Synergistic Antibacterial and Antibiofilm Activity of the MreB Inhibitor A22 Hydrochloride in Combination with Conventional Antibiotics against Pseudomonas aeruginosa and Escherichia coli Clinical Isolates.

In the era of antibiotic resistance, the bacterial cytoskeletal protein MreB is presented as a potential target for the development of novel antimicrobials. Combined treatments of clinical antibiotics with anti-MreB compounds may be promising candidates in combating the resistance crisis, but also in preserving the potency of many conventional drugs. This study aimed to evaluate the synergistic antibacterial and antibiofilm activities of the MreB inhibitor A22 hydrochloride in combination with various antibiotics. The minimum inhibitory concentration (MIC) values of the individual compounds were determined by the broth microdilution method against 66 clinical isolates of Gram-negative bacteria. Synergy was assessed by the checkerboard assay. The fractional inhibitory concentration index was calculated for each of the A22-antibiotic combination. Bactericidal activity of the combinations was evaluated by time-kill curve assays. The antibiofilm activity of the most synergistic combinations was determined by crystal violet stain, methyl thiazol tetrazolium assay, and confocal laser scanning microscopy analysis. The combined cytotoxic and hemolytic activity was also evaluated toward human cells. According to our results, Pseudomonas aeruginosa and Escherichia coli isolates were resistant to conventional antibiotics to varying degrees. A22 inhibited the bacterial growth in a dose-dependent manner with MIC values ranging between 2 and 64 µg/mL. In combination studies, synergism occurred most frequently with A22-ceftazidime and A22-meropemen against Pseudomonas aeruginosa and A22-cefoxitin and A22-azithromycin against Escherichia coli. No antagonism was observed. In time-kill studies, synergism was observed with all expected combinations. Synergistic combinations even at the lowest tested concentrations were able to inhibit biofilm formation and eradicate mature biofilms in both strains. Cytotoxic and hemolytic effects of the same combinations toward human cells were not observed. The findings of the present study support previous research regarding the use of MreB as a novel antibiotic target. The obtained data expand the existing knowledge about the antimicrobial and antibiofilm activity of the A22 inhibitor, and they indicate that A22 can serve as a leading compound for studying potential synergism between MreB inhibitors and antibiotics in the future.

In vitro and in vivo study on the effect of antifungal agents on hematopoietic cells in mice.

Liposomal amphotericin B, voriconazole, and caspofungin are currently used for systemic and severe fungal infections. Patients with malignant diseases are treated with granulocyte-colony stimulating factor (G-CSF) for the recovery of granulocytes after chemotherapy or hematopoietic cell (HC) transplantation. Since they have a high incidence of fungal infections, they inevitably receive antifungal drugs for treatment and prophylaxis. Despite their proven less toxicity for various cell types comparatively with amphotericin B and the decrease in the number of leukocytes that has been reported as a possible complication in clinical studies, the effect of liposomal amphotericin B, voriconazole, and caspofungin on HCs has not been clarified. The present study aimed to examine the in vitro and in vivo effect of these three modern antifungals on HCs. Colony-forming unit (CFU) assays of murine bone marrow cells were performed in methylcellulose medium with or without cytokines and in the presence or absence of various concentrations of liposomal amphotericin B, voriconazole, and caspofungin. In the in vivo experiments, the absolute number of granulocytes was determined during leukocyte recovery in sublethally irradiated mice receiving each antifungal agent separately, with or without G-CSF. In vitro, all three antifungal drugs were nontoxic and, interestingly, they significantly increased the number of CFU-granulocyte-macrophage colonies in the presence of cytokines, at all concentrations tested. This was contrary to the concentration-dependent toxicity and the significant decrease caused by conventional amphotericin B. In vivo, the number of granulocytes was significantly higher with caspofungin plus G-CSF treatment, higher and to a lesser extent higher, but not statistically significantly, with voriconazole plus G-CSF and liposomal amphotericin B plus G-CSF treatments, respectively, as compared with G-CSF alone. These data indicate a potential synergistic effect of these antifungals with the cytokines, in vitro and in vivo, with subsequent positive effect on hematopoiesis.

Germinant generation from δ-endotoxin of Bacillus thuringiensis strain 1.1.

The novel finding of this study is that the δ-endotoxin present in the spore coat of Bacillus thuringiensis strain 1.1 (Bt1.1), plays a central role in spore germination by generation of germinant via its ß-glucosidase activity and is based on the following: (i) the crystals of Bt1.1 consist of the 140 kDa δ-endotoxin which exhibits ß-glucosidase enzymatic activity. Besides crystals, δ-endotoxin is also located in the spore coat and at this site displays ß-glucosidase activity, resulting in glucose production; (ii) glucose is an efficient germinant of both Bt1.1 and acrystalliferous Bt4.1 strain; (iii) substrates of ß-glucosidase can activate the germination of Bt1.1 spores, but not those of the acrystalliferous Bt4.1 sister strain that do not contain the 140 kDa δ-endotoxin; (iv) Reduction or enhancement of enzymatic activity of δ-endotoxin, results in retardation or acceleration of germination and outgrowth, respectively. Bt1.1 cells secrete a 60 kDa polypeptide which displays ß-glucosidase activity as indicated by zymogram analysis and which is immunologically related to the 140 kDa δ-endotoxin.

Microstructural characterization and comparative evaluation of physical, mechanical and biological properties of three ceramics for metal-ceramic restorations.

OBJECTIVES: A wide variety of dental ceramics compositions have been introduced in dental clinical practice in order to combine desired aesthetics with superior mechanical performance. The aim of the present study was to investigate the microstructural changes in three dental ceramics after their sintering according to manufacturers' instructions and to comparatively evaluate some of their physical, mechanical and biological properties. METHODS: The analysis of the phases present in each material before and after sintering was performed with scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction analysis (XRD). The thermal properties of ceramic specimens were evaluated with differential thermal and thermogravimetric analysis (TG-DTA). The mechanical properties evaluated were fracture toughness, Young's modulus and microhardness with the Vickers indentation method. MTT assay was used for cell proliferation assessment. One-way analysis of variance (ANOVA) with Bonferroni multiple comparisons tests was used to determine statistically significant differences (significance level of p<0.05). RESULTS: Results showed a remarkable variation among the three ceramic compositions of leucite content in the starting unheated ceramic powders ranging between 14 and 32 wt.% and in the respective sintered powders ranging between 15 and 41 wt.% The low fusing glass-ceramic and the high fusing leucite-based ceramic presented significantly higher fracture toughness (p<0.001) and microhardness and lower modulus of elasticity (p<0.05) compared to the low fusing feldspathic ceramic. The three ceramics were almost equivalent concerning their in vitro biological behavior. SIGNIFICANCE: Variations in crystal structure, distribution and composition are related to differences concerning mechanical properties of dental ceramics.