Farnesol inhibits planktonic cells and antifungal-tolerant biofilms of Trichosporon asahii and Trichosporon inkin.

Trichosporon species have been considered important agents of opportunistic systemic infections, mainly among immunocompromised patients. Infections by Trichosporon spp. are generally associated with biofilm formation in invasive medical devices. These communities are resistant to therapeutic antifungals, and therefore the search for anti-biofilm molecules is necessary. This study evaluated the inhibitory effect of farnesol against planktonic and sessile cells of clinical Trichosporon asahii (n = 3) andTrichosporon inkin (n = 7) strains. Biofilms were evaluated during adhesion, development stages and after maturation for metabolic activity, biomass and protease activity, as well as regarding morphology and ultrastructure by optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy. Farnesol inhibited Trichosporon planktonic growth by 80% at concentrations ranging from 600 to 1200 µM for T. asahii and from 75 to 600 µM for T. inkin. Farnesol was able to reduce cell adhesion by 80% at 300 µM for T. asahii and T. inkin at 600 µM, while biofilm development of both species was inhibited by 80% at concentration of 150 µM, altering their structure. After biofilm maturation, farnesol decreased T. asahii biofilm formation by 50% at 600 µM concentration and T. inkin formation at 300 µM. Farnesol inhibited gradual filamentation in a concentration range between 600 and 1200 µM. Farnesol caused reduction of filament structures of Trichosporon spp. at every stage of biofilm development analyzed. These data show the potential of farnesol as an anti-biofilm molecule.

Antifungal susceptibility of Sporothrix schenckii complex biofilms.

Sporotrichosis, caused by species of Sporothrix schenckii complex, is the most prevalent subcutaneous mycosis in many areas of Latin America. The aim of this study was to evaluate the ability of Sporothrix spp. to form biofilms in vitro and to characterize the growth kinetics, morphology, and antifungal susceptibility of biofilms against classical antifungals. We investigated the ability of strains to produce biofilms in vitro and determined the effects of exposure to amphotericin B, itraconazole, caspofungin, ketoconazole, voriconazole, and fluconazole at minimum inhibitory concentration (MIC) against planktonic form and at 10× MIC and 50× MIC on the biomass and metabolic activity of these biofilms. Biofilm structure was analyzed by optical microscopy using Congo-red staining, confocal and scanning electron microscopy. Strains were classified for biofilm-forming ability, through the analysis of absorbance of crystal violet retained by biomass of mature biofilms. We found that all S. brasiliensis (n = 10), S. schenckii sensu stricto (n = 2), S. globosa (n = 2), and S. mexicana (n = 4) strains were strong biofilm-producers. The analyzed biofilms had dense network of hyphae and conidia immersed in extracellular matrix, with presence of water channels. Antifungal drugs at the three tested concentrations showed different effects on biomass and metabolic activity of biofilms. However, the best inhibitory response was observed with 50× MIC of amphotericin B and caspofungin, which reduced these parameters. Furthermore, high drug concentrations, especially amphotericin B and caspofungin, showed antifungal activity against these biofilms, probably because they damaged the architecture and extracellular matrix, allowing diffusion of the drugs.

Inhibitory effect of a lipopeptide biosurfactant produced by Bacillus subtilis on planktonic and sessile cells of Trichosporon spp.

The present study aimed to investigate the inhibitory effect of a bacterial biosurfactant (TIM96) on clinical strains of Trichosporon. Additionally, the effect of TIM96 on the ergosterol content, cell membrane integrity, and the hydrophobicity of planktonic cells was assessed. The inhibitory activity of TIM96 against Trichosporon biofilms was evaluated by analyzing metabolic activity, biomass and morphology. MIC values ranged from 78.125 to 312.5 µg ml-1 for TIM96; time-kill curves revealed that the decline in the number of fungal cells started after incubation for 6 h with TIM96 at both MIC and 2×MIC. The biosurfactant reduced the cellular ergosterol content and altered the membrane permeability and the surface hydrophobicity of planktonic cells. Incubation at 10×MIC TIM96 reduced cell adhesion by up to 96.89%, thus interfering with biofilm formation. This concentration also caused up to a 99.2% reduction in the metabolic activity of mature biofilms. The results indicate potential perspectives for the development of new antifungal strategies.

Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei.

