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Mechanisms of Resistance

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

Naoki Kato, Kikuo Yamazoe, Chang-Gyun Han, Eiichi Ohtsubo
Naoki Kato
1Institute of Anaerobic Bacteriology, Gifu University School of Medicine
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  • For correspondence: nk19@cc.gifu-u.ac.jp
Kikuo Yamazoe
2Department of Pharmacy, Murakami Memorial Hospital, Asahi University, Gifu
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Chang-Gyun Han
3Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan
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Eiichi Ohtsubo
3Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan
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DOI: 10.1128/AAC.47.3.979-985.2003
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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 cfiA1 to cfiA10) 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 IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, 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, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 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.

Bacteroides fragilis is a strict anaerobe that inhabits the intestines of humans and is the most common anaerobe recovered from various infections, such as intra-abdominal infection, foot ulcer, and sepsis. This organism shows inherently poor susceptibility to most β-lactam antibiotics (10, 13, 30). Carbapenems such as imipenem (15), panipenem (18), meropenem (6), biapenem (36), CS-834 (12), and ertapenem (13, 11) are powerful antimicrobial agents with broad spectra that cover most aerobes and anaerobes, including B. fragilis.

The existence of imipenem-resistant B. fragilis strains was reported nearly 2 decades ago (5), and these strains were found to produce metallo-β-lactamase, which hydrolyzes the four-membered-ring C—N bond of various β-lactam antibiotics to inactivate them (not only carbapenems but also other classes of β-lactams) (1, 35). B. fragilis metallo-β-lactamase requires metal ions for activity and exhibits broad-spectrum resistance to β-lactamase inhibitors, including clavulanic acid, sulbactam, and tazobactam (1, 35). This metallo-β-lactamase is classified into class B (subclass B1) based on its primary genetic structures (3, 27) or group 3 (subclass 3a) based on its functional characteristics and inhibitor profile (3, 23). The di-zinc β-lactamase of B. fragilis is one of the most powerful metallo-β-lactamases in terms of its hydrolytic activity, based on its kcat and kcat/Km values for various β-lactam compounds (8, 23). B. fragilis metallo-β-lactamase is encoded by the gene designated cfiA (or ccrA), which has been sequenced and characterized (24, 31).

Podglajen et al. (20) reported that approximately 3% of 500 randomly selected strains of B. fragilis strains carry cfiA, regardless of whether they express it. We have previously reported similar results. cfiA was found in 1.9 and 4.1% of imipenem-susceptible B. fragilis strains isolated in Japan from 1987 to 1988 and from 1992 to 1994, respectively, and insertion sequence (IS)-like elements adjacent to cfiA were found in metallo-β-lactamase-producing, imipenem-resistant strains but not in cfiA-carrying strains with no detectable metallo-β-lactamase activity (34).

In 1991 an IS element designated IS942 was found to be present in the region immediately upstream of cfiA in a carbapenem-resistant B. fragilis strain (25), and this IS element was suggested to provide a promoter for transcription of the cfiA gene (25). Following that report, two IS elements, IS1186 (21) and IS4351 (20), were found to be inserted just upstream of cfiA. Very recently, other IS elements associated with cfiA, IS1187 and IS1188, were reported (22). IS1186 has been shown to provide a promoter to express the cfiA gene (21). The other IS elements found in the upstream region of cfiA are thought to also provide a promoter to express cfiA, as does IS1186 (20, 22).

In this study, we analyzed the cfiA gene and the regions flanking it in six metallo-β-lactamase-producing imipenem-resistant B. fragilis strains as well as four imipenem-susceptible strains by PCR and DNA sequencing. We show that the strains tested have some cfiA sequence variation but can produce metallo-β-lactamase and that the imipenem-resistant strains have new IS elements immediately upstream of cfiA, providing a promoter to express the cfiA gene.

MATERIALS AND METHODS

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.

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TABLE 1.

