Antifungal susceptibility testing following the CLSI M27 document, along with the measurement of MFC/MIC ratio, could be the optimal approach to detect amphotericin B resistance in Clavispora (Candida) lusitaniae. Susceptibility patterns of contemporary isolates of this species.

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.

Black grain eumycetoma due to Diaporthe ueckerae. Taxonomical update of previous agents of infections due to Diaporthe spp.

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.

Candida auris and some Candida parapsilosis strains exhibit similar characteristics on CHROMagarTMCandida Plus.

Candida auris is considered a public health problem due to its resistance and its tendency to cause nosocomial outbreaks. CHROMagarTMCandida Plus has recently been marketed as capable of presumptively identifying C. auris. The objective of this work was to analyze the ability of this new chromogenic medium to differentiate C. auris from other members of the C. haemulonii complex and from other yeasts commonly isolated in clinical practice. A collection of 220 strains including species of the C. haemulonii (n = 83) and C. parapsilosis (n = 80) complexes was studied. The strains were identified by molecular methods and cultured as individual or as mixed aqueous inoculum on CHROMagarTMCandida Plus plates. Colony morphotypes were evaluated at 5 time points. CHROMagarTMCandida Plus was a helpful tool for presumptive identification for C. auris. Better reading results were obtained after 48 hours of incubation at 35°C. It is able to easily differentiate C. auris from other closely related species of the C. haemulonii complex and other yeasts. This chromogenic medium would be also useful as screening and surveillance tool for C. auris colonization. However, we demonstrated that it would be a possible misidentification of C. parapsilosis as C. auris (44.3% showed similar morphotypes). To reduce false positives when it is used in a context of a C. auris outbreak, we propose to supplement the chromogenic medium with 8 µg/ml fluconazole. This modified medium was tested and it clearly differentiate C. parapsilosis from C. auris.

CHROMagarTMCandida Plus is able to differentiate C. auris from other Candida spp., including other species of the C. haemulonii complex. However, 44.3% of the tested C. parapsilosis strains would be misidentified as C. auris. We propose the addition of 8 µg/ml fluconazole to solve this issue.

Functional characterization of monothiol and dithiol glutaredoxins from Leptospira interrogans.

Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans.

SARS-CoV-2 and Aspergillus section Fumigati coinfection in an immunocompetent patient treated with corticosteroids / Coinfección de SARS-CoV-2 y Aspergillus sección Fumigati en un paciente inmunocompetente tratado con corticosteroides

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

ANTECEDENTES: Los pacientes con neumonía viral grave reciben altas dosis de fármacos inmunomoduladores para prevenir el empeoramiento clínico. Los pacientes con influenza grave que reciben esteroides tienen neumonías secundarias causadas por Aspergillus con una frecuencia relativamente alta. Los pacientes con COVID-19 ingresados en la unidad de cuidados intensivos (UCI) reciben dicha medicación como parte de su tratamiento, y comparten con otro tipo de pacientes muchas de las características clínicas de otras neumonías virales graves. Estos pacientes deberían considerarse como un grupo de riesgo de aspergilosis invasiva. CASO CLÍNICO: Se presenta un caso de coinfección por SARS-CoV-2 y Aspergillus de la sección Fumigati en un paciente intubado de edad avanzada con antecedentes de embolia pulmonar y tratado con corticosteroides. El diagnóstico siguió las definiciones ad hoc descritas para pacientes ingresados en la UCI con gripe grave. El paciente cumplía varios criterios clínicos (fiebre durante 3 días refractaria al tratamiento antibiótico apropiado, disnea, fricción pleural, empeoramiento del estado respiratorio a pesar del tratamiento antibiótico y la necesidad de soporte respiratorio), el criterio radiológico (infiltrado pulmonar) y un criterio micológico (test de galactomanano positivo en suero, (ratio ≥0,5). Además, se detectó ADN de Aspergillus de la sección Fumigati en muestras de suero y sangre del paciente. Estas pruebas fueron positivas 4 semanas después de que el paciente ingresara en la UCI. El paciente recibió tratamiento con voriconazol, y después de 2 meses en la UCI mejoró su estado pulmonar; fue dado de alta después de 6 semanas de tratamiento antifúngico. CONCLUSIONES: Los pacientes gravemente enfermos con COVID-19 deberían considerarse un nuevo grupo de riesgo para la aspergilosis. La detección de galactomanano y ADN de Aspergillus son métodos útiles para el diagnóstico de infección por este hongo al evitar los problemas de bioseguridad en estos pacientes

SARS-CoV-2 and Aspergillus section Fumigati coinfection in an immunocompetent patient treated with corticosteroids.

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.

Functional characterization of methionine sulfoxide reductases from Leptospira interrogans.

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.

First evidence of glutathione metabolism in Leptospira interrogans.

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.

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi.

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.

Functional thioredoxin reductase from pathogenic and free-living Leptospira spp.

Low molecular mass thiols and antioxidant enzymes have essential functions to detoxify reactive oxygen and nitrogen species maintaining cellular redox balance. The metabolic pathways for redox homeostasis in pathogenic (Leptospira interrogans) and free-living (Leptospira biflexa) leptospires species were not functionally characterized. We performed biochemical studies on recombinantly produced proteins to in depth analyze kinetic and structural properties of thioredoxin reductase (LinTrxR) and thioredoxin (LinTrx) from L. interrogans, and two TrxRs (LbiTrxR1 and LbiTrxR2) from L. biflexa. All the TrxRs were characterized as homodimeric flavoproteins, with LinTrxR and LbiTrxR1 catalyzing the NADPH dependent reduction of LinTrx and DTNB. The thioredoxin system from L. interrogans was able to use glutathione disulfide, lipoamide disulfide, cystine and bis-γ-glutamyl cysteine and homologous peroxiredoxin as substrates. Classic TrxR activity of LinTrxR2 had not been evidenced in vitro, but recombinant Escherichia coli cells overexpressing LbiTrxR2 showed high tolerance to oxidative stress. The enzymatic systems herein characterized could play a key role for the maintenance of redox homeostasis and the function of defense mechanisms against reactive oxidant species in Leptospira spp. Our results contribute to the general knowledge about redox biochemistry in these bacteria, positioning TrxR as a critical molecular target for the development of new anti-leptospiral drugs.

On the Ancestral UDP-Glucose Pyrophosphorylase Activity of GalF from Escherichia coli.

In bacteria, UDP-glucose is a central intermediate in carbohydrate metabolism. The enzyme responsible for its synthesis is encoded by the galU gene and its deletion generates cells unable to ferment galactose. In some bacteria, there is a second gene, galF, encoding for a protein with high sequence identity to GalU. However, the role of GalF has been contradictory regarding its catalytic capability and not well understood. In this work we show that GalF derives from a catalytic (UDP-glucose pyrophosphorylase) ancestor, but its activity is very low compared to GalU. We demonstrated that GalF has some residual UDP-glucose pyrophosphorylase activity by in vitro and in vivo experiments in which the phenotype of a galU (-) strain was reverted by the over-expression of GalF and its mutant. To demonstrate its evolutionary path of "enzyme inactivation" we enhanced the catalysis by mutagenesis and showed the importance of the quaternary structure. This study provides important information to understand the structural and functional evolutionary origin of the protein GalF in enteric bacteria.

Kinetic and structural characterization of a typical two-cysteine peroxiredoxin from Leptospira interrogans exhibiting redox sensitivity.

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.