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Antimicrobial Agents and Chemotherapy, August 2005, p. 3590-3592, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3590-3592.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Genetic Structure Associated with an Extended-Spectrum ß-Lactamase blaVEB Gene in a Providencia stuartii Clinical Isolate from Algeria

Daniel Aubert, Thierry Naas, Marie-Frédérique Lartigue, and Patrice Nordmann*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 K.-Bicêtre, France

Received 18 February 2005/ Returned for modification 23 March 2005/ Accepted 18 April 2005


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ABSTRACT
 
A ceftazidime-resistant Providencia stuartii isolate from Algeria harbored a ca. 160-kb conjugative plasmid that contained a truncated blaVEB-1b gene flanked by three 135-bp repeated elements. This work gives further evidence of the worldwide spread of blaVEB genes that are associated with genetic structures other than class 1 integrons.


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TEXT
 
Providencia stuartii is frequently isolated from urinary tract infections of hospitalized patients (15). P. stuartii is naturally resistant to aminopenicillins and narrow-spectrum cephalosporins due to a chromosomally expressed Ambler class C cephalosporinase (AmpC) (1, 4). Resistance to expanded-spectrum cephalosporins in P. stuartii clinical isolates results mostly from overexpression of AmpC, but acquisition of extended spectrum ß-lactamases (ESBL) such as TEM-, SHV-, or CTX-M-type enzymes have also been reported (3, 6, 19, 20).

The ESBL blaVEB-1 gene has been reported as part of class 1 integrons in several gram-negative rods from France (16), the Near East (Kuwait [18]), and Far East (Asia [5, 7, 8, 10, 11, 13, 17]). However, a novel genetic environment of a blaVEB-1a gene has been characterized in a Pseudomonas aeruginosa clinical isolate from India (2). This blaVEB-1a gene was chromosomally located, and, instead of being surrounded by a class 1 integron structure, it was flanked by two 135-bp sequences, named repeated elements (Re), that were bracketed themselves by two truncated 3'-conserved sequences (3'-CS) (9) of class 1 integrons in direct-repeat orientation (2). These Re carried a strong promoter that drove the expression of the downstream blaVEB-1a gene (2).

In the present work, a peculiar genetic environment of the blaVEB-1-like gene was characterized from a multidrug-resistant P. stuartii clinical isolate from a patient coming from Algeria.

Clinical P. stuartii isolate BI was recovered in 2004 from rectal and nasal swabs performed at admission at the Bicêtre Hospital of a 65-year-old patient that was directly transferred from a hospital located in Alger (Algeria). This patient suffered from septic shock with multiorgan failure. P. stuartii BI was identified by standard biochemical techniques (API-20E; bioMérieux, Marcy-l'Etoile, France).

A routine antibiogram, determined by disk diffusion method on Mueller-Hinton (MH) agar as previously described (17), revealed that P. stuartii BI was resistant to most ß-lactams except imipenem and to chloramphenicol, fosfomycin, amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin, tetracycline, and trimethoprim-sulfamethoxazole and was susceptible to ciprofloxacin, nalidixic acid, and rifampin. A synergy image between cefepime- and clavulanate-containing disks on MH agar plates suggested the presence of an ESBL (Fig. 1A). In addition, a marked synergy image between cefepime-, aztreonam-, and cefoxitin- or imipenem-containing disks suggested the presence of a VEB-type ß-lactamase, since VEB-type enzymes have an unusual synergy pattern due to very low Km values for cefoxitin and imipenem (Fig. 1B) (13, 17).



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  		FIG. 1. Double-disk synergy test with blaVEB-1-positive P. stuartii strain BI on MH agar plates with clavulanate (A) or imipenem and cefoxitin (B) as inhibitors. The disks tested contained ticarcillin plus clavulanate (TCC), imipenem (IMP), cefoxitin (FOX), cefepime (FEP), and aztreonam (AZT).

Mating-out experiments, performed in liquid medium as previously described (17), between the P. stuartii BI isolate and a laboratory-obtained streptomycin- and rifampin-resistant Escherichia coli DH10B strain (Life Technologies, Eragny, France) yielded ticarcillin-resistant transconjugants at a frequency of ca. 2 x 10–4. The transconjugants had identical resistance profiles, and E. coli DH10B(pNat-BI) was retained for further analysis. Plasmid DNA extraction as previously described (17) identified a plasmid in P. stuartii BI and in E. coli DH10B (pNat-BI) of ca. 160 kb (data not shown).

This plasmid conferred resistance to amino-, carboxy-, and ureidopenicillins; to narrow- and expanded-spectrum cephalosporins; to aztreonam; and to chloramphenicol, gentamicin, kanamycin, tetracycline, and trimethoprim-sulfamethoxazole. MICs of ß-lactams for P. stuartii BI and E. coli DH10B(pNat-BI), determined and interpreted as described previously (14, 17), mirrored the results obtained with disk diffusion susceptibility testing (Table 1). The resistance to ß-lactams was partially reduced by tazobactam and clavulanic acid addition.


