β-lactam susceptibility testing of ampicillin-susceptible non- faecalis enterococcal clinical isolates in France

OBJECTIVES: To assess the epidemiological susceptibility distribution of five β-lactam antibiotics for contemporary clinical isolates of non-faecalis Enterococcus species categorized as susceptible to ampicillin and to evaluate the performance of gradient diffusion (GD) tests compared to broth microdilution (BMD), the reference method for antimicrobial susceptibility testing. METHODS: A total of 455 non-duplicate non-Enterococcus faecalis clinical isolates reported as ampicillin susceptible were collected from 43 French hospital laboratories. Identification to the species level was performed using MALDI-TOF mass spectrometry. MICs of amoxicillin, piperacillin, imipenem, meropenem and ceftobiprole were determined by BMD and GD. Results were interpreted according to the 2025 EUCAST recommendations. Agreement between GD and BMD was assessed using ISO and FDA criteria. RESULTS: All isolates were susceptible to amoxicillin, 94% (428/455) to ceftobiprole, 88% (401/455) to imipenem at high dose and 88% (402/455) to piperacillin. Enterococcus casseliflavus displayed the lowest MIC values across all β-lactams, followed by Enterococcus avium. In contrast, Enterococcus faecium, Enterococcus lactis, Enterococcus gallinarum and Enterococcus hirae exhibited right-shifted MIC distributions. Most species showed high MICs for meropenem, indicating low intrinsic activity. GD methods did not achieve FDA or ISO criteria for most species-antibiotic combinations. CONCLUSIONS: Overall, ampicillin may predict amoxicillin categorization for frequent non-faecalis enterococci, and with the exception of meropenem, most β-lactams may still have clinical value against these species. The different species exhibit distinct MIC distribution patterns for the tested β-lactams. Furthermore, substantial discrepancies observed between GD and BMD methods highlight the limited reliability of GD as an alternative to BMD.