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Antimicrobial Agents and Chemotherapy, May 2002, p. 1288-1294, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1288-1294.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization and Movement of the Class 1 Integron Known as Tn2521 and Tn1405

Sally R. Partridge,^1,2 Heidi J. Brown,^1,2 and Ruth M. Hall^1*

CSIRO Molecular Science, North Ryde, New South Wales 2113,¹ Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia²

Received 27 August 2001/ Returned for modification 25 November 2001/ Accepted 9 February 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two putative transposons, Tn2521 and Tn1405, carrying determinants for the PSE-4 ß-lactamase and for resistance to streptomycin, spectinomycin, and sulfonamides were previously isolated from the chromosome of Pseudomonas aeruginosa Dalgleish. Detailed mapping and determination of the complete sequence of Tn2521 revealed that it is a class 1 integron, here renamed In33, with a backbone structure identical to that of In4 from Tn1696. In33 contains two gene cassettes, blaP1 and aadA1, replacing the aacC1-orfE-aadA2-cmlA1 cassette array in In4. Although In33 does not include any transposition genes, movement of In33 (Tn2521) targeted to a single location in the IncP-1 plasmid R18-18 has been reported previously (M. I. Sinclair and B. W. Holloway, J. Bacteriol. 151:569-579, 1982). A 5-bp duplication of the target, which lies within the res site recognized by the ParA resolvase of R18-18, was present, indicating that the mechanism of movement was transposition. Together, these data indicate that class 1 integrons that are defective in self-transposition can move under appropriate circumstances. The Tn1405 isolate studied was found to represent only the cassette array of In33, which had replaced the cassette array in the recipient plasmid R388, probably by homologous recombination.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Class 1 integrons predominate among the integrons found in clinical isolates of both gram-negative and gram-positive bacteria (12, 14, 15, 38). Generally, they contain one or more integrated cassettes, each of which includes an antibiotic resistance gene. A large number of gene cassettes, each containing a different resistance gene, have been identified (12, 14, 15, 32). Integrons belonging to class 1 have an identical or nearly identical 5' conserved segment (5'-CS) (38) but exhibit variation in both the number and the identity of the cassettes they contain. The 5'-CS includes the intI1 gene, encoding the site-specific recombinase responsible for cassette insertion (7-9, 12, 16, 26). The 5'-CS also includes the attI1 site (29), into which the cassettes are incorporated (7), and a promoter, P_c, that directs transcription of all of the cassette-encoded genes (15). The 5'-CS is bounded at the inner end by attI1 and at the outer end by a 25-bp sequence, IRi, that is found as an inverted repeat, IRt, at the other end of class 1 integrons (5, 16, 31, 38).

To the right of the last gene cassette, or adjoining the 5'-CS if no cassettes are present, three different sequence configurations have been identified (Fig. 1). Tn402, which is both an active transposon (19, 34) and a class 1 integron (31), includes a set of four genes, tniA, -B, -Q, and -R, that are involved in transposition (20, 31). This backbone type is likely to represent the ancestor of other class 1 integron types. However, most of the class 1 integrons studied to date lack a complete tni gene module. Most of them contain at least part of a region known as the 3'-CS (5, 13, 28, 30, 38) that includes the sulfonamide-resistance gene sul1 (38, 39). They appear to have arisen from a Tn402-like ancestor by incorporation of the genes (qacE1, sul1, orf5, and orf6) that make up the 2,384-bp region defined as belonging to the 3'-CS (5). However, they have also undergone further rearrangements leading to two distinct branches. Both branches have lost part or all of the tni gene module to become transposon derivatives that are defective in self-transposition. Many of them have also lost some part of the 3'-CS (5, 13, 28), and a few have lost all of it (30). One group, the In5 type, were originally identified as containing IS1326 (5), but further members of this group have lost IS1326 (23, 31). The second backbone structure, which is characterized by the presence of IS6100, was recently found in the integron In4, which constitutes the central region of the transposon Tn1696 (accession no. U12338) (28). In4 contains one complete copy of IS6100 and an adjacent partial copy in the same orientation. This configuration is flanked by short segments of the sequence found at the outer right-hand end (IRt end) of other class 1 integrons. The outer and inner copies of the IRt end correspond to the last 152 and 123 bp of the tni module, respectively, and are in inverse orientation.

