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Antimicrobial Agents and Chemotherapy, August 2005, p. 3289-3296, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3289-3296.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Tn5393d, a Complex Tn5393 Derivative Carrying the PER-1 Extended-Spectrum ß-Lactamase Gene and Other Resistance Determinants

Elisabetta Mantengoli and Gian Maria Rossolini*

Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100 Siena, Italy

Received 13 December 2004/ Returned for modification 9 March 2005/ Accepted 4 May 2005


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ABSTRACT
 
In Alcaligenes faecalis FL-424/98, a clinical isolate that produces the PER-1 extended-spectrum ß-lactamase, the blaPER-1 gene was found to be carried on a 44-kb nonconjugative plasmid, named pFL424, that was transferred to Escherichia coli by electroporation. Investigation of the genetic context of the blaPER-1 gene in pFL424 by means of a combined cloning and PCR mapping approach revealed that the gene is associated with a transposonlike element of the Tn3 family. This 14-kb element is a Tn5393 derivative of original structure, named Tn5393d, which contains the transposition module and the strAB genes typical of other members of the Tn5393 lineage plus additional resistance determinants, including the blaPER-1 gene and a new allelic variant of the aphA6 aminoglycoside phosphotransferase gene, named aphA6b, whose product is active against kanamycin, streptomycin, and amikacin. Tn5393d apparently originated from the consecutive insertion of two composite transposons into a Tn5393 backbone carrying the aphA6b and the blaPER-1 genes, respectively. The putative composite transposon carrying blaPER-1, named Tn4176, is made of two original and nonidentical insertion sequences of the IS4 family, named IS1387a and IS1387b, of which one is interrupted by the insertion of an original insertion sequence of the IS30 family, named IS1066. In pFL424, Tn5393d is inserted into a Tn501-like mercury resistance transposon. Transposition of Tn5393d or modules thereof containing the blaPER-1 gene from pFL424 to small multicopy plasmids or to a bacterial artificial chromosome was not detected in an E. coli host harboring both replicons.


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INTRODUCTION
 
Class A serine ß-lactamases with an extended substrate specificity, including specificity for oxyiminocephalosporins and monobactams, also referred to as extended-spectrum ß-lactamases (ESBLs), are among the most important resistance determinants emerging in gram-negative bacterial pathogens (4). Acquisition of similar enzymes by members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and other nonfastidious gram-negative nonfermenters greatly reduces the repertoire of active ß-lactams, often leaving a narrow choice of therapeutic options (6, 41).

Several types of class A ESBLs are encountered in the clinical setting, including the TEM- and SHV-type derivatives and the so-called "unconventional" ESBLs, such as the CTX-M-, PER-, VEB-, and GES/IBC-type enzymes (4, 41). The TEM-, SHV-, and CTX-M-type enzymes are mostly or exclusively found in the Enterobacteriaceae, while the PER-, VEB-, and GES/IBC-type enzymes are apparently more promiscuous and can be encountered both in the Enterobacteriaceae and in gram-negative nonfermenters (2, 4, 41).

PER-1 is an enzyme of notable clinical importance due its powerful ESBL activity (3, 26) and dissemination in various geographic areas of Europe and Asia (see references 27 and 41 and references therein, as well as reference 42). It has been detected in several microbial species including P. aeruginosa (see reference 41 and references therein), Acinetobacter spp. (40, 42), Alcaligenes faecalis (29), and Enterobacteriaceae (Salmonella enterica serovar Typhimurium [39], Proteus mirabilis [28], Providencia rettgeri [1], and Providencia stuartii and Escherichia coli [M. Perilli, F. De Santis, B. Caporale, F. Luzzaro, S. Stefani, A. Toniolo, G. M. Rossolini, and G. Amicosante, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1330, 2004]). PER-1-producing isolates of P. aeruginosa and Acinetobacter spp. were also shown to be associated with a poor clinical outcome (38).

In PER-1-producing P. aeruginosa, the ESBL gene was found to be either inserted in the chromosome (27, 41) or carried on large conjugative plasmids (11). A plasmid location of the blaPER-1 gene was also reported in Salmonella (7), while a chromosomal location was reported in Acinetobacter baumannii (42). The relatively broad host range exhibited by the blaPER-1 gene and its heterogeneous genetic location underscore a notable potential for the mobility of this resistance determinant. However, the nature of the genetic element(s) carrying the blaPER-1 gene has not been investigated in detail.