Efflux pumps are important defense mechanisms against antimicrobial drugs and maintenance of Burkholderia pseudomallei biofilms. This study evaluated the effect of the efflux pump inhibitor promethazine on the structure and antimicrobial susceptibility of B. pseudomallei biofilms. Susceptibility of planktonic cells and biofilms to promethazine alone and combined with antimicrobials was assessed by the broth microdilution test and biofilm metabolic activity was determined with resazurin. The effect of promethazine on 48 h-grown biofilms was also evaluated through confocal and electronic microscopy. The minimum inhibitory concentration (MIC) of promethazine was 780 mg l-1, while the minimum biofilm elimination concentration (MBEC) was 780-3,120 mg l-1. Promethazine reduced the MIC values for erythromycin, trimethoprim/sulfamethoxazole, gentamicin and ciprofloxacin and reduced the MBEC values for all tested drugs (p<0.05). Microscopic analyses demonstrated that promethazine altered the biofilm structure of B. pseudomallei, even at subinhibitory concentrations, possibly facilitating antibiotic penetration. Promethazine improves antibiotics efficacy against B. pseudomallei biofilms, by disrupting biofilm structure.

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth, biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 µg ml-1) inhibited Trichosporon growth. RIT (100 µg ml-1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 µg ml-1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.

Inhibition of heat-shock protein 90 enhances the susceptibility to antifungals and reduces the virulence of Cryptococcus neoformans/Cryptococcus gattii species complex.

Heat-shock proteins (Hsps) are chaperones required for the maintenance of cellular homeostasis in different fungal pathogens, playing an important role in the infectious process. This study investigated the effect of pharmacological inhibition of Hsp90 by radicicol on the Cryptococcus neoformans/Cryptococcus gattii species complex--agents of the most common life-threatening fungal infection amongst immunocompromised patients. The influence of Hsp90 inhibition was investigated regarding in vitro susceptibility to antifungal agents of planktonic and sessile cells, ergosterol concentration, cell membrane integrity, growth at 37 °C, production of virulence factors in vitro, and experimental infection in Caenorhabditis elegans. Hsp90 inhibition inhibited the in vitro growth of planktonic cells of Cryptococcus spp. at concentrations ranging from 0.5 to 2 µg ml(-1) and increased the in vitro inhibitory effect of azoles, especially fluconazole (FLC) (P < 0.05). Inhibition of Hsp90 also increased the antifungal activity of azoles against biofilm formation and mature biofilms of Cryptococcus spp., notably for Cryptococcus gattii. Furthermore, Hsp90 inhibition compromised the permeability of the cell membrane, and reduced planktonic growth at 37 °C and the capsular size of Cryptococcus spp. In addition, Hsp90 inhibition enhanced the antifungal activity of FLC during experimental infection using Caenorhabditis elegans. Therefore, our results indicate that Hsp90 inhibition can be an important strategy in the development of new antifungal drugs.

Synthesis and in vitro antifungal activity of isoniazid-derived hydrazones against Coccidioides posadasii.

Coccidioidomycosis is a potentially severe infection caused by dimorphic fungi Coccidioides immitis and Coccidioides posadasii. Although guidelines are well established, refractory disease is a matter of concern in the clinical management of coccidioidomycosis. In the present study three isoniazid-derived hydrazones N'-[(E)-1-(4-methoxyphenyl)ethylidene]pyridine-4-carbohydrazide, N'-[(E)-1-(4-methylphenyl)ethylidene]pyridine-4-carbohydrazide, and N'-[(E)-1-(phenyl)ethylidene]pyridine-4-carbohydrazide were synthesized and evaluated for antifungal activity against C. posadasii. Susceptibility assays were performed by macrodilution testing. Interactions between the hydrazones and amphotericin B or itraconazole were evaluated by the checkerboard method. We also investigated the impairment of such compounds on cell ergosterol and membrane integrity. The synthesized molecules were able to inhibit C. posadasii in vitro with MIC values that ranged from 25 to 400 µg/mL. Drug interactions between synthesized molecules and amphotericin B proved synergistic for the majority of tested isolates; regarding itraconazole, synergism was observed only when strains were tested against N'-[(E)-1-(phenyl)ethylidene]pyridine-4-carbohydrazide. Reduction of cellular ergosterol was observed when strains were challenged with the hydrazones alone or combined with antifungals. Only N'-[(E)-1-(4-methylphenyl)ethylidene]pyridine-4-carbohydrazide altered membrane permeability of C. posadasii cells. Isoniazid-derived hydrazones were able to inhibit C. posadasii cells causing reduction of ergosterol content and alterations in the permeability of cell membrane. This study confirms the antifungal potential of hydrazones against pathogenic fungi.