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FIG. 1. Structures of the different backbone types of class 1 integrons. Each integron is bounded by the 25-bp inverted repeats IRi and IRt (i and t) and includes the 5'-CS, which begins with IRi, contains the intI1 gene, and ends at the vertical arrow in the attI1 site (narrow open box). This arrow also marks the positions of integrated cassettes, which are shown above it. Parts of the 3'-CS and the tni module are found to the right of this arrow. Tn402 (also called In16 or Tn5090) includes the complete tni module, which contains a full set of transposition genes (tniA, -B, and -Q), a resolvase gene (tniR), and a res site (filled box). In5, representing the In5-like type class 1 integrons, includes the longest version of the 3'-CS and part of the tni module, plus an IS1326 element. The 3'-CS found in found in In5 contains qacE1, a truncated version of the qacE cassette, the sulfonamide resistance determinant sul1, orf5, and orf6. In4, representing the In4-like type class 1 integrons, includes a complete and a partial (; last 321 bp of IS6100) version of IS6100 in the same orientation. This IS6100/ structure is flanked by short inversely oriented regions corresponding to 123 and 152 bp from the IRt end of the tni module.

It seems reasonable to predict that class 1 integrons that lack the complete tni module should be able to move, so long as the two inverted repeats, IRi and IRt, are present and the tni gene products are supplied in trans. Indeed, integrons belonging to both the In5-like and In4-like groups appear to move readily as judged by the fact that they are found in many different locations (5, 13, 16, 28, 30, 31, 38), indicating past movement, and some of them are flanked by a 5-bp direct duplication, consistent with movement by a transpositional mechanism. However, movement has not yet been demonstrated experimentally.

A putative transposon, Tn1405, that carries the determinant for the PSE-4 ß-lactamase together with determinants for resistance to sulfonamides and to streptomycin and spectinomycin was isolated from the Pseudomonas aeruginosa Dalgleish strain (27) by Levesque and Jacoby (24), and movement of this group of resistance determinants was also reported by Hedges and Matthew (17). However, movement of Tn1405 into the IncP-1 plasmid pUZ8 occurred only at low frequencies, and subsequent movement from pUZ8 was observed predominantly in recombination-proficient hosts. A second putative transposon, Tn2521, which also includes determinants of resistance to carbenicillin, streptomycin, spectinomycin, and sulfonamides, was isolated from several different clinical P. aeruginosa strains from hospitals in Melbourne, Australia (35). The resistance determinants were found to be present on the chromosomes of these strains (35). Tn2521 was classified as a transposon on the basis of its ability to move from its original location in the chromosome of the clinical Pseudomonas strains to the IncP-1 plasmid R18-18 (35). However, further movement of Tn2521 from R18-18 in recA^- strains of P. aeruginosa was not detected, suggesting that Tn2521 is not able to support its own movement. Further characterization of the P. aeruginosa Dalgleish strain revealed that the resistance determinants carried by Tn1405 are in the same location as Tn2521 on the chromosome, indicating that the Melbourne isolates are equivalent to the Dalgleish strain (M. Sinclair, personal communication) and hence that Tn2521 and Tn1405 are probably identical.

We noticed that the published restriction map of R18-18::Tn2521 (35) includes a configuration of sites that is reminiscent of that found in class 1 integrons and particularly In4, and here we sequenced Tn2521 and the R18-18 flanking region and showed that Tn2521 is a class 1 integron belonging to the In4 group. The structure of R388::Tn1405, for which no map has been published, was also examined. This plasmid is a secondary transposant of Tn1405 that had first moved to pUZ8 and then into a second recipient plasmid, R388. However, as R388 already carries a class 1 integron, designated In3 (38), and the cassette-encoded trimethoprim resistance determinant was lost when Tn1405 was acquired, we explored the possibility that in this case only the cassette array had moved into the recipient plasmid.

