A Novel CTX-M β-Lactamase (CTX-M-8) in Cefotaxime-ResistantEnterobacteriaceae Isolated in Brazil

ABSTRACT To estimate the diversity of extended-spectrum β-lactamases in Brazil, 18 strains from different species of the familyEnterobacteriaceae exhibiting a positive double-disk synergy test were collected by a clinical laboratory from several hospitals in Rio de Janeiro, Brazil, in 1996 and 1997. Four strains (Proteus mirabilis, Enterobacter cloacae,Enterobacter aerogenes, and Citrobacter amalonaticus) hybridized with a 550-bp CTX-M probe. The P. mirabilis strain produced a CTX-M-2 enzyme. The E. cloacae, E. aerogenes, and C. amalonaticus isolates harbored a bla gene which was identified by cloning and sequencing as ablaCTX-M gene. E. coli HB101 transconjugants and the E. coli DH5α transformant harboring a recombinant plasmid produced a CTX-M β-lactamase with an isoelectric point of 7.6 conferring a resistance phenotype characterized by a higher level of resistance to cefotaxime than to ceftazidime, as observed with the other CTX-M enzymes. The deduced protein sequence showed a novel Ambler class A CTX-M enzyme, named CTX-M-8, which had 83 to 88% identity with the previously described CTX-M enzymes. The phylogenic study of the CTX-M family including CTX-M-8 revealed four CTX-M types, CTX-M-8 being the first member of a new phylum of CTX-M enzymes. The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.

Shortly after the introduction of the broad-spectrum cephalosporins such as cefotaxime, aztreonam, and ceftazidime, extended-spectrum ␤-lactamases (ESBLs) were isolated, first in Europe (24,40) and then worldwide. According to the structural classification of Ambler et al. (1) and the latest function scheme of Bush et al., these ESBLs are generally class A enzymes of the 2be group, arising subsequently to a few amino acid substitutions, from the common plasmid-mediated TEM and SHV-1 ␤-lactamases (12,20).
The CTX-M ␤-lactamases, a new family in class A ESBLs, were characterized at the beginning of the 1990s in the first reports of the MEN-1 (CTX-M-1) enzyme (4,6). In contrast to TEM and SHV type cefotaxime-hydrolyzing ESBLs, CTX-Ms are much more active against cefotaxime than against ceftazidime. The amino acid residues critical for their extendedspectrum activity are distinct from those of TEM-and SHV-1-derived ESBLs (4, 14-16, 18, 27).
To estimate the diversity of ESBLs in Brazil, clinical strains that exhibited ESBL phenotypes in different species were collected in hospitals of Rio de Janeiro in 1996 and 1997. In this report, we describe a novel CTX-M type enzyme, designated CTX-M-8, produced by three different strains of the family Enterobacteriaceae.

MATERIALS AND METHODS
Clinical strains. Table 1 shows the clinical strains and plasmids used in this study. Clinical strains Rio-1, Rio-2, and Rio-3, which produced a novel ␤-lactamase, were isolated from patients hospitalized in intensive care units of three private hospitals of Rio de Janeiro, Brazil. Enterobacter cloacae Rio-1 was isolated in November 1996 from blood. Citrobacter amalonaticus Rio-2 and Enterobacter aerogenes Rio-3 were isolated in March 1997 from a surgical wound and blood, respectively. CTX-M-1-producing E. coli MEN (4), CTX-M-2-producing P. mirabilis Rio-4, and TEM-1-producing E. coli TR4 (38) were used as reference strains.
Mating-out assays. Direct transfers of plasmids carrying resistance genes were performed by mating donor strains with in vitro-obtained rifampin-or nalidixic acid-resistant mutants of E. coli HB101 (35) as the recipient strain at 37°C in solid and liquid Mueller-Hinton medium. Transconjugants were selected on Mueller-Hinton agar containing rifampin (300 g/ml) or nalidixic acid (150 g/ml) and cefotaxime (2 g/ml). The transconjugants E. coli TrRio-2 and TrRio-3 were obtained from clinical strains of C. amalonaticus Rio-2 and of E. aerogenes Rio-3, respectively. Detection of ESBLs was performed with the standard and modified doubledisk synergy tests as described previously (21,41). Antibiotic discs for agar tests were obtained from Sanofi Diagnostics Pasteur.
