Area of technical uncertainty for susceptibility testing of amoxicillin/clavulanate against Escherichia coli: analysis of automated system, Etest and disk diffusion methods compared to the broth microdilution reference

Abstract Objectives EUCAST recently warned about area of technical uncertainty (ATU) of amoxicillin/clavulanate (AMX/C) disk susceptibility testing against Enterobacterales. Thus, we aimed to compare the reliability of three routine methods and to evaluate the ATU impact. Methods 286 Escherichia coli strains (including 159 AMX-resistant) were categorized for the two EUCAST AMX/C breakpoints by disk diffusion (Bio-Rad), Phoenix automate (Becton Dickinson) and Etest (AES) compared to broth microdilution reference method. Results By microdilution, 84.2% of strains were AMX/C susceptible using urinary breakpoint (MIC ≤32 mg/L) and 62.2% using systemic breakpoint (MIC ≤8 mg/L), with 63.6% of MICs between 4 and 16 mg/L. For systemic breakpoint, category agreement (CA) and very major error (VME) were unacceptable for Etest (71.7% and 27.3%), disk (73.1% and 23.4% at 19 mm cut-off) and in a lesser extent for Phoenix (83.6% and 10.5%). For disks, unacceptable VME rate was observed for diameters up to 22 mm, probably due to overcharged disks. For Etest, VME were high at 6 mg/L (46/63) and 8 mg/L (22/29). For urinary breakpoint, CA was more acceptable for disk (88.9%) and Etest (84.3%) (unevaluable for Phoenix). Conclusion AMX/C susceptibility testing of E. coli for systemic breakpoint was unreliable for the three routine methods, mainly explained by the high prevalence (∼60%) of strains with microdilution MICs around the breakpoint (8 mg/L). Our data confirmed the EUCAST 19-20 mm ATU for disk and suggested to introduce ATU for Etest MIC values of 6 and 8 mg/L.