In this report we describe the structure of a plasmid-borne transposonlike element of the Tn3 family carrying the blaPER-1 gene from an A. faecalis clinical isolate. The element is a Tn5393 derivative of original structure, named Tn5393d, which also contains additional determinants for resistance to aminoglycosides.

(These results were presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 2004.)


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MATERIALS AND METHODS
 
Bacterial strains. A. faecalis FL-424/98 was a clinical isolate from Varese University Hospital (northern Italy) and was isolated in 1998. The strain was resistant to several antimicrobial agents, including expanded-spectrum cephalosporins and aminoglycosides, and it was found to produce the PER-1 ESBL (29). E. coli ATCC 25922 was used for quality control of susceptibility testing. E. coli DH5{alpha} (33) was used as the host for plasmids. E. coli MKD135 (argH rpoB18 rpoB19 rpsL; kindly provided by G. Kholodii, Institute for Molecular Genetics, Russian Academy of Sciences, Moscow, Russia) and P. aeruginosa 10145/3 (an rpoB his derivative of the reference strain ATCC 10145T) (27) were used as the recipients in the conjugation experiments.

In vitro susceptibility testing. In vitro susceptibility testing was carried out by a broth microdilution method, as recommended by the CLSI (formerly the National Committee for Clinical Laboratory Standards) (23, 24), by using cation-supplemented Mueller-Hinton (MH) broth (Difco Laboratories, Detroit, Mich.) and a bacterial inoculum of 105 CFU per well. Antibiotics were from Sigma Chemical Co. (St. Louis, Mo.). Resistance to mercury ions was assessed by growing the bacteria on Luria-Bertani medium (Difco Laboratories) containing HgCl2 (10 µg/ml).

DNA analysis methodology. Extraction of genomic DNA from A. faecalis was carried out as described previously (13). Extraction of plasmid DNA from E. coli was carried out by the alkaline lysis method (33). Southern blot analysis was carried out directly on dried gels as described previously (37). A probe containing the entire blaPER-1 open reading frame (ORF), generated by PCR with primers BLAPER/f and BLAPER/r (Table 1) as described previously (29) and labeled with 32P by the random priming technique with a commercial kit (RediPrimer I; Amersham Biosciences, Uppsala, Sweden), was used in Southern blot experiments. The structure of Tn5393d was determined by cloning and PCR mapping experiments, as follows. A library of plasmid pFL424 was constructed in the E. coli plasmid vector pBC-SK (Stratagene Inc., La Jolla, Calif.) by partial digestion of pFL424 with Sau3AI and cloning of the restriction fragments (fragments of 2 to 8 kb, which were purified by agarose gel electrophoresis) in pBC-SK digested with BamHI. The library, transformed in E. coli DH5{alpha}, was screened for clones containing the blaPER-1 gene by using the same probe used for the Southern blot experiments (see above). Two hybridization-positive clones (pEM-9 and pEM1.2) containing partially overlapping DNA inserts were used for analysis of the sequences of the regions flanking blaPER-1 (Fig. 1). A probe generated by PCR with primers BLAPER/r and aphA6/f (Table 1) was then used for a second screening of the pFL424 library, yielding two additional clones (pEM-51 and pEM-43) containing DNA inserts from the region upstream of blaPER-1 (Fig. 1). PCR mapping with pFL424 as the template and direct sequencing of PCR products were then used to confirm the authenticity of the assembled sequences and to determine the complete transposon structure. The primers used for PCR mapping of Tn5393d are listed in Table 1, and their locations are shown in Fig. 1. Long PCR for amplification of the entire Tn5393d sequence with primer IR5393/U was carried out in a 25-µl volume with the Expand 20-kbPLUS PCR system (Roche Biochemicals, Mannheim, Germany) and with the buffer system provided by the manufacturer, 1 enzyme unit, 10 pmol of primer, 10 ng of plasmid template, and the following reaction conditions: initial denaturation at 93°C for 3 min; annealing at 60°C for 30 s, extension at 68°C for 18 min, and denaturation at 92°C for 30 s, repeated for 30 cycles; and then annealing at 60°C for 1 min and extension at 68°C for 30 min. PCR amplifications of smaller fragments of Tn5393d were carried out in a 50-µl volume with the Expand PCR System (Roche Biochemicals) and with the buffer system provided by the manufacturer, 3 enzyme units, 40 pmol of primer, 30 ng of plasmid template, and the following reaction conditions: initial denaturation at 96°C for 3 min and then annealing at 50°C for 1 min, extension at 72°C for 1 min/kb, and denaturation at 94°C for 45 s, repeated for 35 cycles. The following primer combinations were used: IR5393/u plus aphA6/r (amplification product, 5,065 bp), aphA6/f plus BLAPER/r (amplification product, 5,162 bp), and BLAPER/f plus IR5393/u (amplification product, 4,978 bp) (Fig. 1). The complete sequences of the inverted repeats (IRs) and the sequences of the regions flanking Tn5393d were determined directly on pFL424 as the template. DNA sequences were determined on plasmid templates or PCR products, as described previously (31), by using custom sequencing primers. Both strands were sequenced. Analysis and comparison of the sequence data were carried at the ExPASy server (http://us.expasy.org/) and at the BLAST interface of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). Plasmid pEM-9/rH (Fig. 1), which contained a complete copy of the aphA6b gene and which was used for expression experiments with this gene, was constructed by digestion of plasmid pEM-43 (Fig. 1) with HindIII and self-ligation.


