Multifocal diffusion of KPC-3-producing ST512 Klebsiella pneumoniae in Italy

Introduction. The dissemination of carbapenemase-producing Klebsiella pneumoniae strains is an increasing problem worldwide. KPC ß-lactamases are Ambler class A enzymes mostly plasmid-encoded; their global spread represents a threat to clinical patients care and public health. Multi locus sequence type (ST)258 is currently the most spread K. pneumoniae clone associated with KPC enzymes. Here we report the first identification and multifocal spread of KPC-3 producing K. pneumoniae clinical strains belonging to ST512 in Italy. Materials and Methods. Fifty six carbapenem-resistant K. pneumoniae isolates were collected from 7 Italian hospitals during the period June 2009-May 2011. Isolates were obtained from different wards (spinal unit, medicine, hematology, etc.) and biological samples (mostly rectal swabs, urine and blood). Species identification and antimicrobial susceptibilities were obtained by NBC46/NM40 Microscan panels (Siemens). MICs values were interpreted according to EUCAST 2011 breakpoints. Modified Hodge test and combined disk test with phenyl-boronic acid (BOR) and EDTA were performed.The presence of blaKPC genes were confirmed by PCR and sequencing. A complete characterization of the produced ß-lactamases (BLs) was obtained by IEF followed by PCR experiments using primers specific for the detection of blaCTX-M-, blaTEM- and blaSHV type genes. PFGE and multilocus sequence typing (MLST) were both used to investigate clonal isolates relatedness. Results. All 56 isolates resulted positive for the presence of KPC-type carbapenemases by both phenotypical and molecular analysis. Fifteen isolates, chosen as representative, were further investigated. Ten out of 15 isolates harboring the blaKPC-2 gene clustered with the known ST258, while the remaining 5/10 belonged to the newly described ST512 and harbored the blaKPC-3 gene. ST512 isolates, from 3/7 hospitals, were collected from rectal swabs (40%), blood (20%), endotracheal aspirate (20%) and urinary (20%) samples. Carbapenem MICs ranged from 2 mg/L to more than 16 mg/L; the isolates were characterized by the same multi-drug-resistant (MDR) profile. Only in 1/5 cases such antimicrobial resistance profile was extended also to colistin (>2 mg/L).Three different PFGE clones were identified (A, B and C); one resulting characteristic of each hospital. Coherently with pIs obtained by IEF, the presence of additional BLs were investigated by PCR; clones B and C resulted positive for blaCTX-M and blaSHV genes, while blaSHV and blaTEM genes were found in clone A. Conclusion.Intra- and inter-hospital spread of KPC-positive strains was demonstrated.The heterogeneity of PFGE profiles showed the spread of different K. pneumoniae ST258 clones and the emergence of the new ST512 coupled with blaKPC-3 genes in Italian clinical settings. Strategies for carbapenemase detection and appropriate hospital hygiene precautions are indicated to limit the spread of the here described ST512 clone appears essential.