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Journal of Bacteriology, September 2003, p . 5314-5319, Vol . 185, No . 17

IS1999 Increases Expression of the Extended-Spectrum ß-Lactamase VEB-1 in Pseudomonas aeruginosa

Daniel Aubert, Thierry Naas,* and Patrice Nordmann

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre Cedex, France

Received 2 April 2003/ Accepted 5 June 2003


   ABSTRACT

 
The integron-borne blaVEB-1 gene encodes an extended-spectrum ß-lactamase . This gene was associated mostly with IS1999 and rarely with an additional IS2000 element in Pseudomonas aeruginosa isolates from Thailand, whereas IS1999 was only very rarely associated with blaVEB-1 in Enterobacteriaceae . Expression experiments and promoter study identified promoter sequences in IS1999 that increased the expression of VEB-1 in P . aeruginosa .


   TEXT

 
Antibiotic resistance in many gram-negative clinical isolates is due to resistance genes that are captured and expressed in class 1 integrons . These integrons possess two conserved regions located on each side of integrated gene cassettes (14) . The 5' conserved segment (5'-CS) includes a gene encoding an integrase, intI1, the cassette integration site, attI1, and the promoter Pant, sometimes associated with a second promoter P2, which are responsible for the expression of gene cassettes (6, 14) . The 3' conserved segment (3'-CS) includes, along with another open reading frame of unknown function, the disinfectant (qacE{Delta}1) and the sulfonamide (sul1) resistance determinants (14) . The expression of the inserted gene cassettes depends not only on sequences of promoters Pant/P2 but also on the gene cassette position relative to the 5'-CS (2) .

The integron-borne blaVEB-1 gene encodes the extended-spectrum ß-lactamase VEB-1 (Vietnamese extended-spectrum ß-lactamase) found initially in an Escherichia coli clinical isolate from Vietnam (13) . Subsequently, the veb-1 gene cassette was identified in two Pseudomonas aeruginosa clinical isolates from Thailand (9, 18) . In those strains, blaVEB-1 was associated with either one (IS1999) or two (IS1999/IS2000) insertion sequences (IS) that were inserted upstream of blaVEB-1 in the integron-specific recombination site, attI1 . Bacterial IS may bring promoters located in or near their inverted-repeat sequences (IR) that are capable of modulating the expression of neighboring antibiotic resistance genes (7) .

Previous studies performed with ceftazidime-resistant Enterobacteriaceae and P . aeruginosa strains isolated in 1999 from the Siriraj Hospital, Bangkok, Thailand (3, 4), resulted in reports that out of 37 enterobacterial isolates, 18 were blaVEB-1 positive (10 E . coli, 4 Enterobacter cloacae, 1 Enterobacter sakazakii, and 3 Klebsiella pneumoniae) and 19 were blaVEB-1 negative . Out of 33 ceftazidime-resistant P . aeruginosa isolates, 31 were blaVEB-1 positive . blaVEB-1 was mostly plasmid located in Enterobacteriaceae, whereas this gene was mostly chromosome encoded in P . aeruginosa (3, 4) . Moreover, spreading of blaVEB-1-containing P . aeruginosa strains was detected in several unrelated isolates carrying different integrons of various sizes and structures (3, 4) .

Distribution of IS1999 and IS2000 in blaVEB-1-positive isolates. The distribution of IS1999, an IS10-like element, and IS2000, which belongs to the IS5 family (7, 9), was investigated using the same blaVEB-1-positive isolates (Table 1) . Dot blot hybridizations were performed using whole-cell DNAs (12, 15) of the 18 Enterobacteriaceae- and the 31 P . aeruginosa-positive isolates (Table 1) . Using ECL nonradioactive labeling and detection kits (Amersham Biosciences, Orsay, France), hybridizations were performed under high-stringency conditions . The probes consisted of PCR-generated fragments internal to IS1999 and IS2000 (15) (primer sequences are available upon request) .


TABLE 1 . Bacterial strains and plasmids used in this study

 
Out of 18 blaVEB-1-positive enterobacterial isolates, none possessed IS2000 and only one K . pneumoniae isolate carried IS1999 . In contrast, IS1999 was found in 28 out of 31 (90%) blaVEB-1-containing P . aeruginosa isolates and 2 out of these 28 isolates had an additional IS2000 inserted within the IS1999 sequence .

