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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2001, p. 616-620, Vol. 45, No. 2
Servicio de Microbiología, Hospital
Ramón y Cajal, Madrid, Spain
Received 19 May 2000/Returned for modification 21 August
2000/Accepted 28 October 2000
A cefotaxime-resistant, ceftazidime-susceptible Escherichia
coli isolate was obtained from a patient with sepsis in 1997, from which a Plasmid-mediated
expanded-spectrum In 1997 a cefotaxime-resistant, ceftazidime-susceptible
Escherichia coli isolate (97/38582) was obtained from blood
and urine cultures from a patient with sepsis. DNA sequencing,
isoelectric focusing (IEF), and determination of the resistance
phenotype and antibiotic hydrolysis profile were performed. The strain
harbored a novel CTX-M Identification and initial antibiotic susceptibility testing were
performed with the semiautomatic PASCO system (Difco, Detroit, Mich.).
MICs of amoxicillin, amoxicillin-clavulanate, ticarcillin, piperacillin, piperacillin-tazobactam, cefazolin, cefuroxime, cefpodoxime, cefoxitin, cefotaxime, ceftazidime, cefepime,
aztreonam, and imipenem were determined by agar dilution
according to the NCCLS guidelines (16). Additionally, MICs
of cefuroxime, cefpodoxime, cefotaxime, and ceftazidime plus a
4-µg/ml fixed concentration of clavulanate were determined. The
inoculum effect on antibiotic MICs was determined using a
103-CFU/ml inoculum (low inoculum) and a
107-CFU/ml inoculum (high inoculum) in addition to the
105-CFU/ml standard inoculum. The manufacturers provided
all antibiotics as powders.
Conjugation experiments were performed by filter mating using E. coli JMG21 (MC4100 with kanamycin and nalidixic acid resistance markers) (12) as the recipient. Transconjugants were
selected on Mueller-Hinton agar plates (MHAP) with 100 and 50 µg of
ampicillin and kanamycin per ml, respectively. Plasmid DNA from
E. coli clinical strain 97/38582 (High Pure plasmid
isolation kit; Boehringer Manheim, Indianapolis, Ind.) was transformed
into E. coli MC4100 competent cells. Selection of
transformants was performed both on MHAP with 100 µg of ampicillin
per ml and on MHAP with 2 µg of cefotaxime per ml. Plasmid DNAs
obtained from strain 97/38582 and plasmid pBGS18 IEF was performed by applying the supernatant of a crude sonic extract
to Phast gels (pH gradient of 3 to 9) in a PhastSystem apparatus
(Pharmacia AB, Uppsala, Sweden). IEF of the E. coli clinical strain 97/38582 crude sonic
extract revealed two MICs of several antibiotics against the E. coli clinical
strain 97/38582, the transconjugant, and the MC4100 strain with the pI
8.1 cloned
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.616-620.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nucleotide Sequence and Characterization of a Novel
Cefotaxime-Hydrolyzing
-Lactamase (CTX-M-10) Isolated in
Spain
ABSTRACT
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Abstract
Text
References
-lactamase with a pI of 8.1 was cloned. Cephaloridine and cefotaxime relative hydrolysis rates were 167 and 81, respectively (penicillin G rate = 100), whereas ceftazidime hydrolysis was not
detected. The nucleotide sequence revealed a bla gene
related to that coding for CTX-M-3. Despite 21 nucleotide
substitutions, only 2 determined amino acid changes (Ala27Val and
Arg38Gln). The amino acid sequence identity between this enzyme,
designated CTX-M-10, and the chromosomal -lactamase of
Kluyvera ascorbata was 81%.
TEXT
Top
Abstract
Text
References
-lactamases (ESBLs) have been responsible
for several outbreaks of enterobacteria resistant to expanded-spectrum
cephalosporins since they were first reported in 1987 (7,
21). To date, the ESBLs found most frequently are those derived
from the common TEM-1 and SHV-1 broad-spectrum -lactamases (8,
24). These enzymes are characterized as conferring resistance,
to a greater or lesser extent, to all expanded-spectrum cephalosporins
but are most frequently associated with a higher level of resistance to
ceftazidime than to cefotaxime. In 1992, a novel type of ESBL was
reported, which in contrast to those found previously, determined
high-level resistance to cefotaxime but did not significantly affect
ceftazidime (3). ESBLs belonging to this new type, named
cefotaximases (CTX-M), have caused outbreaks of cefotaxime-resistant
enterobacteria mainly in South America, Eastern Europe, and Japan
(6, 11, 14, 15; M. F. Galas, M. J. Rapoport,
F. G. Pasteran, R. G. Melano, A. E. Petroni, P. G. Ceriana, W. Group, and M. A. Rosi, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1474, 1999).
-lactamase closely related to CTX-M-3, which
was previously found in Citrobacter freundii
(11).
