Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Resistance to the expanded-spectrum cephalosporins can
occur in Escherichia coli (E. coli)
and Klebsiella species via the production of expanded-spectrum
β-lactamases (ESBL) that are capable of hydrolyzing
oxyimino-cephalosporins and monobactams[1,
2]. The CTX-M-β-lactamases are a group of molecular class A ESBL that exhibit an overall
preference for hydrolysis of cefotaxime (hence the CTX-M
name) and ceftriaxone and a higher susceptibility to
tazobactam than to clavulanic acid. CTX-M-β-lactamases
have been recognized and reported in the literature with
increasing frequency[2, 3]. This resistance mechanism is
widespread throughout the world, with reports of clinical isolates
producing these β-lactamases from Europe, Africa, Asia,
South America, and most recently in North
America[3, 4].
The blaCTX-M genes are often carried on transferable
plasmids [5]. Two of them
(blaCTX-M-2 and
blaCTX-M-9) were found to be associated with complex class 1 integrons related to
In6 and In7, although they are not found on typical gene
cassettes[5]. CTX-M-β-lactamases are not closely related
to Temoniera (TEM) or Sulphydryl variable (SHV)
ESBL but share high amino acid identity with chromosomal
β-lactamases from Kluyvera georgiana, Kluyvera
cryocrescens, and Kluyvera
ascorbata[6-9]. According to a recent review and new data within GenBank,
CTX-M-β-lactamases can be divided into 5 groups based on their amino
acid sequence identities[3]. Group I includes CTX-M-1, -3,
-10 to -12, -15 (UOE-1), -22, -23, -28, -29, and -30. Group II
includes CTX-M-2, -4 to -7, and -20 and Toho-1. Group III
includes CTX-M-8. Group IV includes CTX-M-9, -13,
-14, -16 to -19, -21, and -27 and Toho-2. Finally, group V
includes CTX-M-25 and -26 (http: // www. lahey. org/
studies/). The members of these groups exhibit >94% amino
acid identity within the group and members of different
lineages differ at 10%_30% of the amino acid
residues[3].
In our previous report, 5 strains of Klebsiella pneumoniae
(K. pneumoniae) and 3 strains of
E. coli produced 5 novel CTX-M enzymes were identified in our
area[10]. These novel enzymes came from CTX-M-14 with
1-3 amino acid substitution by sequencing and the BLAST program
(http://www.ncbi.nlm.nih.gov/BLAST/). We suspected that clonal
outbreak had occurred among those isolates, but did not
have adequate evidence to confirm this hypothesis. Thus,
in the present study, we sought to further characterize the
novel CTX-M enzymes involved and determine the
molecular epidemiology of these strains.
Materials and methods
Bacterial strains Five K. pneumoniae
isolates and 3 E coli isolates were collected from 3 hospitals; 1 hospital
was a tertiary-care teaching hospital with over 1000 beds,
and the other two were tertiary-care hospitals with more than
500 beds in Hefei, Anhui province, between September 1999
and September 2000. Eight strains were from the
hospitalized patients, including 5 from sputum and 3 from urine
samples (Table 1). The isolates were identified by using the
Microscan Walkaway-40 System (Dade Behring, West Sacramento, California, USA). All of the strains were
confirmed non-repeated and clinical significance isolates.
E. coli C600, E. coli
DH5a, and E. coli JM109 were stored by the
Anhui Center for Surveillance of Bacterial Resistance (Hefei,
China).
Conjugation experiment To determine whether
resistance was transferable, conjugations were performed
with a streptomycin-resistant recipient, E. coli
C600 (lac-) as the recipient. Donor strains in the logarithmic phase of
growth were mixed with recipients in the early stationary
phase in a 1:10 ratio in Muller-Hinton broth (Oxiod,
Basingstoke, Hampshire, UK), and the mixture was
incubated at 37oC for 14
h[11]. Conjugation mixtures were plated
on MacConkey agar (Tianhe, Hangzhou, China) containing
streptomycin (500 µg/mL) and cefotaxime (2 µg/mL) and
then incubated for approximately 20 h at
37oC.
