Establishing clonal relationships between VIM-1-like metallo-beta-lactamase-producing Pseudomonas aeruginosa strains from four European countries by multilocus sequence typing

Abstract

Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-beta-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE>RAPD>MLST>fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying blaVIM-1-like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different blaVIM-containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

FIG. 1.

Dendrogram generated by PFGE. The cutoff at the 80% level of genetic similarity is indicated by a vertical line. The CCC value for the dendrogram is 82%.

FIG. 2.

Dendrograms generated by RAPD with primer 208 in three different laboratories (A to C). The cutoff at the 80% level of genetic similarity is indicated by vertical lines. The CCC values for the dendrograms are 89% (laboratory A), 94% (laboratory B), and 87% (laboratory C).

FIG. 3.

Phylogenetic analysis (UPGMA dendrogram) based on findings with MLST. BG1 to BG10 are indicated by dotted circles, while the novel groups BG11, comprising ST227 (VR143/97, PPV97, 105MG, 67MG, and 85MG) and ST230 (Ps100 and PA66), and BG12, comprising ST228 (isolate 134MG) and other STs with at least five common alleles, are circled with continuous lines. ST229 (PA396, PA555, and AK5493) can be found in the previously described BG4.

FIG. 4.

Structures of variable regions of integrons carrying VIM determinants. Triangles indicate the attI primary insertion sites, ellipses indicate the attC recombination sites, and question marks indicate genes for which the full sequence was not determined.