New Insertion Sequence Elements in the Upstream Region of cfiA in Imipenem-Resistant Bacteroides fragilis Strains

Abstract

ABSTRACT The 747-bp cfiA gene, which encodes a metallo-β-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA 1 to cfiA 10 ) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-β-lactamases with full catalytic activities. PCR assay indicated that six metallo-β-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA . Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS 612 , IS 613 , IS 614 , IS 615 , and IS 616 , with a size range of 1,594 to 1,691 bp), of which only IS 616 was found to be almost identical to IS 1188 , one of the IS elements previously identified in the upstream region of cfiA . These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA . A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS 612 , IS 613 , IS 614 , and IS 615 belong to the IS 4 family, which includes IS 942 , previously found in some drug-resistant B. fragilis strains, but that IS 616 belongs to the IS 1380 family. All the IS elements appear to have putative promoter motif sequences (the −7 region's TAnnTTTG motif and the −33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.