Molecular and Biochemical Characterization of the Natural Chromosome-Encoded Class A β-Lactamase from Pseudomonas luteola

The bacterial strains and plasmids used in this study are described in Table 1. P. luteola was identified with the API-20 NE system (bioMérieux, Marcy-l'Etoile, France) and by sequencing PCR-amplified rrs (16S rRNA gene) and rpoB (RNA polymerase beta subunit), as previously described (2).

Antibiotic susceptibility was assessed by the disk diffusion method for 32 antimicrobial drugs (Bio-Rad, Marnes La Coquette, France), as previously described (43). The MIC of each β-lactam antibiotic was determined by Etest (AB Biodisk, Solna, Sweden). Susceptibility was classified according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (CA-SFM) (http://www.sfm.asso.fr/nouv/general.php?pa=2 ). Escherichia coli ATCC 25922 was used as the control for disk diffusion analyses and MIC determinations.

Genomic DNA of the P. luteola strain LAM was partially digested with Sau3AI restriction enzyme, ligated into the BamHI-restricted phagemid pBK-CMV, and electroporated into E. coli strain DH10B, as previously described (34). Antibiotic-resistant colonies were selected on Mueller-Hinton (MH) agar (Bio-Rad) containing kanamycin (30 μg/ml) and cefamandole (5 μg/ml).

The 1.5-kb cloned DNA fragment from the recombinant plasmid pBK-L3 was sequenced on both strands by Cogenics (Meylan, France). Analyses of nucleotide sequences and deduced amino acid sequences were performed with EditSeq and Megalign software (DNAstar, Madison, WI). The BLAST programs available from the NCBI were used for database searches (http://www.ncbi.nlm.nih.gov/BLAST/ ).

We searched for a divergently transcribed regulator gene upstream for the bla_LUT-1 gene in various recombinant plasmids shown by PCR to contain the longest DNA sequences upstream from the bla_LUT-1 gene. The primers used were either T3 or T7 (binding to the multicloning site of pBK-CMV) and REG (5′-CTTTTGTGACTTGAGGAGATCGCA-3′) (binding 300 bp upstream from the ATG initiation codon of the LUT-1 gene). The amplification conditions were as described below, except that an annealing temperature of 47°C was used.

Isoelectric focusing was performed with polyacrylamide gels containing ampholines with a pH range of 3.5 to 10, as previously described (7, 38). β-Lactamases of known pIs (in parentheses) were used as standards: TEM-1 (5.4), TEM-2 (5.6), TEM-6 (5.9), TEM-24 (6.5), and TEM-28 (6.1).

LUT-1-producing E. coli DH5α(pBK-L3) was grown in 6 liters of 2× yeast-tryptone broth supplemented with 20 μg/ml kanamycin and 32 μg/ml amoxicillin for 18 h at 37°C. The β-lactamase LUT-1 was purified as previously described (8, 37), by ion-exchange chromatography with a Q Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) and gel filtration chromatography with a Superose 12 column (Amersham Pharmacia Biotech). Total protein concentration was estimated with the Bio-Rad protein assay (Bio-Rad, Richmond, CA) with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) used as the standard. The purity of LUT-1 extracts was estimated as previously described (9), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie blue R-250 (Sigma Chemical Co.).

The kinetic constants K_m and k_cat for β-lactamases were obtained by a computerized microacidimetric method, as previously described (26). The concentrations of the inhibitors (clavulanate and tazobactam) required to inhibit enzyme activity by 50% (IC₅₀s) were determined with penicillin G (9). Penicillin G (100 mM) was used as the reporter substrate for IC₅₀ monitoring. The kinetic constants were determined three times. The coefficients of variation did not exceed 15%.

The genetic diversity of P. luteola was assessed by pulsed-field gel electrophoresis (PFGE) of genomic DNA digested with XbaI or SpeI (Roche), as previously described (43).

