A bla(TEM-1b)-derived TEM-6 beta-lactamase: a case of convergent evolution.

Since the first communication describing an organism that produces an enzyme capable of hydrolyzing extended-spectrum cephalosporins (5, 6), several outbreaks of Enterobacte-riaceae resistant to oxyimino-␤-lactams have been reported. In these cases, the adopted survival strategy of bacteria challenged by the introduction of aztreonam, cefotaxime, ceftazi-dime, and other oxyimino-␤-lactams was to expand the ␤-lac-tamase spectrum of activity by the mutational alteration of previously existing TEM or SHV enzymes (4). The genes encoding these enzymes have also acquired silent mutations that do not affect the amino acid sequence but are useful in tracing their evolution. During a previous study, we isolated from a patient with a urinary nosocomial infection a ceftazidime-resistant Esche-richia coli strain (FF683) (7) which produces a ␤-lactamase band with a pI of 5.87. Ceftazidime resistance and the presence of an identical ␤-lactamase band was transferred from E. coli FF683 to E. coli K802N by a 65-kb conjugative plasmid (pLV1), together with chloramphenicol, gentamicin, kanamy-cin, neomycin, netilmicin, trimethoprim, and sulfonamide resistance determinants. A 942-bp fragment of the ceftazidimase gene carried by plasmid pLV1 was amplified from the uncut plasmid DNA, by using PCR and primers specific for the bla TEM gene (nucleotides 128 to 144, nucleotides complementary to positions 1051 to 1069 and with restriction sites for EcoRI and SalI, respectively). Both strands of the ceftazidime-resistant bla gene, cloned in pBGS19, were sequenced by the dideoxynucleotide chain termination method with [ 35 S]dATP and the T7 sequencing kit and bla TEM-specific primers. This ceftazidimase differed from TEM-1 by two amino acid substitutions , glutamic acid to lysine at position 104 and arginine to histidine at position 164, as reported before for TEM-6. In addition to these substitutions, the bla gene encoding the studied ceftazidimase activity was found to carry four silent mutations in comparison with the sequence of the TEM-6 gene described by Goussard et al. (3) (Table 1). These mutations are found at nucleotides 175, 226, 436, and 604, locations which correspond to the bla TEM-1b (Tn2) gene and are known to allow discrimination of bla TEM-1a (Tn3) (2, 8). In contrast with the previously described DNA sequence encoding TEM-6 (3), the gene encoding ceftazidimase activity in our strain was more closely related to bla TEM-1b than to bla TEM-1a , and its designations as bla TEM-6b is here proposed. These results strongly suggest that the enzyme reported in this work has evolved from a genetic lineage different from …