Molecular epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit

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Abstract
Objectives: To investigate the molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit of a university hospital in Italy. Methods: Antibiotic susceptibility was evaluated by disc diffusion and Etest. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by PFGE analysis. Conjugation was performed by broth mating. Results: Molecular typing of K. pneumoniae isolates identified three distinct PFGE patterns. Isolates of PFGE profile A were isolated during an epidemic in 1996, while isolates of PFGE profiles B and C were sequentially isolated from September 2002 to December 2004, when 233 colonizations and 19 infections by K. pneumoniae occurred. All K. pneumoniae strains of different PFGE types were identified as ESBL producers. DNA sequencing of amplified β-lactamase genes identified a novel blaTEM ESBL (blaTEM-136) along with blaSHV-1 in chromosomal and plasmid DNA from K. pneumoniae of PFGE type A, respectively, and blaTEM-1 and blaSHV-12 in plasmid DNA from K. pneumoniae of PFGE types B and C. Conjugation experiments demonstrated that resistance to third-generation cephalosporins, along with an ∼80 kb plasmid containing blaSHV-12 and blaTEM-1, was transferred from K. pneumoniae epidemic strains of PFGE types B and C to a susceptible Escherichia coli host at a frequency of 4 × 10−6 and 1 × 10−6 cfu/recipient cell, respectively. Conclusions: The selection of ESBL-producing clones and the transfer of the blaSHV-12 ESBL gene between different clones were responsible for the spread of K. pneumoniae in the neonatal intensive care unit.