Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum β-lactamase VEB-1 in Pseudomonas aeruginosa

FEMS Microbiology Letters 176 (1999) 411^419

Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum L-lactamase VEB-1 in Pseudomonas aeruginosa Thierry Naas

a;b;

*, Laurent Poirel a , Amal Karim a , Patrice Nordmann

a

a

b

Service de Bacte¨riologie-Virologie, Hoªpital de Biceªtre, Assistance Publique-Hoªpitaux de Paris, Faculte¨ de Me¨decine Paris-Sud, 78 rue du Ge¨ne¨ral Leclerc, 94275 Le Kremlin-Biceªtre, France Service de Bacte¨riologie-Virologie, Hoªpital Antoine Be¨cle©re, Assistance Publique-Hoªpitaux de Paris, Faculte¨ de Me¨decine Paris-Sud, 92141 Clamart, France Received 25 March 1999; received in revised form 17 May 1999; accepted 20 May 1999

Abstract A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram. Preliminary PCR analysis revealed the presence of blaVEBÿ1 , an integron-located gene encoding an extended-spectrum L-lactamase previously identified in Escherichia coli MG-1. Using class 1 integron primers and blaVEBÿ1 intragenic primers, the insert region of the blaVEBÿ1 containing integron along with some flanking sequence from P. aeruginosa JES was amplified and subsequently sequenced. In50 contains within its variable region, in addition to qacEv1 and sul1 genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB. In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000. P. aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries. The finding of blaVEBÿ1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum L-lactamase gene. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Integron; Insertion sequence ; L-Lactamase ; Pseudomonas aeruginosa

1. Introduction Pseudomonas aeruginosa possesses naturally occurring cephalosporinases [1] which confer a low level resistance to aminopenicillins, ¢rst generation cephalosporins such as cefalotin and cephamycins such as

* Corresponding author. Tel.: +33 (1) 45 21 36 24; Fax: +33 (1) 45 21 63 40; E-mail: [email protected]

cefoxitin. These Ambler class C L-lactamases [2] are not inhibited by clavulanic acid. The most common mechanism for acquired resistance to extended-spectrum cephalosporins in P. aeruginosa results from an overproduction of cephalosporinases [3]. However, within the last 4 years, ¢ve clavulanic acid-inhibited extended-spectrum L-lactamases (ESBLs) have been detected in P. aeruginosa. [4]. Among the Ambler class A [2] enzymes, four ESBLs have been reported, PER-1 [5,6], TEM-42 [7], SHV2a [8] and TEM-4 (P. Nordmann, personal commu-

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 6 6 - 9

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nication). PER-1, a non-TEM non-SHV ESBL derivative was recently shown in a Turkish study to be present in 11% of P. aeruginosa hospital isolates and in 43% of Acinetobacter sp. strains, underlining its wide spread in this country [4,9]. TEM-42, SHV-2a and TEM-4 were so far only detected in rare isolates in France. The only oxacillin-hydrolyzing L-lactamase (Ambler class D, [2]) with clavulanic acid-inhibited extended-spectrum properties is OXA-18, identi¢ed in a P. aeruginosa strain in Paris in 1995 [10]. Very recently, the identi¢cation of blaCEFÿ1 in P. aeruginosa strain PaTh2 was presented at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, held in San Diego in 1998 [11]. This enzyme is identical to blaVEBÿ1 , an ESBL which was previously identi¢ed as integron- and plasmidlocated in Escherichia coli MG-1 and Klebsiella pneumoniae MG-2 [12]. Some L-lactamase genes may be encoded in gene cassettes that are present on the variable region of integrons [13]. Gene cassettes are discrete mobile units comprising a gene, usually an antibiotic-resistance gene and a recombination site that is recognized by an integrase [13,14]. The cassette-associated recombination sites, known as 59-base elements (59be), are located downstream of inserted genes and are of variable length [14,15]. Class 1 integrons, which are most commonly isolated from antibioticresistant clinical isolates, commonly possess two conserved regions located on either side of the integrated gene cassettes [13]. The 5P-conserved segment (5P-CS) includes a gene, intI1, encoding the integrase attI1, the cassette integration site and the promoter Pc , formerly Pant , that is responsible for expression of cassette genes [13]. The 3P-conserved segment (3PCS) includes, along with another open reading frame (ORF), the desinfectant (qacEv1) and the sulfonamide (sul1)-resistance determinants [13]. While most class D ESBLs are found on integrons [13], the only class A ESBL present on the variable region of integrons so far is blaVEBÿ1 [12]. In this report, we have analyzed the L-lactamase gene content of a P. aeruginosa JES clinical strain for which a slight synergy image between ceftazidime and clavulanic acid was found in the double-disc synergy test. We have compared the integron, In50, identi¢ed in P. aeruginosa JES to those from E. coli MG-1 [12] and to the yet not published blaCEFÿ1

