RESUMEN

Antifungal susceptibility testing (AST) is crucial in clinical settings to guide appropriate therapy. Nevertheless, discrepancies between treatment response and some results still persist, particularly in detecting resistance to amphotericin B (AMB) in Clavispora (Candida) lusitaniae. This study aimed to assess the susceptibility patterns of 48 recent isolates of C. lusitaniae to 9 antifungal agents and explore the feasibility of using a CLSI reference-based method to identify AMB resistance. Microdilution techniques revealed a wide range of minimal inhibitory concentration (MIC) values for azole antifungals, while echinocandins and AMB exhibited a narrow range of MIC values, with all strains considered wild-type for the tested polyene and echinocandins. However, when agar diffusion (ellipsometry) was employed for AST, certain strains displayed colonies within the inhibition ellipse, indicating potential resistance. Interestingly, these strains did not respond to AMB treatment and were isolated during AMB treatment (breakthrough). Moreover, the evaluation of AMB minimum fungicidal concentrations (MFCs) indicated that only the strains with colonies inside the ellipse had MFC/MIC ratios ≥ 4, suggesting reduced fungicidal activity. In conclusion, this study confirms the effectiveness of ellipsometry with RPMI-1640 2% glucose agar for detecting AMB resistance in C. lusitaniae. Additionally, the proposed approach of culturing "clear" wells in the microdilution method can aid in uncovering resistant strains. The findings highlight the importance of appropriate AST methods to guide effective treatment strategies for deep-seated candidiasis caused by C. lusitaniae. Further collaborative studies are warranted to validate these findings and improve the detection of AMB clinical resistance.

Asunto(s)

Anfotericina B , Antifúngicos , Anfotericina B/farmacología , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candida , Agar/farmacología , Equinocandinas/farmacología , Pruebas de Sensibilidad Microbiana

RESUMEN

A black-grain eumycetoma due to Diaporthe uekerae in a kidney transplant recipient is presented. The isolate was identified by using the newly available NCBI's curated database (rRNA_typestrains/ITS_RefSeq_Fungi) and the NCBI's GenBank + EMBL + DDBI + PDB + RefSeq database. The isolate's antifungal susceptibility was evaluated. The studied isolate showed low MIC values to the eight tested antifungals. Using this updated database, the identities of previous agents of Diaporthe spp. infections were revised.

RESUMEN

BACKGROUND: Patients with severe viral pneumonia are likely to receive high-dose immunomodulatory drugs to prevent clinical worsening. Aspergillus species have been described as frequent secondary pneumonia agents in severely ill influenza patients receiving steroids. COVID-19 patients admitted to Intensive Care Unit (ICU) are receiving steroids as part of their treatment and they share clinical characteristics with other patients with severe viral pneumonias. COVID-19 patients receiving steroids should be considered a putative risk group of invasive aspergillosis. CASE REPORT: We are reporting a SARS-CoV-2/Aspergillus section Fumigati coinfection in an elderly intubated patient with a history of pulmonary embolism treated with corticosteroids. The diagnosis was made following the ad hoc definitions described for patients admitted to ICU with severe influenza, including clinical criteria (fever for 3 days refractory to the appropriate antibiotic therapy, dyspnea, pleural friction rub, worsening of respiratory status despite antibiotic therapy and need of ventilator support), a radiological criterion (pulmonary infiltrate) and a mycological criterion (several positive galactomannan tests on serum with ratio ≥0.5). In addition, Aspergillus section Fumigati DNA was found in serum and blood samples. These tests were positive 4 weeks after the patient was admitted to the ICU. The patient received voriconazole and after two month in ICU his respiratory status improved; he was discharged after 6 weeks of antifungal treatment. CONCLUSIONS: Severely ill COVID-19 patients would be considered a new aspergillosis risk group. Galactomannan and Aspergillus DNA detection would be useful methods for Aspergillus infection diagnosis as they allow avoiding the biosafety issues related to these patients.

Asunto(s)

Aspergillus fumigatus/aislamiento & purificación , Tratamiento Farmacológico de COVID-19 , COVID-19/complicaciones , Coinfección/diagnóstico , Inmunocompetencia , Inmunosupresores/efectos adversos , Aspergilosis Pulmonar Invasiva/complicaciones , Metilprednisolona/efectos adversos , SARS-CoV-2/aislamiento & purificación , Acetaminofén/uso terapéutico , Anciano , Antiinfecciosos/uso terapéutico , Líquido del Lavado Bronquioalveolar/microbiología , COVID-19/diagnóstico , COVID-19/terapia , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19 , Coinfección/microbiología , Coinfección/terapia , Coinfección/virología , Terapia Combinada , Diagnóstico Diferencial , Quimioterapia Combinada , Enoxaparina/uso terapéutico , Galactosa/análogos & derivados , Humanos , Hidroxicloroquina/uso terapéutico , Inmunosupresores/uso terapéutico , Intubación Intratraqueal , Aspergilosis Pulmonar Invasiva/diagnóstico , Aspergilosis Pulmonar Invasiva/microbiología , Aspergilosis Pulmonar Invasiva/terapia , Masculino , Mananos/sangre , Metilprednisolona/uso terapéutico , Nasofaringe/virología , Neumonía por Mycoplasma/diagnóstico , Pseudomonas aeruginosa/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Respiración Artificial , Staphylococcus aureus/aislamiento & purificación , Tráquea/microbiología

RESUMEN

BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.