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  		TABLE 1. Primers used for generation of the probes used for Southern blot experiments for screening the pFL424 library, and/or for PCR mapping of Tn5393da



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  		FIG. 1. Schematic representation of the structure of Tn5393d, the Tn5393 derivative from plasmid pFL424 which carries the blaPER-1 gene. The structures of other known Tn5393 derivatives (9, 18, 35) are shown above for comparison. The compositions of the inserts of plasmids pEM-1.2, pEM-9, pEM-51, and pEM-43, selected from the pFL424 library and used to determine the structure of the genetic element, and that of plasmid pEM-9/rH, constructed to perform expression experiments with the aphA6b gene, are also shown below the Tn5393d map. The positions of the primers used for PCR mapping experiments are indicated by numbered arrows (1, IR5393/u; 2, aphA6/f; 3, aphA6/r; 4, BLAPER/f; 5, BLAPER/r), and the corresponding PCR products are indicated by thin horizontal bars. The primer sequences are reported in Table 1. The positions of the probes used to screen the pFL424 library are shown by hatched bars.

Gene transfer experiments. Conjugation assays were carried out in MH agar for 8 h at 37°C. Donor-to-recipient ratios of 1:10 or 2:5 were used with the E. coli and the P. aeruginosa recipients, respectively. Transconjugants were selected on MH agar containing rifampin (250 µg/ml) plus ceftazidime (50 µg/ml). With either recipient, the detection sensitivity of the conjugation assays was ≥10–8 transconjugants/recipient. E. coli HB101(pRP1.2) (15) and HB101(pRP1.2/pAX22) (pAX22 is a mobilizable plasmid carrying the metallo-ß-lactamase gene blaVIM-1) (30) were used as positive control plasmid donors in conjugation assays with E. coli MKD135 (transfer frequency, approximately 2 x 10–2 transconjugants/recipient, with selection for tetracycline resistance) and P. aeruginosa 10145/3 (transfer frequency, approximately 3 x 10–5 transconjugants/recipient, with selection for tobramycin resistance), respectively. Electroporation of plasmid DNA from FL-424/98 into E. coli DH5{alpha} was carried out with approximately 250 ng of whole genomic DNA of FL-424/98 by using a Gene Pulser apparatus (Bio-Rad, Richmond, Calif.) under the experimental conditions recommended by the manufacturer. Transformants were selected on MH agar containing ceftazidime at 50 µg/ml.