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  		TABLE 1. MICs of ß-lactams for the P. stuartii BI clinical isolate, E. coli DH10B harboring natural plasmid pNat-BI and recombinant plasmid pRec-BI, and E. coli DH10B reference strain

PCR amplification experiments using internal primers specific for blaTEM, blaSHV, blaPER-1, blaVEB-1, and blaGES-1 genes and whole-cell DNA of P. stuartii BI and E. coli DH10B(pNat-BI) as templates were as described previously (16). The blaTEM, blaSHV, and blaVEB-1 gene-specific primers yielded PCR products for P. stuartii BI and E. coli DH10B(pNat-BI). Sequencing of these PCR products revealed internal and partial sequences that were identical to those of the blaTEM-2, blaSHV-2, and blaVEB-1 genes. This result indicated that these ß-lactamase genes were located on the same conjugative plasmid, thus further supporting previous findings that blaVEB-1-like genes are mostly plasmid located in Enterobacteriaceae, whereas they are chromosomally located in P. aeruginosa and Acinetobacter baumannii (7, 8, 16).

ß-Lactamase extracts of cultures of P. stuartii BI and E. coli DH10B(pNat-BI) were prepared and subjected to analytical isoelectric focusing, as previously described (13). P. stuartii BI expressed four ß-lactamases with pI values of 5.6, 7.4, 7.6, and 8.9, consistent with those of ß-lactamases of TEM-2, VEB-1, SHV-2, and AmpC from P. stuartii, respectively (4, 13). The pI values of 5.6, 7.4, and 7.6 were also identified with culture extracts of E. coli DH10B(pNat-BI).

PCR amplification experiments failed using primers located in the blaVEB-1 gene and in the class 1 integron conserved sequences (5'-CS and 3'-CS [9]) or in genes known to be associated with the blaVEB-1 gene (e.g., aadB, cmlA5, and arr-2) (13), thus suggesting a different genetic environment.

To determine the surrounding sequences of the blaVEB-1-like gene in P. stuartii BI, PstI-restricted fragments of whole-cell DNA of P. stuartii BI were ligated into PstI-restricted vector pBBR1MCS.3 (12), followed by electroporation into E. coli DH10B. Recombinant clones were selected on ceftazidime (4 µg/ml)- and tetracycline (15 µg/ml)-containing plates. Recombinant plasmid pRec-BI, expressing the blaVEB-1-like gene, contained a 4.3-kb PstI insert. Sequencing of the entire blaVEB-1-like gene revealed 100% nucleotide identity with the blaVEB-1b gene reported previously in a P. aeruginosa isolate from Kuwait (18) that differed from the blaVEB-1 gene sequence (17) by two nucleotide substitutions that led to two amino acid changes (I18V and V19E) located in the leader peptide sequence.

Sequence analysis on both sides of the blaVEB-1b gene revealed genetic structures identical to those found in P. aeruginosa 10.2 from India (2) (Fig. 2A). Indeed, the blaVEB-1b gene cassette and qacE{Delta}1 from the upstream 3'-CS region were truncated since they were not preceded by their typical recombination core site sequence (Fig. 2B). The breakpoints were at the same location compared to P. aeruginosa 10.2 (Fig. 2A). However, instead of a 336-bp DNA stretch that was flanked by two Re1 sequences in P. aeruginosa 10.2, a single 135-bp Re1 DNA sequence was found in P. stuartii BI (Fig. 2B). The region located downstream of the blaVEB-1b gene in P. stuartii BI was identical to that of blaVEB-1a in P. aeruginosa 10.2, including the truncated tetracycline resistance gene (tetA{Delta}1), the two 135-bp DNA stretches in opposite orientations (Re2 and Re3), and, at the outermost right hand, part of a second 3'-CS region containing a sul1 gene (Fig. 2B). These Re (Re1, Re2, and Re3) were identical to those described in P. aeruginosa 10.2 (2).



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  		FIG. 2. Schematic representations of the genetic environment of the blaVEB-1a gene in P. aeruginosa 10.2 (A) and of the blaVEB-1b in P. stuartii BI (B) (2). The breakpoints and the Re in the blaVEB-1-like gene environment are designated by broken lines and black triangles, respectively. The coding regions are shown as boxes, with an arrow indicating the orientation of transcription and white circles indicating the 59-bp element (59-be). Restriction sites that were used for cloning are indicated. Dashed lines in boldface indicate regions that were identified using PCR (the sequences of primers used are available upon request). The –10 and –35 promoter sequences of Re1 as well as the transcriptional initiation site (+1) are indicated by shaded boxes (2).

This is the first report of a blaVEB-like gene from Africa, further illustrating the worldwide spread of VEB-type ß-lactamases. Moreover, it is the first report of that ESBL in P. stuartii and the second report of blaVEB-like genes being flanked by Re. The function of these Re in gene mobilization will be further investigated. Finally, this report underlines that cassette-associated ß-lactamase genes may be found on genetic structures that are different from typical class 1 integrons and that may contribute to their spread. The blaVEB-1 gene cassette is the first example of an ESBL gene associated with both integrons and Re sequences.


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ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de la Recherche (grant UPRES-EA 3539), Université Paris XI, Paris, France, and the European Community (6th PCRD, LSHM-CT-2003-503-335).


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FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78, rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr. Back


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Antimicrobial Agents and Chemotherapy, August 2005, p. 3590-3592, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3590-3592.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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