Inhibitory activity of isoniazid and ethionamide against Cryptococcus biofilms.

In recent years, the search for drugs to treat systemic and opportunistic mycoses has attracted great interest from the scientific community. This study evaluated the in vitro inhibitory effect of the antituberculosis drugs isoniazid and ethionamide alone and combined with itraconazole and fluconazole against biofilms of Cryptococcus neoformans and Cryptococcus gattii. Antimicrobials were tested at defined concentrations after susceptibility assays with Cryptococcus planktonic cells. In addition, we investigated the synergistic interaction of antituberculosis drugs and azole derivatives against Cryptococcus planktonic cells, as well as the influence of isoniazid and ethionamide on ergosterol content and cell membrane permeability. Isoniazid and ethionamide inhibited both biofilm formation and viability of mature biofilms. Combinations formed by antituberculosis drugs and azoles proved synergic against both planktonic and sessile cells, showing an ability to reduce Cryptococcus biofilms by approximately 50%. Furthermore, isoniazid and ethionamide reduced the content of ergosterol in Cryptococcus spp. planktonic cells and destabilized or permeabilized the fungal cell membrane, leading to leakage of macromolecules. Owing to the paucity of drugs able to inhibit Cryptococcus biofilms, we believe that the results presented here might be of interest in the designing of new antifungal compounds.

Cefepime and Amoxicillin Increase Metabolism and Enhance Caspofungin Tolerance of Candida albicans Biofilms.

It is well known that prolonged antibiotic therapy alters the mucosal microbiota composition, increasing the risk of invasive fungal infection (IFI) in immunocompromised patients. The present study investigated the direct effect of ß-lactam antibiotics cefepime (CEF) and amoxicillin (AMOX) on biofilm production by Candida albicans ATCC 10231. Antibacterials at the peak plasmatic concentration of each drug were tested against biofilms grown on polystyrene surfaces. Biofilms were evaluated for biomass production, metabolic activity, carbohydrate and protein contents, proteolytic activity, ultrastructure, and tolerance to antifungals. CEF and AMOX enhanced biofilm production by C. albicans ATCC 10231, stimulating biomass production, metabolic activity, viable cell counts, and proteolytic activity, as well as increased biovolume and thickness of these structures. Nevertheless, AMOX induced more significant changes in C. albicans biofilms than CEF. In addition, it was shown that AMOX increased the amount of chitin in these biofilms, making them more tolerant to caspofungin. Finally, it was seen that, in response to AMOX, C. albicans biofilms produce Hsp70 - a protein with chaperone function related to stressful conditions. These results may have a direct impact on the pathophysiology of opportunistic IFIs in patients at risk.

Exposure of Candida parapsilosis complex to agricultural azoles: An overview of the role of environmental determinants for the development of resistance.

This work investigated the phenotypic behavior of Candida parapsilosis species complex in response to exposure to agricultural azoles and fluconazole. Three fluconazole-susceptible strains of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were used. Initial minimum inhibitory concentrations (iMICs) for agricultural and clinical azoles were determined by broth microdilution. Then, the strains were exposed to tebuconazole, tetraconazole and fluconazole for 15 days, at concentrations that were two-folded daily, starting at one-eighth the iMIC (iMIC/8) up to 64 times iMIC (64xiMIC). After 15-day-exposure, antifungal susceptibility, biofilm formation, CDR, MDR and ERG expression were evaluated. The three cryptic species developed tolerance to the antifungals they were exposed and presented reduction (P < 0.05) in fluconazole susceptibility. In addition, C. parapsilosis sensu stricto and C. metapsilosis also presented reduced susceptibility to voriconazole, after fluconazole exposure. Azole exposure decreased (P < 0.05) biofilm production by C. parapsilosis sensu stricto and C. orthopsilosis and increased (P < 0.05) the expression of ERG11 in all tested strains. The results show that exposure to agricultural azoles and fluconazole induces changes in the phenotypic behavior and gene expression by the three cryptic species of C. parapsilosis complex, highlighting the importance of environmental determinants for the development of antifungal resistance.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification.