Determination of ␤-lactamases kinetic constants. The K m and V max constants of the ␤-lactamases were obtained by a computerized microacidimetric method as previously described (26) with extracts purified as reported previously (10). The relative V max rates of hydrolysis were compared with that for benzylpenicillin, which was taken as 100%. The concentrations of the inhibitors (clavulanate and tazobactam) required to inhibit enzyme activity by 50% (IC 50 s) were determined as described previously for penicillin G (39). Hybridization. Plasmid DNAs were extracted by the method of Birnboim and Doly (9) and the bromide-CsCl linear gradient method (35). DNA probes used for hybridization were PCR products obtained with primers CTX-MA and CTX-MB. Labeling was performed by random priming with the dinitrophenyl (DNP) DNA-labeling kit purchased from Appligene Oncor (Illkirch, France). Hybridization and revelation were performed with the DNP DNA chemiluminescence detection kit (Appligene Oncor) according to the manufacturer's recommendations on DNA extracts denatured, transferred, and immobilized on Nytran filters.
␤-Lactamase gene cloning. Recombinant DNA manipulation and transformations were performed as described by Sambrook et al. (35). T4 DNA ligase was purchased from Boehringer GmbH, Mannheim, Germany. The CTX-M-encoding gene was cloned as follows: plasmid DNA of strain TrRio-2 was cleaved by EcoRI, and the resultant fragments were ligated in the EcoRI site of pACYC184 (34). E. coli DH5␣ (35) was transformed by electroporation. The transformant C1Rio-2 harboring the CTX-M-8-encoding plasmid pC1Rio-2 was selected on Mueller-Hinton agar supplemented with 2 g of cefotaxime per ml.
DNA sequencing. The sequences were determined by sequencing both strands of recombinant plasmid DNA. The strategies used to establish the nucleotide sequences of the CTX-M type genes are summarized in Fig. 1. The sequence of bla CTX-M-8 was determined from recombinant plasmid pC1Rio-2 with a primer localized on plasmid pACYC184, close to the EcoRI restriction site. It was performed by the dideoxy chain termination procedure of Sanger et al. (36) on an ABI 1377 automatic sequencer using the ABI PRISM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase FS (Perkin-Elmer/Applied Biosystems Division, Foster City, Calif.).
Computer analysis. The nucleotide sequence and the deduced protein sequence were analyzed with the software available over the Internet at the National Center for Biotechnology Information. A hydrophobic plot was obtained by the method of Nielsen et al. (29). Multiple sequence alignment and pairwise comparisons of sequences were carried out with the help of the software ClustalW, version 1.74 (43). Nucleotide sequence accession number. The bla CTX-M-8 gene nucleotide sequence data appear in the GenBank nucleotide sequence database under accession no. AF189721.

Characterization of clinical isolates. The clinical isolates
Rio-1, Rio-2, and Rio-3 (Table 1) exhibited a resistance to broad-spectrum cephalosporins and a positive double-disk synergy test and produced ␤-lactamases of pI 7.6 and 5.4. In addition, the Enterobacter strains Rio-1 and Rio-3 harbored ␤-lactamases with alkaline pI values consistent with cephalosporinase production.
PCR and DNA sequencing identified the ␤-lactamase of pI 5.4 as TEM-1 penicillinase. The enzyme of pI 7.6 was not of the SHV type, and no PCR products were obtained with the CTX-M-1 and CTX-M-2 type gene-specific primers CTX-MA and CTX-MB. In contrast, CTX-M type genes were detected in the three strains by hybridization with a CTX-M type gene probe, suggesting the presence of new CTX-M type genes in these strains.  Transfer of ␤-lactam resistance. Transconjugants TrRio-2 and TrRio-3 were only obtained from C. amalonaticus Rio-2 and E. aerogenes Rio-3 strains. They produced cefotaximehydrolyzing ␤-lactamase of pI 7.6, associated with the TEM-1 penicillinase. The CTX-M gene was carried by large plasmids (Ն75 kb), which also harbored aminoglycoside resistance genes ( Table 1).
Cloning of the ␤-lactamase gene. The genes carried by pRio-2 were cloned in plasmid pACYC184. Transformant C1Rio-2 contained recombinant plasmid pC1Rio-2 of 14 kb, producing only the CTX-M type ␤-lactamase of pI 7.6. The restriction map of the insert and hybridization with CTX-M type gene probe localized gene bla CTX-M close to the cloning site of pACYC184 (Fig. 1).
