In this study, we characterized Cryptococcus gattii biofilm formation in vitro . There was an increase in the density of metabolically active sessile cells up to 72 h of biofilm formation on polystyrene and glass surfaces. Scanning electron microscopy and confocal laser scanning microscopy analysis revealed that in the early stage of biofilm formation, yeast cells adhered to the abiotic surface as a monolayer. After 12 h, extracellular fibrils were observed projecting from C. gattii cells, connecting the yeast cells to each other and to the abiotic surface; mature biofilm consisted of a dense network of cells deeply encased in an extracellular polymeric matrix. These features were also observed in biofilms formed on polyvinyl chloride and silicone catheter surfaces. We used RNA-Seq-based transcriptome analysis to identify changes in gene expression associated with C. gattii biofilm at 48 h compared to the free-floating planktonic cells. Differential expression analysis showed that 97 and 224 transcripts were up-regulated and down-regulated in biofilm, respectively. Among the biological processes, the highest enriched term showed that the transcripts were associated with cellular metabolic processes, macromolecule biosynthetic processes and translation.
Streptococcus agalactiae or Group B Streptococcus (GBS) remains a leading cause of neonatal infections worldwide; and the maternal vaginal-rectal colonization increases the risk of vertical transmission of GBS to neonates and development of infections. This study reports the in vitro antibacterial effect of the oleoresin from Copaifera officinalis Jacq. L. in natura (copaiba oil) and loaded into carbomer-hydrogel against planktonic and sessile cells of GBS. First, the naturally extracted copaiba oil was tested for the ability to inhibit the growth and metabolic activity of planktonic and sessile GBS cells. The time-kill kinetics showed that copaiba oil exhibited a dose-dependent bactericidal activity against planktonic GBS strains, including those resistant to erythromycin and/or clindamycin [minimal bactericidal concentration (MBC) ranged from 0.06 mg/mL to 0.12 mg/mL]. Copaiba oil did not inhibit the growth of different Lactobacillus species, the indigenous members of the human microbiota. The mass spectral analyses of copaiba oil showed the presence of diterpenes, and the kaurenoic acid appears to be one of the active components of oleoresin from C. officinalis related to antibacterial activity against GBS. Microscopy analyses of planktonic GBS cells treated with copaiba oil revealed morphological and ultrastructural alterations, displaying disruption of the cell wall, damaged cell membrane, decreased electron density of the cytoplasm, presence of intracellular condensed material, and asymmetric septa. Copaiba oil also exhibited antibacterial activity against established biofilms of GBS strains, inhibiting the viability of sessile cells. Low-cost and eco-friendly carbomer-based hydrogels containing copaiba oil (0.5% – CARB-CO 0.5; 1.0% – CARB-CO 1.0) were then developed. However, only CARB-CO 1.0 preserved the antibacterial activity of copaiba oil against GBS strains. This formulation was homogeneous, soft, exhibited a viscoelastic behavior, and showed good biocompatibility with murine vaginal mucosa. Moreover, CARB-CO 1.0 showed a slow and sustained release of the copaiba oil, killing the planktonic and sessile (established biofilm) cells and inhibiting the biofilm formation of GBS on pre-coated abiotic surface. These results indicate that carbomer-based hydrogels may be useful as topical systems for delivery of copaiba oil directly into de vaginal mucosa and controlling S. agalactiae colonization and infection.
Herein, twenty-six benzoylthioureas were evaluated for their antimicrobial activity against different bacterial and fungal species. Two 4-substituted benzoylthiourea, one benzoylurea and one benzoylguanidine derivatives were further synthesized to identify the most promising compound. Eight compounds were active against at least one microbial species tested. N-(butylcarbamothioyl)-benzamide (1 e) exhibited the best antimicrobial activity towards Streptococcus agalactiae (group B Streptococcus-GBS), including clinical isolates susceptible or resistant to clindamycin and/or erythromycin and azithromycin.1 e presented a bacteriostatic effect, causing morphological and ultrastructural alterations on planktonic cells, and decreased the metabolic activity of GBS biofilms. No hemolytic and cytotoxicity to mammalian cells were detected for 1 e, that also displayed drug-likeness properties. Molecular docking was performed on Streptococcus pneumoniae enoyl-ACP reductase obtained by homology modeling. 1 e showed relevant interactions with the GBS enoyl-ACP reductase enzyme. N-(butylcarbamothioyl)-benzamide may be a good starting point for the development of new antimicrobials against GBS.
