Plasmid-linked virulence genes and high-level gentamicin resistance in Enterococcus faecalis from persistent endodontic infections

Abstract Aim: To evaluate the relationship between plasmid-mediated aminoglycoside resistance, including silent resistance to high-level gentamicin resistance (HLGR), and the virulence genes aggregation substance ( asa ), enterococcal surface protein ( esp ), and cytolysin A ( cylA ) in Enterococcus faecalis isolates from persistent endodontic infections (PEIs). Materials and Methods: Antimicrobial identification and susceptibility testing were performed using the MicroScan system. HLGR resistance, including silent phenotypes, was assessed by synergy tests and conventional polymerase chain reaction (PCR) targeting the bifunctional aac(6’)-Ie-aph(2”)-Ia gene. Virulence genes ( asa , esp , cylA ) were detected using conventional PCR. Statistical Analysis: Mixed clustering analysis was applied, combining mixed data factor analysis (MDFA) with hierarchical Ward and K-means methods, to identify recurrent combinations of virulence genes associated with resistance phenotypes. Statistical analysis performed by IBM SPSS Statistics, V 26.0 (IBM Corp., Armonk, NY, USA). Results: E. faecalis was isolated in 12.5% (69/552) of samples, with HLGR resistance observed in 18.8% (13/69), including two strains exhibiting a silent phenotype. The asa + esp − cylA + genotype showed a significant association with HLGR-resistant strains (odds ratio = 15.6, P < 0.001). MDFA revealed a plasmid cluster grouping resistant and silent-phenotype strains with identical virulence profiles. Conclusion: The coexistence of HLGR resistance and virulence genes supports a possible mechanism of plasmid co-selection in E. faecalis from IEPE. Recognition of silent resistance is crucial for improving diagnosis and preventing treatment failure in PEIs.