Transposition assays. Transposition of Tn5393d or modules thereof containing the ESBL determinant was assayed by following a strategy similar to that successfully used to demonstrate the transposition of Tn5393 and Tn5393c (9, 18). Briefly, high-copy-number plasmid pBC-SK, low-copy-number plasmid pACYC184 (8), or the single-copy bacterial artificial chromosome pBeloBAC11 (17) was transformed into E. coli DH5{alpha}(pFL424). The resulting strains were grown aerobically at 25°C either in Super Broth (33) (for strains containing pFL424 plus pBC-SK or pFL424 plus pACYC184) or in MH broth (for strain containing pFL424 plus pBeloBAC11) containing chloramphenicol (85 µg/ml) and ceftazidime (50 µg/ml); and plasmid DNA was extracted after approximately 15, 30, 45, and 60 generations. Plasmid preparations were analyzed by agarose gel electrophoresis to screen for the presence of plasmid bands of modified size. Plasmid preparations were also diluted to a concentration of approximately 10 ng/µl and transformed into E. coli DH5{alpha} by electroporation. Ceftazidime-resistant transformants (selected on MH agar containing ceftazidime 50 µg/ml) were replica plated on MH agar containing chloramphenicol (85 µg/ml), and transformants resistant to both antimicrobial agents were analyzed for their plasmid contents.

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank database and assigned accession number AJ627643.


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RESULTS AND DISCUSSION
 
Characterization of plasmid pFL424 from A. faecalis FL-424/98 carrying the blaPER-1 gene. A. faecalis FL-424/98 is a clinical isolate that was previously shown to produce the PER-1 ESBL (29). Mating experiments, carried out with FL-424/98 as the donor and either E. coli MKD135 or P. aeruginosa 10145/3 as the recipient, failed to demonstrate the transferability of the ESBL determinant by conjugation. However, a blaPER-1-containing plasmid could be transferred to E. coli DH5{alpha} by electroporation with total genomic DNA of FL-424/98. The plasmid was named pFL424, and its size was estimated to be approximately 44 kb according to restriction analysis. Southern blot analysis confirmed the presence of the blaPER-1 gene in pFL424 (data not shown).

Analytical isoelectric focusing of a crude extract of DH5{alpha}(pFL424) revealed a ß-lactamase band of pI 5.4 (data not shown). This finding was consistent with the expression of PER-1 (26).

Compared to DH5{alpha}, DH5{alpha}(pFL424) was resistant to several ß-lactams, including extended-spectrum cephalosporins and aztreonam (Table 2), in agreement with the production of the PER-1 ESBL. It also exhibited reduced susceptibility to some aminoglycosides (streptomycin and kanamycin) (Table 2), suggesting that pFL424 also carried one or more determinants for resistance to these drugs.


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  		TABLE 2. Antimicrobial susceptibilities of A. faecalis FL-424/98, E. coli DH5{alpha}(pFL424), and E. coli DH5{alpha} containing various pFL424 subclonesa

Genetic context of the blaPER-1 gene carried on pFL424, the Tn5393d transposonlike element. The genetic context of the blaPER-1 gene carried on plasmid pFL424 was investigated by a cloning and PCR mapping approach as described in Materials and Methods. The sequence data assembled revealed that blaPER-1 was embedded in a transposonlike element of the Tn3 family derived from Tn5393c (18), which contains a large insertion of genetic material of original structure (Fig. 1). This new Tn5393 derivative is 14,184 bp and was named Tn5393d. Tn5393d has 81-bp IRs and a transposition module identical to those of Tn5393c (18). Compared to Tn3 (12), the tnpA and tnpR genes of Tn5393d exhibit 47.2% and 47.7% sequence identities, respectively, while the IRs are longer (81 versus 38 nucleotides) and are 42% divergent in the homologous region.

