Aims
To synthesize and evaluate the antifungal activity of poly(methacrylic acid)‐silver nanoparticles (PMAA‐AgNPs) against nine Candida albicans isolated from clinical specimens.
Methods and Results
...The effects of PMAA‐AgNPs‐fluconazole combination was analysed by checkerboard methodology. The synergistic potential of PMAA‐AgNPs‐fluconazole was determined by the fractional inhibitory concentration index (FICI). The inhibition of germ tube formation and the determination of PMAA‐AgNPs cytotoxicity were also performed. All C. albicans strains were susceptible to PMAA‐AgNPs and resistant to fluconazole. PMAA‐AgNPs at subinhibitory concentrations restored the susceptibility of resistant C. albicans to fluconazole, whose FICI ranged from 0.3 to 0.5. The synergistic interaction of the combination was observed in eight of nine strains. The PMAA‐AgNPs‐fluconazole combination was also able to inhibit the germ tube formation. PMAA‐AgNPs showed a dose‐dependent decrease in viability for cells tested, with 50% cytotoxic concentration (CC50) values of 6.5, 4.9 and 6.8 μg ml−1 for macrophages, fibroblasts and Vero cells, respectively.
Conclusions
This study demonstrated that, in general, PMAA‐AgNPs acts synergistically in combination with fluconazole, inhibiting fluconazole‐resistant C. albicans strains. PMAA‐AgNPs‐fluconazole combination was also able to inhibit germ tube formation, an important virulence factor. Inhibitory effect of PMAA‐AgNPs alone or in combination was higher in C. albicans than in mammalian cells.
Significance and Impact of Study
This study shows the potential of PMAA‐AgNPs combined with fluconazole to inhibit fluconazole‐resistant C. albicans strains.
This study assessed the efficacy of using the essential oil from Origanum vulgare L. (oregano; OVEO) and carvacrol (CAR) to remove biofilms formed on stainless steel surfaces. The sessile cells ...counts (SSC) in young and mature biofilms formed by Staphylococcus aureus on AISI304 surfaces were assessed after 10- and 15-min exposure to OVEO and CAR. Scanning electron microscopy analysis were performed to assess the ultrastructure of sessile cells. The decrease in the initial SCC varied with the test strain, assayed OVEO or CAR concentration and exposure time, however no differences were observed between young and mature biofilms. A 10-min exposure to 10 μL/mL OVEO or 5 μL/mL CAR caused a decrease ≥2 log CFU/cm2 in the SCC of young and mature biofilms formed by both S. aureus strains tested. No SCC (<1 log CFU/cm2) in young or mature S. aureus LPMA63 biofilms were detected after a 15-min exposure to 10 μL/mL OVEO or 5 μL/mL CAR. Sessile cells exposed to OVEO or CAR presented irregular morphology with bubbles or spots on their surface. Holes on cell membranes were observed in sessile cells exposed to CAR. OVEO and CAR were effective to remove young and mature biofilms on stainless steel surfaces.
•Carvacrol (CAR) eliminates S. aureus young and mature biofilms on stainless steel.•Oregano essential oil (OVEO) and CAR show time-dependent efficacy against biofilms”.•OVEO and CAR induced bubble and spot formations on S. aureus sessile cell surfaces.•CAR induced appearance of holes in membranes of S. aureus sessile cells.
Visceral leishmaniasis caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae) is a major neglected tropical disease and Brazil is the responsible for most cases reported in the Americas. In ...this region, L. infantum is primarily transmitted by Lutzomyia longipalpis and Migonemyia migonei (França) (Diptera: Psychodidae) is considered a permissive vector. We evaluated the susceptibility of Lu. longipalpis and Mg. migonei to Beauveria bassiana and to Eucalyptus globulus (Myrtales: Myrtaceae) essential oil. A spore suspension of B. bassiana was prepared and sand flies divided into five groups: test 1 (107 spores/ml of B. bassiana with E. globulus essential oil at 4 mg/ml), test 2 (107 spores/ml of B. bassiana), test 3 (E. globulus essential oil at 4 mg/ml), positive control (cypermethrin 0.1%), and negative control (sterile distilled water). Scanning electron microscopy (SEM) was performed on specimens from each group. A 50% reduction was recorded in the survival time of Lu. longipalpis in test 1 and 2, where hyphal adhesion and cuticle damage were observed by SEM. No significant differences in the survival time of Mg. migonei were found, probable due to the high mortality rate observed in the negative control group, which may be a result of the greater sensitivity of this species to laboratory conditions. The results obtained herein suggest that B. bassiana may be a potential biological control agent against Lu. longipalpis, the main vector of L. infantum in the Americas.
