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Antimicrobial Agents and Chemotherapy, November 2007, p. 4187-4190, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00542-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Tn6001, a Transposon-Like Element Containing the blaVIM-3-Harboring Integron In450{triangledown}

Sung-Pin Tseng,1 Po-Ren Hsueh,2 Jui-Chang Tsai,3,4 and Lee-Jene Teng1,2*

Department of Clinical Laboratory Sciences and Medical Biotechnology,1 Center for Optoelectronic Biomedicine, National Taiwan University College of Medicine,3 Department of Laboratory Medicine,2 Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan4

Received 24 April 2007/ Returned for modification 10 June 2007/ Accepted 28 August 2007


   ABSTRACT
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 ABSTRACT
TEXT
 REFERENCES
 
We describe the structure of a transposon-like element named Tn6001, which contains a blaVIM-3-harboring integron In450, which was derived from a multidrug-resistant Pseudomonas aeruginosa clinical isolate in Taiwan. The transposon backbone structure is most closely related to those of Tn1404* and Tn1403. Tn6001 was inserted into the chromosome of the clinical isolate.


   TEXT
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 ABSTRACT
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REFERENCES
 
Multidrug-resistant (MDR) Pseudomonas aeruginosa has caused serious problems in many countries, including Taiwan (5, 7, 10). Resistance to carbapenems in MDR P. aeruginosa strains is a major concern. Resistance to carbapenems in P. aeruginosa is usually associated with impermeability that arises via the loss of the oprD-encoded porin, active efflux pump, and/or the production of metallo-ß-lactamases (MBLs), such as the IMP- and VIM-type MBLs (5, 6, 19, 20, 22).

Strains producing the VIM-3 MBL were first identified in Taiwan in 2001 (20). VIM-3 is almost identical to VIM-2, differing in only two amino acids (20). We reported a nosocomial infection caused by MDR P. aeruginosa carrying the blaVIM-3 gene in 2005 in Taiwan (5). Thereafter, Yan et al. described an unusual integron containing the blaVIM-3 gene (21). However, the complete structure of the element containing the blaVIM-3 gene and the mechanism of its spread remain unclear.

In this work, we report the structure of a transposon-like element (named Tn6001) which carries a blaVIM-3-containing integron and exhibits a backbone structure closely related to those of Tn1404* and Tn1403, which belong to the Tn501 family of transposons (15).

NTUH-PA450, a VIM-3-positive MDR P. aeruginosa strain isolated in 2003 at National Taiwan University Hospital, was used as a representative isolate for the subsequent cloning and sequencing of the VIM-3-containing element. Southern blot analysis revealed that a blaVIM-3-specific probe hybridized to a 6.5-kb EcoRI-digested DNA fragment of NTUH-PA450. After the EcoRI-digested DNA fragments were ligated with cassette adapters, amplification was performed with cassette primers supplied by the manufacturer (C1 for the first PCR and C2 for the second PCR) and a target gene-specific primer, blaVIM-3_448R (5'-AGAGTCCTTCTAGAGAGTGCGTGGGAAT-3') under PCR conditions, followed by PCR performed using a long and accurate PCR in vitro cloning kit (Takara Shuzo Co. Ltd., Japan). The amplified product from the second PCR was subsequently sequenced. The sequence data of the 6.5-kb fragment revealed that it contained the blaVIM-3 cassette inserted into a class 1 integron associated with a transposon-like element with putative tnpA and tnpR genes and a potential 38-bp inverted repeat sequence, Tn-IR (5'-GGGGCGAATAGAGAAAACGGAAAAAATCGTACGCTAAG-3') typical of the Tn501 family of transposons (4). Since it was likely that the 3' end of the transposon contained the same terminal 38-bp inverted repeat sequence, we used the blaVIM-3 sequence, vim-outF (5'-GAGCGGATTCAACAACACTACCCG-3') as a forward primer and the Tn-IR sequence as a reverse primer to amplify a ca. 8.5-kb fragment covering the region downstream of blaVIM-3 by PCR.

The assembled sequence was made up of a 14,627-bp-long transposon-like element comprising an integron harboring multiple resistance genes, including blaVIM-3, which was designated Tn6001 (Fig. 1A). The length of Tn6001 was also confirmed by PCR using Tn-IR as the single primer (data not shown). Sequence analysis and comparison with published sequences in the GenBank database revealed 17 putative open reading frames (ORFs) in Tn6001.


