The Synthetic Peptide LyeTx I mn∆K, Derived from Lycosa erythrognatha Spider Toxin, Is Active against Methicillin-Resistant Staphylococcus aureus (MRSA) In Vitro and In Vivo.

The urgent global health challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) infections demands effective solutions. Antimicrobial peptides (AMPs) represent promising tools of research of new antibacterial agents and LyeTx I mn∆K, a short synthetic peptide based on the Lycosa erythrognatha spider venom, is a good representative. This study focused on analyzing the antimicrobial activities of LyeTx I mn∆K, including minimum inhibitory and bactericidal concentrations, synergy and resensitization assays, lysis activity, the effect on biofilm, and the bacterial death curve in MRSA. Additionally, its characterization was conducted through isothermal titration calorimetry, dynamic light scattering, calcein release, and finally, efficacy in a mice wound model. The peptide demonstrates remarkable efficacy against planktonic cells (MIC 8-16 µM) and biofilms (>30% of inhibition) of MRSA, and outperforms vancomycin in terms of rapid bactericidal action and anti-biofilm effects. The mechanism involves significant membrane damage. Interactions with bacterial model membranes, including those with lysylphosphatidylglycerol (LysylPOPG) modifications, highlight the versatility and selectivity of this compound. Also, the peptide has the ability to sensitize resistant bacteria to conventional antibiotics, showing potential for combinatory therapy. Furthermore, using an in vivo model, this study showed that a formulated gel containing the peptide proved superior to vancomycin in treating MRSA-induced wounds in mice. Together, the results highlight LyeTx I mnΔK as a promising prototype for the development of effective therapeutic strategies against superficial MRSA infections.

Lactobacillus delbrueckii CIDCA 133 fermented milk modulates inflammation and gut microbiota to alleviate acute colitis.

Lactobacillus delbrueckii subsp. lactis CIDCA 133 is a health-promoting bacterium that can alleviate gut inflammation and improve the epithelial barrier in a mouse model of mucositis. Despite these beneficial effects, the protective potential of this strain in other inflammation models, such as inflammatory bowel disease, remains unexplored. Herein, we examined for the first time the efficacy of Lactobacillus delbrueckii CIDCA 133 incorporated into a fermented milk formulation in the recovery of inflammation, epithelial damage, and restoration of gut microbiota in mice with dextran sulfate sodium-induced colitis. Oral administration of Lactobacillus delbrueckii CIDCA 133 fermented milk relieved colitis by decreasing levels of inflammatory factors (myeloperoxidase, N-acetyl-ß-D-glucosaminidase, toll-like receptor 2, nuclear factor-κB, interleukins 10 and 6, and tumor necrosis factor), secretory immunoglobulin A levels, and intestinal paracellular permeability. This immunobiotic also modulated the expression of tight junction proteins (zonulin and occludin) and the activation of short-chain fatty acids-related receptors (G-protein coupled receptors 43 and 109A). Colonic protection was effectively associated with acetate production and restoration of gut microbiota composition. Treatment with Lactobacillus delbrueckii CIDCA 133 fermented milk increased the abundance of Firmicutes members (Lactobacillus genus) while decreasing the abundance of Proteobacteria (Helicobacter genus) and Bacteroidetes members (Bacteroides genus). These promising outcomes influenced the mice's mucosal healing, colon length, body weight, and disease activity index, demonstrating that this immunobiotic could be explored as an alternative approach for managing inflammatory bowel disease.

Exploring the anti-inflammatory effects of postbiotic proteins from Lactobacillus delbrueckii CIDCA 133 on inflammatory bowel disease model.

Lactobacillus delbrueckii CIDCA 133 is a promising health-promoting bacterium shown to alleviate intestinal inflammation. However, the specific bacterial components responsible for these effects remain largely unknown. Here, we demonstrated that consuming extractable proteins from the CIDCA 133 strain effectively relieved acute ulcerative colitis in mice. This postbiotic protein fraction reduced the disease activity index and prevented colon shortening in mice. Furthermore, histological analysis revealed colitis prevention with reduced inflammatory cell infiltration into the colon mucosa. Postbiotic consumption also induced an immunomodulatory profile in colitic mice, as evidenced by both mRNA transcript levels (Tlr2, Nfkb1, Nlpr3, Tnf, and Il6) and cytokines concentration (IL1ß, TGFß, and IL10). Additionally, it enhanced the levels of secretory IgA, upregulated the transcript levels of tight junction proteins (Hp and F11r), and improved paracellular intestinal permeability. More interestingly, the consumption of postbiotic proteins modulated the gut microbiota (Bacteroides, Arkkemansia, Dorea, and Oscillospira). Pearson correlation analysis indicated that IL10 and IL1ß levels were positively associated with Bacteroides and Arkkemansia_Lactobacillus abundance. Our study reveals that CIDCA 133-derived proteins possess anti-inflammatory properties in colonic inflammation.

Prevention of bone infection after open fracture using a chitosan with ciprofloxacin implant in animal model

Abstract Purpose To evaluate different concentrations of ciprofloxacin to prevent infection after open fracture contaminated with S. aureus in rats using absorbable local delivery system. Methods Fifty-two Wistar rats were assigned to six groups. After 4 weeks, all animals underwent 99mTc-ceftizoxima scintigraphy evaluation, callus formation measurement and histological analysis. ANOVA, t-Student and Kruskal Wallis were used for quantitative variables statistical analysis, whereas qui square and exact Fisher were used for qualitative variables. Results Treatment using 25% and 50% of ciprofloxacin incorporated at the fracture fixation device were effective in preventing bone infection compared to control group (p<0.05). Chitosan were not effective in preventing bone infection when used alone compared to control group (p>0.05). Histological findings demonstrated bone-healing delay with 50% of ciprofloxacin. No difference in callus formation were observed (p>0.05). Conclusion Local delivery treatment for contaminated open fracture using chitosan with ciprofloxacin is effective above 25%.

Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Background Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc. Methods Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2. 2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values < 0.05). Results Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 μmol.L−1 (S. aureus) and 10.10 μmol.L−1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 μg of LyeTx I-K-HYNIC; 250 μg of SnCl2. 2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n= 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.(AU)

Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc. Methods Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2. 2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values 0.05). Results Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 mol.L1 (S. aureus) and 10.10 mol.L1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 g of LyeTx I-K-HYNIC; 250 g of SnCl2. 2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n= 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.

New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment

Leishmaniasis is one of the six major tropical diseases targeted by the World Health Organization. It is a life-threatening disease of medical, social and economic importance in endemic areas. No vaccine is yet available for human use, and chemotherapy presents several problems. Pentavalent antimonials have been the drugs of choice to treat the disease for more than six decades; however, they exhibit high toxicity and are not indicated for children, for pregnant or breastfeeding women or for chronically ill patients. Amphotericin B (AmpB) is a second-line drug, and although it has been increasingly used to treat visceral leishmaniasis (VL), its clinical use has been hampered due to its high toxicity. This review focuses on the development and in vivo usage of new delivery systems for AmpB that aim to decrease its toxicity without altering its therapeutic efficacy. These new formulations, when adjusted with regard to their production costs, may be considered new drug delivery systems that promise to improve the treatment of leishmaniasis, by reducing the side effects and the number of doses while permitting a satisfactory cost-benefit ratio.