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Bacterial strains and plasmids. Escherichia coli JM109 (lac-proAB) supE thi F' (traD36 proAB⁺ lacI^q lacZ M15) was used to propagate plasmid DNA. Plasmids used in this work are shown in Table 1. R388::Tn1405 was obtained from George Jacoby, and pMO266 (R18-18::Tn2521) was from Bruce Holloway. Fragments from pMO266 (R18-18::Tn2521) or R388::Tn1405 were cloned into either pUC19 (40) or pACYC184 (6) by standard procedures (33). Plasmids containing the appropriate fragments were identified by screening for antibiotic resistance, by restriction mapping, and by sequencing the fragment ends with a universal primer. Subclones were also derived from these primary clones. pRMH162 contains two HindIII fragments that are adjacent in pMO266. pRMH486 was constructed by ligating a KpnI fragment of pRMH164 to KpnI-digested pUC19. Bacteria were routinely cultured at 37°C in Luria-Bertani medium or on Luria-Bertani agar supplemented as appropriate with ampicillin (100 µg ml^-1), chloramphenicol (25 µg ml^-1), streptomycin (25 µg ml^-1), or sulfamethoxazole (25 µg ml^-1). Antibiotics were obtained from Sigma.

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TABLE 1. Plasmids

DNA isolation and restriction mapping. Plasmid DNA for restriction analysis and cloning was isolated by an alkaline lysis method (2). Restriction enzymes were used in accordance with the manufacturers' instructions. Fragments were separated by electrophoresis on 1% (wt/vol) agarose gels and visualized by staining with ethidium bromide. An EcoRI digest of bacteriophage SPP1 (Geneworks) and a HindIII digest of DNA (Progen) were used as size markers. Plasmid DNA for sequencing was purified with a Magic miniprep DNA purification system (Promega) or a Wizard maxiprep kit (Promega).

DNA sequencing and analysis. The DNA sequences of fragments of R18-18::Tn2521, R388::Tn1405, and R388 cloned in plasmid vectors were determined on at least one strand. Where there were differences from standard or prototype sequences, the sequence was determined on both strands. The sequences of the integron boundaries in R388::Tn1405 were obtained by using this plasmid as the template. Manual DNA sequencing was performed as described previously (28). Automated sequencing was performed by SUPAMAC (Sydney University/Royal Prince Alfred Hospital, Sydney, Australia) or by the sequencing facility at the Department of Biological Sciences, Macquarie University, Sydney, Australia, on an ABI-PRISM 377 sequencer using the Big Dye system. DNA sequences were assembled by using MacVector6.5 and AssemblyLIGN (Oxford Molecular). GenBank searches were performed with the BLASTN and FastA programs available through WebANGIS (Australian National Genomic Information Service). Programs in the Genetics Computer Group Wisconsin Package, version 8.1.0, were used via WAG (WebANGIS GCG) to align and analyze DNA sequences.

Nucleotide sequence accession numbers. The sequence for In33 (Tn2521) has been submitted to GenBank under accession no. AF313471, and the compiled R388 sequence has been submitted under accession no. U12441.

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Structure of Tn2521. A detailed map of the appropriate region of pMO266, one of the R18-18::Tn2521 transposants isolated by Sinclair and Holloway (35), was constructed (Fig. 2). Apart from differences due to the presence of different integrated cassettes, the map of the right-hand end of Tn2521 is identical to that of In4 (28). To confirm this, the complete sequence of Tn2521 was determined (GenBank accession no. AF313471) and the backbone of Tn2521 was found to be identical to that of In4 (GenBank accession no. U12338) except for minor differences in the 5'-CS. Tn2521 does not include the duplication of 19 bp of the attI1 region found in In4 (28) and the P_c promoter is weak (TGGACA-17 bp-TAAGCT) as opposed to strong (TTGACA-17 bp-TAAACT). Because Tn2521 is not a self-mobilizing transposon, it was renamed as a class 1 integron, In33.

