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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, July 2000, p. 1936-1942, Vol. 44, No. 7
Laboratoire de Bactériologie,
Faculté de Médecine, 63001 Clermont-Ferrand
Cedex,1 and UMR 175, CNRS-MNHN,
29000 Quimper,3 France, and Setor de
Bacteriologia, Laboratório Lâmina LTDA, 71 Botafogo,
Rio de Janeiro, Brazil 22280-0302
Received 18 October 1999/Returned for modification 25 March
2000/Accepted 12 April 2000
To estimate the diversity of extended-spectrum Shortly after the introduction of
the broad-spectrum cephalosporins such as cefotaxime, aztreonam, and
ceftazidime, extended-spectrum The CTX-M The growing CTX-M family comprises nine members: CTX-M-1 (MEN-1)
(4, 6), CTX-M-2 (5), Toho-1 (19),
CTX-M-3 (17), CTX-M-4 (16), CTX-M-5
(11), Toho-2 (27), CTX-M-7 (15) (previously designated CTX-M-5), and CTX-M-6 (15).
They have high homology with the class A chromosomally encoded
First described in Europe, CTX-M-producing strains have now been
reported over a wide geographic area including the Near East (7), the Far East (19, 27, 44), South America
(5, 7; M. Galas, F. Pasteran, R. Melano, A. Petroni,
G. Lopez, A. Corso, A. Rossi and the WHONET Collaborative Group, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-109, p.
201, 1998), and Europe (4, 6, 11, 15-17). CTX-M enzymes have been observed in Escherichia coli (4, 6, 19,
27, 44; Galas et al. 38th ICAAC, abstr. E-109) and
Salmonella enterica serovar Typhimurium (5, 11, 15,
16) and also less frequently in Klebsiella pneumoniae
(7), Proteus mirabilis (7),
Citrobacter freundii (7), and Vibrio
cholerae El Tor (M. Galas, A. Petroni, R. Melano, A. Corso, M. Rodriguez, M. L. Cacace, A. Bru, and A. Rossi, Abstr. 38th
Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-124, p. 119, 1998).
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.
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
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Novel CTX-M
-Lactamase (CTX-M-8) in Cefotaxime-Resistant
Enterobacteriaceae Isolated in Brazil
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamases in
Brazil, 18 strains from different species of the family
Enterobacteriaceae 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 a
blaCTX-M gene. E. coli HB101
transconjugants and the E. coli DH5
transformant
harboring a recombinant plasmid produced a CTX-M -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.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-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).
-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 extended-spectrum activity are distinct from those of TEM-
and SHV-1-derived ESBLs (4, 14-16, 18, 27).
-lactamases of Proteus vulgaris (31),
Serratia fonticola (30), Citrobacter diversus (32), and Klebsiella oxytoca
(2, 33) and plasmid-mediated -lactamase SFO-1
(28). However, there is no clear direct phylogenic connection between CTX-M enzymes and these -lactamases
(7).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-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.
TABLE 1.
Strains and plasmids used in the study
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 µg/ml) or nalidixic acid (150 µg/ml) and cefotaxime (2 µg/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.
Susceptibility of -lactams.
MICs were determined by a
dilution method on Mueller-Hinton agar (Sanofi Diagnostics Pasteur,
Marnes la Coquette, France) with an inoculum of 104 CFU per
spot. Antibiotics were provided as powders by SmithKline Beecham
Pharmaceuticals (amoxicillin, ticarcillin, and clavulanate), Lederle
Laboratories (piperacillin, tazobactam), Eli Lilly, Paris, France
(cephalothin), Roussel-Uclaf (cefotaxime, cefpirome), Glaxo Wellcome
Research and Development (ceftazidime), Bristol-Myers Squibb
(aztreonam, cefepime), and Merck Sharp & Dohme (imipenem).
Isoelectric focusing.
Isoelectric focusing was performed
with polyacrylamide gels containing ampholines with a pH range of 3.5 to 10 as previously described (37). The following
-lactamases of known pIs were used as standards: TEM-1 (pI 5.4),
SHV-1 (pI 7.6), and CTX-M-1 (pI 8.6).
Determination of -lactamases kinetic constants.
The
Km and Vmax constants of
the -lactamases were obtained by a computerized microacidimetric
method as previously described (26) with extracts purified
as reported previously (10). The relative
Vmax 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% (IC50s) were determined as described
previously for penicillin G (39).
PCR of CTX-M genes. The detection of genes encoding CTX-M-1 and CTX-M-2 type enzymes (CTX-M-1 and CTX-M-2 type genes) and the synthesis of probe CTX-M were performed with the primers CTX-MA (5'-CGCTTTGCGATGTGCAG-3') and CTX-MB (5'-ACCGCGATATCGTTGGT-3') (temperature of annealing, 52°C). An internal fragment of 550 bp was amplified from positions 264 to 814 (blaCTX-M-1 numbering), which correspond to conserved regions of CTX-M-1 and CTX-M-2 type genes.
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 µg 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
blaCTX-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.).
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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). Nine class A CTX-M enzymes were compared to CTX-M-8: CTX-M-1, CTX-M-2, Toho-1, CTX-M-3, CTX-M-4, CTX-M-5, CTX-M-6, CTX-M-7, and Toho-2. A dendrogram was derived from the protein multiple-sequence alignment by the parsimony method using the phylogenetic package PAUP (phylogenetic analysis using parsimony), version 3.0 (42).
