Molecular basis of extended-spectrum beta-lactamase production in nosocomial isolates of Klebsiella pneumoniae from Mérida, Venezuela.

Twelve Klebsiella pneumoniae isolates resistant to expanded-spectrum cephalosporins and aztreonam, from patients with nosocomial septicaemia at the intensive care unit of the Andes University Hospital, Mérida, Venezuela, were studied for production of extended-spectrum beta-lactamase (ESbetaL) activity. All were also resistant to gentamicin, kanamycin, tetracycline and chloramphenicol but sensitive to cefoxitin, imipenem, amikacin and tobramycin. Production of ESbetaL activity was confirmed by restoring susceptibility to ceftazidime in the presence of clavulanic acid. All isolates carried an identical plasmid of approximately 87 kb. Resistance to beta-lactams, aminoglycosides, tetracycline and chloramphenicol was lost en bloc after plasmid curing by treatment with acridine orange and was transferable en bloc to Escherichia coli by conjugation. Transconjugants always showed the same plasmid profile as that of Klebsiella donors. Isoelectric focusing analysis of the crude extracts of transconjugants showed in all cases, the presence of two beta-lactamases of pI 5.4 and 8.2. Analysis of the plasmid carried by one of the transconjugants by means of hybridization assays, revealed the presence of both bla(TEM) and bla(SHV) determinants. Cloning and sequencing of each determinant identified them as bla(TEM-1) and bla(SHV-5), respectively, the latter being responsible for the ESbetaL activity. Results of this study indicate that ESbetaL determinants of the SHV-type carried by transferable elements, are spreading among nosocomial isolates of K. pneumoniae in Mérida, Venezuela.