MALDI-TOF MS based procedure to detect KPC-2 directly from positive blood culture bottles and colonies

Abstract

In this study, we identified specific carbapenemase-producing isolates applying an easy and rapid protocol for the detection of mature KPC-2 β-lactamase by MALDI-TOF MS from colony and positive blood culture bottles. In addition, we evaluated the correlation of the ~11,109 Da signal as a biomarker associated with KPC-2 production. A collection of 126 well-characterized clinical isolates were evaluated (including 60 KPC-2-producing strains).Presence of KPC-2 was assessed by MALDI-TOF MS on protein extracts. Samples were prepared using the double layer sinapinic acid technique. In order to identify mature KPC-2, raw spectra were analyzed focusing on the range between m/z 25,000-30,000 Da. A single distinctive peak, at approximately m/z 28,544 Da was found in all clinical and control KPC-2-producing strains, and consistently absent in the control groups (ESBL producers and susceptible strains). This peak was detected in all species independently of where the gene bla KPC-2 was embedded. Statistical results showed 100% sensitivity, CI95%: [94.0%; 100%] and 100% specificity, CI95%: [94.6%; 100%], indicating a promising test with a high discriminative power. KPC-2 β-lactamase could be directly detected from both colonies and blood culture bottles. On the other hand, the m/z 11,109 Da signal determinant was only associated with 32% of Klebsiella pneumoniae and Escherichia coli KPC positive isolates. This MALDI-TOF MS methodology has the potential to detect directly the widespread and clinically relevant carbapenemase, KPC-2, in Enterobacterales with a straightforward, low cost process, assuming MALDI-TOF MS is already adopted as the main identification tool, with clear clinical implications on antibiotic stewardship for early infection treatment.

Carbapenemase Producing Klebsiella pneumoniae (KPC): What Is the Best MALDI-TOF MS Detection Method

“…It is a question for future analyses to document the differences found by mass spectrometry between KPC producers and non-productive strains of K. pneumoniae in detail. However, Figueroa-Espinosa et al [ 71 ] observed the predicted enzyme KPC-2 β-lactamase (28,544 m/z ) in the mass spectrum of KPC-2 production bacterial strains of the family Enterobacteriaceae , in particular K. pneumoniae , E. cloacae , E. coli , Serratia marcescens, and Citrobacter braakii . Additionally, they were able to detect KPC-2 β-lactamase in P. aeruginosa KPC-2.…”

Using Molecular Diagnostics to Develop Therapeutic Strategies for Carbapenem-Resistant Gram-Negative Infections

“…There are multiple phenotypic and molecular genotypic tests ( Tamma and Simner, 2018 ) in addition to those listed above that can be used to optimize therapeutic regimens. This includes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to identify microorganisms directly in positive blood culture bottles ( Patel, 2015 ) and to identify proteins associated with antimicrobial resistance mechanisms, including carbapenemases, such as KPC-2, in a rapid and cost effective manner ( Gaibani et al., 2016 ; Vrioni et al., 2018 ; Figueroa-Espinosa et al., 2019 ). Earlier differentiation between CPO and non-CPO enhances the ability of antimicrobial stewardship programs to move from empiric to directed antimicrobial therapy to treat infections.…”

Detection of Multidrug-Resistant Enterobacterales—From ESBLs to Carbapenemases

“…[ 158 , 159 ]. The detection of mass spectrometry peaks of some β-lactamases such as TEM-1 [ 160 ] and KPC-2 [ 161 , 162 ] was shown to distinguish reliably between resistant and susceptible isolates. Cordavana and colleagues designed an algorithm integrated into the MALDI Biotyper System (Bruker DALTONICS, Bremen, Germany) that enables the automated detection of KPC harboring Klebsiella pneumoniae from cultured colonies as well as directly from positive blood cultures during the routine identification process (sensitivity 85.1%; specificity 100%) [ 163 ].…”