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Antimicrobial Agents and Chemotherapy, June 2004, p . 2308-2313, Vol . 48, No . 6

High-Level Resistance to Ceftazidime Conferred by a Novel Enzyme, CTX-M-32, Derived from CTX-M-1 through a Single Asp240-Gly Substitution

Monica Cartelle, Maria del Mar Tomas, Francisca Molina, Rita Moure, Rosa Villanueva, and German Bou*

Servicio de Microbiologia, Complejo Hospitalario Universitario Juan Canalejo, 15006 La Coruña, Spain

Received 27 June 2003/ Returned for modification 8 September 2003/ Accepted 13 February 2004


   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


   TEXT

 
The emergence of plasmid-mediated extended-spectrum ß-lactamases in members of the family Enterobacteriaceae has become a worldwide problem (3, 4, 6, 7, 11-13, 16) .

Most extended-spectrum ß-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 ß-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 ß-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 ß-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 . MICs of ß-lactams for the JC19325 clinical strain, E . coli XL1(pMC-1), E . coli TG1, E . coli TG1(pMC-2), and E . coli TG1(pMC-3)

 
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 ß-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 ß-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 ß-lactamase activity band from the E . coli transformants cofocused with the ß-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 ß-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 .


FIG . 1 . Nucleotide sequence of a 2,326-bp DNA fragment of the pMC-2 plasmid . The deduced amino acid sequence is indicated in single-letter code below the nucleotide sequence . Stop codons are indicated by asterisks . The –35 and –10 promoter sequences of the blaCTX-M-32 gene and the IRR sequence of ISEcp1 are underlined and indicated by bold letters, as is the +1 position of the transcriptional start of the blaCTX-M-32 gene (10) . The CTX-M-32 and transposase of IS5 proteins are indicated by arrows . Bold amino acids are those conserved in class A ß-lactamases (15) . Oligonucleotides used for sequencing are indicated by arrows, and KpnI restriction sites delimiting the 4-kbp insert are also underlined.

 
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 ß-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) .


FIG . 2 . Electrophoresis analysis of CTX-M-32 and CTX-M-1 purified extracts in a sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis gel stained with Coomassie brilliant blue R-250 . Lanes: 1, protein molecular markers; 2, purified CTX-M-1 protein used in kinetic experiments; 3, purified CTX-M-32 protein used in kinetic experiments.

 
For kinetic experiments, CTX-M-32 ß-lactamase was used at a 1,800 µM concentration . The ß-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 . Substrate profile of ß-lactamase CTX-M-32

 
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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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) .


   FOOTNOTES

 
* Corresponding author . Mailing address: Servicio de Microbiologia, Complejo Hospitalario Universitario Juan Canalejo, C/Xubias de Arriba s/n, 15006 La Coruña, Spain . Phone: 981-178000, ext . 21144 . Fax: 981-178216 . E-mail: germanbou{at}canalejo.org .


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