Integron-located VEB-1 extended-spectrum ß-lactamase gene in a Proteus mirabilis clinical isolate from Vietnam

Thierry Naasa,*, Farida Benaoudiab, Sandrine Massuarda and Patrice Nordmanna

a Service de Bactériologie–Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre Cedex; b Service de Microbiologie, Centre Hospitalier de Troyes, Troyes, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A clinical isolate of Proteus mirabilis Lil-1 was obtained from a Vietnamese patient hospitalized in Paris, France. This isolate was resistant to cephalosporins, and there was marked synergy between cephalosporins and clavulanic acid together with unusual synergy between cefoxitin and cefuroxime. PCR analysis revealed the presence of blaVEB-1, an integron-located gene coding for an extended-spectrum ß-lactamase (ESBL) identified previously in an Escherichia coli isolate MG-1 from Vietnam. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1-containing integron along with flanking sequences were amplified from P. mirabilis Lil-1 whole-cell DNA. A novel class 1 integron, In55, was identified that contained, in addition to intI1, qacE{Delta}1, sul1 and Orf5 genes, an 8 kb variable region. This region was comparable in size to that found previously in E. coli MG-1, but different from those previously identified in two Pseudomonas aeruginosa isolates from Thailand. In55 was located on a 190 kb self-transferable plasmid, which was different in size and structure from that found in E. coli MG-1. The finding of blaVEB-1 on different plasmids and integrons in enterobacterial isolates underlines the interspecies spread of this novel ESBL gene.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Proteus spp., especially Proteus mirabilis, are common opportunistic pathogens in humans. A recent study showed that P. mirabilis is the second most frequently isolated (8%) enterobacterial species after Escherichia coli (65%) in French hospitals.1 While most P. mirabilis isolates are susceptible to ß-lactams, because they do not express ß-lactamase, some isolates produce a broad-spectrum ß-lactamase (TEM, SHV and CARB types),2 and plasmid-mediated cephalosporinase and several clavulanic acid-inhibited extended-spectrum ß-lactamases (ESBLs) have been identified in this species recently.3–7 The majority of ESBLs arise by mutations that alter the hydrolytic properties of the broad-spectrum penicillinases, TEM-1 and -2 and/or SHV-1.7 These ESBLs are encoded by plasmids that spread readily among Enterobacteriaceae.7,8 Their presence is detected by demonstrating synergy between clavulanic acid and any expanded-spectrum cephalosporins (particularly ceftazidime) in an agar disc diffusion susceptibility assay. In addition to the TEM/SHV-type ESBLs, non-TEM, non-SHV Ambler class A ESBLs have been detected in clinical isolates in a variety of Enterobacteriaceae,9 with, in some cases, a specific geographical distribution; CTX-M derivatives in European, South American and Mediterranean countries; TOHO-1/2 in Japan; PER-1 mostly in Turkey; and PER-2 in South America.7,10–12 The blaVEB-1 gene has been reported from the Klebsiella pneumoniae isolate MG-2 and the E. coli isolate MG-1 from the same Vietnamese patient, where it was plasmid- and integron-located,13 and in two Pseudomonas aeruginosa isolates from Thailand, where it was chromosomally and integron-located.14,15

Some ß-lactamase genes may be encoded in gene cassettes that are present in the variable region of integrons.16 Gene cassettes are discrete mobile units comprising a gene, usually an antibiotic resistance gene, and a recombination site that is recognized by an integrase.16–18 The cassette-associated recombination sites, known as 59 base elements, are located downstream of inserted genes and are of variable length.17–20 Class 1 integrons, which are isolated predominantly from antibiotic-resistant clinical isolates, commonly possess two conserved regions located on either side of the integrated gene cassettes.18 The 5'-conserved segment (5'-CS) includes a gene, intI1, encoding the integrase; attI1, the cassette integration site; and the promoter, Pc (formerly Pant), that is responsible for the expression of inserted cassette genes.18 The 3'-conserved segment (3'-CS) includes, along with another open reading frame (orf5), a disinfectant-resistance determinant (qacEDelta;1) and a sulphonamide-resistance determinant (sul1).18 Primers to these conserved segments are usually used for the amplification of the variable regions of class 1 integrons. In addition, defining a cassette when it is the only integrated cassette is done normally by identifying the known boundaries of the 5' and 3'-CSs. While most class D ESBL genes are found on integrons,21 the only class A ESBL genes present on the variable region of integrons are blaVEB-113 and the blaGES-1 reported recently from K. pneumoniae ORI-1.22

