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

Abstract

The integron-borne bla_VEB-1 gene encodes an extended-spectrum β-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 bla_VEB-1 in Enterobacteriaceae. Expression experiments and promoter study identified promoter sequences in IS1999 that increased the expression of VEB-1 in P. aeruginosa.

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 P_ant, 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 (qacEΔ1) and the sulfonamide (sul1) resistance determinants (14). The expression of the inserted gene cassettes depends not only on sequences of promoters P_ant/P2 but also on the gene cassette position relative to the 5′-CS (2).

The integron-borne bla_VEB-1 gene encodes the extended-spectrum β-lactamase VEB-1 (Vietnamese extended-spectrum β-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, bla_VEB-1 was associated with either one (IS1999) or two (IS1999/IS2000) insertion sequences (IS) that were inserted upstream of bla_VEB-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 bla_VEB-1 positive (10 E. coli, 4 Enterobacter cloacae, 1 Enterobacter sakazakii, and 3 Klebsiella pneumoniae) and 19 were bla_VEB-1 negative. Out of 33 ceftazidime-resistant P. aeruginosa isolates, 31 were bla_VEB-1 positive. bla_VEB-1 was mostly plasmid located in Enterobacteriaceae, whereas this gene was mostly chromosome encoded in P. aeruginosa (3, 4). Moreover, spreading of bla_VEB-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 bla_VEB-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 bla_VEB-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

Strain or isolate(s) or plasmid	Genotype or descriptiona	Source or reference
Strains or isolates
E. coli
DH10B	F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ−rpsL nupG	Life Technologies, Eragny, France
P. aeruginosa
KG2505	P. aeruginosa reference strain that does not express the naturally chromosome-encoded AmpC β-lactamase and is deficient for the multidrug efflux system MexAB-OprM mexA::res-ΩSm ampC::res-ΩSm of PAO1; Smr	11
Isolate JES	Clinical isolates from a French patient hospitalized in Thailand; blaVEB-1 positive; Cazr	9
Isolates 1 to 31	Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr	3
E. coli
Isolates 1 to 10	Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr	4
E. cloacae
Isolates 11 to 14	Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr	4
E. sakazakii
Isolate 15	Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr	4
K. pneumoniae
Isolates 16 to 18	Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr	4
Plasmids
pBK-CMV phagemid	Neor, Kanr	Stratagene, La Jolla, Calif.
pCR-Blunt II-TOPO	Kanr	Invitrogen, Cergy Pontoise, France
pBBR1MCS.3	Low-copy-number broad-host-range cloning vector; Tetr	5
pDA-1	pBK-CMV recombinant plasmid containing genomic HindIII fragment from P. aeruginosa 14, including blaVEB-1	This work
pDA-2	pBK-CMV recombinant plasmid containing genomic HindIII fragment from P. aeruginosa 1, including blaVEB-1 and IS1999	This work
pDA-3	pBK-CMV recombinant plasmid containing genomic HindIII fragment from P. aeruginosa JES, including blaVEB-1 and IS1999/IS2000	This work
pInt-Veb	A 2.4-kb SpeI-XhoI fragment from plasmid pDA-1, containing	This work
	blaVEB-1 inserted in the SpeI-XhoI site of pBBR1MCS.3
pInt-1999-Veb	A 3.7-kb SpeI-XhoI fragment from pDA-2, containing blaVEB-1 and IS1999 inserted in the SpeI-XhoI site of pBBR1MCS.3	This work
pInt-1999-2000-Veb	A 4.9-kb SpeI-XhoI fragment from pDA-3, containing blaVEB-1 and IS1999/IS2000 inserted in the SpeI-XhoI site of pBBR1MCS.3	This work
pTOP1	A 1.1-kb fragment resulting from PCR amplifications using VEBCAS-A/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as template; this fragment, including the veb-1 gene cassette, was inserted in the cloning site of pCR-BluntII-TOPO	This work
pTOP2	A 2.5-kb fragment resulting from PCR amplifications using 5′-CS/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as the template; this fragment, including the veb-1 gene cassette and IS1999, was inserted in the cloning site of pCR-BluntII-TOPO	This work
pTOP3	A 1.1-kb fragment resulting from PCR amplifications using VEBCAS-AStu/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as the template; this fragment, including a StuI restriction site in the veb-1 gene cassette upstream of blaVEB-1, was inserted in the cloning site of pCR-BluntII-TOPO	This work
pTOP4	A 2.2-kb fragment resulting from PCR amplifications using MUTIS1999Stop/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as the template; this fragment includes a site-directed mutation generating after transcription a stopcodon within the IS1999 transposase sequence; this fragment, inserted in the cloning site of pCR-BluntII-TOPO, includes the two XmnI sites that are present in IS1999 and blaVEB-1 sequences and that are located on each side of the mutation	This work
pVeb	A 1.1-kb KpnI-ApaI fragment from pTOP1, containing the veb-1 gene cassette inserted in the KpnI-ApaI site of pBBR1MCS.3	This work
p1999R-Veb	pInt-1999-Veb from which a 1.9-kb SpeI-StuI fragment has been deleted that includes intII and the left end of IS1999	This work
p1999-Veb	A 2.6-kb KpnI-ApaI fragment from pTOP1, containing the veb-1 gene cassette and IS1999, inserted in the KpnI-ApaI site of pBBR1MCS.3	This work
p1999L-Veb	A 2.1-kb KpnI-StuI fragment from pTOP3, containing the veb-1 gene cassette, inserted in the KpnI-StuI site of p1999-Veb, containing the left end of IS1999	This work
pInt-1999*-Veb	A 1.5-kb XmnI fragment from pTOP4, containing parts of the mutated IS1999 and blaVEB-1 sequences inserted in the XmnI site of pInt-1999-Veb	This work
p1999R-2000R-Veb	pInt-1999-2000-Veb from which a 1.8 kb SpeI-PstI fragment has been deleted that includes intII and the right ends of IS1999/IS2000	This work

