Abstract
To detect the outer membrane protein (OMP), which plays a key role in carbapenem resistance, whole-genome and transcriptome analysis of the clinical carbapenem-resistant Klebsiella pneumoniae was carried out. The index strain lacked both OmpK35 and OmpK36, whereas the other strains lacked only OmpK35. After SDS-PAGE, the putative OMP bands were excised and identified as OmpA and OmpK36. MALDI-TOF MS showed peaks at ∼36 and ∼38 kDa that corresponded to OmpA and OmpK36, respectively. In all the strains except YMC2014/03/P345, the ∼38 kDa peaks were present. The K. pneumoniae ATCC 13883 isolate showed three bands on SDS-PAGE and three corresponding peaks on MALDI-TOF MS. The additional third peak at ∼37 kDa corresponding to OmpK35 was observed. To verify OmpK35 peak detection in other K. pneumoniae isolates by MALDI-TOF MS, we analyzed six strains from our laboratory’s strain bank. Whole genome sequence indicated that only two isolates had intact OmpK35. Both MALDI-TOF MS and SDS-PAGE did not show a ∼37 kDa peak or an OmpK35 band as observed in the K. pneumoniae ATCC 13883 isolate. Separation using SDS-PAGE showed a single peak representing OmpA. Therefore, both SDS-PAGE and MALDI-TOF MS were not completely reliable for OMP detection because they fail to detect OmpK35. To the best of our knowledge, this is the first report on the performance of SDS-PAGE and MALDI-TOF MS for the detection of OMP’s using whole-genome and RNA sequencing analyses.
Highlights
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used for rapid bacterial identification in hospital settings [1], and some studies have validated its use for the detection of outer membrane proteins (OMPs) or porins [2, 3]
Sequence analysis indicated that the OmpK36 porin in the strain YMC2014/03/P345 was interrupted by the insertion of a transposon that hindered its expression, whereas no mutations were observed in the other three isolates
While analyzing the RNA data for this study, trimmed mean of M-value (TMM) was used to normalize the transcriptomic gene expression values because the coefficient of variation (CV) was 0.3387, which was lower than the values of 0.342 and 0.3396 for reads per kilobase per million mapped reads (RPKM) and relative log expression (RLE), respectively (Supplementary Table 2)
Summary
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used for rapid bacterial identification in hospital settings [1], and some studies have validated its use for the detection of outer membrane proteins (OMPs) or porins [2, 3]. A recent study indicated discrepancies in the results from K. pneumoniae; the OmpK35 band was not detected by SDS-PAGE, while the corresponding peak was detected using MALDI-TOF MS [3]. The study was confined to the correlation between SDS-PAGE and MALDITOF MS without whole-genome sequence analysis or the examination of protein expression levels. The aim of the present study was to validate the results obtained from SDS-PAGE and MALDI-TOF MS using wholegenome sequencing (WGS) and a transcriptomic analysis of carbapenem-resistant K. pneumoniae strains, and to ascertain the reproducibility of MALDI-TOF MS. We evaluated and compared the performance of MALDITOF MS using two different available instruments, the Microflex LT (Bruker Daltonik GmbH, Bremen, Germany) and Tinkerbell LT (ASTA, Suwon, Korea) mass spectrometers
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