A partial copy of the strAB genes (strAB{Delta}1) is present downstream of tnpR, while a complete copy of the strAB genes is present upstream of IR-5393o, resulting in a partial duplication of the strAB resistance module typical of the Tn5393 elements (Fig. 1). In strAB{Delta}1, most of the strB gene is deleted, and the sequence merges directly into a partially deleted copy of IS1012 (named IS1012L{Delta}1), an insertion sequence of the IS1 family that has previously been described, although partially, in plasmid pKLH205 from an Acinetobacter environmental isolate (EMBL/GenBank accession no. AJ459234) (16). A complete copy of IS1012 (named IS1012R), in the same orientation as IS1012L{Delta}1, is present upstream of the complete set of strAB genes (Fig. 1). IS1012R reveals, for the first time, the complete structure of IS1012 (Table 3). The region between the two copies of IS1012 contains an allelic variant of the aphA6 gene that was previously detected in a plasmid from an A. baumannii clinical isolate and that encodes an APH(3') aminoglycoside phosphotransferase (20). This aphA6 allelic variant, named aphA6b, encodes a protein which is 93% identical to AphA6 and which retains the three highly conserved motifs typical of APH(3') enzymes (Fig. 2). The putative promoter sequences and ribosome-binding site previously identified upstream of aphA6 (20) were found to be fully conserved in the sequence upstream of aphA6b. However, a better putative promoter could be at positions 4819 to 4824 (–35 hexamer) and 4842 to 4847 (–10 hexamer). Whether a similar promoter is also conserved upstream of aphA6 remains unknown, since the sequence upstream aphA6 is not available up to that point (20).


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  		TABLE 3. Features of the original insertion sequences detected in Tn5393d



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  		FIG. 2. Comparison of the amino acid sequences of the aphA6 (20) and the aphA6b gene products. Identical residues are indicated by asterisks. The conserved motifs typical of APH(3') enzymes (20) are boxed.

IS1012R is interrupted by the presence of a putative composite transposon, named Tn4176. Tn4176 is made of two original insertion sequences of the IS4 family, named IS1387a and IS1387b, which bound to a region containing the blaPER-1 gene and an ORF which encodes a protein that exhibits the highest similarity (25% amino acid identity) to a putative glutathione-S-transferase of Ralstonia solanacearum (32). This ORF starts 73 bp downstream the blaPER-1 gene and is truncated at the carboxy terminus by the presence of IS1387b (Fig. 1). IS1387a and IS1387b are not identical to each other (65% nucleotide sequence identity) but exhibit the same size, the same orientation, and very similar terminal IRs (Table 3). Tn4176 is inserted into the putative transposase gene of IS1012R, and an 8-bp duplication (GGCGTAAA) flanks the site of insertion, suggesting insertion by transposition. No direct repeats flanking the individual IS1387a and IS1387b insertion sequences were detectable, a finding which would support the composite transposon nature of Tn4176. IS1387a is interrupted by an original insertion sequence of the IS30 family, named IS1066 (Fig. 1). IS1066 is inserted into the putative transposase gene of IS1387a, and a 4-bp duplication (GTTG) flanks the site of insertion, suggesting insertion by transposition. Interestingly, in PER-1-producing P. aeruginosa isolates from northern Italy (27), the blaPER-1 gene was found to be associated with an element identical to Tn5393d but lacking IS1066 (E. Mantengoli and G. M. Rossolini, unpublished results). It should also be noted that in Tn5393d the sequence upstream of blaPER-1 is identical to that found upstream of blaPER-1 in the RNL-1 PER-1-producing index strain (25) for the first 27 bp, but thenceforth, the two sequences abruptly diverge from each other. The point at which the two sequences diverge falls inside the IRR of IS1387a. Altogether, these findings suggest the existence of some diversity among blaPER-1-containing elements.

A summary of the features of the new insertion sequences associated with Tn5393d is reported in Table 3.

Functional analysis of the aphA6b aminoglycoside phosphotransferase gene product. Compared to E. coli DH5{alpha}, DH5{alpha}(pEM-43) and DH5{alpha}(pEM-9/rH), which contained a cloned copy of the aphA6b gene on a multicopy plasmid vector (Fig. 1), exhibited reduced susceptibilities to kanamycin and amikacin but not to gentamicin, tobramycin, netilmicin, sisomicin, or spectinomycin. Susceptibility to streptomycin was only slightly affected (Table 2). Thus, the spectrum of activity of the enzyme was, overall, consistent with that reported for AphA6 from A. baumannii BM2580 (19).

