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1.
Science ; 371(6534): 1154-1159, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33707263

RESUMO

Alterations of the mycobiota composition associated with Crohn's disease (CD) are challenging to link to defining elements of pathophysiology, such as poor injury repair. Using culture-dependent and -independent methods, we discovered that Debaryomyces hansenii preferentially localized to and was abundant within incompletely healed intestinal wounds of mice and inflamed mucosal tissues of CD human subjects. D. hansenii cultures from injured mice and inflamed CD tissues impaired colonic healing when introduced into injured conventionally raised or gnotobiotic mice. We reisolated D. hansenii from injured areas of these mice, fulfilling Koch's postulates. Mechanistically, D. hansenii impaired mucosal healing through the myeloid cell-specific type 1 interferon-CCL5 axis. Taken together, we have identified a fungus that inhabits inflamed CD tissue and can lead to dysregulated mucosal healing.


Assuntos
Doença de Crohn/microbiologia , Doença de Crohn/patologia , /fisiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Anfotericina B/farmacologia , Animais , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Quimiocina CCL5/metabolismo , Colo/microbiologia , Colo/patologia , Doença de Crohn/imunologia , Feminino , Microbioma Gastrointestinal , Vida Livre de Germes , Humanos , Íleo/microbiologia , Íleo/patologia , Inflamação , Interferon Tipo I/metabolismo , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
2.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33688802

RESUMO

Introduction. Trichosporon asahii has been recognized as an opportunistic agent having a limited sensitivity to antifungal treatment.Hypothesis/Gap Statement. Molecular mechanisms of azole resistance have been rarely reported for Trichosproron asahii. Similar to other fungi, we hypothesized that both ERG11 gene mutation and efflux pumps genes hyper-expression were implicated.Aim. The current work aimed to study the sensitivity of clinical T. asahii isolates to different antifungal agents and to explore their resistance mechanisms by molecular methods including real-time PCR and gene sequencing.Methods. The sensitivity of T. asahii isolates to fluconazole, amphotericin B and voriconazole was estimated by the Etest method. Real-time PCR was used to measure the relative expression of Pdr11, Mdr and ERG11 genes via the ACT1 housekeeping gene. Three pairs of primers were also chosen to sequence the ERG11 gene. This exploration was followed by statistical study including the receiver operating characteristic (ROC) curve analysis to identify a relationship between gene mean expression and the sensitivity of isolates.Results. In 31 clinical isolates, the resistance frequencies were 87, 16.1 and 3.2 %, respectively, for amphotericin B, fluconazole and voriconazole. Quantitative real-time PCR demonstrated that only Mdr over-expression was significantly associated with FCZ resistance confirmed by univariate statistical study and the ROC curve analysis (P <0.05). The ERG11 sequencing revealed two mutations H380G and S381A in TN325U11 (MIC FCZ=8 µg ml-1) and H437R in TN114U09 (MIC FCZ=256 µg ml-1) in highly conserved regions (close to the haem-binding domain) but their involvement in the resistance mechanism has not yet been assigned.Conclusion. T. asahii FCZ resistance mechanisms are proven to be much more complex and gene alteration sequence and/or expression can be involved. Only Mdr gene over-expression was significantly associated with FCZ resistance and no good correlation was observed between FCZ and VCZ MIC values and relative gene expression. ERG11 sequence alteration seems to play a major role in T. asahii FCZ resistance mechanism but their involvement needs further confirmation.


