Nowadays, it is very important to produce new-generation drugs with antimicrobial properties that will target biofilm-induced infections. The first target for combating these microorganisms, which are the source itself. Antimicrobial peptides, which are more effective than antibiotics due to their ability to kill microorganisms and use a different metabolic pathway, are among the new options today. The aim of this study is to develop new-generation antibiotics that inhibit both biofilm-producing bacteria and the biofilm itself. For this purpose, we designed four different peptides by combining two amino acid forms (D- and L-) with the same sequence having alpha helix structures. It was found that the combined use of these two forms can increase antimicrobial efficacy more than 30-fold. These results are supported by molecular modeling and scanning electron microscopy (SEM), at the same time cytotoxicity (IC₅₀) and hemotoxicity (HC₅₀) values remained within the safe range. Furthermore, antibiofilm activities of these peptides were investigated. Since the existing biofilm inhibition methods in the literature do not technically simulate the exact situation, in this study, we have developed a real-time observable biofilm model and a new detection method based on it, which we call the CoMIC method. Findings have shown that the NET1 peptide with D-leucine amino acid in its structure and the NET3 peptide with D-arginine amino acid in its structure are effective in inhibiting biofilm. As a conclusion, our peptides can be considered as potential next-generation broad-spectrum antibiotic molecule/drug candidates that might be used in biofilm and clinical important bacteria. Key points: • Antimicrobial peptides were developed to inhibit both biofilms producing bacteria and the biofilm itself. • CoMIC will fill a very crucial gap in understanding biofilms and conducting the necessary quantitative studies. • Molecular modelling studies, NET1 peptide molecules tends to move towards and adhere to the membrane within nanoseconds.

Antimicrobial peptides (AMPs) are a diverse group of small, naturally occurring molecules that orchestrate the innate immune response of various organisms, from microorganisms to humans. Characterized by their broad-spectrum activity against bacteria, fungi and viruses, AMPs are increasingly recognized for their potential as novel therapeutic agents in the face of rising antibiotic resistance. Here, we present several newly designed AMPs, one of which, DTN6, exerts significant activity against several organisms with MIC values as low as 0.5 µg/mL. The D-TN6 peptide influences both bacteria and yeasts. Scanning electron microscopy and transmission electron microscopy results showed that the bacterial membrane is affected by D-TN6, which is resistant to proteases and is effective against antibiotic-resistant pathogens with hemolytic activity and low toxicity. The D-TN6 peptide is effective in vivo against standard S. aureus strains in wounds. Thus, D-TN6 is a potent antibiotic candidate with a broad spectrum of activity. Overall, AMPs are a promising tool for the development of next-generation antimicrobial agents that could mitigate global health threats posed by multidrug-resistant pathogens.

Antibiotic-resistant microorganisms have become a serious threat to public health, resulting in hospital infections, the majority of which are caused by commonly used urinary tract catheters. Strategies for preventing bacterial adhesion to the catheters’ surfaces have been potentially shown as effective methods, such as coating thesurface with antimicrobial biomolecules. Here, novel antimicrobial peptides (AMPs) were designed as potential biomolecules to prevent antibiotic-resistant bacteria from binding to catheter surfaces. Thiolated AMPs were synthesized using solid-phase peptide synthesis (SPPS), and prep-HPLC was used to obtain AMPs with purity greater than 90%. On the other side, the silicone catheter surface was activated by UV/ozone treatment, followed by functionalization with allyl moieties for conjugation to the free thiol group of cystein in AMPs using thiol-ene click chemistry. Peptide-immobilized surfaces were found to become more resistant to bacterial adhesion while remaining biocompatible with mammalian cells. The presence and site of conjugation of peptide molecules were investigated by immobilizing them to catheter surfaces from both ends (C-Pep and Pep-C). It was clearly demonstrated that AMPs conjugated to the surface via theirN terminus have a higher antimicrobial activity. This strategy stands out for its effective conjugation of AMPs to silicone-based implant surfaces for the elimination of bacterial infections.

Drug-resistant Group A beta-hemolytic streptococci remain significant infectious agents globally. This study investigated the major S. pyogenes strains responsible for infections in Türkiye and their susceptibility to beta-lactam and macrolide antibiotics. We determined the minimum inhibitory concentration using the penicillin gradient test and performed emm typing and DNA fingerprinting via pulsed-field gel electrophoresis (PFGE) to analyze the clonal spread of 92 S. pyogenes strains isolated from two hospitals in Türkiye between 2020 and 2022. Our findings revealed the predominant S. pyogenes strains causing infections in the population and provided insights into the epidemiological relatedness of these drug-resistant strains. This study also evaluated the correlation between emm typing and PFGE in tracking S. pyogenes epidemiology. In this study, the current resistance patterns of S. pyogenes strains in Türkiye identified erythromycin resistance in a few strains, but no resistance to penicillin was detected. This study revealed that emm types 1, 12 and 89 as S. pyogenes strain genotypes were responsible for epidemic infections in Türkiye. PFGE genotyping and emm typing were found to provide better phylogenetic classification in the investigation of S. pyogenes epidemiology.

Antimicrobial Peptides and Their Anti-Leishmanial Efficacies on Leishmania tropica Promastigotes In vitro - Turkish Journal of Parasitology

It is estimated that one in four people worldwide carries Mycobacterium tuberculosis bacteria. MPT64 is a protein exclusively secreted by Mycobacterium tuberculosis complex (MTC) bacteria. It serves as a crucial diagnostic marker and plays a role in the bacterium’s survival by modulating the host immune response. Consequently, the development of innovative diagnostic tools based on MPT64, as well as the production of high-purity MPT64 protein to support research on tuberculosis pathogenesis and the advancement of novel therapeutic strategies, is of great importance. In this study, optimization experiments were conducted to produce this protein in E. coli with high yield and purity. First, a gBlock was designed by codon optimization and then cloned into a plasmid vector using the LIC method. For more efficient production, E. coli BL21(DE3)-R3-pRARE2 strain, which carries rare tRNAs for rare codons, was used as the host. Five different culture media were tested to maximize protein production, with the highest yield obtained in eBHI medium. The resulting protein yield was 4.9 mg/L. To the best of our knowledge, this study provides the most detailed information on the recombinant production and characterization of MPT64 to date. Therefore, these results contribute important data for future studies on the MPT64 protein.