In this report, we have analysed the ß-lactamase gene content of a P. mirabilis clinical isolate for which synergy between ceftazidime and clavulanic acid was found along with unusual synergy between cefoxitin and cefuroxime in a disc diffusion assay. We have compared the integron, In55, identified in P. mirabilis Lil-1 with those of E. coli MG-1,13 P. aeruginosa JES and P. aeruginosa PaTh2,14,15 the latter two being from Thailand.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains, antimicrobial agents and MIC determinations

P. mirabilis Lil-1 and K. pneumoniae Lil-2 were identified with the API-20E system (bioMérieux, Marcy-l'Etoile, France). Electrocompetent E. coli DH10B (Gibco-BRL Life Technologies, Eragny, France) was used as the recipient in electroporation experiments and E. coli JM109, which is naturally resistant to nalidixic acid, was used for the conjugation experiments.23 E. coli MG-1 has been described previously.13 E. coli NCTC 50192 harbouring 154, 66, 38 and 7 kb plasmids was used as a plasmid-containing reference strain.24

Routine antibiograms were determined by the disc diffusion method on Mueller–Hinton agar (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France). The antimicrobial agents and their sources have been described elsewhere.25 MICs of selected ß-lactams were determined by an agar dilution technique on Mueller–Hinton plates with a Steers multiple inoculator and an inoculum of 1 x 104 cfu/spot.25 All plates were incubated at 37°C for 18 h. MICs of ß- lactams were determined alone or in combination with a fixed concentration of clavulanic acid (2 mg/L) or tazobactam (4 mg/L). MIC results were interpreted according to NCCLS guidelines.26

Plasmid content, mating-out assays and electroporation

Plasmid DNA of P. mirabilis Lil-1 was prepared using a Qiagen plasmid DNA maxi kit according to the manufacturer's instructions (Qiagen, Courtaboeuf, France) and according to Kieser's plasmid extraction protocol.27 The P. mirabilis Lil-1 plasmid DNA was analysed by electrophoresis on a 0.8% agarose gel, containing ethidium bromide 0.15 mg/L and was compared with that isolated previously from E. coli MG-1 containing blaVEB-1 and with those extracted from E. coli NCTC 50192.24

The extracted plasmid DNA from P. mirabilis Lil-1 was transferred into E. coli DH10B by electroporation using a Gene Pulser II according to the manufacturer's instructions (Bio-Rad, Ivry/Seine, France). Recombinant bacteria were plated on to Trypticase Soy (TS) agar plates containing amoxycillin 100 mg/L. The plasmids were again extracted using a Qiagen maxi column kit and the sizes were estimated by restriction endonuclease digestion (Amersham Pharmacia Biotech, Orsay, France).

Direct transfer of the ß-lactam resistance phenotype from P. mirabilis Lil-1 into nalidixic acid-resistant E. coli JM109 was attempted by liquid and solid mating-out assays at 37°C.25 Transconjugants were selected on TS agar plates containing nalidixic acid 100 mg/L and amoxycillin 100 mg/L.

Isoelectric focusing

ß-Lactamase extracts were prepared as described13 and were subjected to analytical isoelectric focusing (IEF) on a pH 3.5–9.5 ampholine polyacrylamide gel (Amersham Pharmacia Biotech) for 3 h at 10 W constant power on a flatbed apparatus (FBE-3000, Amersham Pharmacia Biotech). The ß-lactamases were visualized with an overlay of nitrocefin 0.2 g/L in 0.1 M phosphate buffer pH 7.0. The pIs of the ß-lactamases extracted from P. mirabilis Lil-1 and from the E. coli JM109(pLil-1) transconjugant were determined by comparison with those of known ß-lactamases, including VEB-1, TEM-1 and OXA-10 extracted from E. coli MG-1.13

