Evaluation of Phenotypic Tests to Detect Extended-Spectrum β-Lactamase (ESBL)-Producing Klebsiella oxytoca Complex Strains.

Klebsiella oxytoca complex ( Ko C) species may overproduce their chromosomal class A OXY β-lactamases, conferring reduced susceptibility to piperacillin-tazobactam, expanded-spectrum cephalosporins and aztreonam. Moreover, since clavulanate maintains its ability to inhibit these enzymes, the resulting resistance phenotype may falsely resemble the production of acquired extended-spectrum β-lactamases (ESBLs). In this work, a collection of 44 Ko C strains of human and animal origin was characterized with whole-genome sequencing (WGS) and broth microdilution (BMD) susceptibility testing. Comparison of ESBL producers ( n  = 11; including CTX-M-15 [ n  = 6] and CTX-M-1 [ n  = 5] producers) and hyperproducers of OXYs ( n  = 21) showed certain phenotypic differences: piperacillin-tazobactam (MIC 90s : 16 versus >64 μg/mL), cefotaxime (MIC 90s : 64 versus 4 μg/mL), ceftazidime (MIC 90s : 32 versus 4 μg/mL), cefepime (MIC 90s : 8 versus 4 μg/mL) and associated resistance to non-β-lactams (e.g., trimethoprim-sulfamethoxazole: 90.9% versus 14.3%, respectively). However, a clear phenotype-based distinction between the two groups was difficult. Therefore, we evaluated 10 different inhibitor-based confirmatory tests to allow such categorization. All tests showed a sensitivity of 100%. However, only combination disk tests (CDTs) with cefepime/cefepime-clavulanate and ceftazidime/ceftazidime-clavulanate or the double-disk synergy test (DDST) showed high specificity (100%, 95.5%, and 100%, respectively). All confirmatory tests in BMD or using the MIC gradient strip did not perform well (specificity, ≤87.5%). Of note, ceftazidime/ceftazidime-avibactam tests also exhibited low specificity (CDT, 87.5%; MIC gradient strip, 77.8%). Our results indicate that standard antimicrobial susceptibility profiles can raise some suspicion, but only the use of cefepime/cefepime-clavulanate CDT or DDST can guarantee distinction between ESBL-producing Ko C strains and those hyperproducing OXY enzymes.