Nucleotide sequence accession number. The blaCTX-M-8 gene nucleotide sequence data appear in the GenBank nucleotide sequence database under accession no. AF189721.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
-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 cefotaxime-hydrolyzing -lactamase of pI 7.6, associated with the TEM-1 penicillinase. The CTX-M gene was carried by large plasmids (
75 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
blaCTX-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 > 2,048 µg/ml), ticarcillin (MICs > 2,048 µg/ml),
and cephalothin (MICs 1,024 µg/ml) and a low level of resistance to cefotaxime (MICs, 8 to 32 µg/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 µg/ml versus 0.12 µg/ml). In contrast, the activities of
imipenem and cefoxitin were not affected.
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-lactams. All strains were susceptible to associations of clavulanate and broad-spectrum cephalosporins (MICs, 0.06 to
1 µg/ml).
Kinetic parameters.
The substrate and inhibition profiles of
CTX-M-8 are shown in Table 3. The best
affinities were observed with penicillins and cefuroxime
(Km, 11 to 19 µM) 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 Km values (Km, 87 µM).
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1%) and aztreonam (relative Vmax, 9%) were
at least 49-fold and 5-fold lower, respectively, than that for
cefotaxime (relative Vmax, 49%). Aztreonam and
ceftazidime were poor substrates of CTX-M-8. Imipenem and cefoxitin
were not affected by CTX-M-8.
CTX-M-8 was susceptible to tazobactam (IC50, 0.010 µM),
clavulanate (IC50, 0.036 µM), and sulbactam
(IC50, 4.0 µM).
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 
35 (TTGAGA)
and 10 (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 blaCTX-M
gene.
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-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).
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Homology with other -lactamases.
The sequence of the mature
form of the CTX-M type enzyme has less than 37% amino acid identity
with the sequences of TEM-1 and SHV-1 but 75 to 77% amino acid
identity with the sequences of class A -lactamases of P. vulgaris R0104 (31), S. fonticola CUV
(30), C. diversus ULA27 (32), K. oxytoca E23004 (2), and K. oxytoca D488
(33). The previously described CTX-M -lactamases have 83 to 88% amino acid identity with this novel enzyme, which was
designated CTX-M-8.
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 one branch (49%). The dendrogram showed four major branches, whose members were closely related,
separated by large evolutionary distances. Four types of CTX-M were
obtained: the CTX-M-1 type including CTX-M-1 and CTX-M-3; the CTX-M-2
type including CTX-M-2, Toho-1, CTX-M-5, CTX-M-4, CTX-M-6, and CTX-M-7; and Toho-2 and CTX-M-8, both of which had only one member. Doubt over
the length and also the position of the Toho-2 branch persists because
of disagreements relating to its sequence (25).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 enhanced 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 penicillin-recognizing 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) demonstrate that the majority of the substitutions are
localized far from the active site, in weakly conserved zones of class
A -lactamases. This explains the close similarities in the catalytic
activities of CTX-M enzymes.
The phylogenic study of the CTX-M family showed four major types of CTX-M: the CTX-M-1 type, the CTX-M-2 type, Toho-2, and CTX-M-8. The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters of CTX-M branched off early from an unknown protein. Thus, these enzymes could be mutant derivatives from a distant common ancestor. Closely related enzymes of the CTX-M-1 type (M-1, M-3) and of the CTX-M-2 type (M-4, M-5, M-6, M-7) have been observed in a concentrated geographic area where they may have occurred as a result of point mutations, which suggests the existence of many unknown intermediate enzymes. In contrast, CTX-M-2 and Toho-1, classified on the same branch of the CTX-M-2 cluster, have been characterized in geographically distant areas (Japan and South America). The expansion of the CTX-M family may therefore be due to the spread and mutations of the CTX-M-encoding genes, but independent genetic events cannot be excluded.
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 (7; Galas et al., 38th ICAAC, abstr. C-174), the blaCTX-M-8 gene was characterized for three different species of the family Enterobacteriaceae. The transferable capacity of plasmids could explain the in vivo transfer of the blaCTX-M-8 gene in different strains and the spread of this enzyme.
CTX-M-2 was first characterized in Argentina (5, 7), and
Galas et al. (38th ICAAC, abstr. E-109) report that the predominant ESBL types are first CTX-M-2 and then SHV in that country. We report 5 CTX-M-producing strains out of 18 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
blaCTX-M could shed light on the origin and the
intermediate enzymes of the plasmid-mediated CTX-M -lactamases.
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ACKNOWLEDGMENTS |
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We thank Rolande Perroux, Marlène Jan, and Dominique Rubio for technical assistance. We are also grateful to Thierry Naas, Service de Bactériologie-Virologie, Faculté de Médecine Paris-Sud, for assistance in the phylogenic study and for use of the phylogenic package PAUP.
This work was supported in part by a grant from Ministère de l'Education Nationale, de la Recherche et de la Technologie.
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FOOTNOTES |
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* Corresponding author. Mailing address: Faculté de Médicine, Service de Bactériologie-Virologie, 28, Place Henri Dunant, 63001 Clermont-Ferrand Cedex, France. Phone: 33 (0) 4 73 60 80 18. Fax: 33 (0) 4 73 27 74 94. E-mail: Richard.Bonnet{at}u-clermont1.fr.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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