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FIG. 2. Map of In33 (Tn2521) and surrounding regions of R18-18. i and t, inverted repeats of In33 (arrows indicate their orientation). Features of the integron backbone are as in Fig. 1. Each cassette is shown as an open box that includes the gene and an adjacent filled box that represents the 59-base element (59-be). The par genes in the adjacent R18-18 sequence (dotted lines) are also shown. The fragments cloned in various plasmids are shown the map. Restriction enzyme sites: B, BamHI; Bg, BglII; H, HindIII; K, KpnI; P, PstI; S, SalI.

In33 contains two integrated cassettes, blaP1a and aadA1, that replace the four cassettes (aacC1-orfE-aadA2-cmlA1) found in In4. The blaP1a gene (also known as bla_PSE-4) encodes the ß-lactamase designated PSE-4 (17) and confers resistance to carbenicillin and ampicillin. The sequence of the In33 blaP1a cassette is identical to the partial sequence for the blaP1a cassette of Tn1405 (GenBank accession no. J05162) (3). It differs at only one position from the complete blaP1b cassette, which encodes the identical BlaP1 variants called CARB-2 and PSE-1 (GenBank accession no. Z18955 and AF313472) (30), and at one position from the partial sequence of the blaP1c cassette encoding the CARB-3 variant (S46063) (22) (Table 2). The 856-bp aadA1 cassette, which confers resistance to streptomycin and spectinomycin, differs at two positions (G732C and C759T; numbering from base 1 of the cassette) from the prototype aadA1a cassette sequence (GenBank accession no. X12870) (25, 39), which is from Tn21, but these differences do not result in any amino acid changes. Though a C is present at position 759 in the prototype aadA1a cassette, a T is found at this position in all other variant types found in GenBank (Fig. 3). The aadA1a cassette in In33 is identical to that in In28 found in Tn1403, which is also derived from P. aeruginosa (30). The G732C C759T variant of aadA1a is also observed in a few other sequences derived from Pseudomonas species and Acinetobacter baumannii (Fig. 3). These changes are also seen together with C20T in an aadA1a-type cassette from E. coli.

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TABLE 2. Comparison of blaP1 cassette sequences

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FIG. 3. Variants of the aadA1a cassette. The linear form of the 856-bp aadA1a cassette is shown, with the 59-be represented as a filled box and an arrow below indicating the extent of the aadA1a gene. At positions where variations occur, the base found in the prototype aadA1a sequence is shown, except for position 759, which is C in the prototype but T in all of the other known aadA1a sequences. The positions of the variant bases in the cassette sequence are also indicated, where 1 is the first T residue of the 1R site (GTTAAAC) of the 59-be. Available aadA1a sequences are grouped according to base changes, and amino acid changes, GenBank accession numbers, species of origin, and assigned transposon or integron numbers are listed. For simplicity, only changes found in at least two of the known aadA1a variants were included. The sequences marked by asterisks contain additional changes, including differences in the 59-be, which may be due to sequencing errors. The sequences of the Tn21 and Tn7 addA1a cassettes are each found in several GenBank entries, but only one accession number is listed.

Movement of In33/Tn2521. The sequence flanking In33 in pMO266, the R18-18::Tn2521 transposant studied here, revealed that incorporation of In33 into R18-18 created a direct duplication of 5 bp (Fig. 4), as expected for transposition. R18-18 is closely related to RP4 and RP1, and the duplication corresponds to bases 35017 to 35021 of RP1 (GenBank accession no. L27758). This lies within site II of the multimer resolution site (mrs or res site), which is found in the Par region of RP1, and is the site recognized by the ParA resolvase (11). As In33 does not contain any of the tni genes and is thus a defective transposon derivative, its movement from the chromosome of the original clinical Pseudomonas isolate to R18-18 must have been supported by tni genes in another transposon present in that strain.

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FIG. 4. Sequence surrounding In33 in R18-18. Sequence adjacent to the IRi and outer IRt ends of In33 is aligned with the R18-18 sequence. The 5 bp that are duplicated are boxed. The corresponding region of the RP1/RP4 sequence (bases 34951 to 35060 in GenBank accession no. L27758) is also shown, with the extents of the three sites making up the res (mrs) site, as identified by Eberl et al. (11), indicated above it.

