Abstract
A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (blaCTX-M-32) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis
The emergence of plasmid-mediated extended-spectrum β-lactamases in members of the family Enterobacteriaceae has become a worldwide problem (3, 4, 6, 7, 11-13, 16).
Most extended-spectrum β-lactamases are derivatives of TEM-1, TEM-2, or SHV-1 enzymes; however, there are an increasing number of reports that describe the worldwide emergence of β-lactamases belonging to other families, such as CTX-M and/or OXA derivatives (8).
The family of CTX-M enzymes is grouped on the basis of similarities in amino acid sequences into four major phylogenetic trees (6): the CTX-M-1 group (CTX-M-1 or MEN-1, CTX-M-3, CTX-M-10, CTX-M-12, CTX-M-15, and now CTX-M-32), the CTX-M-2 group (CTX-M-2, CTX-M-4, CTX-M-5, CTX-M-6, CTX-M-7, CTX-20, and Toho-1), the CTX-M-8 group, and the CTX-M-9 group (CTX-M-9, CTX-M-13, CTX-M-14, CTX-M-16, CTX-M-18, CTX-M-19, CTX-M-21, and Toho-2). The designation CTX-M refers to a potent activity against cefotaxime and having only a remnant of activity toward ceftazidime.
Here we report the molecular characterization of a new CTX-M β-lactamase derived from CTX-M-1 through a single Asp240-Gly substitution, CTX-M-32. In addition, we report experimental data showing that substitution of this amino acid is itself sufficient to confer hydrolytic activity against ceftazidime.
Patterns of antibiotic susceptibility shown by the clinical strain E. coli JC19325, as well as its transconjugant and transformants, are shown in Table 1. The MICs were determined by E-test and interpreted according to the method of the National Committee for Clinical Laboratory Standards (18). The clinical strain JC19325 showed a high level of resistance to cefotaxime, ceftazidime, and aztreonam (MICs of >256 μg/ml), cefoxitin (MIC of >256 μg/ml), and cefepime (MIC of 64 μg/ml). Moreover, clavulanic acid acted synergistically with amoxicillin, cefotaxime, and ceftazidime (E-test; ABBiodisk, Solna, Sweden), thus indicating the presence of a class A β-lactamase (9). An Escherichia coli TG1 transformant harboring the pMC-2 plasmid showed higher MICs of the affected antibiotics, probably due to more copies of the bla gene.
TABLE 1.
Antibioticsa | MIC (μg/ml) for:
|
||||
---|---|---|---|---|---|
JC19325 (produces CTX-M-32) | XL1(pMC-1b) (produces CTX-M-32) | TG1 | TG1(pMC-2c) (produces CTX-M-32) | TG1(pMC-3d) (produces CTX-M-1) | |
Amoxicillin | >256 | >256 | 3 | >256 | >256 |
Amoxicillin + clavulanate | 12 | 6 | 2 | 4 | 8 |
Piperacillin | >256 | >256 | 0.38 | >256 | >256 |
Cephalothin | >256 | >256 | 3 | >256 | >256 |
Cefuroxime | >256 | >256 | 1.5 | >256 | >256 |
Cefoxitin | >256 | 3 | 2 | 2 | 2 |
Cefotaxime | >256 | >256 | 0.02 | >256 | >256 |
Cefotaxime + clavulanate | >1 | 0.03 | 0.02 | 0.03 | 0.06 |
Ceftazidime | 128 | 96 | 0.06 | >256 | 6 |
Ceftazidime + clavulanate | >4 | 0.25 | 0.06 | 0.19 | 0.25 |
Cefepime | 64 | 16 | 0.02 | 64 | 48 |
Aztreonam | >256 | >256 | 0.03 | >256 | 48 |
Imipenem | 0.19 | 0.25 | 0.12 | 0.19 | 0.19 |
Meropenem | 0.047 | 0.03 | 0.008 | 0.02 | 0.02 |
Clavulanate was used at 4 μg/ml.
Transconjugant harboring CTX-M-32.