Abstract Polymyxins have become drugs of last resort for treatment of multi-drug resistant (MDR) Gram-negative infections. However, the mechanisms of resistance to this compound have not been ...completely elucidated. In this study, we evaluated the mechanisms of resistance to this antimicrobial in two A. baumannii clinical isolates, respectively, susceptible (A027) and resistant (A009) to polymyxin B before and after polymyxin B exposure (A027ind and A009ind ). The pmrAB and lpxACD were sequenced and their transcriptional levels were analyzed by qRT-PCR. The bacterial cell morphology was evaluated by transmission electronic microscopy (TEM) and the membrane potential was measured using Zeta-potential analyzer. The virulence of strains was studied using a Caenorhabditis elegans model. Both clinical isolates exhibited an elevation of the polymyxin B MIC after exposure to this compound. On the other hand, A027ind showed decreased values of MIC for β-lactams, aminoglycosides, vancomycin, teicoplanin, oxacillin and erythromycin. A027ind harbored two mutations in pmrB and the IS Aba125 disrupting the lpxA. In contrast, A009ind strain exhibited increase of pmrB transcriptional level, after polymyxin B exposure, despite the absence of mutations in the pmrAB genes. The TEM images revealed a thicker and more electron-dense peptidoglycan layer for A009 than that of A027. The exposure to polymyxin B induced a strong condensation and darkening of intracellular material, mainly in A009ind . In addition, the surface charge of A009 was significantly less negative than the one of A027. Using the C. elegans model, only A027ind strain showed a reduction on virulence. The diversity of polymyxin B resistance mechanisms among A. baumannii strains evaluated in this study confirms the complexity of these mechanisms, which may vary depending of the background of each strain.
This study aimed to investigate the effects of sublethal concentrations of carvacrol (CAR) and 1,8-cineole (CIN) alone and in combination on the morphology, cell viability and membrane permeability ...of Pseudomonas fluorescens ATCC 11253 cultivated in a vegetable-based broth. Transmission and scanning electron microscopy images of bacterial cells exposed to CAR and CIN alone or in combination showed marked ultrastructural changes after 1h of exposure. These changes included shrunken protoplasm, discontinuity of the outer and cytoplasmic membranes and leakage of the intracellular material. Confocal scanning laser microscopy images corroborated the electron microscopy data, showing a decrease in the number of SYTO-9 cells (intact cells) with a concomitant increase in the number of PI-positive cells (dead cells). All of these morphological changes are indicative of increased membrane permeability and the loss of bacterial envelope integrity, which ultimately lead to cell death. The combination of sublethal concentrations of CAR and CIN could be applied to inhibit the growth of P. fluorescens on vegetables.
► Carvacrol and 1,8-cineole caused loss of the viability of P. fluorescens. ► The compounds caused major changes in cell morphology. ► The compounds increased the membrane permeability of P. fluorescens. ► The compounds, singly and at sublethal amounts, revealed similar effects.
The amount of organic material in the cariogenic environment correlates with the amount of organic material incorporated in carious enamel. The incorporated organic material may be expected to reduce ...the pore volumes available for remineralization and resin infiltration, but these expected outcomes have not yet been quantified. We tested the effect of the amount of organic content in the cariogenic agent on remineralization and the resin-occluded pore volume in artificial subsurface enamel caries. An acid gel (organic-rich; G1) and an aqueous solution (organic-poor; G2) were used to induce subsurface lesions in human enamel. Undemineralized histological sections were prepared, microradiographed, and then submitted to resin infiltration in vitro. The enamel component volumes (mineral, organic, remineralizable total water volume, loosely and firmly bound water volumes, and resin-occluded volume) were measured (by microradiography and polarizing microscopy) at histological sites (n = 38, G1; n = 34, G2). The main outcomes were the differences between the experimental and the predicted volumes (Δremineralizable and Δresin-occluded volumes). Resin infiltration was confirmed by confocal scanning laser microscopy. Compared to G2, G1 presented more incorporated organic volume and lower Δremineralizable volume (p = 0.003; Hedges g = 0.66; power = 0.87), a lower increase in loosely bound water volume (p = 0.0013; Hedges g = 0.74; power = 0.93), a lower remineralization volume in the surface layer (p = 0.017; Hedges g = 0.68; power = 0.8), and a lower Δresin-occluded volume (p = 0.0015; Hedges g = 0.73; power = 0.92). In conclusion, the higher amount of organic matter in the cariogenic gel negatively affected remineralization and the resin-occluded volume in subsurface lesions.
