Oryasin, Asude GulceDogan, Hanife SalihSalih Doğan, HanifeBozdogan, Bulent2026-04-212026-04-2120260302-89331432-072X10.1007/s00203-026-04812-x2-s2.0-105033211103https://hdl.handle.net/123456789/9071https://doi.org/10.1007/s00203-026-04812-xEnterococcus faecium, listed by the World Health Organization as a bacterial priority pathogen, poses a growing challenge due to increasing antibiotic resistance, underscoring the need for novel antimicrobial strategies. Single-domain antibodies like variable heavy chain domain (VH) are being developed against various targets, including infectious agents. This study aimed to generate a VH specific to E. faecium Penicillin Binding Protein 5 (PBP5), a key enzyme linked to cephalosporin resistance. The soluble and active part of PBP5 protein was cloned, produced as recombinant and purified via Immobilized Metal Ion Affinity Chromatography. The mice were immunized with the purified recombinant protein, a VH library was constructed, and PBP5-specific binders were selected by phage display, respectively. Sequenced VH candidate, named VH43, was analyzed for complementarity-determining regions (CDRs), germline similarity, and structurally modeled using AlphaFold. VH43, a high-affinity binder to soluble PBP5 (sPBP5), exhibited a beta-sandwich fold characteristic of immunoglobulin domains, with hydrogen-bond interactions concentrated in CDR3. VH43 was cloned, expressed, and purified, and its antibacterial activity against E. faecium HM1070 was assessed. In combination with ceftazidime, VH43 significantly enhanced antibacterial effects compared to antibiotics alone, and it was confirmed by spot/disc diffusion assays. These findings indicate that VH43 is a recombinant antibody fragment with measurable antibiotic-potentiating activity in vitro, capable of enhancing cephalosporin efficacy against Enterococcus faecium.eninfo:eu-repo/semantics/closedAccessVariable Heavy Chain DomainEnterococcus FaeciumPhage Display LibraryRecombinant ProteinArticle