Assuntos
Antifúngicos/farmacologia , Basidiomycota , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Tricosporonose/microbiologia , Anfotericina B/farmacologia , Basidiomycota/efeitos dos fármacos , Basidiomycota/genética , Fluconazol/farmacologia , Humanos , Voriconazol/farmacologia
3.
ABCS health sci ; 46: e021203, 09 fev. 2021. tab
Artigo em Inglês | LILACS | ID: biblio-1147180

RESUMO

INTRODUCTION: The resistance of fungal species to drugs usually used in clinics is of great interest in the medical field. OBJECTIVE: To evaluate susceptibility and in vitro response of species of Trichophyton spp. to antifungal drugs of interest in clinical medicine. METHODS: 12 samples of clinical isolates from humans were used, nine of T. mentagrophytes and three of T. tonsurans. Susceptibility tests were performed according to the agar diffusion (AD) and broth microdilution (BM) methods. RESULTS: In the AD method, the species T. tonsurans presented a percentage of sensitivity of 33% in relation to amphotericin B and 66% to itraconazole, with 100% resistance to ketoconazole and fluconazole. T. mentagrophytes also showed 100% resistance to ketoconazole in this technique, with 11% sensitivity to ketoconazole, 22% to itraconazole and 22% of samples classified as sensitive dose dependent. In the MC method, the species T. tonsurans presented a sensitivity percentage of 66%, 55% and 33% in relation to ketoconazole, fluconazole and itraconazole, respectively. The T. mentagrophytes species presented sensitivity percentages of 11%, 11%, 33% and 55% for amphotericin B, itraconazole, ketoconazole and fluconazole, respectively. CONCLUSION: There was resistance in vitro of the species of T. mentagrophytes and T. tonsurans against the antifungal fluconazole and relative resistance against ketoconazole in the AD method. In BM, however, important percentages of sensitivity were observed for the two species analyzed in relation to the antifungals fluconazole and ketoconazole when compared to itraconazole and amphotericin B.


INTRODUÇÃO: A resistência de espécies fúngicas às drogas usualmente empregadas no meio clínico é motivo de grande interesse na área médica. OBJETIVO: Avaliar susceptibilidade e resposta in vitro de espécies de Trichophyton spp. a drogas antifúngicas de interesse em clínica médica. MÉTODOS: Foram utilizadas 12 amostras de isolados clínicos de humanos, sendo nove de T. mentagrophytes e três de T. tonsurans. Foram realizados testes de susceptibilidade segundo os métodos de difusão em ágar (DA) e microdiluição em caldo (MC). RESULTADOS: No método de DA, a espécie T. tonsurans apresentou percentual de sensibilidade de 33% em relação à anfotericina B e de 66% ao itraconazol, com 100% de resistência frente ao cetoconazol e ao fluconazol. A espécie T. mentagrophytes também apresentou 100% de resistência frente ao cetoconazol nesta técnica, com 11% de sensibilidade ao cetoconazol, 22% ao itraconazol e 22% das amostras classificadas como sensível dose dependente. No método de MC, a espécie T. tonsurans apresentou percentual de sensibilidade de 66%, 55% e 33% em relação ao cetoconazol, fluconazol e itraconazol, respectivamente. A espécie T. mentagrophytes apresentou percentuais de sensibilidade de 11%, 11%, 33% e 55% para anfotericina B, itraconazol, cetoconazol e fluconazol, respectivamente. CONCLUSÃO: Houve resistência in vitro das espécies do T. mentagrophytes e T. tonsurans frente ao antifúngico fluconazol e resistência relativa frente ao cetoconazol no método de DA. Na MC, no entanto, foram observados importantes percentuais de sensibilidade das duas espécies analisadas frente aos antifúngicos fluconazol e cetoconazol quando comparadas ao itraconazol e à anfotericina B.


Assuntos
Trichophyton/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Farmacorresistência Fúngica , Suscetibilidade a Doenças/microbiologia , Antifúngicos/farmacologia , Tinha/microbiologia , Tinha/tratamento farmacológico , Contagem de Colônia Microbiana , Fluconazol/farmacologia , Anfotericina B/farmacologia , Itraconazol/farmacologia , Cetoconazol/farmacologia
4.
Exp Parasitol ; 221: 108059, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33338468

RESUMO

Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 µM and 427.50 ± 17.60 µM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 µM and 1.06 ± 0.23 µM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.