Structure and movement of Tn1405. R388::Tn1405, one of the putative Tn1405 transposants isolated by Levesque and Jacoby (24), conferred resistance to ampicillin, carbenicillin, streptomycin, spectinomycin, and sulfonamides but did not confer the expected trimethoprim resistance determined by the dfrB2 cassette in In3 of the target plasmid R388 (Fig. 5). We reasoned that this indicates either that Tn1405 lies within the dfrB2 cassette or that this cassette has been lost from the transposant. To distinguish these possibilities, BamHI digests of R388, R388::Tn1405, and an R388 derivative, pRMH560, were compared. In R388, all of the BamHI sites lie within In3 and bands of 1.8 and 2.1 kb, which include the bulk of the In3 integron and contain the dfrB2-orfA cassette array, are observed on digestion with BamHI (Fig. 5). In pRMH560, which has lost both the dfrB2 and orfA cassettes, a 2.9-kb band is seen. For R388::Tn1405 (Fig. 5), a 4.8-kb fragment replaces the band(s) seen in the R388 and pRMH560 digests. This indicates that only one integron is present in R388::Tn1405 and, as a 4.8-kb BamHI fragment is also found in In33, that the cassette array of In3 appears to have been replaced by the one from In33. Comparison of the sizes of the fragments obtained when R388 and R388::Tn1405 were digested with other restriction enzymes revealed differences in the plasmid backbones. However, by reference to maps of R388 (1) it was clear that the two plasmids are identical in the region flanking In3 in R388, indicating that the new cassettes lie within the boundaries of In3, and this was confirmed by sequencing (data not shown). The differences between R388::Tn1405 and R388 were not investigated further.

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FIG. 5. Characterization of R388::Tn1405. (A) BamHI digests of pRMH560, R388, R388::Tn1405, and pRM858 are shown. Size markers are SPP1 digested with EcoRI and digested with HindIII, and the sizes of the fragments are given in kilobases. (B) Maps of the integrons in pRMH560, R388, and R388::Tn1405. The features of the integron regions are as in Fig. 2, with the names and positions of the cassette genes indicated. Flanking regions are shown as dashed lines. Note that neither IRt nor any part of the tni module is found in R388. BamHI sites (B) and the sizes of the fragments expected on BamHI digestion are shown.

The 4.8-kb fragment from R388::Tn1405 was cloned into pACYC184 (chloramphenicol resistant) (6) to form pRMH858, which confers resistance to ampicillin, streptomycin, spectinomycin, sulfonamides, and chloramphenicol. The sequence of this fragment was determined and is identical to that of the equivalent region of In33 (Tn2521). The simplest mechanism that explains the properties of R388::Tn1405 is cassette exchange via two homologous recombination events occurring in the conserved regions that flank both cassette arrays, the first in the 5'-CS and the second in the 3'-CS. Hence, R388::Tn1405 did not arise by transposition and is inappropriately named.

To determine if the location of the recombination events that gave rise to R388::Tn1405 could be identified, the regions of In3 which had not previously been sequenced and those where conflicts were apparent in existing GenBank entries (J01773 and V00252) were also sequenced and the sequence was compiled. The only differences between the 5'-CS of In3 and In33 were found in the P_c promoter, with In3 containing the strong version and In33 containing the weak version. Therefore, it appears that the left-hand recombination crossover must have occurred between the IRi boundary of the 5'-CS and P_c. The sequence of the 3'-CS is identical in R388, In33, and pRMH858 and hence provides no further information.

