Antifungal activity of 3,3'-dimethoxycurcumin (DMC) against dermatophytes and Candida species.

Dermatomycosis is an infection with global impacts caused especially by dermatophytes and Candida species. Current antifungal therapies involve drugs that face fungal resistance barriers. This clinical context emphasizes the need to discover new antifungal agents. Herein, the antifungal potential of 10 curcumin analogs was evaluated against four Candida and four dermatophyte species. The most active compound, 3,3'-dimethoxycurcumin, exhibited minimum inhibitory concentration values ranging from 1.9‒62.5 to 15.6‒62.5 µg ml-1 against dermatophytes and Candida species, respectively. According to the checkerboard method, the association between DMC and terbinafine demonstrated a synergistic effect against Trichophyton mentagrophytes and Epidermophyton floccosum. Ergosterol binding test indicated DMC forms a complex with ergosterol of Candida albicans, C. krusei, and C. tropicalis. However, results from the sorbitol protection assay indicated that DMC had no effect on the cell walls of Candida species. The in vivo toxicity, using Galleria mellonella larvae, indicated no toxic effect of DMC. Altogether, curcumin analog DMC was a promising antifungal agent with a promising ability to act against Candida and dermatophyte species.

Virulence potential of Salmonella 1,4, [5],12:i:- strains isolated during decades from different sources in the Southeast region of Brazil.

Salmonella 1,4, [5],12:i:- is one of the most prevalent serovars associated with gastroenteritis in several countries, including Brazil. However, few studies have analyzed the virulence potential of this variant in this country. Therefore, this study aimed to characterize S. 1,4, [5],12:i:- strains isolated in Southeast Brazil. To this end, 113 S. 1,4, [5],12:i:- strains isolated from different sources between 1983 and 2020 were analyzed. For all strains, the frequencies of 11 virulence genes were investigated using PCR and the molecular typing was performed using pulsed-field gel electrophoresis (PFGE). Furthermore, 40 strains isolated from human and non-human sources were characterized by survival under acid and oxidative stress, and virulence analysis in Galleria mellonella was performed for 20 selected strains. All virulence genes were detected in more than 91% of the strains. The studied strains were grouped into four clusters using PFGE. Most strains were present in one cluster, named PFGE-A, with a genetic similarity of ≥ 79.5%. All 40 strains survived acid stress after 10 min and 1 h of exposure. Under oxidative stress, all 40 strains survived after 10 min, and 36 survived after 1 h of exposure. In the G. mellonella assay, nine isolates from non-human sources and six isolates from human showed high-to-intermediate virulence profiles. In conclusion, the pathogenic potential of the strains studied was corroborated by the high frequency of all the virulence genes identified. The PFGE results suggested that most strains belonged to one main cluster that has been prevailing in the São Paulo State, Brazil. The S. 1,4, [5],12:i:- strains isolated from human and non-human sources successfully survived the unfavorable conditions in the human gastrointestinal tract. Finally, strains isolated from non-human sources showed a higher proportion of isolates with high to intermediate virulence profiles in G. mellonella than in human isolates, suggesting a possible difference between isolates from different origins.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction.

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XlnR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xlnA and xlnD mRNA accumulation. The ΔfbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi.

Different virulence genetic context of multidrug-resistant CTX-M- and KPC-producing Klebsiella pneumoniae isolated from cerebrospinal fluid.

Information regarding resistance and virulence traits of meningitis-causing enterobacteria in hospital environment remains scarce. The aim of this study was to characterize virulence and acquired resistance genes of carbapenem-resistant and/or 3rd to 4th generation cephalosporin-resistant Klebsiella pneumoniae isolated from the cerebrospinal fluid of inpatients. Antimicrobial susceptibility testing was performed by disk diffusion. The string test was performed to identify hypermucoviscous phenotype. Galleria mellonella infection model was used to evaluate the virulence profile of the isolates. Screening for virulence determinants and acquired resistance genes were performed by PCR. The blaCTX-M and/or blaKPC and/or rmtG were detected in all the isolates. Genetic virulence determinants, including mrkD, entB, iroD, fecIRA, uge, wabG, fimH, ureA, ybtS, and clb were detected in the majority of multidrug-resistant K. pneumoniae isolates. One isolate presented hypermucoviscous phenotype, and several isolates showed enhanced virulence in G. mellonella infection model. The combination of the virulence genes found here seems to support not only the known virulence genetic context among nosocomial infections-causing K. pneumoniae but also the role that clb and ybtS may play in K. pneumoniae virulence.

