Development and Evaluation of a Novel Protein-Based Assay for Specific Detection of KPC β-Lactamases from <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> Clinical Isolates

Abstract

ABSTRACT Carbapenemases confer resistance to nearly all β-lactam antibiotics. The extensive spread of carbapenemase-producing multidrug-resistant bacteria contributes significantly to hospital-acquired infections. We have developed a novel protein-based binding assay that identifies KPC β-lactamases from clinical isolates. We used the protein-protein interaction between KPCs and a soluble β-lactamase inhibitory protein (BLIP) variant, BLIPK74T/W112D, which specifically inhibits KPCs but not other β-lactamases. In this assay, BLIPK74T/W112D was allowed to form complexes with KPC-2 in bacterial cell lysates and then extracted using His tag binding resins. We demonstrated the presence of KPC-2 by monitoring the hydrolysis of a colorimetric β-lactam substrate. Also, to further increase the accuracy of the method, a BLIPK74T/W112D-mediated inhibition assay was developed. The binding and inhibition assays were validated by testing 127 Klebsiella pneumoniae clinical isolates with known genome sequences for the presence of KPC. Our assays identified a total of 32 strains as KPC-2 producers, a result in 100% concordance with genome sequencing predictions. To further simplify the assay and decrease the time to obtain results, the BLIPK74T/W112D protein was tested in combination with the widely used Carba-NP assay. For this purpose, the genome-sequenced K. pneumoniae strains were tested for the presence of carbapenemases with the Carba-NP test with and without the addition of BLIPK74T/W122D. The test accurately identified carbapenemase-producing strains and the addition of BLIPK74T/W112D allowed a further determination that the strains contain KPC carbapenemase. Thus, the BLIPK74T/W112D protein is an effective sensor to specifically detect KPC β-lactamases produced by clinical isolates. IMPORTANCE Infections caused by carbapenem-resistant Enterobacteriaceae are associated with high therapeutic failure and mortality rates. Thus, it is critical to rapidly identify clinical isolates expressing KPC β-lactamases to facilitate administration of the correct antibiotic treatment and initiate infection control strategies. To address this problem, we developed a protein-based, KPC-specific binding assay in combination with a cell lysate inhibition assay that provided a 100% identification rate of KPC from clinical isolates of known genomic sequence. In addition, this protein sensor was adapted to the Carba-NP assay to provide a rapid strategy to detect KPC-producing isolates that will facilitate informed treatment of critically ill patients.

Keywords

• antibiotic resistance
• beta-lactamase
• carbapenemase
• diagnostic
• protein engineering
• sensor