The entire coding region of the bla_LUT gene of all P. luteola strains was amplified by PCR, using the primers UpPlut (5′-ACCGTCTAGGCTGCTACTTCA-3′) and LoPlut (5′-CCGCTGCGCATGAGCGTA-3′), binding 200 bp upstream from the ATG initiation codon of LUT-1 and 10 bp downstream from the stop codon, respectively. The PCR products (1,100 kb) were sequenced at the Plateforme de Génotypage des Pathogènes et Santé Publique, PF8 (Institut Pasteur).

The ClustalW program (http://infobiogen.fr ) was used to align the amino acid sequences obtained (40). Phylogenetic analysis was carried out with the bioinformatics tool TOPALi v2.5 (28, 29). A phylogenic tree was constructed by the Bayesian method, as implemented in the MRBAYES program (22). LUT-1 was compared with 37 class A β-lactamases. The consensus tree calculated by MRBAYES was imported into MEGA4 for the purposes of displaying and printing the tree (25, 39).

Conjugation and transformation experiments were carried out on the P. luteola LAM isolate, as previously described (43). The β-lactam antibiotic used for selection was cefotaxime with a final concentration of 0.25 μg/ml.

For determination of the chromosomal location of the bla_LUT-1 gene, we digested agarose plugs, prepared as described previously, with the I-CeuI endonuclease (New England Biolabs, Beverly, MA) (27). I-CeuI restriction fragments were subjected to Southern blot hybridization with a PCR-generated probe for the bla_LUT-1 gene and with an rrs (16S rRNA gene) probe, as described above. Hybridization, labeling, and detection were performed according to the manufacturer's recommendations, using a nonradioactive enhanced chemiluminescence kit (ECL; GE Healthcare, United Kingdom).

The nucleotide sequences of the bla_LUT-1 to bla_LUT-6 genes of the P. luteola strains have been deposited in the GenBank database under accession numbers AY695112, FJ750572, FJ750573, FJ750574, FJ750575, and FJ750576, respectively.

The disk diffusion method showed the five P. luteola isolates and the type strain to be resistant to cephalothin, cefamandole, cefoxitin (resistance or intermediate susceptibility), nalidixic acid, trimethoprim, and trimethoprim-sulfamethoxazole. All strains were susceptible to ciprofloxacin and aminoglycosides. Additional resistance to sulfonamides and chloramphenicol was observed only in the LAM isolate. The MICs of the β-lactams determined by Etest are shown in Table 2. The addition of clavulanic acid slightly decreased (by a factor of 2 to 4) the MICs of amoxicillin, ticarcillin, and piperacillin. The MICs of the ESC and also aztreonam were slightly affected within the susceptible or intermediate range. Only the cefotaxime MICs of some P. luteola strains were in the resistant (>2-μg/ml) range according to the guidelines of the CA-SFM. By using the Etest ESBL cefepime/cefepime plus clavulanic acid strips, a deformation of the cefepime inhibition ellipse was observed for two strains with the highest MICs of cefepime (isolate 03-5093 and type strain CIP 102995^T). The MICs of nalidixic acid and ciprofloxacin ranged from 16 to >256 μg/ml and from 0.032 to 0.25 μg/ml, respectively.