containing integron from P. aeruginosa PaTh2 [11] (GenBank accession number AF078527).

2. Materials and methods 2.1. Bacterial strains P. aeruginosa JES was identi¢ed by conventional methods as described [10] with the API system 20NE (BioMe¨rieux, Marcy-l'Etoile, France). 2.2. Anti-microbial agents and minimal inhibitory concentration (MIC) determinations The agents and their sources have been described elsewhere [10]. MICs were determined by an agar dilution technique on Mueller-Hinton plates with a Steers multiple inoculator and an inoculum of 1U104 colony forming units per spot [3,10]. 2.3. DNA extraction Plasmid DNA analysis of P. aeruginosa JES and electroporation techniques were performed as described elsewhere [10]. Genomic DNA of P. aeruginosa JES was extracted as described [8]. 2.4. PCR For each PCR experiment, 500 ng of total DNA of P. aeruginosa JES was used in a standard PCR reaction mixture [16]. All the PCR ampli¢cations were performed using the PCR primers shown in Table 1 and with the following ampli¢cation program: 10 min 94³C, 35 cycles of 1 min 94³C, 1 min 55³C, 3 min 72³C, followed by a ¢nal extension of 10 min at 72³C. VEB-F and VEB-B primers were used as diagnostic primers for the detection of the blaVEBÿ1 gene, while VEBcas-F and VEBcas-B were used to amplify the entire veb1 gene cassette. The 5PCS and the 3P-CS primers were used in combination with VEBINV1 and VEBINV2, respectively, both primers reading outward of blaVEBÿ1 , in order to determine the integron content. All amplicons positive with the primers for antibiotic genes were sequenced. INT1-B with VEBINV1 primers were used to demonstrate the co-linearity of the integrase

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Fig. 1. (A): Schematic representation of a possible ancestor of In50, containing the blaVEBÿ1 and aadB coding regions. The coding regions are represented as boxes and relevant nucleotides for the gene cassettes and integron features are shown. The boxed sequence `TGTTACGCA' represents the nucleotides duplicated upon IS1999 insertion. Part of the 5P-CS and part of the 3P-CS are represented by single arrows while the veb1 and aadB gene cassettes are represented by divergent arrows. The attI1 recombination site and the cassetteassociated recombination sites known as 59-be are also represented by divergent arrows. (B) Schematic representations of the di¡erent veb1 containing integrons. 1: Structure of the naturally occurring class 1 integron In50. The intI1 gene, which encodes the integrase, is contained in the 5P-CS. The 3P-CS found downstream of the integrated gene cassettes includes the sulfonamide-resistance gene, sul1, and the desinfectant-resistance determinant, qacEv1. Inserted genes are indicated by boxes and the arrows indicate their translational orientation. 2: Structure of the veb1 gene cassette containing integron from P. aeruginosa PaTh2, as in GenBank accession number AF078527 and yet not published [11]. 3: Structure of the gene cassettes found in E. coli MG-1 [12].

with the resistance genes, while SUL1-B or ORF5-B primers together with VEBINV2 primer were used to analyze the genetic content of the 3P-CS. 2.5. DNA sequencing After PCR ampli¢cation, the DNA was puri¢ed using the Qiaquick PCR puri¢cation kit (Qiagen, Courtaboeuf, France). Sequencing on both strands was performed using laboratory-designed primers on an Applied Biosystem sequencer (ABI 373). Nucleotide sequence analysis and alignment methods were obtained on-line over internet at the following websites: Pedro's biomolecular research tools website (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 numbers of the published sequences are AF133697 for IS1999, AF133698 for IS2000 and AF133699 for In50. 2.6. Pulse-¢eld gel electrophoresis Agarose plugs containing P. aeruginosa JES DNA were prepared according to the the manufacturer's instructions (Bio-Rad, Ivry-sur-Seine, France). The DNAs were analyzed with 30^50 s pulses for 24 h on a CHEF DRIII pulse-¢eld gel electrophoresis apparatus (Bio-Rad). After migration, the gel was stained in a 0.5 Wg ml31 ethidium bromide solution.

