Carbapenem resistance in Pseudomonas aeruginosa

Carbapenem (imipenem and meropenem) resistance in Pseudomonas aeruginosa is increasing, with global resistance rates approaching 20%. The most common resistance mechanisms are down-regulation of the porin OprD, and increased activity of multi-drug efflux pumps, primarily MexAB-OprM. Down-regulation of OprD, which is the primary porin for carbapenem uptake, confers resistance to both imipenem and meropenem, although the latter is affected less. Only meropenem is a substrate for the efflux pumps, due to the absence of a heterophilic side chain in the chemical structure of imipenem. Apart from alterations of OprD and the efflux pumps, changes of penicillin-binding proteins have been suggested as a third chromosomal mechanism of carbapenem resistance. Transferable carbapenemases, conferring resistance to all â-lactams except aztreonam, have emerged during the last decade, and consist of five groups of enzymes, namely IMP, VIM, SPM, GIM and SIM. These enzymes are designated metallo-â-lactamases (MBL) due to their dependency of zinc for â-lactam hydrolysis. The genes encoding the MBLs are usually found on integrons, often embedded in functional transposons.

The aim of this thesis was to study chromosomal and transferable carbapenemresistance in clinical isolates of P. aeruginosa. Totally 67 isolates with variable levels of carbapenem resistance were characterized regarding transcription of the chromosomal genes encoding OprD, and the efflux pumps MexAB-OprM, MexXY, MexCD-OprJ and MexEFOprN. Transcription of the genes encoding penicillin-binding 2 and 3 (PBP-2 and -3) was studied in some of the isolates. Quantitative reverse transcriptase PCR (qRT-PCR) was used for the quantification of mRNA for the respective resistance genes. A phenotypic efflux inhibition assay was also employed, using the inhibitor Phe-Arg-â-naphtylamide (40 mg/L). DNA sequencing of oprD, pbp-2, pbp-3 and some of the efflux pump regulatory genes was conducted in selected isolates. Transferable carbapenem-resistance was studied in 11 isolates producing MBLs belonging to the VIM-lineage, derived from four European countries. Isolates were characterized by serotyping, pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST) and integron sequencing.

Studies of isolates with decreased susceptibility to imipenem, and retained meropenem susceptibility, confirmed that down-regulation of oprD was the primary mechanism of resistance, although this was not found in all of the isolates. In isolates intermediately susceptible to meropenem and with variable susceptibility to imipenem, we found phenotypic or genotypic evidence of efflux in 5/8 isolates. These isolates are reported as susceptible according to some of the international breakpoint groups, but are regarded intermediately susceptible according to the Swedish Reference Group for Antibiotics (SRGA) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). In isolates with decreased susceptibility to both carbapenems, we found evidence of concomitant downregulation of oprD and up-regulation of mexB in many of the isolates. In five isolates we found evidence of down-regulation of either pbp-2 or pbp-3, although the relative importance of this finding was difficult to ascertain. Studies of transferable carbapenem resistance showed that the serotype O11 isolates from Italy and Greece belonged to the same clonal complex, according to MLST. Also, serotype O12 isolates from Greece harboring genes encoding different VIM-variants were found to belong to the same sequence type. PFGE and RAPD did not cluster all isolates that were found to belong to the same clonal complex according to MLST, and the latter is therefore probably best suited for studies of international epidemiology.

List of scientific papers

I. El Amin N, Giske CG, Jalal S, Keijser B, Kronvall G, Wretlind B. (2005). Carbapenem resistance mechanisms in Pseudomonas aeruginosa: alterations of porin OprD and efflux proteins do not fully explain resistance patterns observed in clinical isolates. APMIS. 113(3): 187-96
https://pubmed.ncbi.nlm.nih.gov/15799762

II. Giske CG, Boren C, Wretlind B, Kronvall G. (2005). Meropenem susceptibility breakpoint for Pseudomonas aeruginosa strains hyperproducing mexB mRNA. Clin Microbiol Infect. 11(8): 662-9
https://pubmed.ncbi.nlm.nih.gov/16008620

III. Giske CG, Rylander M, Kronvall G. (2003). VIM-4 in a carbapenem-resistant strain of Pseudomonas aeruginosa isolated in Sweden. Antimicrob Agents Chemother. 47(9): 3034-5
https://pubmed.ncbi.nlm.nih.gov/12937022

IV. Giske CG, Libisch B, Colinon C, Scoulica E, Pagani L, Fuzi M, Kronvall G, Rossolini GM. (2006). Establishing clonal relationships between VIM-1-like metallo-beta-lactamase-producing Pseudomonas aeruginosa strains from four European countries by multilocus sequence typing. J Clin Microbiol. 44(12): 4309-15
https://pubmed.ncbi.nlm.nih.gov/17021059

V. Giske CG, Buarø L, Sundsfjord A, Wretlind B. (2007). Decreased transcription of penicillin-binding proteins 2 and 3 in carbapenem resistant clinical isolates of Pseudomonas aeruginosa. [Manuscript]