Molecular and in-silico characterization of vancomycin-resistant Enterococcus spp. isolated from clinical urine samples, Lahore, Pakistan

Vancomycin-resistant enterococci are a serious threat to public health because of their multidrug resistance and the limitations in the treatment options available. This study investigated clinical VRE isolates from Lahore, Pakistan, and characterized the vanA gene via in-silico approaches. In this study, urine specimens were collected and cultured on selective media. Morphological and biochemical observation, such as Gram staining and catalase, oxidase, and mannitol salt agar tests, were conducted. Antibiotic susceptibility testing was done via the Kirby-Bauer method. DNA was extracted and the PCR amplified the genomic DNA, vanA , and vanB genes. Protein sequences were obtained from NCBI and analyzed with ProtParam, PSIPRED, and AlphaFold3. olecular docking was carried out using AutoDock Vina, structural dynamics were evaluated with iMODS-based normal mode analysis, and immune simulation conducted with C-ImmSim. Seven clinical VRE isolates were characterized. All isolates showed elevated MICs to vancomycin, ciprofloxacin, and chloramphenicol, meeting CLSI resistance criteria; gentamicin and azithromycin MICs were also elevated but are reported descriptively, as CLSI interpretive breakpoints for these agents against Enterococcus spp. are not established for routine susceptibility categorization. PCR provided evidence for the presence of the vanA gene (732 bp) in all of the isolates. Genomic DNA sequencing of two representative isolates (NK-01, NK-02) yielded partial vanA gene sequence, confirmed via BLASTN analysis against isolate-derived and reference vanA records; these sequences did not provide species-level discrimination, and species identification for all isolates remained at the genus level ( Enterococcus spp.). The VanA protein was relatively stable (aliphatic index 94.69) with a globular fold and showed moderate docking with Vancomycin (− 6.0 kcal/mol). Normal mode analysis showed the domains to be flexible due to low eigenvalues. Immune simulation demonstrated prediction for strong IgM and IgG responses, along with identification of promising B- and T-cell epitopes, though a separate immunogenicity assessment yielded conflicting predictions. The current study confirmed VanA-mediated resistance in multidrug-resistant Enterococcus spp. and gave structural and immunological insight into VanA. These findings support VanA as a potential target for further therapeutic investigation; given that surface exposure, secretion potential, conservation across isolates, and population coverage were not assessed, its vaccine potential remains preliminary and unconfirmed, and the necessity for continued surveillance and development of new inhibitors against VRE is emphasized.