Figure 1
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  		FIG. 1. Genetic organization of transposon Tn6001 in Pseudomonas aeruginosa NTUH-PA450. Genes are shown as arrows with the direction of transcription indicated by the arrowheads. A4, aacA4 gene. (A) Structures of Tn6001 and related backbone transposons (Tn1403 and Tn1404*). Tn6001 contains a blaVIM-3-harboring integron In450. Part of In450 is nearly identical to that in strain N2-1 reported earlier by Yan et al. (21) (GenBank accession no. DQ393784). The GenBank accession no. for Tn1403 is AF313472. The structural information for Tn1404* is from data reported by Stokes et al. (15). TIRL, left-hand transposon inverted repeat; f-TIRR, potential right-hand transposon inverted repeat. Restriction enzyme sites: B, BamHI; E, EcoRI; H, HindIII. IRi, left-hand integron inverted repeat (25 bp); IRt, right-hand integron inverted repeat (25 bp). (B) Comparison of In450 with other In4-like class 1 integrons. Two copies of IRt flanking IS6100 are present in In4. A partial copy ({triangleup}) of IS6100 is shown. The GenBank accession numbers for In4 and InpCG4 are U12338 and AF164956, respectively.

 
The backbone structure of Tn6001 is most closely related to those of Tn1403 (GenBank accession no. AF313472) (15) and Tn1404*, which was deduced from that of Tn1404 (GenBank accession no. AF157797 to AF157801) (14), and gaps were filled in using available sequences (15). The inverted repeat (38 bp) identified on the left end of Tn6001 is identical to that of Tn1403 and Tn1404*. Two ORFs, tnpA (2,810 bp) and tnpR (560 bp), located on the left end of Tn6001, encoding the transposase and resolvase, respectively, are 95.8% and 89.6% identical to those of Tn1403. There are four ORFs to the right of Tn6001 (Fig. 1A). These ORFs include a truncated orfA (which is interrupted by IS6100), orfB (90.5% and 97.0% identity to those in Tn1403 and Tn1404*, respectively), orfC (91.4% and 97.4% identity to those in Tn1403 and Tn1404*, respectively), and orfD (93.0% and 96.3% identity to those in Tn1403 and Tn1404*, respectively) (14, 15). These findings suggest that Tn6001, Tn1403, and Tn1404* may have originated from a common ancestor transposon and acquired different drug resistance genes via the acquisition of different integrons. The significance of the homology of the deduced proteins encoded by orfB, orfC, and orfD genes with the putative nucleotide-binding protein, a DnaK suppressor protein and the sdiA-regulated protein, respectively, is unclear (14, 15).

The blaVIM-3-containing integron identified in P. aeruginosa NTUH-PA450 included a gene cassette array which is nearly identical to that in strain N2-1 (21), with blaVIM-3, orf2 (putative fosfomycin resistance gene), two copies of aacA4 and aadB, and another aacA4 cassette (Fig. 1A). The blaVIM-3-containing integron carried by Tn6001 is related to those of the In4-like family and was designated In450. In450 contains a 1,352-bp 5'-conserved segment (5'-CS), an attI1 site, a 2,239-bp 3'-CS segment, and one copy of IS6100 (Fig. 1B). Unlike In4, In450 does not include a 19-bp duplication in the attI1 site, similar to InpCG4 (16) and In28 (11). The 3'-CS segment includes the qacE{Delta}1 gene, the sul1 gene, and two ORFs of unknown function, orf5 and orf6 (11, 12). The structure of In450 is very similar to those of In4 and InpCG4, but In450 has different downstream sequences adjacent to IS6100. In In4, the IS6100 region (one complete copy and one partial copy) is flanked by two short segments, which are the internal 123-bp fragment with integron inverted repeat (IRt) (on the left) and the outer 152-bp fragment with IRt (on the right). The internal 123-bp fragment is also found in InpCG4 and In450, suggesting that they may be derived from the same ancestor (Fig. 1B). Comparison of In450 with In4 and InpCG4 shows that there is no IRt on the right of IS6100 in InpCG4 and In450 (Fig. 1B).

