INSERTIONAL ACTIVATION OF CEPA LEADS TO HIGH-LEVEL BETA-LACTAMASE EXPRESSION IN BACTEROIDES-FRAGILIS CLINICAL ISOLATES

Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infect ions, including p-lactams. A strain set comprised of B. fragilis isola tes producing either low or high levels of the endogenous cephalospori nase activity, CepA, has been described previously (M. B. Rogers, A. C . Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleot ide sequence comparisons revealed that there were few differences betw een the cepA coding sequences of the low- and high-activity strains. T he cepA coding sequences were cloned into an expression vector, pFD340 , and analyzed in a B.fragilis 638 cepA mutant. The results of P-lacta mase assays and ampicillin MICs showed that there was no significant d ifference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codo n, the sequence for high-activity strains change dramatically. This re gion of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expres sion of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differen tial cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter th e cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mecha nisms of cepA activation are discussed.