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

Bacterial strains, MIC measurement, and metallo-β-lactamase assay.A total of 137 B. fragilis strains, which were isolated from clinical specimens in Japan between 1987 and 1994, were used. MICs of imipenem were determined by an agar dilution method with an inoculum size of 10⁵ CFU per spot as described previously (34). Strains were categorized on the basis of susceptibility to imipenem into three groups, resistant (MIC, ≥16 μg/ml), intermediate (MIC, 8 μg/ml), and susceptible (MIC, ≤4 μg/ml), according to the breakpoints proposed by the National Committee for Clinical Laboratory Standards (17). Metallo-β-lactamase activity was assayed by both a spectrophotometric technique and a biological method as described previously (34).

Presence of cfiA in B. fragilis strains. B. fragilis strains were tested by PCR assay to determine whether they carried cfiA by using the primer set GBI-1 and GBI-2 (Table 1). PCR amplification was run for 35 cycles consisting of 20 s at 95°C and 2 min at 64°C as described previously (14). PCR-amplified fragments were analyzed by electrophoresis on a 5% polyacrylamide gel and then stained with ethidium bromide and visualized with UV light.

DNA sequencing of cfiA and its upstream region from B. fragilis strains.The fragments with the cfiA coding region were obtained by PCR with each of three pairs of primers (Table 1 and Fig. 1A) and with total DNA from each B. fragilis strain as the template, as described elsewhere (34).

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FIG. 1.

(A) Locations of six primers used for PCR and sequencing analyses of the cfiA gene; (B) deduced amino acid sequences of the products from the cfiA genes (cfiA₁ to cfiA₁₀) from 10 B. fragilis strains used in this study. These are compared with the sequence of the product of the standard cfiA gene in B. fragilis TAL 2480 (GenBank accession no. M34831 ) (32). Amino acid residues (His99, His101, Asp103, His162, Cys181, and His223) thought to be involved in the binding of the two Zn²⁺ ions are shown in boldface. Fully conserved amino acid residues are denoted by asterisks.

The region immediately upstream of cfiA along with the portion of the 5′ end of cfiA were obtained by PCR using primers G and E (Table 1 and Fig. 1A), and after sequencing of the amplicon, various primer sets selected from inside this amplified region by primers G and E were used for each strain examined. PCR was performed as described above except that annealing and extension were carried out for 2 min at 62 or 64°C, depending on the primer pair used. The PCR products were purified with Prep-A-Gene DNA purification systems (Bio-Rad Laboratories, Hercules, Calif.). Cycle sequencing was performed with a personal PCR thermal cycler (Takara, Ohtsu, Japan) using a Prism Ready Reaction DyeDeoxy Terminator cycle sequencing kit (PE Applied Biosystems, Foster City, Calif.) according to the manufacturer's protocol. DNA sequencing was carried out with an automated DNA sequencer (model ABI Prism 310 genetic analyzer; PE Applied Biosystems). All nucleotide sequences were determined in duplicate on both strands.

Computer analyses and processing of sequence information were performed using the GENETYX software version 11.2.0. (Software Development, Tokyo, Japan).

Nucleotide sequence accession numbers.The nucleotide sequences of the IS elements (IS612, IS613, IS614, IS615, and IS616) identified in this study were deposited in the DDBJ database (http://www.ddbj.nig.ac.jp ) under accession numbers AB042548 , AB042549 , AB042559 , AB042617 , and AB042560 , respectively. The nucleotide sequences of the cfiA genes (cfiA₁ to cfiA₁₀) were also deposited under accession numbers AB087225 , AB087226 , AB087228 , AB087229 , AB087230 , AB087231 , AB087232 , AB087233 , AB087234 , and AB087227 , respectively.

