Clinical Performance of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Detection of Certain bla _KPC -Containing Plasmids

Abstract

Rapid detection of bla KPC -containing organisms can significantly impact infection control and clinical practices, as well as therapeutic choices. Current molecular and phenotypic methods to detect these organisms, however, require additional testing beyond routine organism identification. In this study, we evaluated the clinical performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to detect pKpQIL_p019 (p019)-an ϳ11,109-Da protein associated with certain bla KPC -containing plasmids that was previously shown to successfully track a clonal outbreak of bla KPC -pKpQILKlebsiella pneumoniae in a proof-of-principle study (A. . PCR for the p019 gene was used as the reference method. Here, blind analysis of 140 characterized Enterobacteriaceae isolates using two protein extraction methods (plate extraction and tube extraction) and two peak detection methods (manual and automated) showed sensitivities and specificities ranging from 96% to 100% and from 95% to 100%, respectively (2,520 spectra analyzed). Feasible laboratory implementation methods (plate extraction and automated analysis) demonstrated 96% sensitivity and 99% specificity. All p019-positive isolates (n ‫؍‬ 26) contained bla KPC and were carbapenem resistant. Retrospective analysis of an additional 720 clinical Enterobacteriaceae spectra found an ϳ11,109-Da signal in nine spectra (1.3%), including seven from p019-containing, carbapenem-resistant isolates (positive predictive value [PPV], 78%). Instrument tuning had a significant effect on assay sensitivity, highlighting important factors that must be considered as MALDI-TOF MS moves into applications beyond microbial identification. Using a large blind clinical data set, we have shown that spectra acquired for routine organism identification can also be analyzed automatically in real time at high throughput, at no additional expense to the laboratory, to enable rapid detection of potentially bla KPC -containing carbapenemresistant isolates, providing early and clinically actionable results. The spread of carbapenemase-producing organisms represents an urgent global public health threat in an era of increased international travel and limited effective antimicrobial options. The genes encoding carbapenemases are commonly carried on plasmids, and it has been hypothesized that an important factor contributing to their spread is the diversity of the plasmid contexts in which they are found. In particular, the bla KPC gene encoding the Klebsiella pneumoniae carbapenemase (KPC) has been identified in a variety of plasmid backbones in association with a mobile Tn4401 transposable element. This association may have contributed to the global dissemination of the bla KPC gene in a variety of genera and species of bacteria (1). Rapid methods to detect bla KPC -carrying plasmids in the clinical microbiology laboratory would be of great clinical and epidemiologic value. Various techniques such as the Carba NP test (2), modified Hodge test (3), and molecular assays (4...

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of KPC-Producing Klebsiella pneumoniae

“…Our data are consistent with previous findings that demonstrated a correlation between the 11.109-Da peak, representing a cleavage product of a hypothetical protein named pKpQIL_p019 (p019), and the bla KPC -harboring pKpQIL-like plasmids (7). Moreover, Youn and colleagues (8) recently proposed an in-house script developed in flexAnalysis software for rapid detection of the 11.109-Da peak in KPC-producing enterobacterial clinical strains.…”

Transplantation of human skin microbiota in models of atopic dermatitis

“…Colonies were then taken for species identification by mass spectrometry using MALDI-TOF analysis. Bacterial protein extraction for MALDI-TOF mass spectrometry using the BioTyper (v3.1, Bruker Daltonics Inc.) was performed by the NIH Clinical Center microbiology lab using previously described methods (46), instrument settings, and calibration (47, 48). BioTyper identification was supplemented by additional mass spectra profiles provided by several NIH-developed databases (46, 49, 50).…”

Drug repurposing screens and synergistic drug‐combinations for infectious diseases

“…Improvement of the current antimicrobial susceptibility testing methods or invention of new methods is needed to meet the challenge of drug‐resistant bacteria. Recent advances include the use of matrix‐assisted laser desorption/ionization time of flight MS and next generation sequencing that enables rapid identification of proteins and plasmids of clinically relevant multidrug‐resistant bacteria in a real time and high‐throughput fashion (Conlan et al , 2014; Dekker and Frank, 2016; Youn et al , 2016). The new and future generations of antimicrobial susceptibility testing methods should be able to rapidly screen hundreds of approved drugs in a concentration–response manner with individual compounds and with hundreds of drug combinations.…”