The amikacin and kanamycin MICs for E. coli DH5{alpha} carrying the cloned aphA6b gene were notably lower that those reported for E. coli HB101 carrying the cloned aphA6 gene (19). This could be the case for one or more of several reasons, including (i) an inefficient activity of the aphA6b promoter in E. coli which could be different from that present upstream of the aphA6 gene (see above), (ii) poor stability of the AphA6b enzyme when it is expressed in this E. coli host, or (iii) low specific phosphotransferase activity of the AphA6b enzyme toward those aminoglycoside substrates. However, given the strong similarity between AphA6b and AphA6 and the possible divergence of the sequences upstream of the two genes, differences at the level of gene expression would seem to be the more plausible reason that accounts for this observation. The fact that DH5{alpha}(pFL424) exhibited kanamycin and amikacin MICs lower than those of DH5{alpha}(pEM-43) and DH5{alpha}(pEM-9/rH) (Table 2) likely reflects the lower gene dosage present in the former strain due to a lower copy number of pFL424. The fact that a similar effect was not observed for the streptomycin MIC [which was actually higher for DH5{alpha}(pFL424)] is likely due to the additional presence of the strAB streptomycin resistance module in the original plasmid.

The insertion site of Tn5393d. Sequence analysis of the regions flanking Tn5393d, spanning 776 bp upstream of IRt and 413 bp downstream of IRo, revealed that the transposon was inserted into a Tn501 backbone (Fig. 3). Tn501 is a mercury resistance transposon of the Tn3 family originally found in a P. aeruginosa plasmid (5, 34). The insertion site of Tn5393d is located in orf2 of Tn501, which encodes a conserved protein of unknown function (EMBL/GenBank accession no. Z00027), and is flanked by a 5-bp direct repeat (Fig. 3), suggesting that the presence of Tn5393d in this position was the result of a transposition event. Sequence analysis confirmed the presence of an orf2, part of orf1, and part of the res site identical to those of Tn501 (EMBL/GenBank accession no. Z00027) (Fig. 3). The structure of the Tn501-like element present in pFL424 was not further investigated in this work. However, the fact that, unlike DH5{alpha}, E. coli DH5{alpha}(pFL424) was able to grow on mercury-containing medium (10 µg/ml) suggested that a mercury resistance module was also present on the plasmid.



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  		FIG. 3. Insertion site of Tn5393d into orf2 of Tn501. The partial Tn5393d IR sequences are boldfaced. The 5-bp direct repeats flanking the insertion site are boxed. The numbering is based on that for the sequence with EMBL/GenBank accession no. Z00027. A map of Tn501 (EMBL/GenBank accession no. Z00027) is also shown. The extents of the sequenced regions flanking the insertion site of Tn5393d are indicated by a dotted line above the map.

The insertion of Tn5393d in orf2 of a Tn501-like backbone, in proximity of the res site and associated with a 5-bp target duplication, could relate to the serine recombinase-targeting mechanism previously described for Mu-related transposons such as Tn402/Tn5090 (14) and Tn5053 (21).

Studies of mobility of Tn5393d. Transposition assays were carried out to detect the mobility of Tn5393d or modules thereof containing the blaPER-1 gene from plasmid pFL424 to plasmid pBC-SK, pACYC184, or pBeloBAC11 by following an experimental strategy that was overall similar to that successfully used to demonstrate the transposition of Tn5393 and Tn5393c (9, 18). By use of this strategy, no transposition events from the donor to the recipient plasmids were detected after up to 60 generations either in E. coli DH5{alpha}(pFL424/pBC-SK), in E. coli DH5{alpha}(pFL424/pACYC184), or in E. coli DH5{alpha}(pFL424/pBeloBAC11).

The apparent lack of mobility of Tn5393d could reflect a low transposition efficiency of Tn5393d due to its complex structure and/or the genetic context in which it is found in pFL424. However, it could also be due to the fact that the experimental conditions adopted (host, recipient plasmids, and/or culture conditions) were not suitable for the detection of transposition events. However, the presence of a full set of elements involved in the transposition machinery, including a transposition module identical to that of Tn5393c, suggests that Tn5393d could retain the potential for transposition. The apparent lack of mobility of Tn4176 could be due to similar reasons, but it could also reflect the insertion of IS1066 into IS1387a, which is one of the insertion sequences that constitute the putative composite transposon.