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Table 1 Primers used in this study Primers

Sequence 5P to 3P

5P-CS 3P-CS VEBcas-F VEBcas-B VEB-F VEB-B VEBINV1 VEBINV2 INT1-B SUL1-B ORF5-B

GGC AAG GTT CGG CGA GGA CAG AGC CAG GCA GCG

ATC CAG AGC TTT CTT CTC TTT GTA CGC AGG TTC

CAA ACT GGT GGG CCA TGC GAG TTT ATC CGG TGC

GCA TGA AAT CTA TTT AAC CAT GTT AAG AAA GAT

GCA CCT TTA TGG CCC AAA TTG GCA CGG CCC GAA

AGC GAT ACC GCA GAT TAC AAT GAG TGA GCG GGT

AGA TAG G GC GC ACA C TCC GC CC TGG

3. Results 3.1. Origin of P. aeruginosa JES isolate P. aeruginosa JES was isolated in August 1998, at the Hoªpital de Biceªtre (Le Kremlin-Biceªtre (suburbs of Paris), France) from a urinary tract infection of a 52-year old woman hospitalized in the orthopaedic ward. She was previously hospitalized in an intensive care unit in Bangkok, Thailand, where she su¡ered from multiple bone fractures that resulted from a tra¤c accident. She was then transferred to the intensive care unit at the Hoªpital de Biceªtre and subsequently to the orthopaedic ward where she developed a P. aeruginosa urinary tract infection. During the entire hospitalization period, no other patient su¡ered from infection with a P. aeruginosa strain presenting a similar antibiotic-resistance pro¢le. Moreover, multi-resistant bacterial rectal swab screening performed on the 35 patients hospitalized at the same time in the orthopaedic ward was negative for the same P. aeruginosa strain, indicating that this patient had likely imported the strain from Thailand. 3.2. Antibiotic-resistance MIC values showed that P. aeruginosa JES was resistant to amino-, carboxy- and ureido-penicillins, to restricted and to extended-spectrum cephalosporins (Table 2). The L-lactam-resistance pattern was partially reduced by clavulanic acid addition. A routine antibiogram revealed that P. aeruginosa JES was

Accession number

Position of primer

Reference

U12338 U12338 AF010416 AF010416 AF010416 AF010416 AF010416 AF010416 AF133699 AF133699 AF071413

1416^1433 4831^4814 128^151 1198^1180 343^362 985^966 239^218 965^985 27^46 6271^6252 8149^8129

[15] [15] [12] [12] [12] [12] [12] [12] This work This work This work

multi-resistant to most antibiotics including chloramphenicol, tetracycline, gentamicin, tobramycin, netilmycin, amikacin, kanamycin, pe£oxacin, cipro£oxacin, spectinomycin, rifampicin, fosfomycin, trimethoprim, sulfonamide and trimethoprim-sulfamethoxazole. 3.3. PCR mapping and sequencing of the integron in P. aeruginosa JES PCR ampli¢cation followed by direct sequencing of the entire blaVEBÿ1 gene revealed 100% identity with blaVEBÿ1 found in E. coli MG-1 [12]. Analysis of the genetic environment of blaVEBÿ1 revealed key signatures of gene cassettes. The veb1 gene cassette in P. aeruginosa JES is identical to that of E. coli MG-1 (Fig. 1A). The length of the veb1 gene cassette is Table 2 MICs of L-lactams for P. aeruginosa JES clinical isolate L-Lactam

MIC (Wg ml

Ticarcillin Ticarcillin+Claa Piperacillin Piperacillin+Cla Ceftazidime Ceftazidime+Cla Cefepime Cefepime+Cla Aztreonam Aztreonam+Cla Imipenem Meropenem

s 512 256 128 32 512 128 128 32 s 256 32 32 8

a

31

)

Cla: clavulanic acid at a ¢xed concentration of 2 Wg ml31 .