Asunto(s)

Citoplasma/química , Leptospira interrogans/genética , Metionina Sulfóxido Reductasas/genética , Metionina/metabolismo , Secuencia de Aminoácidos/genética , Catálisis , Citoplasma/enzimología , Genoma Bacteriano/genética , Humanos , Leptospira interrogans/enzimología , Metionina/química , Metionina/genética , Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/ultraestructura , Oxidación-Reducción , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por Sustrato

RESUMEN

BACKGROUND: Glutathione (GSH) plays a role as a main antioxidant metabolite in all eukaryotes and many prokaryotes. Most of the organisms synthesize GSH by a pathway involving two enzymatic reactions, each one consuming one molecule of ATP. In a first step mediated by glutamate-cysteine ligase (GCL), the carboxylate of l-glutamic acid reacts with l-cysteine to form the dipeptide γ-glutamylcysteine (γ-GC). The second step involves the addition of glycine to the C-terminal of γ-GC catalyzed by glutathione synthetase (GS). In many bacteria, such as in the pathogen Leptospira interrogans, the main intracellular thiol has not yet been identified and the presence of GSH is not clear. METHODS: We performed the molecular cloning of the genes gshA and gshB from L. interrogans; which respectively code for GCL and GS. After heterologous expression of the cloned genes we recombinantly produced the respective proteins with high degree of purity. These enzymes were exhaustively characterized in their biochemical properties. In addition, we determined the contents of GSH and the activity of related enzymes (and proteins) in cell extracts of the bacterium. RESULTS: We functionally characterized GCL and GS, the two enzymes putatively involved in GSH synthesis in L. interrogans serovar Copenhageni. LinGCL showed higher substrate promiscuity (was active in presence of l-glutamic acid, l-cysteine and ATP, and also with GTP, l-aspartic acid and l-serine in lower proportion) unlike LinGS (which was only active with γ-GC, l-glycine and ATP). LinGCL is significantly inhibited by γ-GC and GSH, the respective intermediate and final product of the synthetic pathway. GSH showed inhibitory effect over LinGS but with a lower potency than LinGCL. Going further, we detected the presence of GSH in L. interrogans cells grown under basal conditions and also determined enzymatic activity of several GSH-dependent/related proteins in cell extracts. CONCLUSIONS: and General Significance. Our results contribute with novel insights concerning redox metabolism in L. interrogans, mainly supporting that GSH is part of the antioxidant defense in the bacterium.

Asunto(s)

Proteínas Bacterianas/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Glutatión Sintasa/metabolismo , Glutatión/metabolismo , Leptospira interrogans/metabolismo , Proteínas Bacterianas/genética , Clonación Molecular , Glutamato-Cisteína Ligasa/genética , Glutatión Sintasa/genética , Leptospira interrogans/genética , Leptospira interrogans/crecimiento & desarrollo , Oxidación-Reducción

RESUMEN

BACKGROUND: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids. METHODS AND RESULTS: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000 µM) and heavy metal (1-500 µM) toxicity. Treatment with TryS inhibitors (5-30 µM) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. CONCLUSIONS: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti-trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.

Asunto(s)

Amida Sintasas/metabolismo , Antioxidantes/metabolismo , Cardiomiopatía Chagásica/parasitología , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/fisiología , Amida Sintasas/genética , Animales , Antiprotozoarios/uso terapéutico , Proliferación Celular , Células Cultivadas , Cardiomiopatía Chagásica/tratamiento farmacológico , Resistencia a Medicamentos , Humanos , Oxidación-Reducción , Estrés Oxidativo , Proteínas Protozoarias/genética , Transgenes/genética

RESUMEN

Little is known about the mechanisms by which Leptospira interrogans, the causative agent of leptospirosis, copes with oxidative stress at the time it establishes persistent infection within its human host. We report the molecular cloning of a gene encoding a 2-Cys peroxiredoxin (LinAhpC) from this bacterium. After bioinformatic analysis we found that LinAhpC contains the characteristic GGIG and YF motifs present in peroxiredoxins that are sensitive to overoxidation (mainly eukaryotic proteins). These motifs are absent in insensitive prokaryotic enzymes. Recombinant LinAhpC showed activity as a thioredoxin peroxidase with sensitivity to overoxidation by H2O2 (Chyp 1% ~30 µM at pH 7.0 and 30°C). So far, Anabaena 2-Cys peroxiredoxin, Helicobacter pylori AhpC, and LinAhpC are the only prokaryotic enzymes studied with these characteristics. The properties determined for LinAhpC suggest that the protein could be critical for the antioxidant defense capacity in L. interrogans.

Asunto(s)

Proteínas Bacterianas/química , Leptospira interrogans/enzimología , Peroxirredoxinas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxirredoxinas/biosíntesis , Peroxirredoxinas/genética , Filogenia