Antimicrobial Agents and Chemotherapy, September 2004, p . 3284-3290, Vol . 48, No . 9

The bla_VEB-1 gene was reported first from an Escherichia coli isolate from a Vietnamese patient, in which it was located on a plasmid and an integron (26) . Subsequently, it was detected in two P . aeruginosa isolates from Thailand, in which it was found to be located chromosomally and on an integron (19, 32) . Then, ß-lactamase VEB-1 was identified in a series of gram-negative rods, which underlines its interspecies spread . Several surveys in Thailand and Vietnam emphasized the widespread nature of VEB-1 in Southeast Asia (5, 6, 11, 12, 18) . In addition, VEB-1 had been detected in Kuwait (27) and was recently identified in many Acinetobacter baumannii isolates in France (25) .

Four VEB-1-like ß-lactamases that share 99% amino acid identity with VEB-1 have been characterized: VEB-1a (11), VEB-1b (11), VEB-2 (27), and VEB-3 (GenBank accession number AAS48620; X . Jiang, unpublished data) . Compared to VEB-1, VEB-1a has only one amino acid substitution (I18V), which occurs in the leader peptide . The ß-lactamase specific activities determined for E . coli DH10B clones expressing VEB-1 and VEB-1a were similar for the ß-lactams tested (P . Nordmann, unpublished data) . The hydrolytic properties of the other variants have not been studied .

We analyzed the ß-lactamase content of a multiresistant P . aeruginosa clinical isolate from India . We report on the identification in India of the VEB-1a ß-lactamase, which had been detected in Kuwait (27), further underlining the spread of bla_VEB-1-like genes in Asia . Until now, the bla_VEB-1-like genes had always been found to be inserted within the variable regions of class 1 integrons and were mostly associated with the insertion sequence (IS) IS1999 in P . aeruginosa (2) . In the present study, we characterize a bla_VEB-1-like gene located in a novel and very peculiar genetic environment made up of repeated DNA sequences . These sequences that drive bla_VEB-1-like expression likely represent a novel way for expression of antibiotic resistance genes .

Antimicrobial agents and susceptibility testing. Routine antibiograms were determined by the disk diffusion method on Mueller-Hinton (MH) agar (Sanofi Diagnostics Pasteur) . The antimicrobial agents and their sources have been described elsewhere (17) . The MICs of selected ß-lactams were determined by an agar dilution method on MH plates with a Steers multiple inoculator (31) and an inoculum of 10⁴ CFU/spot . MIC results were interpreted according to NCCLS guidelines (22) . The double-disk synergy test was performed with cefepime and ticarcillin-clavulanic acid disks on MH agar plates . Double-disk tests were also performed on cloxacillin (250 µg/ml)-containing plates to inhibit the activity of the AmpC-type ß-lactamase of P . aeruginosa (10) .

Mating-out assay. Conjugation experiments were attempted between isolate P . aeruginosa 10.2 as the donor and rifampin-resistant strains E . coli C600 and P . aeruginosa PU21 as the recipients in liquid and solid media at 37°C . The mating culture was plated onto TS agar plates containing ticarcillin (100 µg/ml) and rifampin (200 µg/ml) .

Nucleic acid extractions. Recombinant plasmids were extracted by use of Plasmid Mini-Midi kits (Qiagen, Courtaboeuf, France), whereas natural plasmids were extracted as described by Kieser (15) . Whole-cell DNA from P . aeruginosa 10.2 was extracted as described elsewhere (24) . Total RNAs were extracted with an RNeasy Maxi kit (Qiagen), according to the recommendations of the manufacturer .

PCR amplification, cloning experiments, and sequencing. Taq and Pfu DNA polymerases were from Roche Diagnostics (Meylan, France) and Promega Corporation (Madison, Wis.), respectively . Standard PCR amplification experiments (30) were attempted with primers specific for the aadB, arr-2, cmlA5, and aadA1 genes and the genes coding for ß-lactamases OXA-10, TEM, SHV, PER-1, VEB-1, and GES-1, as described previously (12) . The PCR products were purified with Qiaquick columns (Qiagen) .