PCR amplification and bla gene sequencing
Plasmid DNA from different K.
pneumoniae and E. coli strains was used as the template in the PCR amplification. The
oligonucleotides used as primers for amplification and
sequencing are shown in Table 2. A search for
the blaTEM,
blaSHV,
blaCTX-M-9,
blaOXA-1,
blaOXA-2, and
blaOXA-10 genes in the clinical isolates was performed by PCR amplification, as described
previously. The detection of the ampC gene was performed
as described by Bou and
Martínez-Beltrán[12, 13].
The entire CTX-M genes from the screening for the
CTX-M-9 group gene positive strains were sequenced directly
from PCR amplified DNA. Primers were used for the
amplification of 1101 bp products containing the whole CTX-M-9
group open reading frame (ORF). Plasmid DNA extracted
from 8 clinical isolates and their transconjugant by rapid
alkaline lysis protocol was used as the template. PCR
amplification was carried out under the conditions as previously
described[10]. The purified PCR products were ligated with
pGEM-Teasy vectors (Promega, Madison, Wisconsin, USA)
and expressed in E. coli
DH5a. All nucleotide sequences
were determined by the bidirectional sequencing of PCR
production with the 3730 automatic DNA sequencer (Sangon,
Shanghai, China). The sequences were compared with the
sequence of the CTX-M-14 gene (AF252622).
Cloning of the CTX-M gene To ascertain the resistant
characteristic of the novel enzymes, the transform
experiment was performed as described by Ishii et
al[14]. For cloning the novel CTX-M genes, the whole ORF amplicon was
linked into the vector pHSG398 (2227bp) by T4
DNA ligase (TaKaRa, Dalian, China) after cleavage
by EcoRI and BamHI restriction enzymes (TaKaRa, China). Then, the
recombinant plasmid was introduced into E. coli
JM109 made competent by the calcium chloride method. After transformation,
a few clones grew on Luria-Bertani (L-B) agar plates
supplemented with cefotaxime (2 µg/mL) and chloromycetin (50 µg/mL). They harbored an identical plasmid with an insert
of approximately 1101 bp. These plasmids were used as
templates to determine the nucleotide sequence in the 3730
automatic DNA sequencer (Sangon, China).
Antimicrobial susceptibility tested
The minimal inhibitory concentrations (MIC) of antimicrobial agents were
determined by the broth dilution method
according to the guidelines of the Clinical and Laboratory Standards
Institute (CLSI), 2005[15]. The MIC was defined as the lowest
concentration that prevented visible growth
after incubation for 16_20 h at 35
oC. The antimicrobial agents were as follows: penicillin G, piperacillin, cefuroxime,
ceftriaxone, cefotaxime, ceftazidime, cefepime, imipenem,
aztreonam, tazobactam, ciprofloxacin, and levofloxacin
(National Institute for the Control of the Pharmaceutical
and Biological Products, Beijing, China), clavulanic acid
(Glaxo Smith Kline, London, UK), cephalothin (Sigma, St
Louis, MO, USA). The concentration of tazobactam and
clavulanic acid was tested with a fixed concentration of 4
µg/mL, respectively. All antimicrobial agents were
incorporated into cation-adjusted Mueller-Hinton broth in
serial 2-fold concentrations from 0.06 to 256 µg/mL. The
quality control strains were E. coli ATCC 25922 and
E. coli ATCC 35218 with every batch of clinical isolates to ensure
the accurate and comparable performance of
assays[15]. The inoculating concentration of bacteria was approximate
1.5×108 CFU/mL, equivalent to a 0.5 McFarland standard.
The final concentration of inoculum was
5×105 CFU/mL. Then 1 mL of the adjusted inoculum was added to each tube
containing l mL of antimicrobial agents in the dilution
series and mixed.
The ESBL production of the transformants was detected
by phenotypic confirmatory tests (cation-adjusted
Mueller-Hinton broth dilution test) as recommended by the CLSI, 2005.
E. coli ATCC 25922 and K. pneumoniae
ATCC 700603 were used as negative and positive controls,
respectively[15].