Cryptococcosis by Cryptococcus neoformans/Cryptococcus gattii Species Complexes in non-HIV-Infected Patients in Southeastern Brazil.

INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.

Paracoccidioidomycosis due to Paracoccidioides lutzii complicated with adrenal injury and pulmonary arterial hypertension.

Paracoccidioidomycosis caused by Paracoccidioides lutzii is endemic in the Midwest of Brazil and its clinical spectrum is still little known due to the recent identification of this fungal species. A patient resident in Southeast Brazil, but who had lived for many years in the Midwest region, presented with skin injuries, chronic cough and bilateral adrenal involvement. Paracoccidioides spp. was isolated in culture from a skin lesion biopsy. This isolate was later identified as P. lutzii using gene sequencing. A favorable initial response to treatment with itraconazole was observed, but a few weeks later, the patient developed respiratory failure and worsening of lung lesions. Evaluation by computed tomography and echocardiography were suggestive of pulmonary arterial hypertension, and a bronchoscopic biopsy showed peribronchial remodeling. The patient completed the antifungal treatment but maintained the respiratory dysfunction. The reported case shows that P. lutzii can be isolated from patients in a geographic area far from the place of infection acquisition and that, as P. brasiliensis , it can cause adrenal injury and cardio-respiratory complications as a consequence of excessive necrosis and fibrosis.

Virulence traits and expression of bstA, fliC and sopE2 in Salmonella Dublin strains isolated from humans and animals in Brazil.

Salmonella Dublin is a strongly cattle-adapted serovar that has also been responsible for severe invasive infections in humans. Although invasive infections by non-typhoid Salmonella have increased in developed and in developing countries, in sub-Saharan Africa these infections have been frequently related to Salmonella Typhimurium strains from Sequence Type (ST) 313 that harbor a possible virulence marker, the bstA gene, broadly detected in S. Dublin strains. The aims of this study were to verify the frequency of bstA by PCR in 113 Salmonella Dublin strains isolated from humans (83) and animals (30) in Brazil and the expression by RT-PCR of bstA, sopE2 and fliC in six strains isolated from humans (4) and animals (2). Moreover, the invasion capacity in Caco-2 human epithelial cells and U937 human macrophages, plus in vivo virulence analysis in Galleria mellonella and the motility were verified for 20 S. Dublin strains isolated from humans (15) and animals (5). All studied strains presented the bstA gene. The relative expression rates ranged from 0.1 to 2.3 fold change for bstA and from no expression to 16.6 fold change for sopE2, while no expression was detected for fliC. The invasion in Caco-2 cells ranged from 54.0 to 88.9% and in U937 cells from 72.9 to 98.1% in the 20 strains studied. In addition, 17 strains presented a highly virulent profile in the G. mellonella model and 15 strains presented a non-motile profile. In conclusion, the presence and expression of bstA in the S. Dublin strains studied suggested that this gene may influence in the invasive characteristic of this serovar. The low expression of sopE2 in strains from human invasive cases suggested that its expression may not be a limiting factor to the invasion of S. Dublin strains. The absence of fliC expression and the low motility rates observed suggest that the flagella absence may favor the host immune system evasion by S. Dublin and the establishment of infection. Moreover, the high mortality rates observed in vivo in Galleria mellonella reinforce the pathogenic potential of S. Dublin strains.

First Comprehensive Report of Clinical Fusarium Strains Isolated in the State of Sao Paulo (Brazil) and Identified by MALDI-TOF MS and Molecular Biology.

The aim of this study was to compare the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), phenotypic and molecular methods for the identification of Fusarium species complexes isolated from clinical cases in the State of Sao Paulo (Brazil) between the years 2001 and 2017. Sequencing of ITS region of ribosomal DNA and elongation factor 1 alpha gene (ET1α) were used as reference method in the analysis of a total of 108 Fusarium spp. clinical strains isolated from human hosts with superficial and systemic infections. Agreement between MALDI-TOF-MS and molecular data was observed for 97 out of 108 clinical isolates (89.8%), whereas five (4.6%) and six (5.5%) clinical isolates were misidentified and were not identified by MALDI-TOF MS, respectively. ITS region sequences and MALDI-TOF MS mass spectra identified and grouped correctly most of Fusarium clinical isolates at species complex level. This investigation highlights the potential of MALDI-TOF MS technique as a fast and cost-efficient alternative for clinical Fusarium identification. However, MALDI-TOF MS requires a more accurate and larger database. This work is the first comprehensive report for Fusarium population, based on phenotypic analyses, proteomic profile by MALDI-TOF and phylogenetic analyses of Fusarium species complexes isolated from clinical cases in the State of Sao Paulo, Brazil.