Transformant harboring CTX-M-32 β-lactamase gene.
Transformant harboring CTX-M-32mut or CTX-M-1 β-lactamase gene.
Isoelectric focusing was performed using polyacrylamide gels containing Ampholine, within a pH range of 3.5 to 9.5, as previously described (17). The clinical isolate produced one enzyme with a pI of 9.0.
In the present study the E. coli XL1-Blue MRF′Kan strain (Stratagene Europe, Amsterdam, The Netherlands) was used in the conjugation experiments.
The clinical strain JC19325 had one plasmid which harbored a β-lactamase with a pI of 9.0 that was transferred by conjugation into E. coli XL1-Blue MRF′Kan using kanamycin (25 μg/ml) and cefotaxime (2 μg/ml) as selective antibiotics. A few of the transconjugants which grew harbored an identical plasmid of approximately 15 kb, which was named pMC-1.
Plasmid DNA was isolated by the alkaline lysis method (23) from the transconjugant that produced a single β-lactamase with a pI of 9.0. Plasmid DNA was digested with KpnI and ligated to the plasmid vector pBGS18− (25); afterwards, the ligation mixture was introduced into E. coli TG1 cells by transformation with CaCl2, and transformants were detected on Luria-Bertani agar plates with cefotaxime (2 μg/ml) and kanamycin (25 μg/ml). The resulting plasmid, designated pMC-2, carried a bla-producing insert of size circa 4 kb. Double-stranded templates were subjected to nucleotide sequencing by using the method of Sanger et al. (23, 23a).
During isoelectric focusing, the pI 9.0 β-lactamase activity band from the E. coli transformants cofocused with the β-lactamase activity band from the clinical strain JC19325. Nucleotide sequencing of the KpnI insert revealed some interesting features, including (i) a new bla gene. This new bla gene was 876 bp long, initiated with an ATG codon, and ended with a TGA codon (291 amino acids long). The initiation codon was preceded by a Shine-Dalgarno ribosome-binding sequence, AAGGAA. The EMBL and Swiss-Prot database searches for this open reading frame revealed similarities to CTX-M β-lactamases. The deduced amino acid sequence had the closest homology (99%) with the CTX-M-1 enzyme (2, 3), from which it differed by the single amino acid substitution Asp240-Gly (Ambler numbering) (1). (ii) The second interesting feature was the inverted repeat right (IRR) sequence of ISEcp1B 80 bp upstream of the ATG start codon of CTX-M-32. No putative promoter sequences were found in the 80-bp sequence that separated the IRR of ISEcp1B from the ATG site of the blaCTX-M-32 gene; moreover, this IRR provided −35 and −10 promoter sequences, thus probably contributing to the expression of the blaCTX-M-32 gene. (iii) Third, this IRR was downstream of a tnpA gene that encoded the transposase of IS5. Figure 1 shows the 2,326-bp sequence of the original 4-kpb KpnI fragment.
To purify the CTX-M enzyme, the blaCTX-M-32 gene was cloned in the pGEX-6P-1 vector, which allowed a fusion protein between glutathione S-transferase (GST) and the CTX-M enzyme. The β-lactamase was purified to homogeneity following the manufacturer's directions for the GST gene fusion system (Amersham Pharmacia Biotech, Europe GmbH). The purified protein appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a band of 28 kDa (≥99% pure) (Fig. 2).
For kinetic experiments, CTX-M-32 β-lactamase was used at a 1,800 μM concentration. The β-lactamase showed a hydrolytic profile similar to that expected for a molecular class A CTX-M enzyme (6), with the Km for ampicillin lower than the Km for cefalothin, a Km for cefotaxime of <500 μM, and a clear hydrolytic activity towards cefotaxime. Moreover, moderate hydrolytic activity was detected against ceftazidime, as ceftazidime MICs suggested (Table 2).
TABLE 2.
