Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Serratia marcescens in a neonatal intensive care unit

Objectives: To investigate the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae and Serratia marcescens in the neonatal intensive care unit (NICU) of a university hospital in Italy. Methods: Antibiotic susceptibility was evaluated by disc diffusion and E-test. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by pulsed- field gel electrophoresis (PFGE) and dendrogram analysis. Beta-lactamase gene transfer was performed by broth mating. Results: The molecular epidemiology of ESBL-producing K. pneumoniae and S. marcescens was studied in the neonatal intensive care unit of University ‘‘Federico II’’ of Naples, Italy from September 2002 to September 2005. During the study period, ESBL-producing K. pneumoniae and ESBL-producing S. marcescens were responsible for 23 infections (15.6%) and 290 colonizations (37.4%), and 6 infections (4.1%) and 60 (7.7%) colonizations, respectively. The outbreak period began in September 2002 for ESBL-producing K. pneumoniae and in May 2004 for ESBL-producing S. marcescens. Molecular typing identified two distinct PFGE patterns for ESBL-producing K. Pneumoniae isolates, one major PFGE pattern and two subtypes for ESBL-producing S. marcescens. The two K. pneumoniae epidemic clones showed an identical antibiotype, characterized by resistance to penicillins, monobactams, third generation cephems, kanamicin, netilmicin and gentamicin. S. marcescens strains of the major PFGE type and of the two subtypes were all resistant to penicillins, monobactams, third and fourth generation cephems, kanamicin, netilmicin and gentamicin. Molecular analysis of beta-lactamase genes identified blaTEM-1 in chromosomal and plasmid DNA and blaSHV-12 in plasmid DNA from K. pneumoniae strains of both PFGE types. All S. marcescens epidemic strains showed ampC in chromosomal DNA, blaTEM-1 in chromosomal and plasmid DNA and blaSHV-12 in plasmid DNA. Conjugation experiments demonstrated that resistance to third generation cephems, along with blaTEM-1 and blaSHV-12, was transferred from all K. pneumoniae and S. marcescens epidemic strains to a susceptible E. coli host at a frequency of about 1 · 10)6 CFU/recipient cells. Conclusions: The conjugal transfer of blaSHV-12 ESBL gene was responsible for the selection of ESBL-producing K. pneumoniae and S. marcescens epidemic clones in the NICU of our university hospital.