Improved disc diffusion-based detection of extended spectrum β-lactamase production in the Klebsiella oxytoca complex

Accurate discrimination between extended-spectrum β-lactamase (ESBL) production and hyperproduction of the intrinsic OXY-β-lactamase (hyperOXY) in the Klebsiella oxytoca complex remains challenging, as both mechanisms can generate overlapping resistance phenotypes and lead to unnecessary confirmatory testing or misclassification. We analyzed a collection of 123 whole-genome-sequenced clinical K. oxytoca complex isolates (2009-2024), including 33 ESBL producers and multiple species-specific bla_OXY variants, to develop a phenotypic strategy for distinguishing these resistance mechanisms using disc diffusion data. We performed antimicrobial susceptibility testing according to EUCAST guidelines and correlated phenotypic findings with genomic resistance determinants. HyperOXY isolates harbored classical activating mutations in bla_OXY promoter regions and displayed a characteristic resistance pattern marked by high-level resistance to amoxicillin-clavulanic acid and piperacillin-tazobactam, resistance to ceftriaxone and cefpodoxime, preserved or borderline susceptibility to ceftazidime, and variable reductions in cefepime susceptibility. In contrast, ESBL-producing isolates showed reproducible clavulanate synergy and distinct inhibition zone distributions for piperacillin-tazobactam, cefpodoxime, and gentamicin. A cefpodoxime inhibition zone diameter <17 mm identified all ESBL-producing isolates (sensitivity 100%) but showed limited specificity (44%). Combining cefpodoxime <17 mm with additional phenotypic criteria allowed confident ESBL attribution without additional testing (specificity 100%), albeit at reduced sensitivity (72%). EUCAST-recommended combination disc ESBL confirmatory testing using cefotaxime or cefepime with clavulanic acid demonstrated the highest diagnostic performance (sensitivity/specificity ≥ 96%). Thirty-three isolates showed ambiguous phenotypes requiring confirmatory testing. Overall, interpretative reading of routine disc diffusion patterns can reliably classify characteristic hyperOXY and ESBL phenotypes and restrict additional confirmatory testing to ambiguous isolates, thereby improving diagnostic efficiency while maintaining high accuracy.IMPORTANCEMembers of the K. oxytoca complex possess an intrinsic OXY β-lactamase that can be overexpressed and generate resistance patterns closely resembling those of extended-spectrum β-lactamase (ESBL) producers. This phenotypic overlap complicates routine antimicrobial susceptibility testing and frequently leads to unnecessary ESBL confirmatory testing or misclassification, with potential consequences for patient management and infection control. Using a large, genomically characterized strain collection, this study defines reproducible disc diffusion patterns that distinguish hyperOXY-mediated resistance from true ESBL production and evaluates screening and confirmatory strategies based on routinely tested antibiotics. By providing practical, phenotype-based guidance aligned with EUCAST methodology, our findings help clinical laboratories improve diagnostic accuracy, streamline workflows, and avoid ESBL-misidentification in K. oxytoca complex isolates.