Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates

Abstract

Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including P-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400.Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low-and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B.fragilis 638 cepA mutant. The results of j-lactamase assays and ampicillin MICs showed that there was no significant difference 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 codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low-and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.The anaerobe Bacteroides fragilis contains an endogenous, chromosomally encoded P-lactamase which preferentially hydrolyzes cephalosporins and is responsible for the intrinsic resistance to most penicillins and cephalosporins (6, 15). These organisms are generally susceptible to some of the newer ,B-lactams such as cephamycins (cefoxitin) and carbapenems (imipenem), although newly acquired 3-lactamases capable of degrading these antibiotics have been described (18,21,38).The indigenous 3-lactamase is present in between 90 and 99% of B. fragilis strains and at the biochemical level, this P-lactamase has been shown to be species specific (8,16). Recently, the gene for this enzyme, cepA, was cloned and the nucleotide sequence was determined (27). Southern hybridization analyses with a cepA probe showed that there was homology only with other B. fragilis strains, and construction of a cepA mutant provided evidence that this gene did in fact encode for the endogenous ,-lactamase. Comparison of the predicted CepA amino acid sequence with other 3-lactamase sequences indicated that it was not in the Ambler molecular class C like the chromosomal ,-lactamases of most other gram-negative bacteria, but rather CepA belonged to the class A 3-lactamases. The CepA enzyme together with...

Genomic structure of phage B40-8 of Bacteroides fragilis

“…Both the presence o€ the 20 N-terminal residues and the nucleotide combination of the degenerate probe designed were confirmed. The putative RBS sequence €or this gene at 47 nucleotide positions downstream o€ the transcription origin shows a consensus motif (GAGG) common in bacteria and similar to that reported for cepA gene of Bmteroides fragifis (Rogers et al, 1994). This clone could be used in future studies of the expression of the MP2 protein in B. fragilis.…”

Regulation of Bacteriodes fragilis katB mRNA by oxidative stress and carbon limitation

“…Total RNA was isolated by the hot phenol method of Aiba et al (1) as modified previously (27). Cultures were grown to mid-logarithmic phase (A 550 ϭ 0.…”

“…Membrane blots were washed twice in 2ϫ SSC-0.1% SDS for 30 min each at 45°C followed by at least two washes in 0.1ϫ SSC-0.1% SDS at 55°C and sometimes at 65°C. Autoradiographs were obtained by exposing the membranes to X-ray film, and hybridization was quantified by densitometry as described previously (27). All values were normalized by comparison to the 16S and 23S RNAs present in the ethidium bromide-stained gels prior to blotting.…”

Resistance to -Lactam Antibiotics in Bacteroides Spp.

“…In Nottingham, the percentage of clinical Bacteroides isolates displaying raised p-lactamase production increased from 17% to 25% between 1986 and 1995 [30]. In those cases in which elevated levels of typical P-lactamases are found, insertion elements may be responsible for increased expression of the cepA gene that controls production of these enzymes [53].…”