Oligonucleotide primers used to sequence the cfiA gene

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).

FIG. 1.
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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.

RESULTS

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.

FIG. 2.
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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.

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TABLE 2.

Five new IS elements identified from six imipenem-resistant B. fragilis strains isolated in Japana

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).

DISCUSSION

Full catalytic activity of the metallo-β-lactamase depends on the presence of two atoms of Zn2+ in its active site (4). The active site of metallo-β-lactamase encoded by the cfiA gene is defined by the zinc-binding sites, the constituent residues that bind to the zinc atoms, and two loops. The metal ions are bridged by a common water-hydroxide molecule, and additional ligands to the zinc ions include three histidines (His99, His101, and His162) to the first zinc ion and an aspartate carboxyl group (Asp103), a cysteine thiolate (Cys181), a histidine (His223), and an additional water molecule to the second zinc ion (4). It has also been suggested that the hairpin loop region (residues 44 to 52) participates in the binding of the substrate and in the shielding of the zinc sites from solvent (28). Note that the 10 cfiA variants tested in this study, cfiA1 to cfiA10, encode proteins with all the amino acid residues that are involved in the binding of the two Zn2+ ions (His99, His101, Asp103, His162, Cys181, and His223). cfiA1 to cfiA10 have been found to have several substitution mutations compared with the standard cfiA gene from TAL2480, which produces active metallo-β-lactamase, but these mutations did not cause a change of amino acid residues constituting the active site and the hairpin loop region in metallo-β-lactamase. Although in B. fragilis the replacement of Cys104 with Arg104, which is a specific feature seen in Bacillus cereus metallo-β-lactamase and not in the B. fragilis enzyme, was reported to introduce a dramatic decrease in the catalytic activity of metallo-β-lactamase (7), Cys104 was conserved in all the cfiA variants found in this study. These results indicate that metallo-β-lactamases generated from the cfiA variants are as active as that produced by the standard cfiA.

IS elements are mobile bacterial DNA elements which were discovered during investigation of mutations that are highly polar in the galactose and lactose operons of Escherichia coli K-12 and in the early genes of bacteriophage λ (19). The identified functions of IS elements include promoting genomic rearrangements, recruiting a foreign gene(s), and inactivating genes or activating the expression of some genes upon insertion (16, 19). The general features of IS elements are as follows (16, 19): IS elements contain one or more reading frames encoding an enzyme, transposase, which is required for transposition; the majority of known IS elements exhibit terminal inverted repeat sequences of about 10 to 40 bp, which serve as recognition sites for transposase during the transposition process; IS elements can move to new sites by mechanisms largely independent of the homology-dependent recombination pathway; and upon insertion, these elements generate short directly repeated sequences of the target DNA at the insertion point (target site duplication) ranging from 2 to 13 bp, but some show variations in target duplication length. This duplication is presumably due to the staggered cleavage of target DNA by transposases (16, 19).

In this study we have shown that six imipenem-resistant B. fragilis strains tested have five kinds of new IS elements in the region upstream of cfiA, as do carbapenem-resistant B. fragilis strains previously examined (20, 21). Newly identified and characterized IS elements were IS612, IS613, IS614, IS615, and IS616. Among them, IS612, IS613, IS614, and IS615 were related to one another but were different from those previously identified in the region upstream of cfiA. Another element, IS616, was, however, found to be almost identical to IS1188, an element recently identified to be associated with cfiA (22). All the IS elements in the imipenem-resistant B. fragilis strains tested in this study have possible promoter sequences consisting of −7 and −33 regions with the 17-bp spacing, as does IS1186, which has been proved to provide a promoter for the expression of cfiA (20). Although the transcription initiation sites of cfiA in our strains were not explored, our observation supports the idea that each IS element provides a promoter to activate cfiA and render B. fragilis strains drug resistant, as in the case of IS1186 (20). Recently, it was reported that the majority of promoters examined in B. fragilis consist of the −7 region with a consensus sequence, TAnnTTTG, and the −33 region with a sequence motif, TTTG, and that the spacing between the −7 and −33 regions may be the matter that partly explains the differences between the low- and high-level classes in the expression of the cepA gene (2); this gene encodes the class A β-lactamase among those produced by B. fragilis (26). Our data regarding the −7 and −33 motifs and their spacing did not clearly explain the low level of resistance to imipenem in GAI92087 carrying IS615 and GAI20436 carrying IS616. The factor(s) affecting the low and high levels of cfiA expression has yet to be determined.

