Stepwise evolution of tigecycline resistance in Enterococcus hirae through early IS1216E-mediated tet(L)...

Enterococcus spp. from food animals represent important reservoirs of antimicrobial resistance; however, the mechanisms underlying tigecycline resistance remain poorly understood. In this study, we investigated the genetic basis and evolutionary dynamics of tigecycline resistance in Enterococcus hirae. Among 118 Enterococcus isolates recovered from 150 swine-derived samples, E. hirae was the predominant species (68/118, 57.63%). Tigecycline resistance was identified in ten isolates (10/118, 8.47%), all belonging to a single E. hirae clone. Whole-genome sequencing of a representative isolate, EG2361, revealed three plasmids, among which pEG2361-2 harbored an IS1216E-associated multidrug resistance region capable of generating two circular translocatable units (TUs). TU1, carrying the promoter-variant tet(L) locus together with tet(M) and poxtA, underwent tandem amplification, leading to plasmid structural heterogeneity. Functional analyses demonstrated that tet(L) reduced tigecycline susceptibility, whereas enhanced resistance mainly associated with variation in the upstream promoter region rather than Tet(L) amino acid substitutions. Experimental evolution revealed a dynamic resistance trajectory. The initial increase in tigecycline resistance was associated with TU1 amplification and marked upregulation of tet(L) expression. Subsequently, tet(L) expression declined, whereas tigecycline minimum inhibitory concentrations (MICs) remained elevated and stable. Recurrent rpsJ mutations emerged independently across evolved lineages and were associated with high-level resistance. Together, these findings support a stepwise evolutionary model in which insertion sequence (IS)1216E-mediated amplification of the promoter-variant tet(L) locus drives rapid early adaptation, followed by the emergence of chromosomal rpsJ mutations associated with stable high-level resistance. This study expands current understanding of tigecycline resistance evolution in enterococci and highlights the interplay among mobile genetic elements, transcriptional regulation, and chromosomal adaptation in shaping antimicrobial resistance.