Molecular and phenotypic characterization of carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii from bovine mastitis milk with development of a novel optimized spectrophotometric UniCarb assay

Carbapenemase-producing Gram-negative bacteria are increasingly reported in veterinary medicine, posing a serious threat to both animal and public health. This study investigated phenotypic and molecular mechanisms of carbapenem resistance in Klebsiella pneumoniae and Acinetobacter baumannii isolated from bovine mastitis milk. Antimicrobial susceptibility testing, minimum inhibitory concentration (MIC) profiling, and multiplex PCR were performed to characterize resistance. Phenotypic assays, including the Modified Hodge Test (MHT), modified and optimized carbapenem inactivation methods (mCIM/oCIM), Carba NP, and CarbAcineto NP, were evaluated. In addition, a novel PCR-validated, cross-species spectrophotometric assay (UniCarb) was developed and benchmarked. A total of 34 isolates (K. pneumoniae, n = 21; A. baumannii, n = 13) were recovered from 335 milk samples collected in Aydın Province and surrounding regions of Türkiye. All K. pneumoniae isolates exhibited multidrug resistance, with 62% classified as pandrug-resistant (PDR). A. baumannii isolates showed a uniform PDR profile. Molecular analysis revealed high prevalences of blaNDM (90.5%), blaOXA-48 (61.9%), and blaKPC (33.3%) in K. pneumoniae. All A. baumannii carried intrinsic blaOXA-51 together with acquired blaOXA-23, but none harbored blaNDM, blaOXA-48, blaKPC, blaVIM, or blaIMP. Traditional phenotypic methods demonstrated variable sensitivity and specificity, whereas the newly developed UniCarb assay achieved perfect diagnostic accuracy (Area Under the Curve (AUC) = 1.0; Cohen’s kappa coefficient (κ) = 1.0) across both species and carbapenemase classes within 45 min. Spectrophotometric absorbance strongly correlated with pH changes, confirming enzymatic hydrolysis. The UniCarb assay outperformed conventional phenotypic methods, providing rapid, accurate, and species-independent carbapenemase detection. Its PCR-confirmed results and robust cross-species applicability support its use as a practical diagnostic tool in both veterinary and clinical microbiology within a One Health surveillance framework.