␤-Lactam susceptibility. MICs of ␤-lactams for C. amalonaticus Rio-2, its E. coli HB101 transconjugant TrRio-2 harboring plasmid pRio-2, and E. coli DH5␣ harboring recombinant plasmid pC1Rio-2 are listed in Table 2. These CTX-M-producing strains exhibited a high level of resistance to amoxicillin (MICs Ͼ 2,048 g/ml), ticarcillin (MICs Ͼ 2,048 g/ml), and cephalothin (MICs Ն 1,024 g/ml) and a low level of resistance to cefotaxime (MICs, 8 to 32 g/ml). Unlike those for the C. amalonaticus Rio-2 isolate, MICs for E. coli transconjugant TrRio-2 and transformant C1Rio-2 of cefotaxime were 8-to 16-fold higher than those of ceftazidime and 2-to 4-fold higher than those of aztreonam. The same results were observed with strains Rio-1, Rio-3, and TrRio-3 (data not shown). MICs of cefepime and cefpirome, even when they were weak, were appreciably higher than those obtained with TEM-1-producing E. coli (2 to 16 g/ml versus 0.12 g/ml). In contrast, the activities of imipenem and cefoxitin were not affected.
Clavulanate restored partially or totally the activities of the ␤-lactams. All strains were susceptible to associations of clavulanate and broad-spectrum cephalosporins (MICs, 0.06 to 1 g/ml).
Kinetic parameters. The substrate and inhibition profiles of CTX-M-8 are shown in Table 3. The best affinities were ob-served with penicillins and cefuroxime (K m , 11 to 19 M) and led to good catalytic efficiency. However, the best substrate of CTX-M-8 was cephalothin. The catalytic activity of the enzyme against cephalothin was 10-fold higher than that against penicillins despite the fact that cephalothin had higher K m values (K m , 87 M).
Likewise, CTX-M-8 had activity against cefotaxime, cefepime, and cefpirome. The greater catalytic efficiency of CTX-M-8 against cefotaxime than against cefepime, and to lesser extent cefpirome, was due to the higher K m values for the last two substrates (cefepime K m , 990 M; cefpirome K m , 1,200 M; cefotaxime K m , 74 M). In contrast, the catalytic activities for ceftazidime (relative V max Յ 1%) and aztreonam (relative V max , 9%) were at least 49-fold and 5-fold lower, respectively, than that for cefotaxime (relative V max , 49%). Aztreonam and ceftazidime were poor substrates of CTX-M-8. Imipenem and cefoxitin were not affected by CTX-M-8.   DNA sequencing. The nucleotide sequence and the deduced amino acid sequence are given in Fig. 2. There was an open reading frame of 873 nucleotides. This coding region had 73 to 75% identity with the previously described CTX-M-type genes. The initiation codon sequence was preceded by putative Ϫ35 (TTGAGA) and Ϫ10 (TTTTTT) consensus sequences. A terminator hairpin loop was detected 10 nucleotides from the stop codon (Fig. 2). Sequencing of CTX-M type genes of strains Rio-1 and Rio-3 confirmed that these strains harbored the same bla CTX-M gene.
The precursor amino acid sequence deduced from the nucleotide sequence consisted of 291 amino acid residues. On the basis of alignments with the CTX-M peptide sequence previously determined by direct amino acid sequencing (4, 27) (Fig.  3) and hydropathy plots, it is likely that the signal peptide comprised 28 amino acids. Thus, the putative mature CTX-M type enzyme consisted of 263 amino acid residues with a calculated isoelectric point of 7.72 and a calculated molecular weight of 28,039. The four structural elements characteristic of class A ␤-lactamases were found: S-X-X-K at positions 70 to 73, S-D-N at positions 130 to 132, E at position 166, and K-T-G at positions 234 to 236 (Fig. 2).
The peptide sequence alignment shown in Fig. 3 displays the conserved regions and the amino acid substitutions observed in different CTX-M enzymes. Excluding positions 185 to 219 of Toho-2, whose sequences have been discussed elsewhere (25), seven conserved regions were observed. Still excluding positions 186 to 218 of Toho-2, the alignments of CTX-M enzymes showed 72 polymorphic positions, of which 5 were reported solely in CTX-M-8 (positions 92, 103, 109, 158, and 197). Twelve amino acid residues of CTX-M-8 had not previously been observed in the other CTX-Ms (Fig. 3).
