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

Abstract

Objective

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.

Methods

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.

Results

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.

Conclusions

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.