67%) and tacrolimus + caspofungin (73.33%) against fluconazole-susceptible isolates. Combinations with azole antifungal agents resulted in low rates of synergism for this group (fluconazole + tacrolimus = 40% and itraconazole + tacrolimus = 10%). Antagonistic interactions were not observed. For the fluconazole-resistant T. asahii group, all tested combinations showed indifferent interactions. The synergism showed against fluconazole-susceptible T. asahii isolates suggests that the potential antifungal activity of tacrolimus deserves in vivo experimental investigation, notably, the combination of tacrolimus with amphotericin B or caspofungin.

Antifungal activities of tacrolimus in combination with antifungal agents against fluconazole-susceptible and fluconazole-resistant Trichosporon asahii isolates.

The antifungal activity of tacrolimus in combination with antifungal agents against different fungal species has been previously reported. Here we report the in vitro interactions between tacrolimus and amphotericin B, fluconazole, itraconazole, and caspofungin against 30 clinical isolates of both fluconazole-susceptible and fluconazole-resistant Trichosporon asahii. For these analyses, we used the broth microdilution method based on the M27-A3 technique and checkerboard microdilution method. Tacrolimus showed no activity against T. asahii strains (minimal inhibitory concentrations, MICs>64.0µgmL-1). However, a larger synergistic interaction was observed by the combinations tacrolimus+amphotericin B (96.67%) and tacrolimus+caspofungin (73.33%) against fluconazole-susceptible isolates. Combinations with azole antifungal agents resulted in low rates of synergism for this group (fluconazole+tacrolimus=40% and itraconazole+tacrolimus=10%). Antagonistic interactions were not observed. For the fluconazole-resistant T. asahii group, all tested combinations showed indifferent interactions. The synergism showed against fluconazole-susceptible T. asahii isolates suggests that the potential antifungal activity of tacrolimus deserves in vivo experimental investigation, notably, the combination of tacrolimus with amphotericin B or caspofungin.

In Vitro Susceptibility of Pythium insidiosum to Melaleuca alternifolia, Mentha piperita and Origanum vulgare Essential Oils Combinations.

Pythium insidiosum is the etiologic agent of pythiosis, a severe and emerging disease that affects mammals. Failure of conventional antifungal therapies is partially justified by the absence of ergosterol in the plasma membrane of this oomycete. Despite research advancement, the treatment of pythiosis has not been not fully established. The present study investigated the in vitro susceptibility profile of Brazilian isolates of P. insidiosum (n = 20) against Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils, and their combinations. Susceptibility tests were performed according to CLSI M38-A2 protocol, and combinations were evaluated by the microdilution cherkerboard method. All tested essential oils showed antimicrobial activity against P. insidiosum, and the greatest activity of O. vulgare was highlighted. Synergistic and/or indifferent effect was observed for all combinations evaluated, especially the M. piperita and O. vulgare combination, which showed 65 % synergism. This is the first study to report in vitro combinations of essential oils against P. insidiosum indicating the susceptibility of this oomycete to M. alternifolia, M. piperita and O. vulgare essential oils, as well as their combinations.

Antimicrobial Activity and Chromatographic Analysis of Extracts from Tropaeolum pentaphyllum Lam. Tubers.

BACKGROUND: Tropaeolum pentaphyllum Lam. tubers (Tropaeolaceae) are known and used as a condiment and for the treatment of skin infections in Southern Brazil. However, its activity and composition has not yet been investigated. Thus, different extracts and the essential oil from the tubers were tested against a range of microorganisms. The most active extracts were submitted to chromatographic analysis. METHODS: Hydroalcoholic extract (70%), fractions of it, and the essential oil from the tubers were tested against several bacteria, yeasts and molds, furnishing the corresponding inhibitory, bactericidal and fungicidal minimal concentration values. The most active extracts were submitted to GC-MS investigation. RESULTS: The strongest effects against different strains of microorganisms, such as Gram-positive and negative bacteria, Candida spp. and dermatophytes were observed for the essential oil and the chloroform fraction, with minimal inhibitory concentrations (MICs) well below 200 µg/mL. GC-MS analysis revealed that the major essential oil constituent is benzyl isothiocyanate (BITC), while the chloroform fraction is constituted of BITC, amides, sulfur, fatty acids and its esters, all compounds that may be related to the demonstrated activity. CONCLUSIONS: Overall, the results support the popular use of the plant for the treatment of skin infections, and revealed the main active compounds.

Interference of melanin in the susceptibility profile of Sporothrix species to amphotericin B.

BACKGROUND: The presence of melanin in the fungal cell is a major virulence factor of the genus Sporothrix since it protects the fungal cells against the defense systems. AIMS: The present study aimed to investigate the interference of melanin in the susceptibility of Sporothrix brasiliensis and Sporothrix schenckii sensu stricto to amphotericin B and itraconazole, drugs recommended as therapy for disseminated and subcutaneous sporotrichosis, respectively. METHODS: Yeast cells were cultivated in minimal medium with or without l-DOPA in order to induce the production of melanin. Microdilution and killing assay methods were used to determine the antifungal activity against yeast cells with different amounts of melanin. RESULTS: The killing assay showed that melanization protected isolates within the S. schenckii complex from amphotericin B, particularly in the lower concentrations tested. CONCLUSIONS: Studies combining amphotericin B and inhibitors of melanin are required in order to avoid this effect.

Synergistic effects of tacrolimus and azole antifungal compounds in fluconazole-susceptible and fluconazole-resistant Candida glabrata isolates.

In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata , a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro , notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.