Hypothetical origin of Tn5393d. The structure of Tn5393d led us to hypothesize the following steps as an explanation for the genesis of this element. The first step was likely represented by the insertion of a composite resistance transposon (made by two IS1012 insertion sequences that had captured the aphA6b gene) into the region between tnpR and strAB or between strAB and IRo of Tn5393c, apparently accompanied by a duplication of the strAB module, resulting in the presence of two such modules flanking the composite transposon. A deletion involving part of strB and part of IS1012L could have then followed, resulting in the fixation of the aphA6b aminoglycoside resistance gene while leaving the element with a complete set of the strAB module. This hypothesis is consistent with the fact that IS1012R is not flanked by direct repeats. Insertion of Tn4176, which carries the blaPER-1 gene, into IS1012L could have occurred either before or after the deletion involving strB and IS1012L, leading to the additional acquisition of the ESBL determinant on the transposon. Insertion of IS1066 into IS1387a likely represents the most recent event. The finding of a Tn4176 that lacks IS1066 in a Tn5393d-like element from PER-1-producing P. aeruginosa clinical isolates from northern Italy (Mantengoli and Rossolini, unpublished) supports this view. The insertion of IS1066 in this position could affect the mobility of Tn4176 and/or the expression of the blaPER-1 gene. However, the actual role of IS1066 in this sense remains to be established and will be the subject of future work.

Concluding remarks. Mobile DNA plays a major role in the dissemination of antibiotic resistance determinants. Investigation of the genetic elements involved in the flow of resistance genes from the environmental gene pool to that of pathogenic bacteria and among the latter is important to obtain an understanding of the mechanisms and pathways that lead to the dissemination of clinically relevant resistance genes.

In this work we have described a novel derivative of the Tn5393 transposon which, in addition to the strAB genes, which confer resistance to streptomycin (10), also carries the blaPER-1 gene (which encodes a powerful ESBL) and an allelic variant of the aphA6 aminoglycoside phosphotransferase gene (whose product is active against kanamycin, streptomycin, and amikacin). Tn5393d, therefore, can function as a vehicle for at least two clinically important resistance genes; and this association could explain, at least in part, the coresistance to extended-spectrum cephalosporins and amikacin that has often been reported in PER-1-producing clinical isolates (27, 39, 40, 42). To the best of our knowledge, this is the first example of a Tn5393 derivative that carries clinically relevant resistance genes in addition to the strAB module (Fig. 1). Tn5393-like transposons have been detected in animal, plant, and human pathogens (22, 36). The present results and the finding of a very similar element in PER-1-producing clinical isolates of P. aeruginosa from Italy (Mantengoli and Rossolini, unpublished) underscore the notion that these highly promiscuous elements can play an increasingly important role in the dissemination of relevant resistance determinants in the clinical setting.

As a final remark, the fact that the insertion sequences which constitute Tn4176 as well as IS1066 exhibit the strongest similarities with insertion sequences from environmental bacteria living in aquatic environments (Table 3) would suggest, for blaPER-1, a likely origin from some unknown species of bacteria living in similar environments.


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ADDENDUM IN PROOF
 
After the revised version of this paper had been submitted, another paper reporting on the genetic environment of the blaPER-1 gene in some gram-negative bacteria was published (L. Poirel, L. Cabanne, H. Vahaboglu, and P. Nordmann, Antimicrob. Agents Chemother. 49:1708-1713, 2005). The internal region of Tn5393d described in this paper (including the blaPER-1 gene and its genetic environment) is closely related, although not identical, to the genetic environments of blaPER-1 described by Poirel et al. In particular, the insertion sequences IS1012R, IS1387a, and IS1387b are identical to ISPa14, ISPa12, and ISPa13, respectively, described in that paper, while the composite transposon Tn4176 corresponds to the composite transposon Tn1213 described in that paper.


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ACKNOWLEDGMENTS
 
This work was supported in part by a grant from the FPVI of the European Commission (Specific Targeted Research Project COBRA: Combating bacterial resistance to antibiotics, LSHM-CT-2003-503335).

We are grateful to Milva Pepi for help and advice with the testing for mercury resistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, Policlinico Santa Maria alle Scotte, 53100 Siena, Italy. Phone: 39 0577 233326. Fax: 39 0577 233334. E-mail: rossolini{at}unisi.it. Back


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Antimicrobial Agents and Chemotherapy, August 2005, p. 3289-3296, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3289-3296.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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