Emergence of a colistin-resistant KPC-3-producing Klebsiella pneumoniae ST512 clone in an Italian university hospital.

The spread of multidrug-resistant gram-negative pathogens is one of the major hazards for patients requiring long-term hospitalization in intensive care units. We investigated phenotypic and molecular features and clonal relatedness of carbapenem-resistant K. pneumoniae clones circulating in our 1077-beds teaching hospital. Methods: Starting from April 2010, an outbreak of multiresistent Klebsiella pneumoniae occurred in Pisa hospital; after seventeen months (September 2011), 41 patients resulted infected (41/92, 45%), with 17 cases of sepsis (41%) and 51 colonized (51/92, 55%). Lethality was 22%. Clinical strains were isolated from different body sites (blood, urine, rectal swabs or sputum), during the patient hospitalization, in particular when the antibiotic resistance profile changed. Species identification and antimicrobial susceptibilities were obtained by Vitek2 System (bioMérieux). All of isolates were suspected for KPC production based on synergistic activity with phenylboronic acid. Imipenem, meropenem, ertapenem, colistin and tigecycline susceptibility was assessed also by Etest. A total of 52 isolates were chosen as representative and further investigated by genotyping. PCR for blaKPC-like genes and sequencing were performed. PFGE and multilocus sequence typing (MLST) were used to investigate clonal relatedness. Results: All 52 isolates resulted KPC-positive, harbouring blaKPC-3 gene. Carbapenem MICs ranged from 4 mg/L to > 32 mg/L and 18 isolates were resistant to colistin (in the range of 4 to 24 mg/L), whereas only five isolates were still sensitive to tigecycline. PFGE analysis revealed the spread of one prevalent clone belonging to ST512 and showing 7 pulsotypes clonal variants (pt A1-A7), and two sporadic clones, belonging to ST258 (pt A8) and ST101 (pt B). Several isolates with distinct genotypes in the same patient were found, suggesting that the colonizing strain may occasionally be replaced. Conclusions: The phenotypic and molecular heterogeneity of the CC258 circulating in our hospital supports the hypothesis that K. pneumoniae infection and colonization was a very dynamic process, where the ST512 clone could be the result of a single locus mutation occurred in the high risk clone ST258 worldwide distributed. The emergence of colistin-resistant could be attributed to selection pressure from excessive and inadequate colistin use.