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Most of the In4-like class 1 integrons and integron remnants described to date are located on plasmids. In contrast, In33 is located within the bacterial chromosome of the original isolate (35). The translocation of In33 (Tn2521) detected by Sinclair and Holloway (35) is also the only example to date where transposition of an In4- or In5-like integron has been detected experimentally. This movement occurred only in the original clinical isolate, and further movement of In33 could not be detected in recA^- strains of E. coli or P. aeruginosa. These data are readily explained by our finding that In33 lacks transposition genes, if it is assumed that these genes were present elsewhere in the original clinical isolate. Transposition of In33 was targeted in an orientation-specific manner to a single site in the IncP-1 plasmid R18-18 (35), which is a close relative of RP1 and RP4, and this site was known to be the preferred target of a mercury resistance transposon, Tn502 (36). Another mercury resistance transposon, Tn5053, which has ends and tni genes that are closely related to those of the class 1 integron Tn402, was subsequently shown to be preferentially inserted in one orientation at the same position, and the precise location was identified as the res (or mrs) site in the Par region of RP1 (20, 21). Tn402, which is both a class 1 integron and an active transposon, was recently shown to target the same site (19). The location of In33 in one of the R18-18::In33 transposants isolated by Sinclair and Holloway was found here to be the same as that of the Tn5053 transposants and some of the Tn402 transposants. As the original clinical isolate used by Sinclair and Holloway was also resistant to mercury, transposition of In33 may well have been catalyzed by the transposition proteins encoded by a mercury resistance transposon resembling Tn502 or Tn5053. Indeed, transposons of this type may play an important role in relocating In5-like and In4-like integrons.

The ability to detect movement of In33 in the Tn2521 study was undoubtedly facilitated by use of R18-18 as the target, because transposition of Tn5053 and Tn402 is constrained by target site specificity and R18-18 possesses the appropriate target site. As pUZ8 is also an IncP-1 plasmid (17) and may therefore contain the same res site target, it seems reasonable to assume that the initial movement of Tn1405, which is likely to be identical to In33 (Tn2521), to pUZ8 could have occurred by a transpositional mechanism. Transposition of Tn5053 has been studied in detail (20) and shown to proceed by a replicative mechanism that creates a cointegrate between the target plasmid and the molecule that is the source of the Tn5053 (21). This step requires the products of the tniA, -B, and -Q genes. The cointegrate contains two copies of the transposon and is subsequently resolved by the tniR gene product, which is a resolvase that acts on an adjacent res site. In the case of In33, the TniA, -B, and -Q proteins can be supplied in trans, allowing cointegrate formation, but the TniR product cannot act because its cognate res site is not present in In33. Thus, it is likely that to form the R18-18::In33 plasmids, R18-18 was first integrated into the Pseudomonas chromosome and resolution occurred via homologous recombination within the two copies of In33 generated by the transpositional event.

However, it is likely that the plasmid designated R388::Tn1405 arose via two homologous recombination events, one in the 5'-CS and one in the 3'-CS, as the tni gene products would not have been present in the strain in which it arose, precluding transposition. Sinclair and Holloway (35) were also able to detect movement of the resistance determinants from R18-18::Tn2521 to three other plasmids, but only in rec⁺ strains. As these three plasmids contained a sulfonamide resistance determinant, which is suggestive of the presence of a class 1 integron, this movement could also be explained by cassette exchange via homologous recombination. Because transposition of In4- and In5-type integrons can only occur if tni genes are supplied by another transposon and IntI1-mediated cassette insertion occurs at very low frequencies in the wild, swapping of cassette arrays between different class 1 integrons by homologous recombination (15, 37) is likely to be the predominant way in which cassettes and the resistance genes that they contain are exchanged.

 
We thank Bruce Holloway, Martha Sinclair, and George Jacoby for supplying transposants from the original studies. We also thank Diana Brookes for technical assistance and Tina Collis for constructing pRMH858.

S.R.P. was supported by a grant from the Australian National Health and Medical Research Council.

 
* Corresponding author. Mailing address: CSIRO Molecular Science, Sydney Laboratory, P.O. Box 184, North Ryde, NSW 1670, Australia. Phone: 61-2-9490 5162. Fax: 61-2-9490 5005. E-mail: ruth.hall{at}molsci.csiro.au.

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Antimicrobial Agents and Chemotherapy, May 2002, p. 1288-1294, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1288-1294.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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