IS elements are classified into a number of families based on the homology of their transposase genes (16, 19). We have shown by homology search and phylogenetic analysis that IS612, IS613, IS614, and IS615 belong to the IS4 family, together with the two IS elements (IS942 and IS1170) which have been found in drug-resistant B. fragilis strains. This finding may suggest that the elements belonging to the IS4 family tend to contribute to antimicrobial resistance in the species B. fragilis. In this study, we have also shown that IS616 belongs to the IS1380 family, though IS1188 is almost identical to IS616 and has been reported to be a member of the IS5 family (22). The IS1380 family elements, IS1247, IS1380, and IS1412, have been respectively detected from Xanthobacter autotrophicus (33), Acetobacter pasteurianus (29), and Sphingomonas species (9), which are phylogenetically unrelated to B. fragilis. Although the ancestral relationships of these microorganisms are diverse, it may be worthwhile to attempt to detect these IS elements in the B. fragilis group organisms and the closely related anaerobic bacterial genus to appreciate the habitat of this IS family.

IS4351, IS942, and IS1186 have been reported to be confined to the cfiA-carrying group of strains of the species B. fragilis (20). We studied the occurrence of the five IS elements in B. fragilis strains isolated in Japan by PCR assay using primers that amplify the inside region of each IS element and demonstrated that IS615 and IS616 are found in 15 (12.6%) and 26 (21.8%) of the 119 imipenem-susceptible, cfiA-negative strains tested (unpublished data). These results were in disagreement with a previous study (20). Since we did not detect whole IS elements, there is the possibility that the PCR assays detected only a short segment of imperfect IS elements. We need to carry out a more intensive study using several measures to determine the prevalence of IS elements in B. fragilis strains because IS elements in cfiA-negative B. fragilis strains, the majority of B. fragilis strains, can be important reservoirs of the promoter that activates cfiA.

ACKNOWLEDGMENTS

We are grateful to G. E. Killgore from the Centers for Disease Control and Prevention for critical reading of the manuscript. We thank T. Ara and H. Mori from the Nara Institute of Science and Technology for their useful suggestions. We also thank S. Choi from the University of Tokyo for his help in constructing phylogenetic trees.

FOOTNOTES

    • Received 14 August 2002.
    • Returned for modification 8 October 2002.
    • Accepted 30 November 2002.
  • Copyright © 2003 American Society for Microbiology

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New Insertion Sequence Elements in the Upstream Region of cfiA in Imipenem-Resistant Bacteroides fragilis Strains
Naoki Kato, Kikuo Yamazoe, Chang-Gyun Han, Eiichi Ohtsubo
Antimicrobial Agents and Chemotherapy Mar 2003, 47 (3) 979-985; DOI: 10.1128/AAC.47.3.979-985.2003

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New Insertion Sequence Elements in the Upstream Region of cfiA in Imipenem-Resistant Bacteroides fragilis Strains
Naoki Kato, Kikuo Yamazoe, Chang-Gyun Han, Eiichi Ohtsubo
Antimicrobial Agents and Chemotherapy Mar 2003, 47 (3) 979-985; DOI: 10.1128/AAC.47.3.979-985.2003
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KEYWORDS

Bacterial Proteins
Bacteroides fragilis
DNA Transposable Elements
imipenem
Thienamycins
beta-lactamases

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