Milka Malešević

The emergence of multidrug-resistant pathogens has driven the development of novel antimicrobial peptides (AMPs) as therapeutic alternatives. Lactolisterin LBU (LBU) is a bacteriocin with promising activity against Gram-positive bacteria, including Staphylococcus aureus. In this study, we designed and evaluated a panel of amino acid variants of LBU to investigate domain–activity relationships and improve activity. Peptides were commercially synthesized, and their effect was evaluated for minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), hemolytic activity, cytotoxicity, in vivo toxicity, and virulence modulation. AlphaFold3 structural prediction of LBU revealed a four-helix topology with amphipathic and hydrophobic segments. Helical wheel projections identified helices I and IV as amphipathic, suggesting their potential involvement in membrane interaction and activity. Glycine-to-alanine substitutions at helix I markedly increased antimicrobial activity but altered toxicity profiles. In contrast, changes at helix junctions and kinks reduced antimicrobial activity. We also showed differential regulation of virulence genes upon sub-MIC treatment. Overall, rational substitution enabled identification of residues critical for activity and toxicity, providing insights into therapeutic tuning of lactolisterin-based peptides.

Biofilm-associated infections caused by Gram-negative bacteria, especially multidrug-resistant strains, frequently occur in intensive care units and represent a major therapeutic challenge. The economic burden of biofilm-associated infections is considerable, making the search for new treatment approaches a focal point for policymakers and scientific funding bodies. Biofilm formation is regulated by quorum sensing (QS), a population density-dependent communication mechanism between cells mediated by small diffusible signaling molecules. QS modulates various intracellular processes, and some features of QS are common to all Gram-negative bacteria. While there are differences in the QS regulatory networks of different Gram-negative bacterial species, a common feature of most Gram-negative bacteria is the ability of N-acylhomoserine lactones (AHL) as inducers to diffuse across the bacterial membrane and interact with receptors located either in the cytoplasm or on the inner membrane. Targeting QS by inhibiting the synthesis, transport, or perception of signaling molecules using small molecules, quorum quenching enzymes, antibodies, combinatorial therapies, or nanoparticles is a promising strategy to combat virulence. In-depth knowledge of biofilm biology, antibiotic susceptibility, and penetration mechanisms, as well as a deep understanding of anti-QS agents, will contribute to the development of antimicrobial therapies to combat biofilm infections. Advancing antimicrobial therapies against biofilm infections requires a deep understanding of biofilm biology, antibiotic susceptibility, penetration mechanisms, and anti-QS strategies. This can be achieved through in vivo and clinical studies, supported by state-of-the-art tools such as machine learning and artificial intelligence.

Purpose The present study investigated the role of resistance-nodulation-cell division (RND) efflux pumps in tigecycline resistance of Acinetobacter baumannii clinical isolates recovered from three Western Balkan countries (Serbia, Bosnia and Herzegovina and Montenegro). Methods A total of 37 A. baumannii isolates recovered from seven tertiary care hospitals in 2016 and 2022 were tested against tigecycline using broth microdilution method. Then, efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used to determine the involvement of efflux pumps in tigecycline resistance. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multiplex PCR-based determination of clonal lineage. Regulators of efflux pumps were analyzed for amino acid substitutions, while reverse transcription-quantitative PCR (RT-qPCR) enabled quantification of RND efflux pumps expression. Results All tested isolates were interpreted as resistant to tigecycline and showed reduced tigecycline minimum inhibitory concentration (MIC) values in the presence of CCCP. PFGE analysis showed significant diversity among isolates grouped in cluster I including IC2 (n = 32) and IC3 (n = 1) isolates, while cluster II was comprised of four IC1 isolates. The most prevalent substitutions in AdeR were V120I and A136V and in AdeS G186V and N268H (n = 33). The Q262R substitution was detected in AdeL proteins of IC1 isolates, whereas no alterations were observed within AdeN. The expression of the adeB, adeG, and adeJ genes in selected isolates was upregulated in five (1.16- to 3-fold), sixteen (1.35- to 2.82-fold), and twelve isolates (1.62- to 4-fold) compared to ATCC19606, respectively. Conclusion This study revealed that overexpression of RND efflux pumps underlies tigecycline resistance in A. baumannii clinical isolates from the Western Balkans.