Evaluation of agar dilution as an alternative method for antimicrobial susceptibility testing and tentative ECOFF determination for Arcobacter butzleri

Arcobacter butzleri is an emerging pathogen associated with human gastrointestinal illness. Despite its growing significance, information on the resistance of this microorganism is limited due to the lack of practical antimicrobial susceptibility testing (AST) methods and well-defined interpretive criteria. This study evaluates agar dilution as a reliable alternative to the broth microdilution method, the current reference protocol approved by the CLSI Veterinary AST (VAST) subcommittee. A total of 415 A. butzleri isolates were tested against ciprofloxacin, erythromycin, gentamicin, and tetracycline using both agar dilution and broth microdilution methods under aerobic and microaerobic conditions. Additionally, tentative epidemiological cut-off (ECOFF) values of these antimicrobials were determined by visual estimation and the statistical tool, ECOFFinder. Agreement between methods was assessed using Cohen's kappa and Gwet's AC₁. Our results reveal that aerobic agar dilution at 24 h showed the highest agreement with the reference broth microdilution, particularly for ciprofloxacin, erythromycin, and gentamicin. The proposed tentative ECOFFs for A. butzleri against ciprofloxacin, erythromycin, gentamicin, and tetracycline were 0.5 µg/ml, 16 µg/ml, 2 µg/ml, and 16 µg/ml, respectively. The findings from this study represent a critical advancement toward establishing interpretive criteria tailored specifically for Arcobacter spp. This study underscores aerobic agar dilution as a reliable yet scalable approach for AMR surveillance of A. butzleri.