Thus, the frequent association mostly of IS1999, which is sometimes associated with IS2000, with the blaVEB-1 gene in P . aeruginosa and its absence from Enterobacteriaceae, especially from E . coli, led us to study the contribution of these IS on ß-lactamase expression in both bacterial species .

Influence of IS1999/IS2000 on blaVEB-1 expression. Using three blaVEB-1-positive P . aeruginosa isolates as templates (P . aeruginosa 14, 1, and JES), recombinant plasmids were constructed containing blaVEB-1 without any IS (pDA-1), with IS1999 (pDA-2), and with IS1999 and IS2000 (pDA-3) (Fig . 1 and Table 1) . Plasmids were constructed by standard recombinant techniques (15) . The low-copy-number and broad-host-range cloning vector pBBR1MCS.3 that replicates in E . coli and in P . aeruginosa (5) was used for subcloning experiments, generating recombinant plasmids pInt-Veb, pInt-1999-Veb, and pInt-1999-2000-Veb (Fig . 1 and Table 1) . Recombinant plasmids were introduced by electroporation into E . coli DH10B (12) and P . aeruginosa KG2505 (11, 17) . P . aeruginosa KG2505 does not express the naturally chromosome-encoded AmpC ß-lactamase and is deficient for the multidrug efflux system MexAB-OprM (11) .


FIG.1 . Schematic map of the constructs used in this study . Constructs 1 (pDA-1, pInt-Veb, and pVeb), 2 (pDA-2, pInt-1999-Veb, pInt-1999*-Veb, p1999R-Veb, p1999-Veb, and p1999L-Veb), and 3 (pDA-3, pInt-1999-2000-Veb, and p1999R-2000R-Veb) were cloned from genomic DNAs of P . aeruginosa clinical isolates 14, 1, and JES, respectively . The blaVEB-1 gene was inserted in opposite orientation to Plac, thus removing any contribution of promoter Plac in ß-lactamase expression . The stop codon resulting from a site-directed mutagenesis experiment is shown by an asterisk (construct pInt-1999*-Veb) . Restriction sites that were used at each cloning step are underlined . The coding regions are shown as boxes, with an arrow indicating the orientation of their transcription . The IR of IS1999 and IS2000 are shown by filled and empty triangles, respectively . IRL and IRR of IS1999 are indicated for pDA-2 . The broken arrows indicate the promoter Plac. Thin dashed lines indicate ligation in the multiple-cloning site of the shuttle vector pBBR1MCS.3.

 
Sequencing was performed using laboratory-designed primers on an ABI PRISM 3100 automated sequencer (Applied Biosystems, Les Ullis, France) . Sequence analysis of inserts of all the recombinant plasmids revealed the presence of the two previously characterized promoters: (i) the weak Pant promoter and (ii) the P2 promoter that is likely nonfunctional (as previously shown) due to its reduced spacing between the -10 and -35 promoter sequences (14 bp instead of 17 bp) (2, 6) .

Using ß-lactamase extracts from cultures of E . coli DH10B and P . aeruginosa KG2505 harboring recombinant plasmids (Fig . 2) prepared as previously described (10), blaVEB-1 gene expression was then investigated by measuring the specific ß-lactamase activities for cefepime (a ß-lactam antibiotic hydrolyzed specifically by VEB-1) . Total protein contents and the initial rate of cefepime hydrolysis were determined as previously described (13) .


FIG . 2 . Comparative study (using cefepime as the substrate) of the specific activity of ß-lactamase VEB-1 from cultures of E . coli DH10B (left panel) and P . aeruginosa KG2505 (right panel) strains harboring recombinant plasmids . Error bars represent standard deviations calculated from five independent cultures . No measurable activity was detected for E . coli DH10B(pVEB) and for E . coli DH10B(p1999L-VEB).

 
To determine whether IS1999 and IS2000 carry active promoter sequences located in their right ends, Pant was deleted from pInt-Veb, pInt-1999-Veb, and pInt-1999-2000-Veb, generating recombinant plasmids pVeb, p1999R-Veb, and p1999R-2000R-Veb, respectively (Fig . 1 and Table 1) .

