Typing and Species Identification of Clinical Klebsiella Isolates by Fourier Transform Infrared Spectroscopy and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Abstract

Klebsiella pneumoniae and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical Klebsiella isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical Klebsiella strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different Klebsiella species. Phylogenetic analysis revealed that the Klebsiella isolates comprised three different species: K. pneumoniae, K. variicola, and K. quasipneumoniae Genome analysis showed that MALDI-TOF MS can be used to distinguish K. pneumoniae from K. variicola due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of K. pneumoniae and K. variicola by MALDI-TOF MS.

Keywords: Fourier transform infrared spectroscopy; Klebsiella pneumoniae; Klebsiella variicola; MALDI-TOF mass spectrometry; bacterial typing.

Copyright © 2018 American Society for Microbiology.

Figures

FIG 1

SNP-based cluster dendrogram of 68 Klebsiella isolates. A core genome of all genome sequences was used to call SNPs in the isolate genomes. Genomes with distances of

FIG 2

Clustering of FTIR spectra of 68 Klebsiella isolates. (A) The dendrogram was calculated by UPGMA clustering of the pairwise Euclidean distances of the isolate spectra. Clusters are displayed as shaded boxes. The species of each isolate is indicated by a colored circle (blue, K. pneumoniae; red, K. variicola; green, K. quasipneumoniae), and the corresponding WGS cluster of the isolate is derived from Fig. 1. Incongruently clustered isolates are marked with arrows. (B) Frequency distribution of all pairwise FTIR spectrum similarities. The similarity value with the lowest frequency (arrow) between the peaks of the bimodal distribution was used as the cluster cutoff in panel A.

FIG 3

Clustering of MALDI-TOF spectra of 68 Klebsiella isolates. (A) UPGMA clustering resulted in 29 MALDI-TOF clusters (shaded boxes). The species of each isolate is indicated by a colored circle (blue, K. pneumoniae; red, K. variicola; green, K. quasipneumoniae). The corresponding WGS cluster of the isolate is derived from Fig. 1. The presence of a specific MALDI-TOF mass peak in an isolate spectrum is shown by a filled rectangle in the column under the respective m/z ratio. (B) Differences in the adjusted Rand index among two spectrum similarity calculation algorithms (the curve-based Pearson coefficient [circles] and peak-based Dice algorithm [triangles]) and two clustering algorithms (UPGMA [filled symbols] and Ward's algorithm [open symbols]) with different numbers of clusters.

FIG 4

Species-specific MALDI-TOF mass peaks. Shown are MALDI-TOF spectrum peaks for K. pneumoniae (ID 32) and K. variicola (ID 49) that represent the singly (z) or doubly (2z) charged ions of three ribosomal proteins (unmodified or with the leading methionine removed [dMet]) and one stress response protein. The m/z ratios of the peaks are given. The height of each peak reflects the relative intensity (Int.) with respect to the highest peak in the spectrum. Neighboring peaks without relevance for the species delineation are marked by asterisks.