Evolving epidemiology of carbapenem-resistant Enterobacteriaceae: molecular characterisation of KPC-positive strains and circulating plasmids

Objectives: The worldwide dissemination of carbapenemaseproducing MDRO is becoming a crucial public health problem. A large number of plasmid-mediated enzymes with carbapenemase activity has been identified in Enterobacteriaceae, KPC (Klebsiella pneumoniae carbapenemase) being the most frequent. The aim of this study was to characterize both at the phenotypic and molecular level carbapenem-resistant strains that caused outbreaks in the Padua Teaching Hospital from January 2009 to October 2011. In particular, we focused on detection of clonal spreading of resistant strains and on sequence modification of KPC-encoding plasmids, monitoring and updating the epidemic spreading for almost 3 years. Methods: Non-repetitive Enterobacteriaceae isolates with an imipenem MIC ‡ 1 mg/L were identified by automated systems and collected from intensive care units, surgical and medical wards. The presence of ESBL and carbapenemase genes belonging to Ambler class A, B or D was assessed by PCR. Molecular typing of positive strains was performed by PFGE, MLST and ERIC-PCR. Plasmids conferring carbapenem resistance were extracted and compared by RFLP, southern blot and deep sequenced. Results: The initial outbreak in 2009 of carbapenem-resistant strains was caused by KPC-positive K. pneumoniae strains mostly collected from intensive care units. In particular, KPC-3 was associated with ST258 and KPC-2 with ST147. Since the end of 2010, spreading of non-clonally related KPC-3/KPC-2-positive strains (ST37, ST527, ST512, ST554, ST307, ST510 and ST437) was observed in different wards, including general medical ones. All KPC-positive plasmids were collected and sequenced: we found two different plasmid backbones encoding KPC-3 and one presenting KPC-2. In addition, most of the strains presented TEM-1, SHV-11/12 and OXA-9. Interestingly, four KPC-2 isolates were additionally positive for the metallo-betalactamase VIM-1. Conclusion: Our data indicate that few KPC-positive plasmids have rapidly spread in our region with a worrisome highly efficient horizontal transfer. Importantly, carbapenem-resistant strains have not been confined to intensive care units, but have extended to medical wards as well, leading to involvement of the non-hospitalized population with subsequent dangerous community acquisition. Early identification of carbapenemase producers in clinical infections, also at the carriage state, is thus becoming mandatory to prevent and contain further resistance spreading.