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1059 bp and its 59-be is 133 bp long (Fig. 1A). A second antibiotic-resistance gene cassette, aadB, encoding an aminoglycoside adenyl transferase [17,18] which confers resistance to gentamicin, kanamycin and tobramycin, was identi¢ed in the same orientation as blaVEBÿ1 . This cassette had 100% identity with an other sequenced aadB gene cassette [12,13,18]. Furthermore, the sequence upstream of the veb1 gene cassette is identical to part of a sequence submitted to GenBank (AF078527) and not yet published [11]. The sequence identity goes to the end of the integron-speci¢c recombination site attI, which is interrupted by a novel IS element, which we named IS1999. This element, which is 99% identical to the IS element (IS10-like) described under the GenBank accession number AF078527, belongs to the IS4 family of insertion sequences. The transposase of this element shares 71% amino acid identity with the transposase of IS10, the best-characterized IS4 family member [19]. In addition, their terminal inverted repeats (IR) share 90% identity. The 47% GC content of that element is close to that expected for enterobacterial genes (45%). IS1999 has 21-bp imperfect terminal repeats and generates a 9-bp target site duplication (Fig. 2). While the sequence of the IS element described under GenBank accession number AF078527 is intact, IS1999 is itself interrupted by another novel IS element that we named IS2000. In contrast, this IS element has a GC content of 58% which lies close to the one expected for P. aeruginosa genes (60%) [5]. IS2000 generates, upon insertion, a 4-bp target site duplication and has 17-bp imperfect inverted repeats. It contains an ORF of 981 bp which encodes a putative IS2000 transposase of 327 amino acids. The transposase of IS2000 shares 80% amino acid identity with the transposase of IS1384, an IS element which was isolated from a plasmid of Pseudomonas putida strain H (not published, GenBank accession number AF052751). IS2000 belongs to the IS5 family of insertion sequences based on transposase identities (55% amino acid identity with the IS5 transposase), on the length of the terminal inverted repeats (17 bp), on the number of base pairs duplicated upon insertion (4 bp) and on the nature of the two nucleotides present at the end of the element (for IS5 family members, it ends with `GG') [19,20].

415

Sequencing of the PCR-ampli¢ed product using integrase and blaVEBÿ1 -speci¢c primers revealed the presence of an intact integrase gene in the 5P-CS. In the same manner, PCR ampli¢cation using sul1 and blaVEBÿ1 -speci¢c primers indicated that the qacEv1 and sul1 genes were present in the 3P-CS of the integron. However, ampli¢cation of the orf5 which is also commonly present in the 3P-CS of class 1 integrons failed. This blaVEBÿ1 containing integron was designated In50. 3.4. Genetic support of In50 Plasmid DNA extractions from P. aeruginosa JES did not reveal extra chromosomal DNA despite repeated attempts using four di¡erent extraction methods [10]. In addition, pulse-¢eld gel electrophoresis of unrestricted genomic DNA of P. aeruginosa JES revealed only one fragment corresponding to the chromosomal DNA, indicating a possible chromosomal origin for In50 (data not shown).

4. Discussion Many IS elements have been shown to activate the expression of neighboring genes [19,20]. It is known that IS5 possesses an outwardly directed 335 promoter hexamer located in the terminal IRs. When placed, by transposition, at the correct distance from a resident 310 hexamer, new promoters capable of driving the expression of neighboring genes can be created [19,20]. This may be the case for IS2000 which is inserted upstream of blaVEBÿ1 . However, no obvious 310 hexamer was detected immediately downstream of the IS2000 IRL . Other elements, such as IS10, have been reported to modulate the expression of neighboring genes by endogenous transcription escaping the IS element and running through the terminal inverted repeat into a £anking sequence [21]. The IS10 promoter, pOUT, responsible for transcription of the £anking sequences was shown to be a strong promoter [22]. IS1999 is closely related to IS10 and may therefore possess a similar endogenous pOUT promoter that drives the transcription of blaVEBÿ1 . However, no obvious IS1999 pOUT promoter sequence was detected.