β-Lactamase preparation CTX-M mutant-encoding
genes overexpressed in E. coli JM109 were grown in 0.1 L of
L-B broth containing cefotaxime at 2 mg/mL for 18 h at 37
oC. The bacteria collected by centrifugation were suspended
with 0.1 mmol/L phosphate-buffered saline (PBS) and
disrupted by ultrasonic treatment (15 times for 10 s, each time at
20 W). After centrifugation at
20000×g for 60 min at 4 oC, the
CTX-M purification was carried out as previously
described[16] by ion-exchange chromatography with an SP sepharose
column (Amersham Pharmacia Biotech, Piscataway, New Jersey,
USA).
Determination of β-lactamase kinetic
constants β-lactamase activity was determined spectrophotometrically
by measuring the change in absorbency at different
wavelengths for penicillin G (231 nm), cephalothin (266 nm),
cefuroxime (271 nm), cefotaxime (234 nm), and ceftazidime
(258 nm) in a SP-752 type recording spectrophotometer
(Spectrum Instruments, Shanghai, China). The reaction
mixtures were maintained at 30 oC (pH 7.0) by means of a
circulating water bath, and the reaction was started by the
addition of the enzyme. For all of the assays, the controls were
performed by omitting from the reaction mixture the specific
substrate. Kinetic parameters were estimated from a
least-squares fit of Lineweaver-Burk plots with a substrate
concentration ranging from 0.01 to 0.10
mmol/L[17].
Isoelectric focusing and enzyme
inhibition assay Isoelectric focusing (IEF) was carried out with polyacrylamide
gel containing ampholytes with a pH range of 3.0_10
(Amersham Pharmacia Biotech, USA) for the new enzymes
as previously described[18]. The pellet was resuspended in 1
mL phosphate buffer (pH 7.0, 10 mmol/L), and sonicated for
10 min in a sonicator (Sonics & Material, Newtown,
Connecticut, USA) in ice-cold water. After IEF, β-lactamases
were detected by spreading nitrocefin (Oxiod, UK) on the
gel surface. Isoelectric points (pI) were determined by
comparison with those of β-lactamases with known pI: TEM-1
(pI 5.4), SHV-1 (pI 7.6), SHV-5 (pI 8.2), SHV-18 (pI 7.8), and
CTX-M-5 (pI 8.8), and were calculated by using CurveExpert
1.3 software. An inhibition assay was carried out by
overlaying the gels with 0.5 mmol/L nitrocefin with or without
0.3 mmol/L clavulanic acid in 0.1 mmol/L PBS (pH 7.0).
Plasmid profiling and restriction fragment length
polymorphism of plasmid DNA A restriction analysis
of the CTX-M-containing plasmids was carried out on
plasmids extracted from transconjugants by a rapid alkaline
lysis procedure. Transferable plasmids were purified, and
purified plasmid DNA was digested with the
PstI restriction enzyme (TaKaRa, China). DNA restriction fragment
length polymorphisms (RFLP) were analyzed by
electrophoresis on 0.8% agarose gels stained with ethidium bromide at 45
V for 16 h at 20 oC.
Pulsed-field gel electrophoresis analysis
Chromosomal DNA was prepared as previously described and digested with
the XbaI restriction enzyme (TaKaRa,
China)[19, 20]. DNA fragments were separated by electrophoresis in 0.8% agarose gels
(Sangon, China) and 0.5×Tris-Borate-EDTA (0.5×TBE) buffer
by using a contour-clamped homogeneous electric field
(Bio-Rad, Hercules, California, USA) according to the following
electrophoresis conditions: 12 oC at 6 V/cm for 27 h with the
pulse time ranging from 10 to 40 s. The DNA bands were
visualized by staining of the gel with ethidium bromide and
were then photographed. Clonal relationships were
established, a criterion recommended by Tenover et
al[19].
Results
The transferability of the CTX-M determinants was
assayed in conjugation experiments using an E.
coli recipient and selection of transconjugants at similar frequencies of
1×10-3-1×10-8. CTX-M genes were detected by PCR in 8
E coli C600 transconjugants. The results of the DNA
sequencing with the whole ORF primer indicated that production of
the CTX-M-9 group was almost identical to that of
CTX-M-14. One to three point mutations occurred in 8 isolates,
resulting in the amino acid substitutions compared with
CTX-M-14 (AF252622), as shown in Table 3. Five novel CTX-M
enzymes were determined as CTX-M-46 (AY847147),
CTX-M-47 (AY847143), CTX-M-48 (AY847144), CTX-M-49
(AY847145), and CTX-M-50 (AY847146). The
blaTEM genes were amplified from 8 studied isolates. All isolates were found
to carry TEM-1 by nucleotide sequencing, and one was
shown to harbor SHV-12 by sequence analysis. The
genotypes of all transconjugants were consistent with those of
their donors.