Minor differences between In450 and the integron in strain N2-1, previously reported by Yan et al. (21) were also seen in this study of P. aeruginosa NTUH-PA450. The region upstream of the intI1 gene in the integron from strain N2-1 has a 65-nucleotide deletion, while in In450, this region is conserved, as in most other class 1 integrons (3). The intI1 gene in In450 also has a point mutation (underlined) resulting in an amino acid change on Asn39His (AAC->CAC). This mutation is located on the –35 region of a common promoter (Pc) and is responsible for the expression of gene cassettes (3). The promoter sequence in In450 is identical to a "hybrid" promoter in In1; thus, it is predicted to express an intermediate level of resistance (8). The first two copies of aacA4 genes in In450 are identical and are predicted to confer resistance to amikacin but not to gentamicin (17). The third copy of aacA4 has a replacement of Leu102 (TTA) with Ser (TCA), which is associated with a change in the resistance phenotype (from amikacin to gentamicin) (2).

The res site of Tn501-like transposons is usually made up of three resolvase-binding domains (sites I, II, and III) that contain the crossover point for cointegrate resolution (13) and which represents a preferential insertion site for Tn402-like transposons associated with class 1 integrons (9). In Tn1403 (or Tn1404*), the integron is inserted in resI, with part of resI identifiable on each side and with a 5-bp duplication of the backbone sequences (11, 15). In Tn1696, In4 lies between resI and resII sites. Comparing the res site sequence of Tn6001 with those of Tn501 (1), Tn1721 (13), Tn4656 (18), Tn1403 (15), and Tn1696 (11), we found that the res site in Tn6001 contains only resIII and a partial resII (it lacks resI) (Fig. 2). In450 was apparently inserted within resII, while no part of resII and resI sequences is present downstream of IS6100, and no 5-bp duplication flanking the insertion site was detected. Therefore, the insertion within res II by In450 is unlike those found in other transposons.


Figure 2
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  		FIG. 2. Comparison of the res site sequence of Tn6001 with those of some class II transposons. The locations of site I, site II, and site III within the res site are indicated. The arrows indicate the insertion site by integrons. Tn6001 contains resIII and a partial resII region but lacks resI. The predicted start codon (GTG or TTG) of tnpR gene is underlined, and the predicted ribosome-binding site (RBS) for the tnpR gene and the promoter regions (–35 and –10) are indicated. Deleted nucleotides (periods) and nucleotides that are different from those in Tn501 (lowercase letters) are indicated. The GenBank accession numbers for Tn501, Tn1721, Tn4656, Tn1403, and Tn1696 are Z00027, X61367, AJ344068, AF313472, and U12338, respectively.

 
We used I-CeuI restriction and hybridization to determine the location of the blaVIM-3-genetic element in P. aeruginosa. One band was hybridized by both the blaVIM-3 probe and 16S rRNA gene probe (Fig. 3), indicating the chromosomal location of the blaVIM-3 gene.


Figure 3
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  		FIG. 3. Localization of blaVIM-3 on I-CeuI-generated chromosome fragments of P. aeruginosa NTUH-PA450. (A) I-CeuI restriction patterns separated by pulsed-field gel electrophoresis. Lane M, DNA size markers (Saccharomyces cerevisiae chromosomal DNA); lanes 1, P. aeruginosa NTUH-PA450. (B and C) Hybridization with a 16S rRNA gene-specific probe (B) or with a blaVIM-3-specific probe(C). The arrow indicates the band that hybridized with both the 16S rRNA gene and blaVIM-3 probe.

 
Nucleotide sequence accession number. The nucleotide sequence of Tn6001 from NTUH-PA450 isolate was deposited in GenBank under accession number EF138817. The Tn number was requested from the website http://www.ucl.ac.uk/eastman/tn/.


   ACKNOWLEDGMENTS
 
We thank Hsin-Chih Lai and Jun-Rong Wei for providing useful information about Tn501 family transposons and "single-primer PCR."


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, 1, Chang-Te Street, Taipei 100, Taiwan. Phone: 886-2-23123456, ext. 6918. Fax: 886-2-23711574. E-mail: ljteng{at}ntu.edu.tw Back

{triangledown} Published ahead of print on 10 September 2007. Back


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Antimicrobial Agents and Chemotherapy, November 2007, p. 4187-4190, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00542-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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