In E . coli, the highest ß-lactamase activity was measured for E . coli DH10B(pInt-Veb), which had blaVEB-1 located just downstream of the promoter Pant of the class 1 integron (Fig . 1 and 2) . Insertion of IS1999 (pInt-1999-Veb), which moved blaVEB-1 away from Pant, decreased blaVEB-1 expression (50% decrease) . After the removal of the promoter Pant (p1999R-Veb and p1999R-2000R-Veb), significant activity (45%) was still measured, suggesting the presence of an IS1999-located promoter . Similar specific activities were obtained for E . coli DH10B(pInt-1999-Veb), (p1999R-Veb), (pInt-1999-2000-Veb), and (p1999R-2000R-Veb), suggesting that Pant made only a minor contribution to the overall ß-lactamase expression . Thus, the activity measured in E . coli DH10B(pInt-1999-Veb) and (pInt-1999-2000-Veb) is mostly due to the presence of an IS1999-located promoter (and possibly to the presence of IS2000) .

Determination of specific activity of P . aeruginosa KG2505 cultures showed that surprisingly, the highest activity was measured for P . aeruginosa KG2505(pInt-1999-Veb) [a 60% increase compared to the activity of cultures of P . aeruginosa KG2505pInt-Veb)] (Fig . 2) . However, insertion of IS2000 (pInt-1999-2000-Veb) or deletion of the Pant promoter along with the left end(s) of IS1999 and/or IS2000 (p1999R-Veb and p1999R-2000R-Veb) led to a decrease in ß-lactamase activity . Thus, the right end of IS1999, which includes the left IR (IRL), most likely carried a functional outward-directed promoter capable of driving blaVEB-1 transcription in P . aeruginosa.

To determine whether the left end of IS1999 carried additional promoter sequences, plasmid p1999L-Veb was constructed . This plasmid contained two-thirds of the IS1999 sequence, including the right IR (IRR) located upstream of blaVEB-1 (Fig . 1) . Strains harboring p1999L-Veb plasmid had very low levels of ß-lactamase activity similar to those observed for strains harboring pVeb, suggesting the absence of functional promoter sequences in the left end of IS1999 .

To determine whether the functional transposase of IS1999 can influence blaVEB-1 expression, the plasmid pInt-1999*-Veb, which contains an interrupted open reading frame encoding the IS1999 transposase, was generated . No significant modification of blaVEB-1 expression was measured for strains harboring pInt-1999*-Veb compared to those harboring pInt-1999-Veb (Fig . 2) . Thus, the transposase had no or little effect on ß-lactamase expression in E . coli and P . aeruginosa .

Mapping of promoter Pout of IS1999. The precise location of the right-end-located promoter of IS1999 was determined with a primer extension system-AMV reverse transcriptase kit (Promega, Charbonnières, France) . Total RNAs were extracted with a Qiagen RNeasy Maxi kit (Qiagen, Courtaboeuf, France) . cDNAs were synthesized using 32P end-labeled primers Vebprom and Vebprom 1999, which annealed to the left end of veb-1 gene cassette and to the right end of IS1999 . Using a Sequenase version 2 DNA sequencing kit (Amersham Biosciences), manual sequencing was performed with the same primers . Sequencing and extension products were separated on an 8% polyacrylamide gel and were visualized by autoradiography after overnight exposure at -80°C .

Primer extension experiments (performed with Vebprom primer and RNAs from E . coli DH10B and P . aeruginosa KG2505 strains containing recombinant plasmids pInt-1999-Veb and pInt-1999-2000-Veb) generated a cDNA starting at a thymidine at nucleotide position 115 (Fig . 3) . Analysis of the sequence located upstream of bp 115 revealed a putative -35 promoter region (CAGTAT) separated by 17 bp from a -10 region (TAGGAT) (Fig . 3) . This promoter was located close to the IRL of IS1999 at a position similar to that of the promoter Pout (-35 [CAGAAT] and -10 [TAAAAT]) identified in the related IS element, IS10 (16) . Using the primer Vebprom 1999, which annealed upstream of Pout, no extension product was identified, indicating that no promoter sequence was located further inside of IS1999 or in IS2000 .