In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

ß-lactam antibiotics & vancomycin increase the growth & virulence of Candida spp.

AIM: To investigate the direct effect of antibiotics on growth and virulence of the major Candida species associated with invasive infections. MATERIALS & METHODS: Cefepime, imipenem, meropenem, amoxicillin and vancomycin were tested at twofold the peak plasma concentration (2× PP) and the peak plasma concentration (PP). The effects of antibiotics on Candida albicans, Candida parapsilosis, Candida krusei and Candida tropicalis were investigated by colony counting, flow cytometry, proteolytic activity and virulence in Caenorhabditis elegans. RESULTS: Antibiotics increase growth and proteolytic activity of Candida spp; In addition, amoxicillin potentiates virulence of C. krusei and C. tropicalis against Caenorhabditis elegans. CONCLUSION: These results suggest that antimicrobial therapy may have a direct effect on the pathophysiology of invasive fungal infections in patients at risk.

Candida tropicalis from veterinary and human sources shows similar in vitro hemolytic activity, antifungal biofilm susceptibility and pathogenesis against Caenorhabditis elegans.

The aim of this study was to evaluate the in vitro hemolytic activity and biofilm antifungal susceptibility of veterinary and human Candida tropicalis strains, as well as their pathogenesis against Caenorhabditis elegans. Twenty veterinary isolates and 20 human clinical isolates of C. tropicalis were used. The strains were evaluated for their hemolytic activity and biofilm production. Biofilm susceptibility to itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin was assessed using broth microdilution assay. The in vivo evaluation of strain pathogenicity was investigated using the nematode C. elegans. Hemolytic factor was observed in 95% of the strains and 97.5% of the isolates showed ability to form biofilm. Caspofungin and amphotericin B showed better results than azole antifungals against mature biofilms. Paradoxical effect on mature biofilm metabolic activity was observed at elevated concentrations of caspofungin (8-64µg/mL). Azole antifungals were not able to inhibit mature C. tropicalis biofilms, even at the higher tested concentrations. High mortality rates of C. elegans were observed when the worms were exposed to with C. tropicalis strains, reaching up to 96%, 96h after exposure of the worms to C. tropicalis strains. These results reinforce the high pathogenicity of C. tropicalis from veterinary and human sources and show the effectiveness of caspofungin and amphotericin B against mature biofilms of this species.

Effect of shading and nitrogen fertilization on nitrogen metabolism, essential oil content and antimicrobial activity of Achillea millefolium

The Achillea millefolium L. is a perennial herb with important antibacterial, antifungal, anti-inflammatory, antitumoral, and antioxidant properties. This research aimed to investigate the effect of shading (75%; black net) and nitrogen fertilization (0, 75 and 150 kg urea ha-1) on the nitrogen metabolism, essential oil yield and antimicrobial activity of A.millefolium at vegetative- and reproductive-stage. The evaluated parameters varied depending on the organ and the phenological stage of the plant considered. Overall, our findings indicated that shading decreased nitrogen assimilation. Decreased activities of nitrate reductase and glutamine synthetase were observed on shaded plants during reproductive and vegetative stages, respectively. Nitrate and total amino acid levels increased in shaded plants at the vegetative stage. Regarding nitrogen supply, the improved nitrogen metabolism and essential oil yield values were accompanied by intermediate concentrations of urea (75 kg ha-1). Plants fertilized with 75 kg urea ha-1 produced the highest amino acids concentration (vegetative stage), ammonium concentration (vegetative stage) and essential oil yield (reproductive stage). Shading or nitrogen supply did not influence the microbial activity of A. millefolium essential oil.However, the essential oil of leaves and flowers were highly effective against fungi and bacteria, especially gram-positive bacteria. In conclusion, the current study showed that full light and 75 kg urea ha-1 enhanced the nitrogen metabolism of A. millefolium in both vegetative and reproductive stages.

Trichosporon inkin biofilms produce extracellular proteases and exhibit resistance to antifungals.