Regulation of hyphal morphogenesis and the DNA damage response by the Aspergillus nidulans ATM homolog AtmA.

Ataxia telangiectasia (A-T) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. The most prominent clinical feature observed in A-T patients is the degeneration of Purkinje motor neurons. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. However, in Purkinje cells, the bulk of ATM localizes to the cytoplasm and may play a role in vesicle trafficking. The nature of this function, and its involvement in the pathology underlying A-T, remain unknown. Here we characterize the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we find that AtmA is also required for polarized hyphal growth. We demonstrate that an atmA mutant fails to generate a stable axis of hyphal polarity. Notably, cytoplasmic microtubules display aberrant cortical interactions at the hyphal tip. Our results suggest that AtmA regulates the function and/or localization of landmark proteins required for the formation of a polarity axis. We propose that a similar function may contribute to the establishment of neuronal polarity.

Cryptococcus neoformans and C. gattii isolates from both HIV-infected and uninfected patients: antifungal susceptibility and outcome of cryptococcal disease.

One of the factors causing treatment failure in cryptococcosis is the resistance of Cryptococcus spp. to antifungal drugs, which has motivated the susceptibility assessment of isolates from patients with cryptococcosis, different clinical conditions and infections outcomes. Clinical isolates of Cryptococcus spp. from three different groups of patients were studied in the present investigation: 19 HIV-positive patients with relapsing and/or refractory meningitis (Group 1), 30 HIV-positive patients who experienced a single and limited episode of cryptococcosis (Group 2), and 19 HIV-negative patients with cryptococcosis (Group 3). Eighty C. neoformans var. grubii isolates and 7 C. gattii isolates were studied. The minimum inhibitory concentration (MIC) of amphotericin B, azole drugs and flucytosine was determined for Cryptococcus spp. by broth microdilution test and E-test. The MIC50 and MIC90 were 0.25 and 0.50 µg/mL for amphotericin B, 4.0 and 8.0 µg /mL for fluconazole, 0.06 and 0.25 µg/mL for itraconazole, 0.25 and 0.50 µg/mL for voriconazole, and 8.0 and 16.0 µg/mL for flucytosine, respectively. Amphotericin B and itraconazole showed higher MICs for C. neoformans var. grubii and C. gattii, respectively. The MICs of fluconazole and itraconazole obtained with the E-test were higher than those obtained with broth microdilution. Isolates from non-HIV coinfected were less sensitive to the azoles. There was no difference in the susceptibility of C. neoformans var. grubii isolates from patients with a favorable or unfavorable outcome or along the episodes of relapsing and/or refractory meningitis.

Cryptococcosis by Cryptococcus neoformans/Cryptococcus gattii Species Complexes in non-HIV-Infected Patients in Southeastern Brazil

Abstract INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.

The immune interplay between the host and the pathogen in Aspergillus fumigatus lung infection.

The interplay between Aspergillus fumigatus and the host immune response in lung infection has been subject of studies over the last years due to its importance in immunocompromised patients. The multifactorial virulence factors of A. fumigatus are related to the fungus biological characteristics, for example, structure, ability to grow and adapt to high temperatures and stress conditions, besides capability of evading the immune system and causing damage to the host. In this context, the fungus recognition by the host innate immunity occurs when the pathogen disrupts the natural and chemical barriers followed by the activation of acquired immunity. It seems clear that a Th1 response has a protective role, whereas Th2 reactions are often associated with higher fungal burden, and Th17 response is still controversial. Furthermore, a fine regulation of the effector immunity is required to avoid excessive tissue damage associated with fungal clearance, and this role could be attributed to regulatory T cells. Finally, in this work we reviewed the aspects involved in the complex interplay between the host immune response and the pathogen virulence factors, highlighting the immunological issues and the importance of its better understanding to the development of novel therapeutic approaches for invasive lung aspergillosis.