Analysis of Klebsiella pneumoniae strains with non-enzymatic carbapenem resistance isolated in the Czech Republic, Poland and Russia
Abstract number: P740
Chudackova E., Bergerova T., Zemlickova H., Edelstein M., Gniadkowski M., Hrabak J.
Objectives: The objectives of this study was to investigate the molecular basis of carbapenem resistance in a set of carbapenemase-negative Klebsiella pneumoniae strains collected in the Czech Republic, Poland and Russia, and to assess their possible clonality in order to check for eventual clone-associated risk factors of the development of this resistance type.
Methods: The study was carried out on 30 K. pneumoniae strains from the Czech Republic (n = 12), Poland (n = 7) and Russia (n = 11). MICs of 24 antimicrobials were determined according to the EUCAST methodology. Imipenem hydrolysis activity was assayed by spectrophotometry in crude protein extracts in order to exclude carbapenemase activity. Isoelectric focusing and bioassay were performed to determine b-lactamase content of the isolates, followed by PCR and sequencing to identify the enzymes hydrolysing newer generation b-lactams. Major outer membrane proteins (OMPs) were purified and separated by SDS-PAGE. The expression of genes encoding OmpK35 and OmpK36 porins was analyzed by RT-PCR with specific primers and probes. Both OmpK genes were amplified and sequenced. The strains were typed by multilocus sequence typing (MLST).
Results: The MICs showed various patterns of resistance with surprisingly good susceptibility of the Russian isolates to quinolones. Each strain produced either an ESBL (of SHV or CTX-M type) or an AmpC-like (DHA-1) b-lactamase. The porin OmpK35 was present in the cell envelope extracts and RT-PCR did not show any significant quantitative changes in its expression. The expression of OmpK36 varied, with a significant decrease in most, though not all of the strains. In one strain, another type of OmpK36 was detected. The MLST analysis is still in progress, showing however high clonal diversity among the strains.
Conclusion: The non-enzymatic mechanisms remain a significant source of carbapenem resistance in enterobacteria. The results of the study indicated the OmpK36 deficiency combined with the ESBL or AmpC expression to be the major resistance mechanism. The non-significant decrease of OmpK36expression in some isolates suggested the rpesence of other mechanism(s).
This work has been supported by the research project grants NS97174/2008 and MSM 0021620819.
|Session name:||Abstracts of 21st ECCMID / 27th ICC|
|Location:||Milan, Italy, 7 - 10 May 2011|
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