Rapid EUCAST disc diffusion testing of MDR Escherichia coli and Klebsiella pneumoniae: inhibition zones for extended-spectrum cephalosporins can be reliably read after 6 h of incubation.

Objectives: The need for rapid antibiotic susceptibility testing increases with escalating levels of antimicrobial resistance in Enterobacteriaceae. Our objective was to evaluate the accuracy of reading EUCAST disc diffusion, ROSCO ESBL and carbapenemase detection kits and the Mast Carbapenemase Activity Test (CAT-ID) disc, after 6 h of incubation.
Methods: We used a collection of 128 isolates of Escherichia coli and Klebsiella pneumoniae with a wide variety of resistance mechanisms. Inhibition zones read from digital photo images with the BD Kiestra™ Total Lab Automation System after 6 h of incubation were compared with standard reading, after 18 h, of the same Mueller-Hinton agar plates.
Results: For WT isolates, zones were generally smaller at 6 h than at 18 h. Cefotaxime had excellent categorical agreement of 99%, despite the high number of challenge isolates. However, for some other antimicrobials, hetero-resistant subpopulations were commonly invisible at 6 h, which resulted in an unacceptable number of errors when using standard EUCAST breakpoints. Accurate ESBL detection was possible at 6 h for isolates lacking other β-lactamases. Carbapenemase detection was not reliable after 6 h. Conclusions: Inhibition zone reading at 6 h is an accurate method for susceptibility testing of extended-spectrum cephalosporins for Enterobacteriaceae. For other antimicrobials, 6 h reading can be used for preliminary reports of clearly resistant or susceptible isolates, preferably with application of adjusted breakpoints including an area of uncertainty between susceptible and resistant values.