Extended lag phases and variable antibiotic tolerance in β-lactam-induced small colony variants of Enterococcus faecalis

Abstract

Abstract Small colony variants (SCVs) of Enterococcus faecalis are associated with persistent infections and have been linked to altered antibiotic susceptibility and tolerance. Here, we investigated SCVs emerging under β-lactam exposure in comparison to corresponding normal colony phenotypes (NCPs). SCVs were generated in vitro under benzylpenicillin, ceftriaxone, and combination therapy and characterized by growth kinetics, antibiotic susceptibility (MIC), killing dynamics (MDK), and bactericidal activity (MBC). Virulence and infection dynamics were further assessed using the Galleria mellonella model. SCVs displayed reduced growth rates and prolonged lag phases, which were partially reversible upon subcultivation. β-Lactam exposure induced substantial MIC increases across multiple lineages, including both SCV and NCP variants, indicating that reduced susceptibility represents a general adaptive response rather than a phenotype-specific trait. In contrast, killing dynamics revealed heterogeneous tolerance profiles that were not consistently associated with SCV formation. While selected SCVs exhibited prolonged MDK values consistent with increased tolerance, other variants showed rapid killing despite elevated MICs. MBC values further supported this dissociation, particularly for ceftriaxone, where bactericidal activity remained limited across variants. In the G. mellonella model, SCVs showed delayed infection onset but did not uniformly result in reduced virulence. Together, these findings indicate that SCVs in E. faecalis represent a heterogeneous and reversible adaptive state. Antibiotic tolerance depends on growth dynamics rather than phenotype alone, while MIC shifts occur independently of SCV formation, highlighting the importance of distinguishing between resistance, tolerance, and phenotypic adaptation. Graphical abstract