ObjectivesTemocillin was introduced in 2015 in the French guidelines for the treatment of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae urinary tract infections. Little susceptibility data is available. We investigated the in vitro activity of temocillin against ESBL-producing Enterobacteriaceae isolated from samples of cytobacteriological examinations of urine. Material and methodsSusceptibility testing was performed on 157 ESBL-producing E. coli and 95 ESBL-producing K. pneumoniae strains using the disk diffusion method. MICs of resistant strains were measured with the Etest method. ResultsUsing current breakpoints, 71.3% of E. coli strains and 77.9% of K. pneumoniae strains were classified as susceptible. However, diameter and MIC breakpoints vary by country, and we reported discordance of clinical categorization between diameters and MIC determination for some strains. The measure of diameters was also sometimes difficult because of contaminating colonies within the inhibition zone. ConclusionWe highlighted difficulties related to the determination of temocillin susceptibility, such as culture of resistant colonies in the inhibition zone and discordance of clinical categorizations obtained with the disk diffusion method or the Etest method. Overall, 42% of tested Enterobacteriaceae had a diameter or MIC close to the current breakpoints; thus, it is necessary to determine the MIC for these strains before considering the clinical use of this molecule.