^aSuperscript “r,” resistance. Abbreviations: Caz, ceftazidime; Kan, kanamycin; Neo, neomycin; Sm, streptomycin; Tet, tetracycline.

Out of 18 bla_VEB-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%) bla_VEB-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 bla_VEB-1 gene in P. aeruginosa and its absence from Enterobacteriaceae, especially from E. coli, led us to study the contribution of these IS on β-lactamase expression in both bacterial species.

Influence of IS1999/IS2000 on bla_VEB-1 expression.

Using three bla_VEB-1-positive P. aeruginosa isolates as templates (P. aeruginosa 14, 1, and JES), recombinant plasmids were constructed containing bla_VEB-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 β-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 bla_VEB-1 gene was inserted in opposite orientation to P_lac, thus removing any contribution of promoter P_lac in β-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 P_lac. 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 P_ant 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 β-lactamase extracts from cultures of E. coli DH10B and P. aeruginosa KG2505 harboring recombinant plasmids (Fig. 2) prepared as previously described (10), bla_VEB-1 gene expression was then investigated by measuring the specific β-lactamase activities for cefepime (a β-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 β-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, P_ant 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 β-lactamase activity was measured for E. coli DH10B(pInt-Veb), which had bla_VEB-1 located just downstream of the promoter P_ant of the class 1 integron (Fig. 1 and 2). Insertion of IS1999 (pInt-1999-Veb), which moved bla_VEB-1 away from P_ant, decreased bla_VEB-1 expression (50% decrease). After the removal of the promoter P_ant (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 P_ant made only a minor contribution to the overall β-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 P_ant promoter along with the left end(s) of IS1999 and/or IS2000 (p1999R-Veb and p1999R-2000R-Veb) led to a decrease in β-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 bla_VEB-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 bla_VEB-1 (Fig. 1). Strains harboring p1999L-Veb plasmid had very low levels of β-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 bla_VEB-1 expression, the plasmid pInt-1999*-Veb, which contains an interrupted open reading frame encoding the IS1999 transposase, was generated. No significant modification of bla_VEB-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 β-lactamase expression in E. coli and P. aeruginosa.

Mapping of promoter P_out 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 P_out (−35 [CAGAAT] and −10 [TAAAAT]) identified in the related IS element, IS10 (16). Using the primer Vebprom 1999, which annealed upstream of P_out, 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, P_out, and the promoter of the transposase gene, P_in, are indicated by broken arrows. The −10 and −35 regions for P_in and P_out are underlined. Nucleotide position 115 (according to the sequence GenBank AF133697) in IS1999 corresponds to the transcription start of P_out. 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 P_out 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, P_out, of IS1999 that was capable of driving bla_VEB-1 expression in E. coli and in P. aeruginosa. The level of expression obtained from P_ant or P_out was about fourfold higher in E. coli than in P. aeruginosa. Furthermore, our results suggest that an association between IS1999 and the P_ant promoter enhances bla_VEB-1 expression by about 60% in P. aeruginosa but not in E. coli. An increase in β-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 β-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 bla_VEB-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 bla_VEB-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 bla_VEB-1 expression in P. aeruginosa KG2505 (pInt-1999-Veb) might be the result of a cooperative effect between P_out and P_ant 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 bla_VEB-1-containing integron remains unclear, but decrease of bla_VEB-1 expression after IS2000 insertion into IS1999 argues against its role in β-lactamase expression.

Most of the genes inserted in class 1 integrons are expressed from a common promoter region (P_ant/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.