Assuntos
Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Anfotericina B/farmacologia , Animais , Antiprotozoários/toxicidade , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Concentração Inibidora 50 , Ivermectina/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Baço/parasitologia
5.
PLoS One ; 15(10): e0240093, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031481

RESUMO

Flowers produce an array of nutrient-rich exudates in which microbes can thrive, making them hotspots for microbial abundance and diversity. During a diversity study of yeasts inhabiting the flowers of Metrosideros polymorpha (Myrtaceae) in the Hawai'i Volcanoes National Park (HI, USA), five isolates were found to represent two novel species. Morphological and physiological characterization, and sequence analysis of the small subunit ribosomal RNA (rRNA) genes, the D1/D2 domains of the large subunit rRNA genes, the internal transcribed spacer (ITS) regions, and the genes encoding the largest and second largest subunits of the RNA polymerase II (RPB1 and RPB2, respectively), classified both species in the family Metschnikowiaceae, and we propose the names Candida metrosideri pro tempore sp. nov. (JK22T = CBS 16091 = MUCL 57821) and Candida ohialehuae pro tempore sp. nov. (JK58.2T = CBS 16092 = MUCL 57822) for such new taxa. Both novel Candida species form a well-supported subclade in the Metschnikowiaceae containing species associated with insects, flowers, and a few species of clinical importance. The ascosporic state of the novel species was not observed. The two novel yeast species showed elevated minimum inhibitory concentrations to the antifungal drug amphotericin B (>4 µg/mL). The ecology and phylogenetic relationships of C. metrosideri and C. ohialehuae are also discussed.


Assuntos
Candida/classificação , Myrtaceae/microbiologia , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candida/genética , Candida/isolamento & purificação , Farmacorresistência Fúngica , Flores/microbiologia , Hawaii , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , RNA Ribossômico/classificação , RNA Ribossômico/genética , RNA Ribossômico/metabolismo
6.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641482

RESUMO

C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry.IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.


Assuntos
Antígenos de Superfície/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus/fisiologia , Interações Hospedeiro-Patógeno , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Internalização do Vírus , Sequência de Aminoácidos , Anfotericina B/farmacologia , Betacoronavirus/efeitos dos fármacos , Linhagem Celular , Coronavirus/efeitos dos fármacos , Infecções por Coronavirus/epidemiologia , Suscetibilidade a Doenças , Evolução Molecular , Proteínas Ligadas por GPI/metabolismo , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Sinais Direcionadores de Proteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
Sci Rep ; 10(1): 12243, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699361

RESUMO

The development of an effective oral therapeutics is an immediate need for the control and elimination of visceral leishmaniasis (VL). We exemplify the preparation and optimization of 2-hydroxypropyl-ß-cyclodextrin (HPCD) modified solid lipid nanoparticles (SLNs) based oral combinational cargo system of Amphotericin B (AmB) and Paromomycin (PM) against murine VL. The emulsion solvent evaporation method was employed to prepare HPCD modified dual drug-loaded solid lipid nanoparticles (m-DDSLNs). The optimized formulations have a mean particle size of 141 ± 3.2 nm, a polydispersity index of 0.248 ± 0.11 and entrapment efficiency for AmB and PM was found to be 96% and 90% respectively. The morphology of m-DDSLNs was confirmed by scanning electron microscopy and transmission electron microscopy. The developed formulations revealed a sustained drug release profile upto 57% (AmB) and 21.5% (PM) within 72 h and were stable at both 4 °C and 25 °C during short term stability studies performed for 2 months. Confocal laser scanning microscopy confirmed complete cellular internalization of SLNs within 24 h of incubation. In vitro cytotoxicity study against J774A.1 macrophage cells confirmed the safety and biocompatibility of the developed formulations. Further, m-DDSLNs did not induce any hepatic/renal toxicities in Swiss albino mice. The in vitro simulated study was performed to check the stability in simulated gastric fluids and simulated intestinal fluids and the release was found almost negligible. The in vitro anti-leishmanial activity of m-DDSLNs (1 µg/ml) has shown a maximum percentage of inhibition (96.22%) on intra-cellular amastigote growth of L. donovani. m-DDSLNs (20 mg/kg × 5 days, p.o.) has significantly (P < 0.01) reduced the liver parasite burden as compared to miltefosine (3 mg/kg × 5 days, p.o.) in L. donovani-infected BALB/c mice. This work suggests that the superiority of as-prepared m-DDSLNs as a promising approach towards the oral delivery of anti-leishmanial drugs.