Partially Sau3AI-digested DNA from the P. luteola clinical isolate LAM was inserted into the BamHI site of pBK-CMV. Ten E. coli DHB10B recombinant clones were obtained after selection on kanamycin and cefamandole (5 μg/ml). The inserts of the recombinant plasmids were between 1.5 and 3.6 kb in size. The pBK-L3 plasmid, which had a 1.5-kb insert, was selected for sequence analysis. An open reading frame (ORF) of 891 bp, preceded by a putative promoter region (339 bp), was identified and shown to encode a 296-amino-acid sequence. These nucleotide and amino acid sequences were absent from databases. However, the deduced protein had amino acid motifs typical of Ambler class A β-lactamases (⁷⁰SXXK⁷³, ¹³⁰SDN¹³², ¹⁶⁶EXXXN¹⁷⁰, and ²³⁴KTG²³⁶) (3). We therefore named this putative novel class A β-lactamase and the corresponding gene LUT-1 and bla_LUT-1, respectively. We used the SignalP 3.0 server (available at: http://www.cbs.dtu.dk/services/SignalP/ ) to determine whether this protein had a putative signal peptide. A putative cleavage site was identified between the 27th and 28th amino acids of the N-terminal region, giving a putative mature protein with a theoretical molecular mass of 28.9 kDa. A phylogenic study was carried out to assess the relationship between LUT-1 and its closest relatives and between this enzyme and members of the major lineages of class A β-lactamases (Fig. 1). The predicted LUT-1 protein showed similarities to several other chromosome-encoded class A β-lactamases identified in beta- and gammaproteobacteria. Figure 2 shows an alignment of the amino acid sequence of LUT-1 with representative members of the various branches of naturally occurring and acquired class A β-lactamases displaying similarity to LUT-1. The LUT-1 β-lactamase was 53 to 59% similar to the chromosomal β-lactamases of Burkholderia cepacia complex (Pen-A to Pen-L), 56% identical to that of Ralstonia eutropha (REUT), 54% identical to that of Citrobacter sedlakii (SED-1), 52% identical to that of Serratia fonticola (FONA-5), and 51% identical to that of Klebsiella oxytoca (OXY-5) (16, 33, 34, 44). LUT-1 also showed similarities with acquired β-lactamases such as KPC-7 from Klebsiella pneumoniae and SFC-1 from S. fonticola (class A carbapenemases) (21, 30, 44). Interestingly, the LUT-1 β-lactamase was also 49 to 52% identical to members of the extended-spectrum β-lactamase (ESBL) CTX-M family (6).

We identified no putative Lys-R-type regulator upstream from the ORF encoding LUT-1 in pBK-L3. The upstream regions of the bla_LUT-1 gene were amplified from nine other recombinant plasmids with the T3 and REG primers. The three plasmids containing the longest regions (750 bp, 600 bp, and 500 bp, respectively) were sequenced, but no regulator gene was identified. However, our results do not completely rule out the possibility that there is a regulator gene.

The recombinant E. coli(pBK-L3) clone had a β-lactam resistance phenotype different from that of the parental strain P. luteola LAM (Table 2). In E. coli, LUT-1 conferred resistance to amoxicillin and ticarcillin and an intermediate level of susceptibility to piperacillin and cephalothin. However, E. coli(pBK-L3) remained susceptible to cefoxitin, ESC, and imipenem. The β-lactamase inhibitors lowered the MICs of amoxicillin, ticarcillin, piperacillin, and cefotaxime by factors of 8 to 32. By using the Etest ESBL cefepime/cefepime plus clavulanic acid strips, a deformation of the cefepime inhibition ellipse was observed indicative of ESBLs. The discrepancy between P. luteola LAM and E. coli(pBK-L3) β-lactam resistance phenotypes may be due to the production of larger amounts of enzyme when the gene is expressed on a high-copy-number plasmid in E. coli. β-Lactam resistance phenotypes of the clinical P. luteola isolates (i.e., susceptibility to hydrolyzable penicillins) suggested a small amount of LUT. This hypothesis is strengthened by the kinetic parameters of LUT described below. Efflux mechanisms and/or differences in outer membrane permeability may also alter periplasmic β-lactam concentrations, thereby affecting apparent enzyme activity.

Both P. luteola isolate LAM and E. coli(pBK-L3) produced a single β-lactamase with an isoelectric point (pI) of approximately 6 (data not shown), consistent with the calculated theoretical pI. The purified LUT-1 protein appeared on SDS-polyacrylamide gels as a single band (≥98% pure) of approximately 29 kDa (data not shown). Kinetic parameters indicated that the LUT-1 enzyme had a broad substrate profile including penicillins and cephalosporins, such as cephalothin, cefuroxime, and cefotaxime in particular (Table 3). As observed for CTX-M, LUT-1 displayed stronger hydrolytic activity against cefotaxime than against ceftazidime or aztreonam (k_cat, 452 versus 1.3 or 1.5 s⁻¹, respectively). It was inhibited by low concentrations of clavulanic acid (IC₅₀, 36 nM) and tazobactam (IC₅₀, 40 nM). Given its susceptibility to clavulanate and its ability to cleave ESC, we can classify LUT-1 as a member of the 2e group in the functional classification of β-lactamases (10). Particular residues of LUT-1 such as Ser104, Thr167, Thr237, and Arg276, identified as important positions for the action of other ESBLs (TEMs and CTX-Ms), may be involved in the activity of LUT-1 against oxyiminocephalosporins (12, 18, 23, 31, 32, 35).