Cryptococcus neoformans is the leading cause of cryptococcosis, an invasive and potentially fatal infectious disease. Therapeutic failures are due to the increase in antifungal resistance, the adverse effects of drugs, and the unavailability of therapeutic regimens in low-income countries, which limit the treatment of cryptococcosis, increasing the morbidity and mortality associated with these infections. Thus, new antifungal drugs and innovative strategies for the cryptococcosis treatment are urgently needed. The aim of the present study was to evaluate the effect of ethyl acetate fraction (EAF) of Poincianella pluviosa stem bark on planktonic and biofilm mode of growth of C. neoformans. Furthermore, the interaction between the EAF and amphotericin B (AmB) was evaluated in vitro and in Galleria mellonella infection model. Minimal inhibitory concentrations (MICs) of EAF ranged from 125.0 to >1,000.0 μg/ml and >1,000.0 μg/ml for planktonic and sessile cells, respectively. The combination between EAF and AmB exhibited a synergistic fungicidal activity toward C. neoformans, with a fractional inhibitory concentration index (FICI) ranging from 0.03 to 0.06 and 0.08 to 0.28 for planktonic and sessile cells, respectively. Microscopy analyses of planktonic C. neoformans cells treated with EAF, alone or combined with AmB, revealed morphological and ultrastructural alterations, including loss of integrity of the cell wall and cell membrane detachment, suggesting leakage of intracellular content, reduction of capsule size, and presence of vacuoles. Moreover, EAF alone or combined with AmB prolonged the survival rate of C. neoformans-infected G. mellonella larvae. These findings indicate that P. pluviosa may be an important source of new compounds that can be used as a fungus-specific adjuvant for the treatment of cryptococcosis.
IntroductionCryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity.MethodsIn the present study, the effect of the synthetic thiourea derivative N-(butylcarbamothioyl) benzamide (BTU-01), alone and combined with amphotericin B (AmB), was evaluated in planktonic and sessile (biofilm) cells of C. neoformans.ResultsBTU-01 alone exhibited a fungistatic activity with minimal inhibitory concentrations (MICs) ranging from 31.25 to 62.5 μg/mL for planktonic cells; and sessile MICs ranging from 125.0 to 1000.0 μg/mL. BTU-01 caused a concentration-dependent inhibitory activity on cryptococcal urease and did not interfere with plasma membrane fluidity. Molecular docking was performed on Canavalia ensiformis urease, and BTU-01 showed relevant interactions with the enzyme. The combination of BTU-01 and AmB exhibited synergistic fungicidal activity against planktonic and sessile cells of C. neoformans. Microscopic analysis of C. neoformans treated with BTU-01, alone or combined with AmB, revealed a reduction in cell and capsule sizes, changes in the morphology of planktonic cells; a significant decrease in the number of cells within the biofilm; and absence of exopolymeric matrix surrounding the sessile cells. Neither hemolytic activity nor cytotoxicity to mammalian cells was detected for BTU-01, alone or combined with AmB, at concentrations that exhibited antifungal activity. BTU-01 also displayed drug-likeness properties.ConclusionThese results indicate the potential of BTU-01, for the development of new strategies for controlling C. neoformans infections.
This study aimed to evaluate the antibacterial activity of the dichloromethane/ethyl acetate fraction (named F4a), obtained from the culture of Pseudomonas aeruginosa LV strain in presence of copper chloride, against planktonic and sessile cells of Staphylococcus aureus, including those presenting multidrug resistance. First, the minimal inhibitory concentrations (MIC) of F4a for twenty-six clinical isolates were determined and the values ranged from 1.56 to 6.25 µg/mL. Minimal bactericidal concentration (MBC) of 3.13 µg/mL was detected in 84.6% of the isolates. The time-kill curve analysis revealed a significant decreased in colony-forming unit counts after 4 h of treatment with the MIC/MBC of F4a. Moreover, the MIC/MBC of the fluopsin C, a copper-containing compound present in F4a, were 1.56/3.13 µg/mL, indicating that this compound seems to be one of the active components related to the antibacterial activity against S. aureus. Images of transmission electron microscopy showed significant ultrastructural alterations in planktonic cells treated with the MIC/MBC of F4a. A significant reduction in the metabolic activity of established biofilms of all S. aureus isolates was observed after treatment with F4a. No hemolytic activity to human erythrocytes was detected for F4a, and the cytotoxic concentration to LLC-MK2 cells was 3.44 µg/mL. In conclusion, F4a exhibited a bactericidal activity against planktonic cells and inhibited the metabolic activity of biofilms of S. aureus. F4a can be promising for the development of new strategies for the treatment of infections caused by S. aureus.
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