Evolution and spread of a multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinases in Europe

Abstract

Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla(CMY) genes from a Citrobacter freundii origin (bla(CMY-2)-like genes). Isolates from Poland harbored several bla(CMY) genes (bla(CMY-4), bla(CMY-12), bla(CMY-14), bla(CMY-15), and bla(CMY-38) and the new gene bla(CMY-45)), while isolates from Italy and Greece harbored bla(CMY-16) only. Earlier isolates with bla(CMY-4) or bla(CMY-12), recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla(CMY) genes resided within ISEcp1 transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1 and bla(CMY), and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying ISEcp1 with similar C. freundii DNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis. In addition, isolates with bla(CMY-12), bla(CMY-15), and bla(CMY-38) genes harbored a second bla(CMY) copy within a shorter ISEcp1 module (Tn6113), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla(CMY-2)-like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla(CMY) genes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.

Fig. 1.

Schematic representation of ISEcp1 transposition modules containing the C. freundii-derived bla_CMY genes, enzyme restriction sites, and primer hybridization sites used in the analysis of the Tn6093 and Tn6113 structures. (A) Tn6114 module present in the pH 205 plasmid; (B) Tn6093 module inserted into the P. mirabilis pepQ gene (“main locus”); (C) Tn6113 module inserted between ppiD and PMI0120 ORF in the P. mirabilis chromosome (“additional locus”). The schemes are aligned according to the ISEcp1 position. EcoRI and HindIII sites, used in cloning experiments, are indicated by dashed vertical lines. Black arrows below the Tn6093 and Tn6113 schemes represent primers used for PCR mapping and sequencing.

Fig. 2.

Sequence comparison of bla_CMY-2-like genes. (A) Dendrogram of all mobile bla_CMY-2-like gene sequences obtained with the MEGA v3.1 software (19); (B) consensus network of the bla_CMY-2-like genes located in ISEcp1 modules with a 110-bp distance between ISEcp1 and bla_CMY (plasmidic Tn6114-like, and P. mirabilis Tn6093-like modules). The consensus network was generated with the SplitsTree 4.10 software (17), after prealignment in Mauve 2.3.0 (10) and analysis by the CloneFrame 1.1 software (12).

Fig. 3.

Dendrogram based on NotI+SfiI PFGE patterns, indicating clusters of the resulting types, as well as ribotypes. The bands arising after NotI+SfiI PFGE for each isolate are shown. The number next to the isolate code denotes the CMY variant. PFGE type clusters (>80% similarity, as per the percent scale at top left) are labeled A to E; ribotypes 1 to 7 are shown to the right of these.