OBJECTIVES: Global dissemination of KPC-type carbapenemases is mainly associated with the spread of high-risk clones of Klebsiella pneumoniae and of KPC-encoding plasmids. In this study, we explored the population structure of KPC-encoding plasmids from the recent epidemics of KPC-producing K. pneumoniae (KPC-Kp) in Greece and Italy, the two major European endemic settings. METHODS: Thirty-four non-replicate clinical strains of KPC-Kp representative of the early phases (2008-11) of the Greek (n = 22) and Italian (n = 12) epidemics were studied. Isolates were typed by MLST, and blaKPC-carrying plasmids were characterized by S1 profiling, PCR-based replicon typing and RFLP. Transfer experiments by conjugation or transformation were carried out with Escherichia coli recipients. Eleven plasmids, representative of all different restriction profiles, were completely sequenced. RESULTS: The representative Greek strains belonged to 14 sequence types (STs), with a predominance of ST258. The representative Italian strains belonged to three STs, with a predominance of clonal complex 258 (ST258, ST512). The 34 strains carried plasmids of variable size (78-166 kb), either with blaKPC-2 or blaKPC-3 gene embedded in a Tn4401a transposon. Plasmids from Greek strains were mostly of a single RFLP type (A) and resembled the archetypal pKpQIL KPC-encoding plasmid, while plasmids from Italian strains belonged to a more heterogeneous population, showing five RFLP profiles (A, C-F). Types A and C resembled pKpQIL or deletion derivatives thereof, while types D-F included plasmids with hybrid structures between pKpQIL, pKPN3 and pKPN101-IT. CONCLUSIONS: pKpQIL-like plasmids played a major role in the dissemination of blaKPC in Greece and Italy, but evolved with different dynamics in these endemic settings.

Department of Medical Biotechnologies University of Siena Siena Italy

Department of Medical Biotechnologies University of Siena Siena Italy Department of Experimental and Clinical Medicine University of Florence Florence Italy

Department of Medical Biotechnologies University of Siena Siena Italy Department of Experimental and Clinical Medicine University of Florence Florence Italy Microbiology and Virology Unit Careggi University Hospital Florence Italy Don Carlo Gnocchi Foundation Florence Italy

Department of Medical Biotechnologies University of Siena Siena Italy Department of Surgery and Translational Medicine University of Florence Florence Italy

Department of Microbiology National School of Public Health Athens Greece

Department of Microbiology National School of Public Health Athens Greece Central Public Health Laboratory Hellenic Centre of Disease Control and Prevention Vari Greece

Department of Microbiology National School of Public Health Athens Greece Department of Microbiology Faculty of Medicine and University Hospital in Plzen Charles University Prague Plzen Czech Republic

Laboratory of Bacteriology Hellenic Pasteur Institute Athens Greece

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