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

Abstract

dWe evaluated a real-time single-peak (11.109-Da) detection assay based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae. Our results demonstrated that the 11.109-Da peak was detected in 88.2% of the KPC producers. Analysis of bla KPCproducing K. pneumoniae showed that the gene encoding the 11.109-Da protein was commonly (97.8%) associated with the Tn4401a isoform. In the last decade, an emerging spread of carbapenemase-producing Enterobacteriaceae (CPE) was observed worldwide, especially in Klebsiella pneumoniae strains. As a result, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has become endemic in several countries, including Italy, Greece, and the United States (1).Rapid detection of KPC producers is mandatory to limit the spread of this pathogen and for clinical management of complicated infection due to KPC-producing K. pneumoniae. Numerous phenotypic (e.g., Carba NP, modified Hodge test, disk-diffusion synergy test) and genotypic (e.g., single and multiplex PCR) assays were developed for rapid identification of CPE (2, 3). Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was proposed to detect carbapenemase production in Gram-negative organisms (4).Here, we propose a simple approach based on MALDI Biotyper RTC software to identify KPC-producing K. pneumoniae clinical strains. The aim of this study was to evaluate the sensitivity and specificity of an innovative MALDI-TOF approach using a large population of well-characterized unrelated K. pneumoniae strains collected in Italy.␤-Lactamase genes and the clonal relatedness of bacterial isolates were investigated by PCR and multilocus sequence typing (MLST) analysis (3, 5). For MALDI-TOF MS analysis, strains were evaluated as previously described (6).Dendrogram analysis revealed two main separate clusters, representing KPC-producing and non-KPC-producing K. pneumoniae strains, which included extended-spectrum ␤-lactamase (ESBL) producers and wild-type strains (Fig. 1). In order to evaluate the major significant differences between the protein patterns of these two groups, spectra were analyzed by ClinProtTools software (v3.0; Bruker Daltonics, Inc.). Single-peak analysis identified a total of 10 peaks (m/z of 5. 555, 6.082, 6.152, 7.384, 7.663, 7.704, 7.763, 7.851, 11.109, and 11.185) that potentially differentiate between KPC-positive and KPC-negative K. pneumoniae strains. In order to identify group-specific peaks as candidate biomarkers for KPC-producing K. pneumoniae strains, the presence of different peaks were manually investigated by ClinProtTools software. Our analysis demonstrated that an 11.109-Da peak was detected in most of the spectra obtained from KPC-positive strains, whereas it was absent in all of the KPC-negative isolates. At the same time, the remaining eight peaks (excluding the 5.555-Da peak as a double charged 11.109-Da peak) were found...

A Full MALDI-Based Approach to Detect Plasmid-Encoded KPC-Producing Klebsiella pneumoniae

“…Recently, a specific peak at 11,109 m/z, related to a pKpQIL plasmid carrying bla KPC was discovered in the MALDI-TOF mass spectra of KPC-producing Klebsiella pneumoniae ( Lau et al, 2014 ). Different approaches to seek for this peak have been described ( Lau et al, 2014 ; Gaibani et al, 2016 ; Youn et al, 2016 ). Nevertheless all of them required a second step after routine species identification process like visual inspection or an additional software analysis.…”

“…This specific peak at 11,109 m/z is clearly detectable in bacterial MALDI-TOF mass spectra. Different approaches to seek for this peak have been used and evaluated ( Lau et al, 2014 ; Gaibani et al, 2016 ; Youn et al, 2016 ). Until recently, the detection of this peak required a manual operation, with visual analysis or additional software in a second step after the routine identification process, and therefore impeding a real-time detection of KPC strains ( Lau et al, 2014 ; Gaibani et al, 2016 ; Youn et al, 2016 ).…”

Management of KPC-producing Klebsiella pneumoniae infections

“…Recently the identification of a 11 109 Da MS peak corresponding to a gene product of the bla KPC pKpQIL plasmid was found to be useful in rapid tracking KPC-producing strains [13].…”

Susceptibility Testing of Bacteria Using Maldi-Tof Mass Spectrometry

“…Further genetic analysis revealed that the 4 strains negative for the 11.109 m/z peak could be explained by different isoforms of Tn 4401 . Only TN 4401a is commonly associated with the 11.109 m/z peak (Gaibani et al, 2016 ). In 2018 the same group published a study on 140 well-characterized K. pneumoniae strains collected between 2011 and 2017 and found an overall accuracy of 98%, a positive predictive value of 98% and a negative predictive value of 97% (Gaibani et al, 2018 ).…”