PCR analyses and sequencing

Standard PCR experiments were performed as described.14,28 The PCR amplification and the primers (TEM-F, TEM-B, SHV-F, SHV-B, PER-1/2F, PER-1/2B, TOHO-1F, TOHO-2B, OXA-10-A, OXA10-B, CTXM-F and CTXM-B) used to search for known ß-lactamase genes (blaTEM, blaSHV, blaPER-1/2, blaTOHO-1/2 and blaCTX-M-1/6) in P. mirabilis Lil-1 and K. pneumoniae Lil-2 have been described previously.22 For PCR mapping of the integron, 500 ng total DNA from P. mirabilis Lil-1 and from E. coli MG-1 were used in standard PCR mixtures.14 All the PCR amplifications were performed using previously described PCR primers14,25 and those shown in Table 1 and in Figure 1Go, lane A, with the following amplification programme: 10 min, 94°C; 35 cycles of 1 min at 94°C, 1 min at 55°C, 3 min at 72°C; followed by a final extension of 10 min at 72°C. In some cases, long-range PCR conditions were used in order to increase the yield of the large PCR products (Long Range PCR kit, Perkin–Elmer, Les Ullis, France). VEB-F and VEB-B internal primers for blaVEB-1 were used for the detection of the blaVEB-1 gene, while VEBcas-F and VEBcas-B, located at each end of the blaVEB-1 cassette, were used to amplify the entire blaVEB-1 gene cassette.10 The AACA1 and AADB primers were used to study the immediate genetic environment of blaVEB-1. The 5'- and 3'-CS primers were used in combination with VEBINV1 and VEBINV2, respectively, both primers reading outwards from blaVEB-1, in order to determine the size of the variable region of the integron.14 INT1-B, INT2-B and INT3-B, intragenic primers specific for the three integrase gene types (1, 2 and 3),29 together with VEBINV1 primers were used to demonstrate the collinearity of the integrase with blaVEB-1, while primers QACE{triangleup}1-B, SUL1-B and ORF5-B together with VEBINV2 were used to analyse the genetic content of the 3'-CS.10 INT-FLANK, a primer that hybridizes to the 25 bp inverted repeat IRi and IRt sequences found at the boundaries of several class 1 integrons,30 and 5'-CSINV, a primer hybridizing to the complementary sequence of the 5'-CS primer, were used to amplify a conserved flanking sequence of the class 1 integrase gene.



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Figure 1. Analytical isoelectric focusing of ß-lactamase extracts of several enterobacterial cultures. Lane A, E. coli JM109(pNLT-1);13 lane B, E. coli JM109(pRLT-8) expressing OXA-10 (T. Naas, unpublished); lane C, E. coli MG-1;13 lane D, E. coli JM109(pRLT-1) expressing only VEB-1;13 lane E, P. mirabilis Lil-1; lane F, E. coli JM109(pLil-1) transconjugant; lane G, E. coli JM109(pRLT-50) expressing only TEM-1;13 lane G, IEF protein standards (Bio-Rad). The pIs of the standard are indicated on the right side of the gel.

 
After PCR amplification, the DNA was purified using a Qiaquick PCR purification kit (Qiagen). The blaVEB-1 amplicon was sequenced on both strands using laboratory-designed primers on an Applied Biosystems sequencer (ABI 373). Nucleotide sequence analysis and alignment methods were obtained from the following websites: Pedro's biomolecular research tools (http://www.fmi.ch/biology/research_tools.html) and the National Center of Biotechnology Information website (http://www.ncbi.nlm.nih.gov). The GenBank accession number of the published sequence is AF220757.

Restriction digestion and hybridization experiments

HindIII- and BamHI-digested whole-cell DNA of P. mirabilis Lil-1, E. coli MG-1 and their electrotransformants were analysed on a 0.7% agarose gel. After electrophoresis, the gels were stained in ethidium bromide 0.5 mg/L and the DNA fragments were transferred on to Hybond N+ membrane (Amersham Pharmacia Biotech) using a vacuum blotting system (Amersham Pharmacia Biotech). Southern hybridizations were performed using an enhanced chemiluminescence (ECL) non-radioactive kit as described by the manufacturer (Amersham Pharmacia Biotech). The probe consisted of a 650 bp intragenic blaVEB-1 PCR fragment amplified from plasmid pRLT-1,13 a recombinant plasmid containing the entire blaVEB-1 gene.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Origin of the P. mirabilis Lil-1 isolate and its antimicrobial susceptibility