T4 DNA ligase and restriction endonucleases were used according to the recommendations of the manufacturer (Amersham Pharmacia Biotech) . Briefly, the cloning procedure resulted in the ligation of either PstI-, BamHI-, or HindIII-digested whole-cell DNA of P . aeruginosa 10.2 into the PstI-, BamHI-, or HindIII-restricted pBK-CMV vector, respectively . The ligation products were electroporated into E . coli DH10B, and selection was performed on TS agar plates containing amoxicillin (100 µg/ml) and kanamycin (30 µg/ml) . Cloning of repeated element (Re) Re1, associated with the bla_VEB-1a gene, was performed as follows: P . aeruginosa 10.2 whole-cell DNA and primer pair Re1F (5'-GCTTTGTCATGTCGACGCGCC-3') and VebcasB (18) were used to amplify a 1.2-kb fragment that was cloned into SmaI-restricted vector pBBR1MCS.3 . The ligation products were electroporated into E . coli DH10B, and selection was performed on TS agar plates containing amoxicillin (100 µg/ml) and tetracycline (30 µg/ml) . One recombinant plasmid, pRe1-Veb, that possessed bla_VEB-1a immediately downstream of element Re1 and in the orientation opposite that of P_lac was retained for further analysis .

Sequencing was performed with laboratory-designed primers on an ABI PRISM 3100 automated sequencer (Applied Biosystems, Les Ullis, France) .

Transcription initiation. Reverse transcription and rapid amplification of cDNA ends (RACE) were performed with the 5'RACE system (version 2.0; Invitrogen, Life Technologies, Cergy Pontoise, France) . Five micrograms of total RNA extracted from the culture of P . aeruginosa 10.2 and gene-specific primers bla_VEB-1GSP1 (5'-TCAAAGTTGTTCCATTAGGGAATTCC-3'), bla_VEB-1GSP2 (5'-GAAAGATTCCCTTTATCTATCTCAGACAA-3'), and bla_VEB-1GSP3 (5'-TCAGTTTGAGCATTTGAATACAC-3') were used to determine the bla_VEB-1a transcription initiation site .

IEF analysis. ß-Lactamase extracts were prepared as described previously (11) and subjected to analytical isoelectric focusing (IEF) on a pH 3.5 to 9.5 ampholine polyacrylamide gel (Amersham Pharmacia Biotech), as described elsewhere (17) . The pI values of the ß-lactamases extracted from P . aeruginosa 10.2 and the E . coli DH10B clones were determined by comparison with those of known ß-lactamases (14) .

Kinetic measurements. ß-Lactamase extracts from cultures of E . coli DH10B harboring recombinant plasmid pRe1-Veb, pInt-Veb, or pVeb were prepared as described previously (21) . Enzyme extracts were used for determination of ß-lactamase activity with cefuroxime (a good substrate for measurement of hydrolysis by VEB-1) as the substrate, and kinetic measurements were performed at 30°C in 100 mM Tris-HCl buffer (pH 7) . The initial rates of cefuroxime hydrolysis were determined with an ULTROSPEC 2000 UV spectrophotometer (Amersham Pharmacia Biotech), and total protein contents were measured by the DC protein assay (Bio-Rad), as described previously (26) .

Nucleotide sequence accession numbers. The nucleotide sequences of the bla_OXA-2a and the bla_VEB-1a environments reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database under accession numbers AY444814 and AY444815, respectively .

 
A synergy image was found between cefepime- and clavulanate-containing disks, which was best shown by using cloxacillin-containing MH agar plates (which inhibit cephalosporinase activity), which suggested the presence of an ESBL .