Assuntos
Anfotericina B/química , Anfotericina B/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Nanopartículas/química , Paromomicina/química , Paromomicina/farmacologia , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular , Emulsões/química , Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão/métodos , Tamanho da Partícula , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologia
8.
Exp Parasitol ; 216: 107940, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32562606

RESUMO

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.


Assuntos
Alternativas aos Testes com Animais/métodos , Antiprotozoários/toxicidade , Citometria de Fluxo/métodos , Leishmania/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico , Adulto , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Leucócitos/parasitologia , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , Antimoniato de Meglumina/toxicidade , Microscopia Confocal , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/parasitologia , Fatores de Tempo , Adulto Jovem
9.
PLoS One ; 15(5): e0233102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392266

RESUMO

Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and overexpression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. All the isolates were found capable enough to form biofilms on 96-well flat bottom microtiter plate that was further confirmed by MTT reduction assay. In addition, these strains have active drug efflux mechanism which was supported by rhodamine-6-G extracellular efflux and intracellular accumulation assays. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. We further studied the role of farnesol, an endogenous quorum sensing molecule, in modulating development of biofilms and drug efflux in C. auris. The MIC for planktonic cells ranged from 62.5-125 mM, and for sessile cells was 125 mM (4h biofilm) and 500 mM (12h and 24h biofilm). Furthermore, farnesol (125 mM) also suppresses adherence and biofilm formation by C. auris. Farnesol inhibited biofilm formation, blocked efflux pumps and downregulated biofilm- and efflux pump- associated genes. Modulation of C. auris biofilm formation and efflux pump activity by farnesol represent a promising approach for controlling life threatening infections caused by this pathogen.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Farneseno Álcool/farmacologia , Anfotericina B/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Fúngica , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana , Percepção de Quorum , África do Sul
10.
J Med Microbiol ; 69(6): 830-837, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32459616

RESUMO

Introduction. Cryptococcus species are pathogens commonly associated with cases of meningoencephalitis in individuals who are immunosuppressed due to AIDS.Aim. The aim was to evaluate the effects of the antiretroviral darunavir alone or associated with fluconazole, 5-flucytosine and amphotericin B against planktonic cells and biofilms of Cryptococcus species.Methodology. Susceptibility testing of darunavir and the common antifungals against 12 members of the Cryptococcus neoformans/Cryptococcus gattii species complex was evaluated by broth microdilution. The interaction between darunavir and antifungals against planktonic cells was tested by a checkerboard assay. The effects of darunavir against biofilm metabolic activity and biomass were evaluated by the XTT reduction assay and crystal violet staining, respectively.Results. Darunavir combined with amphotericin B showed a synergistic interaction against planktonic cells. No antagonistic interaction was observed between darunavir and the antifungals used. All Cryptococcus species strains were strong biofilm producers. Darunavir alone reduced biofilm metabolic activity and biomass when added during and after biofilm formation (P<0.05). The combination of darunavir with antifungals caused a significant reduction in biofilm metabolic activity and biomass when compared to darunavir alone (P<0.05).Conclusion. Darunavir presents antifungal activity against planktonic cells of Cryptococcus species and synergism with amphotericin B. In addition, darunavir led to reduced biofilm formation and showed activity against mature biofilms of Cryptococcus species. Activity of the antifungals against mature biofilms was enhanced in the presence of darunavir.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Darunavir/farmacologia , Anfotericina B/farmacologia , Células Cultivadas , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana/métodos , Plâncton/microbiologia
11.
Sao Paulo Med J ; 138(1): 40-46, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32321104