P. mirabilis Lil-1 was isolated in July 1999, at the hôpital Antoine Béclère, Clamart (a suburb of Paris), France, from the pus of a 2 month old Vietnamese boy adopted by a French family. The child had previously been hospitalized at the Saigon General Hospital in Vietnam, where he was treated for diarrhoea and dehydration. Before his arrival in France, he had a history of scabies and he developed erythematous and maculopapular lesions, which became vesicular and pustular, all over his body. Upon arrival in France, pus samples from his skin lesions were analysed. Four bacterial strains were recovered: a methicillin-resistant strain of Staphylococcus aureus, an ESBL-producing K. pneumoniae Lil-2 isolate, a P. aeruginosa with a wild-type ß-lactam resistance profile and an ESBL-producing P. mirabilis isolate Lil-1 that showed unusual synergy between cefoxitin and cefuroxime on a routine antibiogram, suggestive of an uncommon ESBL. This child probably acquired these bacteria in Vietnam since no other isolate with a similar resistance pattern had been identified in the same hospital during the same time period.

A routine antibiogram revealed that P. mirabilis Lil-1 was resistant to multiple antibiotics, including chloramphenicol, fosfomycin, amikacin, gentamicin, kanamycin, spectinomycin, streptomycin, tobramycin, sulphonamides, tetracycline, trimethoprim and trimethoprim–sulphamethoxazole. MICs showed that P. mirabilis Lil-1 was resistant to amino-, carboxy- and ureidopenicillins, and to restricted and extended-spectrum cephalosporins (Table IIGo). This ß-lactam resistance pattern was antagonized by addition of clavulanic acid and tazobactam. MICs of broad-spectrum penicillins for E. coli MG-1 were higher, probably because this isolate expressed, in addition to VEB-1, a TEM-1 ß-lactamase (Figure 1Go).13


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Table II. MICs of ß-lactams for P. mirabilis Lil-1 clinical isolate, its transconjuguants [E. coli JM109(pLil-1)], E. coli MG-1 clinical isolate, its transconjugants [E. coli JM109(pNLT-1)] and E. coli JM109
 
Preliminary PCR and IEF analysis

In order to investigate the molecular basis of the ß-lactam resistance observed in P. mirabilis Lil-1, PCR was performed. Negative results were obtained with TEM-, SHV-, PER-1/2-, CTXM-1/6- or TOHO-1/2-specific primers. However, blaVEB-1- and blaOXA-10-specific primers gave PCR products of the expected sizes (650 and 760 bp, respectively; data not shown). PCR amplification followed by direct sequencing of the entire blaVEB-1 gene revealed 100% identity with blaVEB-1 found in E. coli MG-1.13 Similarly, sequencing of the blaOXA-10 PCR product revealed 100% identity with blaOXA-10 from P. aeruginosa POW151.31 These results were confirmed by IEF experiments, which showed that P. mirabilis Lil-1 produced two ß-lactamases, with pIs of 7.4 (which corresponds to the pI of VEB-1 ß-lactamase) and 6.1 (which corresponds to the expected value for OXA-10) (Figure 1Go). Interestingly, both ß-lactamases were also present in E. coli MG-1 in addition to an enzyme of pI 5.4, characterized previously as TEM-1.13 The level of expression of these ß-lactamases, as deduced from the relative ß-lactamase intensities on the IEF gel, was different in E. coli MG-1 and P. mirabilis Lil-1, which may explain why the MICs of oxyimino-cephalosporins are higher for P. mirabilis Lil-1 than for E. coli MG-1. However, identical ESBL genes usually provide lower MICs for P. mirabilis than for E. coli.3,8 When a second ESBL-producing isolate, K. pneumoniae Lil-2, was isolated from the same patient, PCR experiments with primers specific for ß-lactamases were also performed. Despite several attempts, no positive PCR signal was obtained with blaVEB-1 primers, while blaTEM and blaSHV primers gave positive PCR results. Therefore it is likely that the ESBL present in K. pneumoniae Lil-2 was not a VEB-1-type ß-lactamase. However, the K. pneumoniae Lil-2 ß-lactamases were not characterized further.