Preliminary PCR screening. PCR experiments with primers specific for bla_TEM, bla_SHV, bla_GES-1, and bla_PER-1 were negative . A PCR product of 650 bp was obtained by using bla_VEB-1-specific internal primers . Sequencing of the PCR product revealed 100% identity with a bla_VEB-1-like gene . The bla_VEB-1 gene had been described (19) as a gene cassette located within the variable region of class 1 integrons and often associated with the aadB, arr-2 (rifampin resistance marker), cmlA5, bla_OXA-10, and aadA1 gene cassettes . No PCR product was obtained by using a series of combinations of primers whose sequences are specific for regions located in class 1 integron structures and the antibiotic resistance genes, suggesting that the genetic environment of bla_VEB-1 in P . aeruginosa 10.2 could be different from that of known bla_VEB-1-containing integrons .

Cloning of ß-lactamase genes. Several E . coli transformants were obtained in each cloning experiment . Disk diffusion antibiograms revealed three distinct ß-lactam resistance profiles . (i) The first phenotype showed resistance to amino-, carboxy-, and ureido-penicillins; restricted- and expanded-spectrum cephalosporins; and aztreonam . A synergy between clavulanic acid and expanded-spectrum cephalosporins suggested the presence of an ESBL that was consistent with the expression of the clavulanate-inhibited VEB-1 ß-lactamase . (ii) The second phenotype showed resistance to amino-, carboxy-, and ureido-penicillins as well as to cephalothin, with a slight synergy between piperacillin and clavulanic acid that indicated the presence of an oxacillinase . (iii) The third phenotype was consistent with expression of a cephalosporinase, since it showed resistance to amoxicillin, amoxicillin-clavulanate, and cephalothin and reduced susceptibility to cefuroxime .

The smallest recombinant plasmid for strains expressing each resistance profile was retained for further analysis . The plasmids were designated (i) p10.2-Veb, (ii) p10.2-Oxa, and (iii) p10.2-Ceph and contained a 4.5-kb PstI insert, a 4.6-kb BamHI insert, and a 9.3-kb HindIII insert, respectively .

IEF analysis. IEF analysis with E . coli DH10B harboring p10.2-Veb, p10.2-Ceph, and p10.2-Oxa showed that each isolate expressed one ß-lactamase with a pI value of 7.4 (consistent with the presence of VEB-1 [18]), 8.6 (consistent with AmpC from P . aeruginosa [11]), or 8.5 (a third ß-lactamase expressed by p10.2-Oxa [data not shown]) . The same pI values, 7.4, 8.5, and 8.6, were observed with the ß-lactamase extract from P . aeruginosa 10.2 (data not shown) .

Genetic locations of ß-lactamase genes. No plasmid DNA was detected in P . aeruginosa 10.2, despite repeated extraction attempts (15) . Transfer of the ß-lactam resistance markers from P . aeruginosa 10.2 to rifampin-resistant E . coli C600 and P . aeruginosa PU21 by mating-out experiments and electroporation of E . coli DH10B cells with the extracted DNA failed . Thus, the genetic support for the locations of the ß-lactamase genes indicated that they are likely chromosomal .

Characterization of bla_VEB-1-like gene and its genetic environment. The sequence of the bla_VEB-1-like gene from p10.2-Veb differed by 1 bp from that of the bla_VEB-1 gene (26) . This nucleotide substitution led to an amino acid change (I18V) in the putative leader peptide sequence, resulting in the VEB-1a enzyme that had been reported from a P . aeruginosa isolate in Kuwait (27) . Sequence analysis of both sides of the bla_VEB-1a gene revealed an atypical genetic environment . Whereas the bla_VEB-1a gene was followed by a 59-base element (59-be) that shared 100% nucleotide sequence identity with that previously described for bla_VEB-1 gene cassettes (11, 12, 18, 19), it was not preceded by the typical sequence for the recombination core site (Fig . 1) (28) . This bla_VEB-1a gene cassette was truncated at its left-hand side just before the cassette-specific ribosomal binding site, which was still present (Fig . 1 and 2A) . Further upstream of bla_VEB-1a a qacE1 gene followed by a sul1 gene, both in the opposite orientations compared to that in bla_VEB-1a, were found . The recombination core site of qacE1 that is usually present at the beginning of a 3' conserved sequence (3'-CS) region was absent due to truncation (Fig . 1 and 2A) . Analysis of the sequence between the beginning of the truncated bla_VEB-1a gene cassette and 3'-CS region revealed the presence of two identical 135-bp sequences in direct orientation, named Re1 . This Re sequence did not share any sequence identity with any known DNA sequences available in the GenBank database . Thus, the bla_VEB-1a gene cassette and the 3'-CS region were truncated and separated by a 336-bp DNA stretch that contained identical 135-bp DNA sequences at each end (Fig . 1 and 2A) .

