Porin expression in clinical isolates of Klebsiella pneumoniae: a comparison of SDS-PAGE and MALDI-TOF/MS and limitations of whole genome sequencing analysis Porin expression in clinical isolates of Klebsiella pneumoniae: a comparison of SDS-PAGE and MALDI-TOF/MS and limitations of whole genome sequencing analysis

ABSTRACT: Precise definition of porin profiles is of critical importance to understand the role of porins in antimicrobial resistance. In this study, the outer membrane proteins (OMP) profiles of 26 clinical isolates of Klebsiella pneumoniae and of strain ATCC 13883 (wild-type) and ATCC 700603 (producing SHV-18) have been determined using both sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization–time of flight/mass spectrometry (MALDI-TOF/MS). SDS-PAGE was performed using both homemade and commercial gels, and protein bands were identified by liquid chromatography coupled to mass spectrometry. A rapid extraction method was used to analyse OMPs by MALDI-TOF/MS. The sequences of porin genes were obtained by whole genome sequencing (WGS) and mutations were defined by BLAST. Same results were obtained for all strains either using SDS-PAGE or MALDI-TOF/MS. SDS-PAGE showed protein bands of ~35, ~36, and ~37 KDa, identified as OmpA, OmpK36 and OmpK35, respectively. By MALDI-TOF/MS, peaks at ~35700 (OmpA), ~37000 (OmpK35), and ~38000 (OmpK36) m/z were detected. ompK35 was intact in nine wild-type isolates and was truncated in 13 isolates, but OmpK35 was not observed in 3 isolates without mutations in ompK35. One point mutation was detected in another isolate and multiple mutations were detected in the remaining isolate. ompK36 was truncated in two isolates lacking this protein and presented one point mutation (n=1) or multiple mutations in the remaining isolates. In conclusion, MALDI-TOF/MS was reliable for porin detection, but because of the complex regulation of porin genes, WGS cannot always anticipate protein expression, as observed with SDS-PAGE and MALDI-TOF/MS.

INSTRUMENT(S):

ORGANISM(S): Klebsiella Pneumoniae Subsp. Rhinoscleromatis Atcc 13884 Bacteria

SUBMITTER: Cristina Elías López  

LAB HEAD: Luis Martínez Martínez

PROVIDER: PXD053879 | Pride | 2025-05-07

REPOSITORIES: Pride