PCR mapping of the integron from P. mirabilis Lil-1 and E. coli MG-1

Analysis of the genetic environment of blaVEB-1 revealed key signatures of gene cassettes. The veb1 gene cassette in P. mirabilis Lil-1 was identical to that in E. coli MG-1. The veb1 gene cassette was 1059 bp long and its 59 base element was 133 bp long. Using primers specific for aadB and aacA1, the known immediate genetic environment of blaVEB-1 in E. coli MG-113 (Figure 2bGo), a 2.7 kb DNA fragment was amplified and subsequently sequenced from both isolates (Figure 2aGo). Sequence analysis revealed identical sequences from both strains. The aadB gene cassette, which encodes an aminoglycoside adenyl transferase that confers resistance to gentamicin, kanamycin and tobramycin, was identical to other sequenced aadB gene cassettes.13,32,33 The AacA1–orfG fusion cassette was first described as integron-located on an E. coli plasmid, pNR79 (GenBank accession number AF047479). AacA1 codes for an aminoglycoside 6'-N-acetyltransferase which confers resistance to amikacin, tobramycin and netilmicin.33



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Figure 2. Schematic representation of the different veb1-containing integrons. The coding regions are represented as boxes, the arrows indicate their translational orientation and the gene names are indicated above the boxes. The 5'-CS contains the intI1 gene, which encodes the integrase, and the 3'-CS found downstream of the integrated gene cassettes includes sul1, qacEDelta;1 and an unknown open reading frame, orf5. Unsequenced parts are represented by dotted lines. Restriction sites used for the PCR restriction mapping or for Southern blotting experiments are represented by vertical arrows. Horizontal and divergent arrows indicate the approximate sizes (in kb) of the restriction products, as seen in Figure 3Go. Small arrows with numbers correspond to the primers, described in Table IGo, that were used for the PCR experiments. The thick, bold lines represent the major PCR products obtained from the variable region using 3'- and 5'-CS primers along with VEB-specific primers with P. mirabilis Lil-1 or E. coli MG-1 whole-cell DNA as template.14 The estimated sizes of the different PCR products are indicated between divergent arrows. (a) Structure of In55, containing the blaVEB-1, aacA1–orfG fusion and aadB gene cassettes. (b) Structure of the integron found in E. coli MG-1.13 The ‘//’ sign represents an interruption of the sequence. No intI1 gene was found in the 5'-CS of this integron. (c) Structure of In50 from P. aeruginosa JES.14 (d) Structure of the blaVEB-1 gene cassette containing integron from P. aeruginosa PaTh2.15

 
Since the full structure of the veb-1 integron in E. coli MG-1 is not known, a PCR approach was used to compare the structures of the two integrons. Using class 1 integron primers and blaVEB-1 intragenic primers,14 the variable region of the blaVEB-1-containing integron from P. mirabilis Lil-1 and from E. coli MG-1 were amplified and their sizes compared (Figure 2aGo). The amplimers were similar in size (Figure 2aGo) and restriction analysis did not detect differences in the structures between the two variable regions. For instance, HindIII cut the left-hand DNA sequence of blaVEB-1 (Figure 2aGo) from both strains into two fragments of 1.9 kb and 0.5 kb, while BamHI cut the right-hand DNA sequence of blaVEB-1 from both strains into two bands of 2.3 and 2.7 kb. These data indicate that the variable regions of both integrons are identical in size and thus very probably identical in sequence. The deduced size of the variable region of both integrons was 8 kb. Sequencing of the 2.4 kb PCR product from both strains, generated with the 5'-CS and the VEBINV1 primers, revealed perfect sequence identity. In addition to the aacA1–orfG fusion cassette, two additional cassettes were found (Figure 2Go). The first cassette contained an ORF of 345 bp (Figure 2Go). The coding sequence, designated qacH, which may direct the synthesis of a 110 amino acid protein, had 90% identity with qacF from Enterobacter aerogenes34 and 67.8% identity with the qacE gene.35 The qacE gene specifies an exporter protein mediating resistance to intercalating dyes and quaternary ammonium compounds.36 The second cassette was an additional aadB cassette, which differed from the first by only one base pair substitution that had no effect on the deduced amino acid sequence32,33 (Figure 2Go). The presence of two almost identical cassettes in integrons has been reported previously for other gene cassettes such as oxa2, but still remains rare.18