RESUMO

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Cryptococcus neoformans , Sinvastatina/farmacologia , Brasil , Sinergismo Farmacológico , Fluconazol , Humanos , Testes de Sensibilidade Microbiana , Estudos Prospectivos
12.
Free Radic Res ; 54(4): 231-243, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32295440

RESUMO

Amphotericin B has been the gold standard for the treatment of invasive mycosis for many years. Its resistance mechanisms are reported to be mainly related to the decrease of ergosterol content or the changes of cell wall. Previous study has shown that Saccharomyces cerevisiae strain lack of BSC2 was sensitive significantly to Amphotericin B. In the present study, the role of BSC2 on Amphotericin B resistance were investigated. We found that BSC2 enhanced the resistance of yeast cells to Amphotericin B, which was not related to cellular ergosterol content. BSC2 can maintain the permeability of mitochondrial membrane and cell membrane integrity by inhibiting the accumulation of intercellular reactive oxygen species and alleviating the production of lipid peroxidation and superoxide radical. These alterations were attributed to the enhancement of the activities of superoxide dismutase, catalase and glutathione peroxidase, and the increased glutathione content. Taken together, BSC2 inhibits oxidative damage induced by Amphotericin B through increasing activities of antioxidant enzymes and levels of GSH to alleviate the accumulation of reactive oxygen species, lipid peroxidation and superoxide radical, resulting in the maintenance of mitochondrial membrane potential and cell membrane integrity. However, Amphotericin B resistance mediated by BSC2 is independent of Yap1p, GSH1 and Hog1p. The results demonstrate for the first time that BSC2 enhances cell resistance to Amphotericin B by inhibiting oxidative damage in yeast. Our findings improve current understanding of the mechanism of Amphotericin B resistance and provide potential strategy for reducing Amphotericin B resistance.


Assuntos
Anfotericina B/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Antifúngicos/farmacologia , Antioxidantes/farmacologia , Farmacorresistência Fúngica , Ergosterol/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/sangue , Membro 2 da Família 12 de Carreador de Soluto/genética , Fatores de Transcrição/metabolismo
13.
PLoS Negl Trop Dis ; 14(4): e0008188, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275665

RESUMO

Leishmaniasis is one of the Neglected Tropical Diseases (NTDs) which is closely associated with poverty and has gained much relevance recently due to its opportunistic coinfection with HIV. It is a protozoan zoonotic disease transmitted by a dipteran Phlebotomus, Lutzomyia/ Sergentomyia sandfly; during blood meals on its vertebrate intermediate hosts. It is a four-faceted disease with its visceral form being more deadly if left untreated. It is endemic across the tropics and sub-tropical regions of the world. It can be considered the third most important NTD after malaria and lymphatic filariasis. Currently, there are numerous drawbacks on the fight against leishmaniasis which includes: non-availability of vaccines, limited availability of drugs, high cost of mainstay drugs and parasite resistance to current treatments. In this study, we screened the antileishmanial activity, selectivity, morphological alterations, cell cycle progression and apoptotic potentials of six Pathogen box compounds from Medicine for Malaria Venture (MMV) against Leishmania donovani promastigotes and amastigotes. From this study, five of the compounds showed great promise as lead chemotherapeutics based on their high selectivity against the Leishmania donovani parasite when tested against the murine mammalian macrophage RAW 264.7 cell line (with a therapeutic index ranging between 19-914 (promastigotes) and 1-453 (amastigotes)). The cell cycle progression showed growth arrest at the G0-G1 phase of mitotic division, with an indication of apoptosis induced by two (2) of the pathogen box compounds tested. Our findings present useful information on the therapeutic potential of these compounds in leishmaniasis. We recommend further in vivo studies on these compounds to substantiate observations made in the in vitro study.


