The cfiA gene is clustered in a bicistronic operon encoding an N-acetyltransferase and an O-acetyltransferase related to resistance markers. This genetic context, exclusively found in strains of Bacteroides fragilis division II, has been highly rearranged by the successive integration of two new mobile sequences, a miniature element and ISBf9. Besides that, among the DNA polymorphisms detected in the cfiA locus, only the integration of IS942 at its promoter was a determinant for expression of carbapenemase activity.The cfiA gene, encoding the unique carbapenemase enzyme found in Bacteroides, is restricted to the division II group of Bacteroides fragilis strains (5). Bacteria belonging to this group were isolated in previous studies of human (3) or animal (unpublished data) infections, where identification of the cfiA genes was performed by PCR with primers P1 and P4 (Table 1) and confirmed by Southern hybridization on genomic DNA digestions (not shown).Imipenemase activity. Division II strains were cultivated under anaerobic conditions (3), and enzyme activity was determined at 37°C with 0.1 mM imipenem and 50 mM phosphate buffer (pH 7.0) by using an ε 299 (extinction coefficient at 299 nm) value of 9,670 M Ϫ1 cm
Ϫ1. Only strains MN11 and MN32 expressed imipenemase activity and were resistant to imipenem (Fig. 1).Allelic polymorphism. The sequences of cfiA genes revealed that they encode previously described protein variants (4). The allele from strain MN5 (cfiA11) corresponds to protein polymorphisms in residues 79 (T), 85 (T), 113 (K), 188 (T), and 225 (N), referred to as TTKTN; alleles from strains MN11 (cfiA12), MN32 (cfiA13), and 4a (cfiA14) encode the protein variant MTRAD; and those from strains MN36 (cfiA15) and MN41 (cfiA16) presented the polymorphisms TAKAD and TTKAD, respectively. Only MTRAD alleles were presented in the two strains, MN11 and MN32, expressing imipenemase activity (Fig. 1). However, the occurrence of a particular protein variant does not correlate with the metallolactamase phenotype, since the 4a strain (allele cfiA14; protein variant MTRAD) is sensitive to carbapenems. Accordingly, a previous work reported TAKTN and TAKAD alleles in strains sensitive to carbapenem, while TAKAD, TTKTN, and TAKTD alleles were found in resistant strains, where the expression of imipenemase activity was determined by the integration of IS elements near the promoter of the gene (4).Upstream sequences. Inverse PCR (iPCR) was performed to isolate the DNA regions upstream from the cfiA genes. Genomic DNAs were digested by HaeIII, and after ligation (3), the R1 and R2 primers (Table 1) were used in a PCR of 35 cycles with annealing/extension at 68°C. A fragment of 0.7 kb was expected from the HaeIII restriction site placed 157 bp upstream of the cfiA start codon (Fig. 2C), but the display of iPCR fragments revealed the polymorphism of that region ( Fig. 2A). Thus, strains MN5, MN35, MN41, and 4a presented