The level of hydrolysis of oxyiminocephalosporins observed, particularly for cefotaxime, was surprisingly high given their low MICs (Table 2). However, low levels of expression in P. luteola LAM may be responsible for these low MICs, suggesting a possible chromosomal location for the bla_LUT gene. The presence of the natural promoter region (in silico analysis [data not shown]) may partly account for the low level of resistance observed in E. coli(pBK-L3).

No biochemical characterization of the five variants of the LUT-1 β-lactamase (LUT-2 to LUT-6) identified on the basis of the bla_LUT gene sequences (see below) was performed. The effects of the amino acid substitutions on the hydrolytic profile of the protein therefore remain unclear.

We used PCR with the UpPlut and LoPlut primers to determine whether the bla_LUT-1 gene was present in the six P. luteola strains and in type strains of P. aeruginosa, B. cepacia, S. fonticola, Yersinia enterocolitica, C. sedlakii, and K. oxytoca. All the P. luteola strains yielded a PCR product of the expected size (1,100 bp). No amplification was observed for the other species. However, the specificity of the PCR requires confirmation through testing in several other rare Pseudomonas species closely related to P. luteola in the phylogenetic tree (e.g., P. anguilliseptica, P. pertucinogena, and P. lundensis) (1). Sequencing of the PCR products on both strands of the DNA revealed that the six bla_LUT genes had nucleotide sequences 98.1 to 99.5% identical to that of the bla_LUT-1 gene. Two to four nonsynonymous single nucleotide polymorphisms with respect to the LUT-1 β-lactamase were observed. These five variants of the bla_LUT-1 gene were named bla_LUT-2 to bla_LUT-6 and deposited in the GenBank database.

The genomic diversity of the six P. luteola strains was assessed by PFGE of XbaI- or SpeI-digested whole-cell DNA. The XbaI enzyme did not appear suitable for this species due to the presence of numerous compressed bands of small molecular size (in the range of 0 to 100 kb) (data not shown). However, the P. luteola strains were found to be genetically unrelated, as each strain harbored an SpeI profile differing from those of the other strains by at least 7 bands (Fig. 3).

β-Lactam resistance could not be transferred by conjugation or by electroporation from the P. luteola LAM isolate to E. coli by using cefotaxime at 0.25 μg/ml for selection. These results suggested that the bla_LUT-1 gene might be located on the chromosome. We tested this hypothesis, by digesting high-molecular-weight P. luteola DNAs embedded in agarose plugs with I-CeuI, separating the digestion products by PFGE and Southern blotting, and hybridizing them with a PCR-generated bla_LUT-1 probe and an rrs (16S rRNA gene) probe. The bla_LUT genes were assigned to a single large I-CeuI fragment, between 700 and 800 kb in size, depending on the strain, which also hybridized with the rrs probe (Fig. 4B and C). All the P. luteola strains harbored 6 chromosomal I-CeuI fragments which hybridized with the rrs probe (Fig. 4C). These results demonstrated a chromosomal location for bla_LUT genes.

In conclusion, we have identified LUT-1, a class A β-lactamase naturally occurring in P. luteola. P. luteola has a weak narrow-spectrum β-lactam-resistant phenotype, but this environmental species may act as a reservoir of β-lactam resistance determinants. Provided that these findings are confirmed with a larger number of P. luteola (sensitivity) and Pseudomonas sp. (specificity) isolates, two practical applications of this study would be the use of the bla_LUT gene as a molecular identification marker for P. luteola species and the use of the DNA sequence microvariation of this gene as an alternative to PFGE for strain differentiation during investigations of outbreaks.