Assuntos
Antiprotozoários/farmacologia , Desenvolvimento de Medicamentos , Leishmania donovani/efeitos dos fármacos , Anfotericina B/farmacologia , Animais , Apoptose/efeitos dos fármacos , Concentração Inibidora 50 , Cinética , Leishmania donovani/crescimento & desenvolvimento , Macrófagos/parasitologia , Camundongos , Microscopia de Fluorescência , Células RAW 264.7
14.
Chem Biol Interact ; 320: 109026, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32112863

RESUMO

Leishmaniasis is a widespread tropical infection caused by different species of Leishmania protozoa. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The discovery of drugs for the treatment of leishmaniasis is a pressing concern. In the present work, we describe in vitro studies of the phenolic compound methyl gallate (MG) against Leishmania (Leishmania) amazonensis and its possible mechanisms of action. The in vitro activity of MG was assayed against L. amazonensis (promastigotes, axenic amastigotes, and intramacrophagic amastigotes). Cytotoxicity tests were performed with J774A.1 macrophages and THP-1 cell derived macrophages. To evaluate mechanisms of action, we analyzed cellular TNF-α, IL-12, IFN-γ, IL-10, IL-6, NO, ROS levels, arginase activity, and structural mechanisms (phagocytic and lysosomal activities) involving macrophage activation. Meglumine antimoniate and amphotericin B were used as reference drugs. It was observed that MG effectively inhibited the growth of both promastigote (IC50 5.71 µM) and amastigote-like forms (EC50 5.39 µM), with much higher selectivity indexes than the reference drugs, being more benign towards J774A.1 macrophages than meglumine antimoniate and amphotericin B, at 1631- and 70.92-fold respectively, with respect to the promastigote form. Additionally, MG proved to be even more active against intracellular amastigotes of the parasite (EC50 4.24 µM). Our results showed that antileishmania activity was associated with increased TNF-α, IL-12, NO and ROS levels, as well as decreased IL-6 and decreased arginase activity. In addition, MG induced increased phagocytic capability, and lysosomal volume in macrophages; structural parameters of microbicidal activity. Taken together, our results suggest that MG may be a promising candidate for new drug development against leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Ácido Gálico/análogos & derivados , Leishmania/efeitos dos fármacos , Anfotericina B/farmacologia , Antiprotozoários/química , Ácido Gálico/efeitos adversos , Ácido Gálico/química , Ácido Gálico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Antimoniato de Meglumina/farmacologia , Estrutura Molecular , Espécies Reativas de Oxigênio
15.
Int J Mol Sci ; 21(5)2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32150884

RESUMO

The enantiomers (R)-(+)-ß-citronellol and (S)-(-)-ß-citronellol are present in many medicinal plants, but little is understood about their bioactivity against Candida yeasts. This study aimed to evaluate the behavior of positive and negative enantiomers of ß-citronellol on strains of Candida albicans and C. tropicalis involved in candidemia. The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined. The evaluation of growth kinetics, mechanism of action, and association studies with Amphotericin B (AB) using the checkerboard method was also performed. R-(+)-ß-citronellol and S-(-)-ß-citronellol presented a MIC50% of 64 µg/mL and a MFC50% of 256 µg/mL for C. albicans strains. For C. tropicalis, the isomers exhibited a MIC50% of 256 µg/mL and a MFC50% of 1024 µg/mL. In the mechanism of action assay, both substances displayed an effect on the fungal membrane but not on the fungal cell wall. Synergism and indifference were observed in the association of R-(+)-ß-citronellol and AB, while the association between S-(-)-ß-citronellol and AB displayed synergism, additivity, and indifference. In conclusion, both isomers of ß-citronellol presented a similar profile of antifungal activity. Hence, they can be contemplated in the development of new antifungal drugs providing that further research is conducted about their pharmacology and toxicity.