Most of the oxacillinase genes so far identified are located on the variable region of integrons.21 Since blaOXA-10 was identified by PCR and confirmed by IEF, a PCR approach was chosen to see whether it was located on In55 or another integron. Using OXA10-F and 3'-CS primers, a 1.7 kb PCR fragment was generated, indicating the presence of the oxa10 cassette on a class 1 integron. Using VEBINV2 and OXA10-B, a 4.2 kb fragment was amplified, indicating that the two genes are collinear on the same integron.

PCR amplification using type 1 integrase gene- and blaVEB-1-specific primers revealed the presence of an intact type 1 integrase gene in the 5'-CS of P. mirabilis Lil-1. The same PCR remained negative, despite several attempts using primers specific for type 1, but also for type 2 and type 3 integrase genes with E. coli MG-1 DNA as template.29 To test whether the integrase gene in E. coli MG-1 had a deletion of part of the integrase gene or a mutation in the primer-binding site, PCR using INT-FLANK and 5'-CSINV primers was performed. INT-FLANK recognizes the inverted repeat IRi sequence that is conserved and found at the boundaries of most class 1 integrons characterized so far,30 while 5'-CSINV binds to a complementary sequence of the 5'-CS primer-binding site. As expected, a 1.3 kb amplimer was obtained with P. mirabilis Lil-1, while no amplification was observed for the E. coli MG-1 DNA. These data indicate that E. coli MG-1 may carry a different integrase gene, or may have an integrase gene located too far from the 5'-CS to be detected by PCR amplification even under long-range PCR conditions, or may lack an integrase gene. Similarly, PCR amplification using, qacEDelta;1-, sul1-, orf5- and blaVEB-1-specific primers indicated that the qacEDelta;1, sul1 and orf5 genes were present in the 3'-CS of both integrons, typical of sul1-associated integrons.18

Taken together, these results indicate that the variable regions of both integrons containing blaVEB-1 are similar in size but differ at least by the absence of a type 1 integrase gene in E. coli MG-1 and by differences within the integrase flanking sequences. There were major differences between the structures of the P. mirabilis Lil-1 and E. coli MG-1 integrons and those found in the two P. aeruginosa isolates from Thailand (Figure 1Go, lanes A–D). The latter possess either one15 or two14 insertion sequences in their 5'-CS and in both cases, the blaVEB-1 gene cassette is located at the first position in the integron (Figure 1Go, lanes C and D). Interestingly, the gene cassette immediately downstream of blaVEB-1, aadB, was identical in the four integrons (Figure 1Go, lanes A–D). This observation strengthened the hypothesis that these integrons may derive from a common ancestor14 and that they may have evolved according to either geographical or species specificities.

Genetic support of In55

Plasmid DNA preparation from P. mirabilis Lil-1 revealed the presence of three large plasmids of 35, 150 and 190 kb. The largest plasmid, pLil-1, coded for blaVEB-1, as shown by hybridization experiments (data not shown). This plasmid was transferred by electroporation into E. coli DH10B, resulting in the transfer of resistance to chloramphenicol, extended-spectrum ß-lactams, gentamicin, kanamycin, streptomycin, spectinomycin, sulphonamides and tobramycin. These results indicated that blaVEB1 was plasmid-borne. pLil-1 was also transferred by conjugation at low frequency (10–6) into an E. coli JM109 recipient strain. E. coli JM109 transconjugants harbouring the natural plasmid from P. mirabilis Lil-1 or that from E. coli MG-1 displayed similar ß-lactam MICs, except the ticarcillin MIC, which was twice that for E. coli JM109(pNLT-1) (Table IIGo). This difference may reflect differences in expression or differences in plasmid structure and/or replication. When the associated antibiotic markers were considered, one major difference between bacteria carrying the two plasmids was observed: E. coli JM109(pLil-1) remained susceptible to trimethoprim and trimethoprim–sulphamethoxazole, while E. coli JM109(pNLT-1) was resistant (data not shown). Both plasmids encoded resistance to chloramphenicol, gentamicin, kanamycin, spectinomycin, streptomycin, sulphonamides and tobramycin.