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Fig. 2. Nucleotide sequence of a 2720-bp fragment of In50 containing the sequence of the two novel insertion sequences IS1999 and IS2000, part of intI1, attI1 and blaVEBÿ1 . The deduced amino acid sequence is designated in a single-letter code below the nucleotide sequence. Double slashes (`//') indicate break points within the sequences due to IS insertions. Facing arrows (` s 6 ') indicate attI1/veb1 gene cassette boundaries. The start and stop codons of the various genes are underlined. Gene names, followed by an arrow indicating their translational orientation, are indicated above their initiation codon. The position and designation of the ORFs are as follows : 16^1, beginning of intI1, encodes the integrase of the integron; 1221^142, IS1999 3P-end of tnpA, encodes the C-terminal part of IS1999 transposase ; 2312^1338, IS2000 tnpA, encodes the IS2000 transposase ; 2582^2408, IS1999 5P-end of tnpA, encodes the N-terminal part of IS1999 transposase ; 2716^2720, beginning of blaVEBÿ1 , encodes VEB-1 extended-spectrum L-lactamase. RBS indicates the ribosomal binding site of blaVEBÿ1 . IS-related inverted repeat right and left (IRR and IRL ) are shown by gray boxes and by two divergent arrows containing its appropriate label. IS-related target site duplications (TSD) are double underlined. (GenBank accession number AF133699).

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The veb1 gene cassette was ¢rst isolated in E. coli MG-1 where it was plasmid borne. The GC content of blaVEBÿ1 (45%) indicates a likely enterobacterial origin. Similarly, a GC content of 45% for IS1999 and its insertion into the integron recombination site, attI1, indicates that this element may have been cotransferred with the entire In50 integron (Fig. 1A). Since In50 may be located in the P. aeruginosa JES chromosome, the integration of In50 into the chromosome may have been the result of either the integration of the entire plasmid used in the process of transfer or the integration of a transposon carrying In50. IS2000, in contrast, is likely to be present on the chromosome of P. aeruginosa JES since it shares typical P. aeruginosa features. This element, through insertion into IS1999, may enhance the expression of blaVEBÿ1 in P. aeruginosa or, simply, may inactivate IS1999, preventing this element from hoping in a species where it is normally absent. In50 has the fewest cassettes of blaVEBÿ1 containing integrons. Comparisons of the three blaVEBÿ1 containing integrons (Fig. 1B) reveals that the veb1 gene cassette is always associated with the aadB cassette, indicating that they may all derive from a common ancestor. Fig. 1A displays the structure of such a possible common ancestor. Two additional steps are necessary to generate In50. First, an IS1999 insertion into attI1 while the integron was still in an enterobacterial environment and second, an IS2000 insertion into IS1999 once the integron was transferred into P. aeruginosa. In50 is therefore a unique integron because it contains two insertion sequences inserted within its attI1 site. It is not known whether the resulting pseudo-attI1, where the 5P-end of the attI1 site is replaced by the sequence of the 3P-end of IS1999, can still function as a recombination site for cassette integration. This work gives further insight into the complex genetic variety of L-lactamase genes and of their potential interspecies spreading. The presence of blaVEBÿ1 in three di¡erent bacterial species (E. coli MG-1 [12], K. pneumoniae MG-2 [12] and two P. aeruginosa strains, this work and [11]) isolated from patients of the same geographical location is a good illustration of how resistance genes may spread in natural conditions using conjugative plasmids and/or integrons. VEB-1 epidemiology studies among various Gram-negative bacteria in south east

Asian countries should be undertaken. Spread of different class A ESBLs in geographically de¢ned areas signals the ongoing evolution of novel enzymes beyond the TEM or SHV derivatives.

Acknowledgements This work was funded by the Universite¨ Paris XI, Faculte¨ de Me¨decine Paris-Sud, France (UPRES-JE 2227). We are also grateful to the Microbiology undergraduate students of the University of Paris VI/VII for their contributions in the preliminary PCR experiments.

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