Assuntos
Monoterpenos Acíclicos/farmacologia , Anfotericina B/farmacologia , Candida/efeitos dos fármacos , Candidemia/tratamento farmacológico , Antifúngicos/farmacologia , Candida/isolamento & purificação , Candidemia/microbiologia , Quimioterapia Combinada , Humanos
16.
Nat Commun ; 11(1): 127, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913284

RESUMO

Patients infected with the fungal pathogen Cryptococcus are most effectively treated with a combination of 5-fluorocytosine (5FC) and amphotericin B. 5FC acts as a prodrug, which is converted into toxic 5-fluorouracil (5FU) upon uptake into fungal cells. However, the pathogen frequently develops resistance through unclear mechanisms. Here we show that resistance to 5FC in Cryptococcus deuterogattii is acquired more frequently in isolates with defects in DNA mismatch repair that confer an elevated mutation rate. We use whole genome sequencing of 16 independent isolates to identify mutations associated with 5FC resistance in vitro. We find mutations in known resistance genes (FUR1 and FCY2) and in a gene UXS1, previously shown to encode an enzyme that converts UDP-glucuronic acid to UDP-xylose for capsule biosynthesis, but not known to play a role in 5FC metabolism. Mutations in UXS1 lead to accumulation of UDP-glucuronic acid and alterations in nucleotide metabolism, which appear to suppress toxicity of both 5FC and its toxic derivative 5FU.


Assuntos
Antifúngicos/farmacologia , Cryptococcus/efeitos dos fármacos , Cryptococcus/genética , Farmacorresistência Fúngica , Flucitosina/farmacologia , Polissacarídeos/biossíntese , Anfotericina B/farmacologia , Cryptococcus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Mutação
17.
J Infect Public Health ; 13(5): 753-758, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31899133

RESUMO

BACKGROUND: Bloodstream Candida infection is a life-threatening event among ICU admitted patients. This infection is caused by a diverse range of Candida species having varied minimum inhibitory concentrations. OBJECTIVES: To identify Candida species causing bloodstream infections with their antifungal susceptibility determination. METHODS: Candida species isolated from the blood of ICU admitted patients were identified by phenotypic as well as by molecular methods including PCR-RFLP using MspI restriction enzyme and MALDI TOF MS. The minimum inhibitory concentration of fluconazole, voriconazole, amphotericin B and caspofungin was determined against isolated Candida species by CLSI M27A3 guidelines. RESULTS: A total of 119 Candida species were isolated. Among them, C. tropicalis(n=29) was the predominant isolate followed by C. parapsilosis(n=18), C. glabrata (n=12), C. krusei (n=11) and C. albicans(n=11). Uncommon Candida species isolated were; Wickerhamomyces anomalus(n=15), Kodaemia ohmeri(n=8), C. lusitaniae (n=5) and C. auris (n=2). A varied antifungal MIC values were observed. Caspofungin had the lowest MIC among the tested antifungals. Increased fluconazole MIC was observed against the isolated Candida species including C. tropicalis. All the isolated C. lusitaniae and C. auris strains have ≥1mcg/ml amphotericin B MIC. In comparison to fluconazole, voriconazole was more effective when tested in vitro. CONCLUSION: Emergence of uncommon Candida species having varied antifungal MIC warns the physicians to have a prompt, accurate identification with antifungal MIC determination in each case of bloodstream Candida infections.


Assuntos
Antifúngicos/farmacologia , Bacteriemia/diagnóstico , Candida/isolamento & purificação , Candidíase/diagnóstico , Anfotericina B/farmacologia , Bacteriemia/microbiologia , Candida/efeitos dos fármacos , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/isolamento & purificação , Candidíase/microbiologia , Caspofungina/farmacologia , Estudos Transversais , Fluconazol/farmacologia , Humanos , Unidades de Terapia Intensiva , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Voriconazol/farmacologia
18.
PLoS One ; 15(1): e0227876, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935275