Antibiotic | Km (μM) | Kcat (s−1) | Kcat/Km (μM−1) · (s−1) |
---|---|---|---|
Ampicillin | 8 ± 2.6 | 3 ± 1.1 | 0.4 |
Cephalothin | 211 ± 0.5 | 928 ± 16.4 | 4.4 |
Cefuroxime | 261 ± 0.2 | 162 ± 11.6 | 0.6 |
Cefotaxime | 322 ± 2.7 | 320 ± 10.4 | 1 |
Ceftazidime | 271 ± 0.2 | 0.91 ± 0.5 | 0.003 |
Cefepime | 1,287 ± 4.1 | 43 ± 8.8 | 0.03 |
Aztreonam | 31 ± 0.7 | 1 ± 0.4 | 0.03 |
The CTX-M-32 enzyme is derived from CTX-M-1 by a single amino acid replacement, Asp240-Gly. To confirm the importance of the Asp-Gly substitution in the hydrolysis of ceftazidime, we replaced the Gly240 with Asp in CTX-M-32 by using site-directed mutagenesis as previously described (14). The CTX-M-32mut or CTX-M-1 gene was then cloned into the pBGS18− vector, yielding the pMC-3 plasmid. The mutagenesis was confirmed by nucleotide sequencing. The MICs for E. coli TG1 harboring pMC-2 and pMC-3 are shown in Table 1. The MICs of ceftazidime corresponding to E. coli TG1 harboring CTX-M-1 β-lactamase were clearly lower than those corresponding to E. coli TG1 carrying CTX-M-32. To confirm this result, the substrate profile of the CTX-M-1 β-lactamase was determined with the enzyme purified as mentioned above for CTX-M-32 (GST gene fusion system) (Fig. 2). For kinetic experiments, CTX-M-1 β-lactamase was used at a 1,670 μM concentration. Kcat/Km (in micromolar per second) values for ceftazidime and cefotaxime were 0.0001 and 1.5; therefore, a lower catalytic efficiency with respect to ceftazidime was detected with CTX-M-1, according to the differences in ceftazidime MICs between CTX-M-32 and CTX-M-1 enzymes (Table 1).
Three different enzymes, CTX-M-15, -16, -19 and, recently, CTX-M-27 have been reported to be associated with ceftazidime hydrolysis (4, 5, 20, 21). The amino acid changes associated with the phenotype of ceftazidime hydrolysis were a Pro-to-Ser substitution at position 167 in CTX-M-19 with respect to CTX-M-18 (20) and an Asp-to-Gly substitution at position 240 in CTX-M-16 with respect to CTX-M-9 (4) and in CTX-M-27 with respect to CTX-M-14 (5). In agreement with these previous results, we also report that the Asp240 substitution is a key factor in the evolution of CTX-M β-lactamases, as it increases their hydrolytic activity toward ceftazidime.
Regarding the CTX-M enzymes, to our knowledge only six different enzymes have been published in the group 1 CTX-M enzymes: CTX-M-1, -3, -10, -12, -15, and -32 (3, 16, 19, 20). Among these, only CTX-M-15 and -32 showed more efficient ceftazidime hydrolysis than their parental enzymes, CTX-M-3 and CTX-M-1, respectively. The two former enzymes share the same amino acid substitution, although CTX-M-15 differs from CTX-M-32 in four additional amino acid changes. In terms of evolution, CTX-M-32 is probably an ancestor between CTX-M-1 and CTX-M-15 and constitutes a step forward in the evolution of β-lactamase in broad-spectrum hydrolysis of antibiotics such as ceftazidime.
In summary, we report the genetic and biochemical characterization of a new CTX-M enzyme, CTX-M-32. This is the fourth report of a CTX-M β-lactamase isolation in Spain, as CTX-M-9, CTX-M-10, and CTX-M-14 have previously been isolated in this country (7, 19, 22, 24).
Nucleotide sequence accession number.
The GenBank accession number for the CTX-M-32 β-lactamase is AJ557142.
..
Acknowledgments
This work was supported by Direccion Xeral de I+D, Xunta de Galicia (PGIDT01PXR90101PR and PGIDIT02SAN91604PR) and Fondo de Investigaciones Sanitarias (PI021415).
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