Phylogenic analysis. A dendrogram (Fig. 4) was constructed on the basis of the peptide alignment shown in Fig. 3. Bootstrapping gave a high degree of resolution for internal nodes (greater than 50% majority consensus confidence) in all but

DISCUSSION
The starting point of this work was the observation of three clinical strains that exhibited a positive double-disk synergy test, resistance to broad-spectrum cephalosporins, and a ␤-lactamase of pI 7.6. A distinctly higher level of resistance to cefotaxime than to ceftazidime was observed with the transconjugants obtained. However, C. amalonaticus Rio-2, unlike transconjugant TrRio-2, exhibited an ESBL phenotype of the ceftazidimase type. These differences of behavior between the wild-type strain Rio-2 and its transconjugant suggest an additional resistance mechanism directed mainly against ceftazidime in C. amalonaticus Rio-2. A chromosomally mediated cephalosporinase, which has mainly cefotaximase activity, in C. amalonaticus has been previously reported (8), but no ␤-lactamase of corresponding pI (5.5 and 6.05) was detected in our strain. Decreased permeability might also explain the en-hanced resistance to ceftazidime. However, such a mechanism in this species has not been reported.
In the course of cloning, a novel CTX-M type gene was characterized. The deduced enzyme, designated CTX-M-8, had 80 to 88% identity with previously described CTX-M enzymes. The conserved regions are known to have a critical role in the catalytic activity of active-site serine penicillinrecognizing enzymes (23). In addition, CTX-M-8 harbors amino acid residues Ser-237 and Arg-276, which have been suspected of playing a part in the cefotaxime-hydrolyzing activity of CTX-M enzymes (4, 14-16, 18, 27). The alignments of CTX-M enzymes showed the high level of polymorphism of the CTX-M family compared to those of the TEM and SHV families. The amino acid substitutions are generally conservative or semiconservative (43). Crystallographic data (18,22)  The catalytic properties of CTX-M-8 are close to those previously reported for CTX-M enzymes. Cephalothin is the best substrate. Catalytic efficiency against cefotaxime is better than that against ceftazidime. CTX-M-8 is slightly more susceptible to the inhibitors than TEM penicillinases (10). Tazobactam is the best inhibitor, as previously described (5,15,16,27).
The strains studied in this report were selected from 18 strains of the family Enterobacteriaceae chosen to characterize the different ESBLs present in Brazil. The majority of these strains (10 of 18) produced SHV type ESBLs. Two TEM type ESBLs were also identified. Four strains produced CTX-M type enzymes: E. cloacae Rio-1, C. amalonaticus Rio-2, and E. aerogenes Rio-3 (CTX-M-8) and P. mirabilis Rio-4 (CTX-M-2). Another CTX-M-like enzyme produced by an E. coli strain and a broad-spectrum cephalosporin-hydrolyzing enzyme not related to the CTX-M, SHV, and TEM type enzymes produced by a Serratia marcescens strain are being studied. These data show the diversity of the ESBLs in Brazil and the spread of CTX-M enzymes.
Like CTX-M-2, which is observed in a large variety of species such as E. coli, P. mirabilis, K. pneumoniae, and V. cholerae ESBL-producing strains isolated in Brazil. Thus, like eastern Europe (11,16,17), South America could be an important source of CTX-M type ␤-lactamases. The spread of CTX-M enzymes and the paucity of TEM type mutants seem therefore to be a regional phenomenon in South America.
The first CTX-M-producing strains were sporadic isolates. Recently, outbreaks involving S. enterica serovar Typhimurium (11) and V. cholerae El Tor (Galas et al., 38th ICAAC, abstr. C-174) have been recently described. Nosocomial infections of the urinary tract induced by CTX-M-producing C. freundii have also been reported (17). In this study, E. aerogenes and E. cloacae, two species known to be responsible for nosocomial infections (3,13) and isolated from patients hospitalized in separate intensive care units of different hospitals, produced CTX-M-8, suggesting that they are involved in nosocomial infections.
Since the first report of the MEN-1 (CTX-M-1) in the 1990s (4, 6), a great variety of CTX-M enzymes have been observed, all of which belong to a new group among the ESBL enzymes. In this study, we report a novel member of the CTX-M family, CTX-M-8, which is not directly related to other CTX-M enzymes. Hence, CTX-M-8 constitutes a novel potential phylum of the CTX-Ms. The intensive use of broad-spectrum cephalosporins such as cefotaxime could account for the emergence and spread of the CTX-M plasmid-mediated enzymes among enteric pathogens. The analysis of novel bla CTX-M could shed light on the origin and the intermediate enzymes of the plasmid-mediated CTX-M ␤-lactamases.