Characterisation and clonal analysis of Klebsiella pneumoniae sequence type 258 KPC‐2 positive isolates responsible for a nosocomial outbreak in northern Italy

Objectives: KPC are an important group of β-lactamases given their spreading in nosocomial settings among Enterobacteriaceae. KPC-2 and -3 are the wider disseminated, found mostly on plasmids in K. pneumoniae. KPC-positive K. pneumoniae (KPC-KP) have been reported in many European countries. After the first description of a KPC-KP in Italy in 2009 only few reports has been published. Here we report on the second Italian outbreak, discussing on clinical and molecular features. Methods: 22 K. pneumoniae isolates collected during 2010 at the San Gerardo Hospital in Monza were analyzed as representative of 58 isolates involved in the outbreak. Antibiotic susceptibility tests were performed with Vitek 2. MICs for imipenem and meropenem were confirmed by Etest for isolates that were positive for carbapenemase production (detected by phenyl boronic acid test). Clonal distribution were assessed by rep-PCR, RAPD, PFGE and MLST. Selected isolates were subjected to PCR analysis and sequencing to detect the pattern of resistance determinants. Results: The 22 investigated isolates showed a MDR phenotype being susceptible only to colistin, gentamicin and tigecycline. Results of genotyping techniques were all concordant and showed that all but one isolates were clonally related and belonged to ST 258. All isolates were confirmed to be blaKPC-2 positive by sequencing. In addition the investigated strains were positive for blaCTX-M-15 and all but one to blaSHV-12 ESBLs. 36 additional isolates showing the same phenotypic pattern were presumed to be related to the epidemic cluster. Conclusion: Before November 2009 at the San Gerardo Hospital no KPC-KP strains has been isolated. During the studied period, 22 clonally related KPC-KP were obtained. Isolates belonged to ST 258 as the first described Italian KPC-KP. This clone is the major responsible of worldwide spreading of these resistance determinants. Molecular analysis demonstrated the presence in all isolates of blaKPC-2, and blaCTX-M-15 and in all but one of blaSHV-12 β-lactamase. The only KPC-KP isolate not clonally related to the outbreak clone, was positive for blaSHV-1 determinant and was obtained from a patient transferred from another Hospital. This different clone represent an exception in the Italian epidemiological scenario where all described KPC-KP belonged to ST258. The rep-PCR approach here described appear to be a rapid and robust technique to investigate clonal relationship of KPC-KP.