The aim of this study was to determine experimental conditions for in vitro biofilm formation of clinical isolates of Trichosporon inkin, an important opportunistic pathogen in immunocompromised patients. Biofilms were formed in microtitre plates in three different media (RPMI, Sabouraud and CLED), with inocula of 104, 105 or 106 cells ml- 1, at pH 5.5 and 7.0, and at 35 and 28 °C, under static and shaking conditions for 72 h. Growth kinetics of biofilms were evaluated at 6, 24, 48 and 72 h. Biofilm milieu analysis were assessed by counting viable cells and quantification of nucleic acids released into biofilm supernatants. Biofilms were also analysed for proteolytic activity and antifungal resistance against amphotericin B, caspofungin, fluconazole, itraconazole and voriconazole. Finally, ultrastructural characterization of biofilms formed in microtitre plates and catheter disks was performed by scanning electron microscopy. Greater biofilm formation was observed with a starter inoculum of 106 cells ml- 1, at pH 7.0 at 35 °C and 80 r.p.m., in both RPMI and Sabouraud media. Growth kinetics showed an increase in both viable cells and biomass with increasing incubation time, with maximum production at 48 h. Biofilms were able to disperse viable cells and nucleic acids into the supernatant throughout the developmental cycle. T. inkin biofilms produced more protease than planktonic cells and showed high tolerance to amphotericin B, caspofungin and azole derivatives. Mature biofilms were formed by different morphotypes, such as blastoconidia, arthroconidia and hyphae, in a strain-specific manner. The present article details the multicellular lifestyle of T. inkin and provides perspectives for further research.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

In vitro antifungal activity of the flavonoid baicalein against Candida species.

The aim of the present study was to evaluate the in vitro activity of baicalein, the flavone constituent of Scutellaria baicalensis, and synergism of the combination of baicalein and fluconazole against Candida albicans, Candida tropicalis and Candida parapsilosis. The MIC(50) (lowest concentration at which there was 50 % inhibition of growth) of baicalein alone against six Candida strains ranged from 13 to 104 µg ml(-1). For the three species tested, exposure to baicalein at the MIC(50) concentrations obtained for each strain resulted in a high loss of viability. The fluconazole plus baicalein combination markedly reduced the MICs of both drugs for all three strains analysed. In addition, a synergistic effect between baicalein and fluconazole was observed for C. parapsilosis in terms of MIC(50) (fractional inhibitory concentration index = 0.207). Scanning electron microscopy analysis revealed that yeast cells exposed to baicalein at MIC(50) produced a profusely flocculent extracellular material, resembling a biofilm-like structure. In conclusion, these results showed the antifungal capability of baicalein against Candida species and highlight a promising role of baicalein when used in combination with fluconazole against Candida infections.

Ultrastructural architecture of colonies of different morphologies produced by phenotypic switching of a clinical strain of Candida tropicalis and biofilm formation by variant phenotypes.

Candida tropicalis has been identified as one of the most prevalent pathogenic yeast species of the Candida-non-albicans (CNA) group. Study of switching in C. tropicalis has not been the subject of extensive research. Therefore, we investigated switching event and characterized the ultrastructural architecture of different phenotypes and biofilm produced in a C. tropicalis clinical strain. Cells switched heritably, reversibly, and at a high frequency between four phenotypes readily distinguishable by the shape of colonies formed on agar at 25°C. SEM analysis was used to verify the architecture of whole Candida colonies at ultrastructural level. The smooth phenotype (parental phenotype) colony showed a hemispherical shape character, while the semi-smooth was characterized by the presence of shallow marginal depressions. The ring and rough phenotypes exhibited more complex architecture and were characterized by the presence of deep central and peripheral depressions areas. The biofilm-forming ability varied among the switch phenotypes. After 12h incubation, the smooth phenotype formed less biofilm compared to the other phenotypes (P<0.05). The electron microscopy analysis revealed that filamentation (pseudohyphae) was associated with ring and rough colonies. The ultrastructural analysis allowed the observation of the arrangement of individual cells within the colonies. At the deep central and peripheral depressions areas of the ring and rough colonies extracellular material was seen in different arrangements. The data presented here open new avenues to study a possible role for extracellular material in the formation and maintenance of the architecture of switch phenotypes in C. tropicalis. It is therefore essential that more strains be investigated to determine the biological significance of extracellular material in C. tropicalis phenotypic switching phenomenon.

Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period.

INTRODUCTION: In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. METHODS: Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. RESULTS: Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. CONCLUSIONS: Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.