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

Twelve Klebsiella pneumoniae isolates resistant to expanded-spectrum cephal osporins and aztreonam, from patients with nosocomial septicaemia at the in tensive care unit of the Andes University Hospital, Merida, Venezuela, were studied for production of extended-spectrum beta-lactamase (ES beta L) act ivity. All were also resistant to gentamicin, kanamycin, tetracycline and c hloramphenicol but sensitive to cefoxitin, imipenem, amikacin and tobramyci n. Production of ES beta L activity was confirmed by restoring susceptibili ty to ceftazidime in the presence of clavulanic acid. All isolates carried an identical plasmid of approximately 87 kb. Resistance to beta-lactams, am inoglycosides, tetracycline and chloramphenicol was lost en bloc after plas mid curing by treatment with acridine orange and was transferable en bloc t o Escherichia coli by conjugation. Transconjugants always showed the same p lasmid 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 b y one of the transconjugants by means of hybridization assays, revealed the presence of both bla(TEM) and bla(SHV) determinants. Cloning and sequencin g of each determinant identified them as bln(TEM-1) and bla(SHV-5), respect ively, the latter being responsible for the ES beta L activity. Results of this study indicate that ES beta L determinants of the SHV-type carried by transferable elements, are spreading among nosocomial isolates of K. pneumo niae in Merida, Venezuela. (C) 2000 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.