 
Sequencing of the region located downstream of the bla_VEB-1a gene cassette revealed another gene cassette that was truncated at both ends . This gene cassette was made up of a part of a tetracycline resistance gene (tetA), named tetA1, and part of its 59-be, which contained the inverse core site (Fig . 1 and 2A) . The bla_VEB-1a and tetA1 gene cassettes were in opposite orientations and were separated by a 566-bp DNA sequence that did not contain any open reading frames (ORFs) . Surprisingly, analysis of the sequences that immediately followed the breakpoints located on each side of the tetA1 gene cassette identified two 135-bp DNA stretches in opposite orientations (Fig . 1 and 2A) . These DNA sequences, named Re2 and Re3, respectively, shared 71 and 98% nucleotide identities with the Re1 sequence, respectively (Fig . 3) . A second partial 3'-CS region containing a sul1 gene was also present at the outermost right hand (Fig . 1 and 2A) .

 
The presence of two truncated 3'-CSs has already been described in In34 by Partridge and Hall (23) . The central region of In34 contained an ORF (orf513), designated conserved region 1 (CR1), and an antibiotic resistance gene (dfrA10) that may have been acquired by homologous recombination, generating two truncated 3'-CSs, called 3'-CS1 and 3'-CS2, one on each side (23) . However, in In34, no Re-like sequences had been characterized between the 3'-CS1 and 3'-CS2, and no CR-like sequences were found in the vicinity of the bla_VEB-1a gene in P . aeruginosa 10.2 .

Characterization of other ß-lactamase genes and their genetic environments. Sequence analysis of inserts of recombinant plasmids p10.2-Oxa, p10.2-Oxa2 (a recombinant plasmid with a 10-kb PstI insert), and p10.2-Ceph was performed . Plasmid p10.2-Oxa2 contained five gene cassettes that were inserted into a class 1 integron named In78 (Fig . 2B): (i) aacA8, which codes for a protein sharing 85% amino acid identity with 6'-N-aminoglycoside acetyltransferase Ib; (ii) bla_OXA-2a, which encodes a class D ß-lactamase sharing 99% amino acid identity with OXA-2 (pI 8.5), with the amino acid change occurring in the putative leader peptide sequence (1, 9); (iii) aacA7, which encodes another 6'-N-aminoglycoside acetyltransferase type I; (iv) aadA6, which encodes an aminoglycoside adenylyltransferase (20); and (v) orfD, an ORF of unknown function . At the left hand, the 5'-CS of In78 was bounded by a 25-bp inverted repeat left (IRi) sequence identical to that associated with other integrons (13) . However, the nucleotide sequence flanking IRi was different from those of other known integrons, and no similarity with sequences available in the GenBank database was found . Moreover, the recently identified IS ISpa7 of 1.6 kb was inserted within the 5'-CS region upstream of the integrase gene, as found in In70.2 (GenBank accession number AJ505012; M . L . Riccio, unpublished data) (Fig . 2B), indicating a possible spread of the ISpa7 sequence in P . aeruginosa . The 3'-CS of In78 contained a truncated tni module with no ISs in the 3'-CS-tniB junction (Fig . 2B) . Therefore, In78 is another member of the group of class 1 integrons associated with a defective transposon originating from Tn402-like elements and shares the closest ancestry with In70 and In70.2, which are members of the In0-In2 lineage (3, 29) . PCR experiments with combinations of primers specific for the environments from bla_VEB-1a and bla_OXA-2a were attempted; however, no PCR product was obtained, which suggests that these genes are not located next to each other .

