Combined disk-based tests for phenotypic identification of KPC variants in Enterobacterales

The identification of Klebsiella pneumoniae carbapenemase (KPC) variants is challenging due to their atypical resistance profiles, especially in resource-limited settings where molecular diagnostics are not readily available.

In this study, we developed a combination of disk-based phenotypic assays to identify KPC variant-positive Enterobacterales, including standard disk diffusion, the 3-aminophenylboronic acid (APB)/EDTA inhibition test, and the ESBL confirmatory test.

In the disk diffusion test, all 18 KPC variant-producing strains analysed in this study were resistant to ceftazidime-avibactam and ertapenem, and 22.2 % (4 of 18) were resistant to imipenem, including those producing KPC-79, KPC-93, KPC-112, and KPC-157. For the APB/EDTA inhibition test, all strains were non-susceptible to meropenem (17 resistant and 1 intermediate) but showed no ≥5 mm increase in inhibition zone diameter with APB, EDTA, or both, thus yielding negative results according to criteria for wild-type carbapenemases. For the ESBL confirmatory test, four strains (producing KPC-79, KPC-93, KPC-112, and KPC-157) were negative, consistent with their imipenem resistance in disk diffusion, suggesting a potential phenotypic linkage. Based on these findings, two distinct disk-based phenotypic patterns of the KPC variants were identified. Both patterns shared resistance to ceftazidime-avibactam and ertapenem, along with a negative APB/EDTA inhibition result but differed in imipenem susceptibility and ESBL test results: one showed imipenem susceptibility with a positive ESBL test, whereas the other showed imipenem resistance with a negative ESBL test.

In conclusion, this study proposes a simple disk-based approach for the phenotypic identification of KPC variants in Enterobacterales, which may aid preliminary screening in clinical laboratories and warrants further validation.