(kanamycin resistance marker) (22) were digested with
EcoRI and BamHI. After enzyme inactivation, the
digestion products were mixed, ligated overnight with T4 DNA ligase,
and transformed into MC4100 competent cells. E. coli MC4100
transformants harboring the pBGS18 plasmid containing the
cloned -lactamase gene were selected on MHAP with 2 and 50 µg of
cefotaxime and kanamycin per ml, respectively. This plasmid was used as
the DNA template, which was sequenced on both strands by the method of
Sanger et al. (19). Sequencing was performed with a Big
DyeDeoxyTerminator cycle sequencing kit (Perkin-Elmer, Foster City,
Calif.) and specific primers for the coding sequence. The DNA sequence
was analyzed in an automatic DNA sequencer (377 Abi-Prism:
Perkin-Elmer).
-Lactamases with known pIs were
focused in parallel as controls. Gels were stained with 500 µg of
nitrocefin (Oxoid, Basingstoke, Hampshire, England) per ml to identify
the -lactamase bands. An exponentially growing culture of the
E. coli MC4100 strain harboring the cloned -lactamase was
sonicated, partially purified through a Sephadex G 100 column (Pharmacia AB), and concentrated with a Minicon concentrator (Amicon, Danvers, Mass.). Hydrolysis rates were determined
spectrophotometrically (UVIKON 940; Kontron, Schlieren, Switzerland) in
duplicate experiments at 25°C in 0.05 M phosphate buffer (pH 7.4)
during 10 min using a 100 µM concentration of each antibiotic.
Kinetic parameters were determined in duplicate experiments based on
the initial steady-state rates using at least five different substrate
concentrations (Lineweaver-Burk transformation). Fifty percent
inhibitory concentrations (IC50s) were determined for
clavulanic acid, sulbactam, and tazobactam. The enzyme and inhibitor
were preincubated for 10 min at 25°C before addition of nitrocefin
(100 µM, final concentration). Hydrolysis was measured
spectrophotometrically at 482 nm, and IC50s were determined graphically.
-lactamases with pIs of 5.4 and 8.1. Both
-lactamases were cotransferred by conjugation into the JMG21
E. coli strain. A few transformants obtained on MHAP with
ampicillin carried only the pI 5.4 -lactamase (suggestive of TEM-1),
and those selected on MHAP with cefotaxime carried only the pI 8.1 -lactamase, suggesting that the two -lactamases were encoded by
different plasmids. Enzymatic restriction of the pBGS18
plasmid harboring the pI 8.1 -lactamase revealed an insert of approximately 3 kb.
-lactamase are shown in Table
1. -Lactam MICs against the
transformant carrying only the pI 5.4 -lactamase resembled those
expected against a TEM-1 -lactamase-producing E. coli
strain (data not shown). Interestingly, a high resistance level was
found for cefotaxime, while MICs of ceftazidime were only slightly
increased. Clavulanate restored the MIC to the basal value in all
combinations tested. A high inoculum effect was observed with all
cephalosporins except ceftazidime. For example, when the inoculum was
increased from 103 to 107 CFU/ml, the cefepime
MICs increased from 2 to 512 µg/ml. Hydrolysis rates and kinetic
parameters for penicillin G, cephaloridine, cefuroxime, cefotaxime,
ceftazidime, and cefepime are shown in Table
2. The hydrolysis rates for
cephalosporins were high compared to that for penicillin G, with the
exception of ceftazidime, for which hydrolysis could not be detected.
TABLE 1.
MICs of different antibiotics for E. coli
97/38582, E. coli MC4100, the E. coli JMG21
(modified MC4100) transconjugant, and E. coli MC4100 with
the cloned -lactamase
TABLE 2.
Kinetic parameters of CTX-M-10
DNA sequencing of the 3-kb fragment carrying the -lactamase gene
revealed an 873-bp open reading frame. The deduced amino acid sequence
revealed a 291-amino-acid protein with the typical motifs of serine
-lactamases. Nucleotide sequence analysis showed that this
bla gene was related to those coding for the CTX-M-3 (11) and CTX-M-1 (4) -lactamases, with 21 and 23 nucleotide substitutions, respectively. The deduced amino acid
sequence of this new -lactamase, designated CTX-M-10, revealed that
despite the high number of nucleotide substitutions, only two and six of them determined amino acid changes compared with the CTX-M-3 (Ala27Val and Arg38Gln) and the CTX-M-1 (Ala27Val, Arg38Gln, Asp114Asn, Ser140Ala, Val177Ala, and Asn288Asp) enzymes, respectively. Amino acid
numbers were assigned as described by Ambler et al. (1). Sequence identity with other CTX-M -lactamases is far lower than that with CTX-M-1 or CTX-M-3; CTX-M-2, CTX-M-5, and CTX-M-8 were the
next three most related CTX-M -lactamases, and each had only 81%
amino acid identity to CTX-M-10 (4, 5, 6). This result suggests the existence of different subgroups with different
phylogenetic origins within the CTX-M -lactamase group.