Finally, plasmid p10.2-Ceph contained a 1.2-kb ORF coding for a ß-lactamase that shared 98% amino acid sequence identity with the naturally occurring AmpC-type cephalosporinase from P . aeruginosa reference strain PAO1 .

Control of bla_VEB-1a gene expression. bla_VEB-1 gene expression in P . aeruginosa has been shown to be driven either by the P_ant promoter, located in the 5'-CS, or by the P_ant promoter together with the P_out promoter located on IS1999 (2); IS1999 is mostly located upstream of bla_VEB-1 in P . aeruginosa . The lack of a 5'-CS region upstream of bla_VEB-1a in P . aeruginosa 10.2 and the lack of IS1999 raised the question of the means by which bla_VEB-1a is expressed . In order to determine whether the Res were capable of driving bla_VEB-1a gene expression, recombinant plasmid pRe1-Veb was constructed (Fig . 2C) . Measurement of the specific ß-lactamase activities of culture extracts of E . coli DH10B(pRe1-Veb) indicated that Re1 likely contained a promoter responsible for bla_VEB-1a gene expression . Comparison of the specific activities of the ß-lactamase extracts from E . coli DH10B(pRe1-Veb) and E . coli DH10B(pInt-Veb) (Fig . 2C) (2) showed that the Re1 promoter was threefold stronger than the P_ant promoter (–35 [TGGACA] and –10 [TAAGCT]) located in the 5'-CSs of class 1 integrons (Fig . 2C) . In contrast, pVeb (Fig . 2C) (2), which lacks any promoter sequence, did not provide detectable ß-lactamase activity, suggesting that the activities measured with pRe1-Veb were not the result of the activities of nonspecific promoters located somewhere in the cloning vector . The strength of the Re1 promoter and its in vivo effect were confirmed by the MIC results . Indeed, the MICs of most ß-lactams for E . coli DH10B(pRe1-Veb) were two- to threefold higher than those for E . coli DH10B(pInt-Veb) (Table 1) . By using 5'RACE, P_Re1, the promoter located on Re1, was precisely mapped in P . aeruginosa 10.2 . Transcription initiation occurred at a thymidine (nucleotide position 405; Fig . 1) . Upstream of base pair 405, a –35 promoter region (TTTTCA) separated by 18 bp from a –10 promoter region (TAAGAT) was found . This promoter sequence is also present in the first Re1 and Re3, whereas Re2 contained a related sequence (Fig . 2A and 3) . It is likely that a second P_Re1 located in the other Re1 sequence may also contribute to bla_VEB-1a gene expression in P . aeruginosa 10.2; however it was not evidenced under our experimental conditions with 5'RACE (data not shown) .

Conclusion. This is the first report describing a bla_VEB-1-like gene in a P . aeruginosa clinical isolate from the Indian subcontinent and thus further underlined its spread in Asia . In addition, this report indicated that a bla_VEB-1-like gene may be located outside a class 1 integron structure . From a more general point of view, this work indicated that integrons may also constitute a reservoir for the spread of antibiotic resistance genes that may be located and expressed either inside or outside an integron structure . The presence of four 135-bp Re sequences surrounding this bla_VEB-1a gene was very peculiar . The question of the origins of these Res and their functions in the mobilization of bla_VEB-1a remain to be determined . These sequences might play a role in homologous recombination events . Finally, we have shown that these sequences carry active promoters for bla_VEB-1a gene expression, which also seems to be a novel feature for expression of antibiotic resistance genes .

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