Susceptibility to imipenem, metallo-β-lactamase production, and the presence of cfiA.Of 137 B. fragilis strains tested, 7 were resistant to imipenem, 2 were intermediate, and 128 were susceptible. Six of the seven imipenem-resistant strains were found to be metallo-β-producers, and none of the other strains produced detectable metallo-β-lactamse. Of 137 B. fragilis strains, 16 possessed cfiA; all 6 imipenem-resistant, metallo-β-lactamase-producing strains harbored cfiA, and the remaining 10 strains were susceptible to imipenem.

Sequence variability of the metallo-β-lactamase gene, cfiA, in B. fragilis strains.To determine whether all the strains have an intact cfiA coding sequence (747 bp in size), the nucleotide sequences of the cfiA genes from 10 strains were determined. The imipenem-resistant B. fragilis strains used were GAI30079 (imipenem MIC, 256 μg/ml), GAI92082 (MIC, >256 μg/ml), GAI92084 (MIC, >256 μg/ml), GAI20264 (MIC, 32 μg/ml), GAI92087 (MIC, 16 μg/ml), and GAI20436 (MIC, 16 μg/ml), and the imipenem-susceptible strains used were GAI20270, GAI2570-22, GAI93311, and GAI93316, which were randomly selected. PCR was performed using three pairs of primers, G and E, GBI-1 and GBI-2, and GBI-3 and GBI-6 (Table 1 and Fig. 1A), which amplify a portion of the cfiA gene and its flanking regions. Gel electrophoresis analysis of the fragments amplified by PCR using primers G and E revealed that the imipenem-resistant strains generated fragments from the region upstream of the cfiA gene with a length of about 1.6 or 1.7 kb but that the imipenem-susceptible strains yielded fragments with a length of about 350 bp (Fig. 1A, upper diagram and lower diagram, respectively). PCR amplifications of 10 strains tested with other primer sets generated amplicons of the same size, regardless of susceptibility to imipenem.

The 10 cfiA genes (designated cfiA₁ to cfiA₁₀) obtained from the 10 strains were all 747 bp in length, the length of the standard cfiA from strain TAL2480 (31). The nucleotide sequences of cfiA₁ to cfiA₁₀ were compared with that of the standard cfiA gene and showed high identities of between 97.9 and 98.5%, and there was no stretch of mutations longer than 3 nucleotides (data not shown). Comparison of the nucleotide sequences of cfiA₁, cfiA₂, cfiA₃, cfiA₄, cfiA₅, cfiA₆, cfiA₇, cfiA₈, cfiA₉, and cfiA₁₀ revealed that cfiA₄ from GAI20264, cfiA₅ from GAI92087, and cfiA₇ from GAI20270 were 100% identical but that others were between 97.9 and 99.9% identical (data not shown).

Comparison of each of the amino acid sequences of the putative proteins encoded by cfiA₁ to cfiA₁₀ with that encoded by cfiA of B. fragilis TAL2480 are given in Fig. 1B. The standard cfiA gene showed strong homologies with cfiA₁ to cfiA₁₀; cfiA shared 98.8% identity with cfiA₁ to cfiA₃ and cfiA₆, with three amino acid substitutions out of 249 amino acid residues, and 98.4% identity with cfiA₄, cfiA₅, and cfiA₇ to cfiA₁₀, with four amino acid substitutions. Common substitutions were Met to Thr at position 79, Thr to Ala at position 85, and Arg to Lys at position 113. With cfiA₁ to cfiA₁₀, 100% identity was found among cfiA₁, cfiA₂, and cfiA₃ and among cfiA₄, cfiA₅, and cfiA₇.

