DEVELOPMENT OF OLIGOTYPING FOR CHARACTERIZATION AND MOLECULAR EPIDEMIOLOGY OF TEM BETA-LACTAMASES IN MEMBERS OF THE FAMILY ENTEROBACTERIACEAE

Based on the DNA sequences of bla(TEM-1) and bla(TEM-2), which encode parental penicillinases TEM-1 and TEM-2, respectively, and of bla(TEM-3), bla(TEM-4), bla(TEM-5), bla(TEM-6), and bla(TEM-7), which encode extended-spectrum β-lactamases, we designed heptadecanucleotides to discriminate point mutations in five loci. Determination of the hybridization profiles by colony hybridization with this selection of probes, termed 'oligotyping', allowed characterization of the TEM variants present in 265 clinical isolates of the family Enterobacteriaceae that exhibit synergism between a penicillinase inhibitor and broad-spectrum cephalosporins. Among the 222 strains harboring TEM enzymes, Klebsiella pneumoniae (48%) and Escherichia coli (21%) were predominant, and TEM-3 was the most common enzyme (60%). Penicillinases TEM-1 and TEM-2 were detected alone (15 and 1%, respectively), combined (1%), or associated with another TEM β-lactamase (17 and 6%, respectively). Fourteen variants, including seven new enzymes, were detected. One, TEM-13, was a new penicillinase with the same isoelectric point and substrate range as TEM-2 but differed by a single amino acid substitution, whereas the others, TEM-14 to TEM-19, were extended-spectrum β-lactamases that consisted of novel combinations of known amino acid substitutions. Different TEM variants were found to coexist within the same cells. A patient could harbor two or three different strains that encoded the same enzyme or two indistinguishable isolates that produced distinct TEM β-lactamases.