Investigation of carbapenem-hydrolysing Klebsiella oxytoca strains lacking detectable carbapenemase genes

<b>Introduction.</b> Carbapenemase-producing <i>Enterobacterales</i> (CPE) pose a clinical concern due to limited treatment options and plasmid-mediated spread of carbapenemase genes. The modified carbapenem inactivation method (mCIM) is increasingly used in clinical settings to detect CPE.<b>Gap Statement.</b> Interpretation of mCIM results for isolates with reduced carbapenem susceptibility remains challenging.<b>Aim.</b> To assess the interpretation of positive mCIM results in <i>Klebsiella oxytoca</i> isolates lacking detectable carbapenemase genes and exhibiting reduced carbapenem susceptibility and to propose a phenotypic testing strategy for accurate evaluation.<b>Methodology.</b> Antimicrobial susceptibility testing with clavulanic or dipicolinic acid supplementation, phenotypic carbapenemase assays, carbapenem hydrolysis assays and whole-genome sequencing (WGS) were conducted on <i>K. oxytoca</i> strains.<b>Results.</b> <i>K. oxytoca</i> strains were isolated from both blood and urine samples of a patient in a Japanese hospital. Although classified as susceptible by clinical breakpoints, these isolates exhibited reduced susceptibility to carbapenems. Although these strains were mCIM-positive, the zinc-supplemented carbapenem inactivation method did not support the presence of metallo-<i>β</i>-lactamase (MBL) activity. MIC reductions in the presence of CVA were consistent with inhibition of a class A <i>β</i>-lactamase. In contrast, dipicolinic acid supplementation did not reduce the meropenem MIC, ruling out MBL production. Furthermore, WGS revealed that both <i>K. oxytoca</i> strains lacked known carbapenemase genes, with <i>bla</i> _OXY-2-4 being the only identifiable <i>β</i>-lactamase gene encoding a class A enzyme. No novel <i>β</i>-lactamase genes were identified; however, an enhanced <i>bla</i> _OXY-2-4 expression-associated point mutation was found in the promoter region. Both <i>K. oxytoca</i> strains exhibited carbapenem-hydrolysing activity, although to a lesser extent than that of the MBL-producing strain.<b>Conclusion.</b> We identified <i>K. oxytoca</i> strains that lacked known carbapenemase genes but that yielded positive mCIM results, possibly due to OXY-2 overproduction. Our study findings emphasize the need for cautious mCIM result interpretation for <i>K. oxytoca</i> and highlight the diagnostic value of combined susceptibility testing with clavulanic and dipicolinic acids to distinguish the underlying resistance mechanisms. Combining complementary clinical laboratory tests will enable clarification of the factors underlying mCIM positivity and guide clinical decision-making.