Sequence analysis of the 1.6- or 1.7-kb fragments upstream of cfiA.Since the previous studies demonstrated that an IS element immediately upstream of cfiA provided the promoter sequence for cfiA expression (21, 25), we attempted to determine the presence of IS elements flanking cfiA₁ to cfiA₆, which were detected in the six imipenem-resistant B. fragilis strains. To identify such an element(s), the nucleotide sequences of the PCR fragments (about 1.6 or 1.7 kb in size) amplified by using primers G and E were determined. Sequence analysis revealed that their DNA sequences differed from one another, except with two strains, GAI92087 and GAI20436, which were identical. These sequences appeared to be IS elements because each sequence had imperfect terminal inverted repeats and a duplication of a target site sequence of a few base pairs in the flanking regions (Table 2 and Fig. 2A) and each encoded a putative transposase (see below). Here, these IS elements are designated IS612, IS613, IS614, IS615, IS616-1, and IS616-2 (Table 2) and were found in strains carrying cfiA₁, cfiA₂, cfiA₃, cfiA₄, cfiA₅, and cfiA₆, respectively. All these elements were upstream of cfiA and all were within 16 bp of the gene except for IS616, which was located about 30 bp upstream of cfiA (Fig. 2B). Each IS element had one large open reading frame whose orientation was opposite to that of cfiA. The numbers of their predicted amino acids were 428, 429, and 448 (Table 2). The open reading frames are expected to code for transposase.

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FIG. 2.

Features of new IS elements and related IS elements. (A) Nucleotide sequences of terminal regions of IS elements and flanking regions. The sequences of IS elements, including IS942 (25), IS1170 (32), IS1380 (29), IS1247 (33), and IS1412 (9), and flanking sequences are shown in uppercase and lowercase letters, respectively. IRL and IRR indicate imperfect terminal inverted repeat sequences at the left and right end regions of each IS element, respectively. IRL and IRR sequences, which are underlined, are located in the upstream and downstream regions of an open reading frame, respectively. The target site sequences possibly duplicated upon the transposition of each element are shown in boldface. (B) Locations of the target sites for the new IS elements IS1186 (21) and IS942 in the upstream region of cfiA. The target sites are shown by bars above or below the nucleotide sequence. (C) Putative promoter sequences in new IS elements. Sequences homologous to consensus sequences of B. fragilis promoters, TAnnTTTG in the −7 region and TnTG, TTG, or TG in the −33 region (2), are underlined. Putative ribosome-binding sites, cfiA start codons, and target site sequences are indicated in boldface, double underlining, and italics, respectively.

Sequence comparisons.IS612, IS613, IS614, and IS615 had similar terminal inverted repeats (Fig. 2A, upper panel). Comparison of the nucleotide sequences of the five IS elements or of the amino acid sequences of putative transposases encoded by them revealed that these four elements were related to one another. A computer-aided homology search with each amino acid sequence as a query revealed that the four elements were related to the two known IS elements, IS942 and IS1170, which have been found in drug-resistant B. fragilis strains (25, 32). These two elements belong to the IS4 family (16, 19), indicating that the four IS elements identified in this study also belong to the IS4 family (Fig. 2). Phylogenetic relationships of all the IS4 family elements demonstrated a close relatedness of these six IS elements (data not shown).

Another element, IS616, was, however, found to be almost identical in its nucleotide sequence to IS1188, an element recently identified as being associated with cfiA (22), except at one nucleotide at position 1384: IS616 has a T, whereas IS1188 has a C. This mutation was located within the region encoding a putative transposase, but no mutation was found at the amino acid level. IS616 was related to IS1380, the representative element of the IS1380 family (16, 19), and thus, IS616 belongs to the IS1380 family, which includes IS1247 and IS1412. IS616 was phylogenetically related to IS1380 and to other IS1380 family elements, IS1247 and IS1412 (data not shown).

Putative promoter sequences and the ribosome-binding site.Sequences homologous to consensus sequences of B. fragilis promoters, TAnnTTTG in the −7 region and TTTG in the −33 region (2), were found in all new IS elements at the end of the transposase gene (Fig. 2C). The −7 consensus sequences were almost identical, while mutations within the −33 motif, namely, TXTG, TG, and TTG, were found in all the IS elements. In all the IS elements, the distance between the −7 and −33 regions was 17 bp and the ribosome-binding site of a tetranucleotide AGAA sequence (21) lay 4 bp upstream of the ATG start codon of the cfiA gene (Fig. 2C).