To date, CTX-M enzymes have been thought to be derived from the
chromosomal -lactamase of Klebsiella oxytoca, although a direct relationship is not likely, given a sequence identity lower than
80%. The amino acid sequence identities between this new -lactamase
and chromosomal enzymes from Kluyvera ascorbata (C. Humeniuk
et al., unpublished data [GenBank accession number CAB59824]), K. oxytoca (2), and Citrobacter
diversus (17) were 81, 71, and 69%, respectively
(Fig. 1). Therefore, if this
-lactamase is derived from one of the known sequenced chromosomal
enzymes, K. ascorbata is the most likely candidate. In fact,
the CTX-M-2 -lactamase differs from that of K. ascorbata
in only two amino acids, making the direct relationship clearly
established.
|
The -lactam resistance phenotypes conferred by CTX-M-10 and the
closely related CTX-M-3 -lactamase (11) in their
respective conjugation recipient strains are very similar. As found for
CTX-M-3, for CTX-M-10, MICs of cefotaxime are at least 8 times greater than those of ceftazidime, which in both cases remain in the
susceptible category. Both amino acid substitutions (Ala27Val and
Arg38Gln) are located in the mature protein that starts at residue 25 (4). Residue 27 has been found to be nonvisible in the
recently crystallized Toho-1 -lactamase, but residue 38 was found to
be solvent oriented (13). Both amino acids are divergent
in CTX-M -lactamases. Alanine 27 in CTX-M-1 and CTX-M-3 is replaced
by an aspargine in CTX-M-2 and closely related CTX-M -lactamases
such as TOHO-1, CTX-M-4, CTX-M-5, and CTX-M-6, whereas it is replaced
by a serine in CTX-M-9 and by a valine in CTX-M-10 (4, 6, 9, 10, 14, 18). On the other hand, arginine 38 of CTX-M-1 and CTX-M-3 is replaced by a lysine in CTX-M-2 and closely related CTX-Ms and by a
glutamine in CTX-M-10 (4, 11).
Several E. coli, Klebsiella pneumoniae, and
Enterobacter cloacae isolates from different patients at our
hospital, as well as five isolates (four E. coli and one
Salmonella) with similar susceptibility profiles from four
other Spanish hospitals, were found to harbor a -lactamase with the
same 8.1 pI value. Moreover, in a 10-year survey approximately 50% of
E. coli isolates harboring ESBLs at our institution produced
a CTX-M-like -lactamase with the same 8.1 pI value (data not shown),
suggesting that CTX-M-10 may be widely distributed. Different outbreaks
involving CTX-M -lactamase-producing Enterobacteriaceae
have been reported. CTX-M -lactamases are now recognized as an
important cause of cefotaxime resistance in
Enterobacteriaceae. In Poland, several E. coli
and C. freundii isolates from the same institution were
found to harbor the CTX-M-3 -lactamase (11). In
Argentina, the CTX-M-2 -lactamase is widely distributed and is
currently the ESBL most frequently found in K. pneumoniae
(69% of all ESBLs found) (Galas et al., 39th ICAAC) as well as in
Enterobacter, Citrobacter, Serratia, Morganella, and Providencia (E. G. Pasteran,
R. G. Melano, M. F. Galas, M. M. Rodriguez, W. Group,
and M. A. Rosi, Abstr., 39th Intersci. Conf. Antimicrob. Agents
Chemother., abstr 1475, 1999).
It is presently not clear whether isolates harboring CTX-M
-lactamases should be considered resistant to all cephalosporins as
recommended for ESBLs (16). In vitro susceptibility
testing shows that MICs of ceftazidime are not much increased in
isolates harboring these -lactamases, and a significant inoculum
effect is not found (Table 1). Moreover, the enzymatic hydrolysis
profile demonstrates that ceftazidime is highly stable to hydrolysis by CTX-M -lactamases. Finally, the chromosomal -lactamases from which CTX-M is thought to be derived (from K. ascorbata,
K. oxytoca, and C. diversus), even when
hyperproduced, hardly affect ceftazidime and are routinely reported
with the NCCLS criteria as being susceptible to this antibiotic.
Determination of whether to report isolates harboring CTX-M
-lactamases as susceptible or resistant to ceftazidime can only be
done with clinical experience.
In summary, we describe the characterization of a novel
cefotaxime-hydrolyzing -lactamase, CTX-M-10, that is closely related to CTX-M-3 and is widely distributed in our institution.
Nucleotide sequence accession number.
The GenBank accession
number for the CTX-M-10 -lactamase is AF255298.
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ACKNOWLEDGMENTS |
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We thank L. de Rafael for English correction and critical reading and J. Blázquez for suggestions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Ramón y Cajal, Cta. Colmenar Km 9.1, Madrid 28034, Spain. Phone: 34 91 3368330. Fax: 34 91 3368809. E-mail: rcanton{at}hrc.insalud.es.
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