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

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 β-lactamases (ESBL) such as TEM-, SHV-, or CTX-M-type enzymes have also been reported (3, 6, 19, 20).

The ESBL bla_VEB-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 bla_VEB-1a gene has been characterized in a Pseudomonas aeruginosa clinical isolate from India (2). This bla_VEB-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 bla_VEB-1a gene (2).

In the present work, a peculiar genetic environment of the bla_VEB-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 β-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 β-lactamase, since VEB-type enzymes have an unusual synergy pattern due to very low K_m values for cefoxitin and imipenem (Fig. 1B) (13, 17).

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 × 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 β-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 β-lactams was partially reduced by tazobactam and clavulanic acid addition.

PCR amplification experiments using internal primers specific for bla_TEM, bla_SHV, bla_PER-1, bla_VEB-1, and bla_GES-1 genes and whole-cell DNA of P. stuartii BI and E. coli DH10B(pNat-BI) as templates were as described previously (16). The bla_TEM, bla_SHV, and bla_VEB-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 bla_TEM-2, bla_SHV-2, and bla_VEB-1 genes. This result indicated that these β-lactamase genes were located on the same conjugative plasmid, thus further supporting previous findings that bla_VEB-1-like genes are mostly plasmid located in Enterobacteriaceae, whereas they are chromosomally located in P. aeruginosa and Acinetobacter baumannii (7, 8, 16).

β-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 β-lactamases with pI values of 5.6, 7.4, 7.6, and 8.9, consistent with those of β-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 bla_VEB-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 bla_VEB-1 gene (e.g., aadB, cmlA5, and arr-2) (13), thus suggesting a different genetic environment.

To determine the surrounding sequences of the bla_VEB-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 bla_VEB-1-like gene, contained a 4.3-kb PstI insert. Sequencing of the entire bla_VEB-1-like gene revealed 100% nucleotide identity with the bla_VEB-1b gene reported previously in a P. aeruginosa isolate from Kuwait (18) that differed from the bla_VEB-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 bla_VEB-1b gene revealed genetic structures identical to those found in P. aeruginosa 10.2 from India (2) (Fig. 2A). Indeed, the bla_VEB-1b gene cassette and qacEΔ1 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 bla_VEB-1b gene in P. stuartii BI was identical to that of bla_VEB-1a in P. aeruginosa 10.2, including the truncated tetracycline resistance gene (tetAΔ1), 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).

This is the first report of a bla_VEB-like gene from Africa, further illustrating the worldwide spread of VEB-type β-lactamases. Moreover, it is the first report of that ESBL in P. stuartii and the second report of bla_VEB-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 β-lactamase genes may be found on genetic structures that are different from typical class 1 integrons and that may contribute to their spread. The bla_VEB-1 gene cassette is the first example of an ESBL gene associated with both integrons and Re sequences.

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FIG. 1.

Double-disk synergy test with bla_VEB-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).

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FIG. 2.

Schematic representations of the genetic environment of the bla_VEB-1a gene in P. aeruginosa 10.2 (A) and of the bla_VEB-1b in P. stuartii BI (B) (2). The breakpoints and the Re in the bla_VEB-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).

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