In this work we aimed to perform an in silico predictive screening, docking and molecular dynamic study to identify 1,2,3-triazole-phthalimide derivatives as drug candidates against SARS-CoV-2. The ...in silico prediction of pharmacokinetic and toxicological properties of hundred one 1,2,3-triazole-phtalimide derivatives, obtained from SciFinder® library, were investigated. Compounds that did not show good gastrointestinal absorption, violated the Lipinski's rules, proved to be positive for the AMES test, and showed to be hepatotoxic or immunotoxic in our ADMET analysis, were filtered out of our study. The hit compounds were further subjected to molecular docking on SARS-CoV-2 target proteins. The ADMET analysis revealed that 43 derivatives violated the Lipinski's rules and 51 other compounds showed to be positive for the toxicity test. Seven 1,2,3-triazole-phthalimide derivatives (A7, A8, B05, E35, E38, E39, and E40) were selected for molecular docking and MFCC-ab initio analysis. The results of molecular docking pointed the derivative E40 as a promising compound interacting with multiple target proteins of SARS-CoV-2. The complex E40-M
pro
was found to have minimum binding energy of −10.26 kcal/mol and a general energy balance, calculated by the quantum mechanical analysis, of −8.63 eV. MD simulation and MMGBSA calculations confirmed that the derivatives E38 and E40 have high binding energies of −63.47 ± 3 and −63.31 ± 7 kcal/mol against SARS-CoV-2 main protease. In addition, the derivative E40 exhibited excellent interaction values and inhibitory potential against SAR-Cov-2 main protease and viral nucleocapsid proteins, suggesting this derivative as a potent antiviral for the treatment and/or prophylaxis of COVID-19.
Communicated by Ramaswamy H. Sarma
Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the ...world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.
The endoplasmic reticulum (ER) of higher eukaryotic cells forms an intricate membranous network that serves as the main processing facility for folding and assembling of secreted and membrane ...proteins. The ER is a highly dynamic organelle that interacts with other intracellular structures, as well as endosymbiotic pathogenic and non-pathogenic microorganisms. A strict ER quality control (ERQC) must work to ensure that proteins entering the ER are folded and processed correctly. Unfolded or misfolded proteins are usually identified, selected, and addressed to Endoplasmic Reticulum-Associated Degradation (ERAD) complex. Conversely, when there is a large demand for secreted proteins or ER imbalance, the accumulation of unfolded or misfolded proteins activates the Unfold Protein Response (UPR) to restore the ER homeostasis or, in the case of persistent ER stress, induces the cell death. Pathogenic trypanosomatids, such as
Trypanosoma cruzi
,
Trypanosoma brucei
and
Leishmania spp
are the etiological agents of important neglected diseases. These protozoans have a complex life cycle alternating between vertebrate and invertebrate hosts. The ER of trypanosomatids, like those found in higher eukaryotes, is also specialized for secretion, and depends on the ERAD and non-canonical UPR to deal with the ER stress. Here, we reviewed the basic aspects of ER biology, organization, and quality control in trypanosomatids. We also focused on the unusual way by which
T. cruzi
,
T. brucei
, and
Leishmania
spp. respond to ER stress, emphasizing how these parasites’ ER-unrevealed roads might be an attractive target for chemotherapy.
•PgTeL is an antifungal agent against C. albicans and C. krusei.•The lectin caused energy collapse and oxidative stress in yeast cells.•Treatment with PgTeL led to damage of cell wall and rupture of ...yeast cells.•PgTeL showed antibiofilm effect on C. albicans at sub-inhibitory concentrations.•PgTeL effects can be linked to the bioactivities attributed to P. granatum fruit.
The pomegranate (Punica granatum) sarcotesta contains a chitin-binding lectin (PgTeL) with antibacterial activity against human pathogenic species. In this work, the structural stability of PgTeL was evaluated by fluorimetric analysis and the lectin was evaluated for cytotoxicity to human peripheral blood mononuclear cells (PBMCs) and antifungal activity against Candida albicans and Candida krusei. PgTeL folding was impaired when lectin was incubated at pH≥6.0. On the other hand, the lectin did not undergo unfolding even when heated at 100°C. PgTeL (1, 10, and 100μg/mL) was not cytotoxic to PBMCs. Antifungal activity was detected for C. albicans (MIC: 25μg/mL; MFC: 50μg/mL) and C. krusei (MIC and MFC of 12.5μg/mL). Treatment of yeast cells with PgTeL resulted in decrease of intracellular ATP content even at sub-inhibitory concentrations (½MIC and ¼MIC) and induced lipid peroxidation. In addition, PgTeL damaged the integrity of fungal cell wall of both species, with more pronounced effects in C. krusei. The lectin showed significant antibiofilm activity on C. albicans at sub-inhibitory concentrations (0.195 and 0.39μg/mL). In conclusion, PgTeL is an anti-Candida agent whose action mechanism involves oxidative stress, energetic collapse, damage to the cell wall and rupture of yeast cells.
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