RESUMO

Candida krusei is one of the most common agents of invasive candidiasis and candidemia worldwide, leading to high morbidity and mortality rates. This species has become a problem due to its intrinsic resistance and reduced susceptibility to azoles and polyenes. Moreover, the number of antifungal drugs available for candidiasis treatment is limited, demonstrating the urgent need for the discovery of novel alternative therapies. In this work, the in vivo and in vitro activities of a new oxadiazole (LMM11) were evaluated against C. krusei. The minimum inhibitory concentration ranged from 32 to 64 µg/mL with a significant reduction in the colony forming unit (CFU) count (~3 log10). LMM11 showed fungicidal effect, similar to amphotericin, reducing the viable cell number (>99.9%) in the time-kill curve. Yeast cells presented morphological alterations and inactive metabolism when treated with LMM11. This compound was also effective in decreasing C. krusei replication inside and outside macrophages. A synergistic effect between fluconazole and LMM11 was observed. In vivo treatment with the new oxadiazole led to a significant reduction in CFU (0.85 log10). Furthermore, histopathological analysis of the treated group exhibited a reduction in the inflammatory area. Taken together, these results indicate that LMM11 is a promising candidate for the development of a new antifungal agent for the treatment of infections caused by resistant Candida species such as C. krusei.


Assuntos
Antifúngicos/química , Candida/efeitos dos fármacos , Candidíase/tratamento farmacológico , Oxidiazóis/química , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida/patogenicidade , Candidíase/microbiologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Oxidiazóis/farmacologia , Células-Tronco/efeitos dos fármacos
19.
Nat Commun ; 11(1): 427, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969561

RESUMO

The frequency of antifungal resistance, particularly to the azole class of ergosterol biosynthetic inhibitors, is a growing global health problem. Survival rates for those infected with resistant isolates are exceptionally low. Beyond modification of the drug target, our understanding of the molecular basis of azole resistance in the fungal pathogen Aspergillus fumigatus is limited. We reasoned that clinically relevant antifungal resistance could derive from transcriptional rewiring, promoting drug resistance without concomitant reductions in pathogenicity. Here we report a genome-wide annotation of transcriptional regulators in A. fumigatus and construction of a library of 484 transcription factor null mutants. We identify 12 regulators that have a demonstrable role in itraconazole susceptibility and show that loss of the negative cofactor 2 complex leads to resistance, not only to the azoles but also the salvage therapeutics amphotericin B and terbinafine without significantly affecting pathogenicity.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Anfotericina B/farmacologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Azóis/farmacologia , Proteínas Fúngicas/genética , Testes de Sensibilidade Microbiana
20.
J Antibiot (Tokyo) ; 73(4): 211-223, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31974520

RESUMO

Eight new potentiators of antifungal amphotericin B (AmB) activity, phialotides A to H, were isolated from the fermentation broths of the rare fungus Pseudophialophora sp. BF-0158. The structures of phialotides were elucidated by spectroscopic analyses, including NMR and MS, and degradation studies. Phialotides were novel polyketide glycosides consisting of a 1,3-dimethylbut-1-ene (C6-unit) repeating substructure and one to three hexopyranoses. None of the phialotides exhibited antifungal activity, whereas all potentiated AmB activity against several fungi. Phialotide F was the most effective potentiator of AmB activity against Candida albicans, with a decrease in the MIC from 0.50 to 0.016 µg ml-1 being observed in combination with phialotide F at 1.0 µg ml-1.


Assuntos
Anfotericina B/química , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Glicosídeos/farmacologia , Policetídeos/farmacologia , Antifúngicos/química , Antifúngicos/isolamento & purificação , Ascomicetos/química , Candida albicans/efeitos dos fármacos , Sinergismo Farmacológico , Glicosídeos/administração & dosagem , Glicosídeos/química , Glicosídeos/isolamento & purificação , Testes de Sensibilidade Microbiana , Policetídeos/administração & dosagem , Policetídeos/química , Policetídeos/isolamento & purificação
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