Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin ( mec A), gentamicin ( aac A), and ciprofloxacin ( grl A, grl B) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mec A, a 5.5-fold decrease in grl A, a 6-fold decrease in grl B, and a 4-fold decrease in aac A expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grl B and aac A genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.

Candida spp. are clinically important pathogens that cause difficulties for treatment by biofilm formation. Considering antifungal resistance rates and the limitations in the discovery of new antifungals, the antifungal and antibiofilm effects of various drugs used for different therapeutic purposes are becoming more important. The goal of our study was to determine the antifungal and antibiofilm effects of the selective serotonin reuptake inhibitors (SSRIs), namely sertraline (SRT), paroxetine (PRX), and fluoxetine (FLX) alone and in combination with fluconazole (FLC) against Candida spp. Twenty Candida spp. strains isolated from clinical samples from Ege University Hospital were identified by the Dalmau method and matrix-assisted laser desorption ionization time of flight mass spectrometry. The minimum inhibitory concentrations (MICs) of the SSRIs and FLC were detected by broth microdilution method. Synergistic interactions between the SSRIs and FLC were investigated by checkerboard assay. The antibiofilm effects of the SSRIs were determined by spectrophotometric microplate method. Among the isolates, five different Candida spp. (C. albicans, C. glabrata, C. krusei, C. tropicalis, and C.parapsilosis) were identified. The MICs of the SSRIs ranged between 16-512 μg/mL. While SRT showed the highest antifungal effect, the antibiofilm efficacy of FLX was higher than that of the other agents. Moreover, FLX and PRX showed a synergistic effect with FLC in 13 and 19 isolates, respectively. Four isolates were strong biofilm producers while nine isolates were moderate biofilm producers. C. parapsilosis strains showed higher biofilm production than the other species. At MIC/2 concentration, FLX and SRT alone inhibited mature biofilms in six and five isolates, respectively, while PRX caused increases biofilm formation in seven isolates. This study revealed that MIC/2 concentrations of SSRIs could have antifungal and antibiofilm effects. SRT and FLX alone or in combination with antifungals may possibly have therapeutic potential for combating fungal infections.

Antibiotic resistance poses a global health crisis, limiting the efficacy of available therapeutic agents and hindering the development of new antibiotics. The pharmaceutical industry’s waning interest in antibiotic production further exacerbates this challenge. Addressing antibiotic resistance demands innovative solutions. Here, we explore the application of CRISPR-Cas-based antimicrobials as a pioneering approach to combat multidrug resistance. Our study focuses on the methicillin-resistant clinical Staphylococcus aureus (MRSA), a significant clinical threat. Utilizing CRISPR/Cas9 technology, we aimed to concurrently target methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes, thereby altering the resistance profile and enhancing sensitivity to antibiotics. We engineered CRISPR plasmids containing sgRNAs specific to the target regions, which were subsequently electroporated into MRSA strains. Real-time Polymerase Chain Reaction (RT-PCR) assessed changes in resistance gene expression, while disk diffusion and broth microdilution methods determined alterations in resistance status. Western blotting analyzed changes in PBP2a expression, and Sanger sequence analysis confirmed mutations in target regions. Results revealed a statistically significant 1.5-fold decrease in mecA gene expression, 5.5-fold decrease in grlA gene, 6-fold decrease in grlB gene, and 4-fold decrease in aacA gene compared to the wild type strain, as determined by RT-PCR. Antibiotic susceptibility tests demonstrated the suppression of mecA, grlA, grlB, and aacA genes, resulting in the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis showed 70% decrease in PBP2a expression, indicating the breakdown of beta-lactam resistance. Sanger sequence analysis confirmed point mutations in grlB and aacA genes, along with three-base mutations in grlA and mecA genes. Our findings underscore the potential of CRISPR/Cas9 technology to mitigate antibiotic resistance, offering a transformative strategy to restore the efficacy of existing antibiotics in the face of multidrug-resistant pathogens.

The main purpose of this study is to design Cefpodoxime proxetil (CFP) loaded Eudragit RS 100 (ERS) nanoparticles to provide prolonged drug release, the enhanced oral efficacy of the drug at a lower oral dose. CFP-loaded nanoparticles are prepared by the single-emulsion solvent evaporation method with different drug:polymer ratios, F1 (CFP:ERS=1:1) and F2 (CFP:ERS=1:2). Physicochemical characterization, antibacterial activity, cytotoxicity profiles of nanoparticles are examined. F1 and F2 nanoparticles have a particle size of 236.70±3.05 and 258.87±11.32 nm, spherical shape, and a porous surface under SEM. The zeta potential values of F1 and F2 nanoparticles were 22.13±1.01 and 25.80±2.01 mV, and the encapsulation efficiencies were 34.37±3.71% and 55.78±3.52%, respectively. Since F2 nanoparticles have a smaller particle size, higher zeta potential, and encapsulation efficiency, this formulation was chosen as superior, and the subsequent experiments were continued with this formulation. The antibacterial activity of F2 nanoparticles against E.coli was found to be 2 times more effective than CFP. Cytotoxicity studies revealed that the cell viabilities in BEAS-2B and HepG2 cells were higher with the exposure to CFP nanoparticles (200 µg/ml) compared to CFP (100 µg/ml) alone. CFP nanoparticles have excellent efficacy and a cytocompatible profile in inhibiting the growth of bacteria compared to free CFP.

Herein we synthesized 6 new N,N-disubstituted taurinamidobenzensulfonamide derivatives and characterized their structures by means of 1H and 13C NMR, HR-MS analysis. In addition, their in vitro antibacterial and antifungal activities were tested against two gram-positive, two gram-negative bacteria, and two fungal strains by using broth microdilution method. Compounds 1 (methoxy substitution) and 2 (methyl substitution) displayed the best antibacterial activity against Escherichia coli and Staphylococcus aureus, respectively. E. faecalis was affected by compounds 1, 2, 4, and 6, becoming the most susceptible pathogen compared to other tested bacterial and fungal strains. Interestingly, changing fluoro atom in compound 6 with the chloro atom, as in compound 5, deteriorated the antibacterial activity against all bacterial strains. As a result, these results provide us to investigate the relationship between structural changes and antibacterial/antifungal activity, which can be further used to develop more effective taurine derivatives.