FIG . 3 . (A) Structure of IS1999 . The IS1999 IR are shown by filled triangles . The arrows indicate the orientation of transcription . The outward-directed promoter, Pout, and the promoter of the transposase gene, Pin, are indicated by broken arrows . The -10 and -35 regions for Pin and Pout are underlined . Nucleotide position 115 (according to the sequence GenBank AF133697) in IS1999 corresponds to the transcription start of Pout . The IRL sequence is boxed . (B) Mapping of transcription initiation . Primer Vebprom was extended using RNAs from cultures of E . coli DH10B(pInt-1999-Veb) (lane 1) or P . aeruginosa KG2505(pInt-1999-Veb) (lane 2) as the templates . Equal volumes (2 µl) of the extension product obtained from P . aeruginosa and E . coli were loaded onto the gel . Size markers were from sequencing reactions generated from pInt-1999-Veb DNA primed with Vebprom . G-, A-, T-, and C-specific lanes are indicated . The nucleotide sequences on the left side correspond to that of the complementary strand, which was deduced from the sequencing reaction . The -10 and -35 promoter sequences of Pout regions are shown, and the +1 transcriptional initiation site is indicated by an arrowhead . Similar results were obtained for RNA extracted from E . coli DH10B and P . aeruginosa KG2505 harboring pInt-1999-2000-Veb recombinant plasmid (data not shown).

 
Conclusions. This work identified a functional outward-directed promoter, Pout, of IS1999 that was capable of driving blaVEB-1 expression in E . coli and in P . aeruginosa . The level of expression obtained from Pant or Pout was about fourfold higher in E . coli than in P . aeruginosa . Furthermore, our results suggest that an association between IS1999 and the Pant promoter enhances blaVEB-1 expression by about 60% in P . aeruginosa but not in E . coli . An increase in ß-lactamase expression may change bacteria from being susceptible to being intermediate or even resistant . This is the case for P . aeruginosa harboring plasmid pInt-1999-Veb, for which certain ß-lactams, such as piperacillin and cefepime, that are extensively prescribed in hospitals display a twofold increase in MICs (data not shown) . Thus, in a hospital environment, where bacteria may be under constant antibiotic pressure, IS1999 (by enhancing the blaVEB-1 gene expression) might bring a selective advantage to P . aeruginosa, at least when it is present on a low-copy-number plasmid (pBBR1MCS) . Future experiments will be directed towards determination of blaVEB-1 gene expression in its native environment, i.e., the chromosome of P . aeruginosa isolates, to see whether our plasmid-mediated system mimics the chromosomal situation .

An increase of blaVEB-1 expression in P . aeruginosa KG2505 (pInt-1999-Veb) might be the result of a cooperative effect between Pout and Pant promoters . Indeed, the maximum amount of expression was obtained only when both promoters were present . In E . coli(pInt-1999-Veb), however, both promoters were present and still the expression decreased compared to that seen with E . coli(pInt-Veb) . These data may reflect major differences in transcriptional properties between P . aeruginosa and E . coli . The role of IS2000 in the blaVEB-1-containing integron remains unclear, but decrease of blaVEB-1 expression after IS2000 insertion into IS1999 argues against its role in ß-lactamase expression .

Most of the genes inserted in class 1 integrons are expressed from a common promoter region (Pant/P2) . In a few cases, however, other promoters of the expression of gene cassettes have been reported (1, 8) . This work identified for the first time an IS-located promoter capable of driving expression of downstream-located gene cassettes in an integron structure .

 


   ACKNOWLEDGMENTS

 
We thank N . Gotoh for providing P . aeruginosa strain KG2505 .

This work was funded by a grant from the Ministère de la Recherche (grant UPRES-EA 3539), Université Paris XI, Paris, France .


   FOOTNOTES

 
* Corresponding author . Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France . Phone: 33-1-45-21-29-86 . Fax: 33-1-45-21-63-40 . E-mail . thierry.naas{at}bct.ap-hop-paris.fr .


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