pNLT-1 was 180 kb in size, i.e. 10 kb smaller than pLil-1 (data not shown). Thus different plasmids were present in these enterobacterial strains. In addition, BamHI-digested total genomic DNA from both strains and from their electrotransformants, when hybridized with a blaVEB-1- specific probe, revealed two hybridization signals of different size (Figure 3Go), indicating again that the genetic environment of the two integrons is different, at least in their 5'-CS region. While an expected fragment of 6.5 kb was observed with P. mirabilis Lil-1 (Figures 2a and 3GoGo) a fragment of 10 kb was observed with E. coli MG-1 (Figure 1Go, lane B, and Figure 2Go). When their genomic DNA was digested with HindIII, an enzyme that cuts within the integron of both isolates, the expected 6.3 kb fragment was observed for both isolates (Figures 2a and b and 3GoGo), underlining the identity of the variable regions of both integrons.



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Figure 3. blaVEB-1-specific hybridization of HindIII-digested (lanes 1, 2, 3 and 4) and BamHI-digested (lanes 5, 6, 7, and 8) whole-cell DNAs from P. mirabilis Lil-1, E. coli MG-1 and their electroporants. The sizes to the left represent the 1 kb molecular weight marker (Gibco-BRL Life Technologies). Lanes 1 and 5, E. coli MG-1 DNA; lanes 2 and 6, E. coli DH10B(pNLT-1) DNA; lanes 3 and 7, P. mirabilis Lil-1 DNA; lanes 4 and 8, E. coli DH10B(pLil-1) DNA.

 
The presence of blaVEB-1 in four different bacterial species (E. coli, K. pneumoniae, P. aeruginosa and now P. mirabilis) from patients in the same geographical region (south-east Asia) illustrates how resistance genes may spread using conjugative plasmids and/or integrons. The integron-located blaIMP-1 gene, which codes for a class B carbapenem-hydrolysing ß-lactamase, has spread among several Gram-negative rods in Japan,30,37 and recent findings concerning blaVEB-1 may indicate a similar spread of an integron-located ß-lactamase gene but, in this case, of Ambler class A. Thus, extended VEB-1 epidemiological studies among various Gram-negative species in south-east Asian countries would be interesting. So far, in that part of the world, TOHO-1 and TOHO-2 ESBLs have been isolated from Japan and SHV and TEM derivatives from Taiwan.12,38 The spread of VEB-1 in a geographically defined area signals the ongoing evolution of novel enzymes beyond the TEM or SHV derivatives. Finally, this study underlines the possibility that P. mirabilis may be a reservoir of class A ESBLs other than TEM and SHV derivatives.


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Table I. blaVEB-1- and integron-specific primers used in this study
 

    Acknowledgments
 
This work was funded by a grant from the Ministères de la Recherche et de l'Enseignement, Université Paris XI (grant UPRES-JE 2227), France.


    Notes
 
* Corresponding author. Tel: +33-1-45-21-36-24; Fax: +33-1-45-21-63-40; E-mail: thierry.naas{at}kb.u-psud.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Nicolas-Chanoine, M. H., Chardon, H., Avril, J. L., Cattoen, Y., Croix, J. C., Dabernat, H. et al. (1997). Susceptibility of Enterobacteriaceae to ß-lactams and fluoroquinolones: a French multicentre study. Clinical Microbiology and Infection 3, Suppl. 2, 74–5.

2 . Chanal, C., Bonnet, R., De Champs, C., Romaszko, J. P., Sirot, D. & Sirot, J. (1998). Surveillance de la résistance aux ß-lactamases et diversité moléculaire des enzymes de type pénicillinase chez Proteus mirabilis. In Program and Abstracts of the Eighteenth Interdisciplinary Meeting on Anti-Infection Chemotherapy. Société Française de Microbiologie, Paris.

3 . Mariotte, S., Nordmann, P. & Nicolas, M. H. (1994). Extended-spectrum ß-lactamase in Proteus mirabilis. Journal of Antimicrobial Chemotherapy 33, 925–35.[Abstract]

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Received 27 January 2000; returned 26 April 2000; revised 19 June 2000; accepted 20 July 2000