All of the wild-type strains and the transconjugants had
the same resistance spectrum, which exhibited the same high
resistant rate to piperacillin, ceftriaxone, and cefotaxime. All
of the strains were susceptible to imipenem, but compared
with the wild-type strains, the transconjugants decreased
the resistant ability to all of the antimicrobial agents and
obviously enhanced the susceptibility to fluoroquinolones.
Five recombinant plasmids containing novel
blaCTX-M were confirmed to clone into the competent cell
(E. coli JM109) successfully by PCR methods. In the
E. coli transformants, the MIC for ceftazidime were low (ranging from 1 to 4 µg/mL), which is characteristic for most CTX-M enzymes,
and significant synergy with β-lactamases inhibition was
observed with β-lactam antimicrobial agents (piperacillin,
cefotaxime, and ceftazidime; Table 4).
Phenotypic confirmatory testing for the ESBL
production of transformants requires use of both cefotaxime and
ceftazidime, alone and in combination with clavulanic acid.
There was a ¡Ý3 2-fold concentration decrease in a MIC for
either antimicrobial agent tested in combination with
clavulanic acid versus its MIC when tested alone. The
results of the antimicrobial test showed that 5 novel CTX-M
enzymes were typical for class A ESBL (Table 4).
The kinetic parameters of 5 novel CTX-M-β-lactamases
were determined for a representative set of β-lactam
antimicrobial agents (Table 5). The results showed that the
β-lactamase exhibited a broad-spectrum activity profile,
although with notable differences for different substrates.
Common enzymatic features included better affinities for
penicillin G than for cephalothin, cefuroxime, cefotaxime, and
ceftazidime. Cephalothin was the best substrate; and the
rate of hydrolysis of ceftazidime was too slow to obtain an
accurate Km value.
On the IEF gels, all β-lactamases produced by the 8
classical ESBL-producing isolates were inhibited by 0.3 mmol/L
clavulanic acid, so the CTX-M-containing isolates that had
pI 8.0 enzymes were represented CTX-M-14-derivative
enzymes. Most isolates expressing CTX-M enzyme were
found to produce additional β-lactamases (Table 3). The
enzymes with a pI of 5.4 detected in the 8 isolates were not
inhibited by clavulanic acid and thus were tentatively
classified as broad-spectrum β-lactamases. A further enzyme with
a pI of 8.2 was inhibited by clavulanic acid. Thus, it was
considered to carry classical ESBL. The genotypes of all
transconjugants were consistent with those of their donors.
The pI of all transconjugants were consistent with 8
wild-type strains, and the pI of 8.0 were observed in transformants.
Purified plasmids from 8 transconjugants of strains were
digested with PstI, and the resulting fragments were
separated on 0.8% agarose gels (Figure 1). The overall results
showed a similarity between plasmids in E. coli
269 and E coli 182, thus suggesting identity in the plasmids
harboring CTX-M-47 on these strains. However, the other 6
transconjugants did not show similarity in their RFLP
patterns. Therefore, the CTX-M-46, CTX-M-48, CTX-M-49,
and CTX-M-50 genes were harbored in different plasmids
on these strains, and so the possibility that a mobile genetic
element might be involved in the dissemination of the
CTX-M-14-derivation gene in these plasmids cannot be ruled out.
Pulsed-field gel electrophoresis analysis (PFGE) was
performed to determine whether clonal spreading was
responsible for the dissemination of CTX-M-14 derivatives. Based
on the criteria previously described by
Tenover et al[15], that
is, differences of no more than 3 bands belonged to the
epidemiologically-related strains, and differences of more than
6 bands belonged to the epidemiologically-unrelated strains.
The 8 CTX-M-14-derivative strains isolated from 3 hospitals
belonged to different clones, except for E. coli
269 and E. coli 182, which exhibited closely-related isolates (Figure 2).
The sources from which the 8 isolates producing 5 novel
CTX-M enzymes were recovered, as well as the selected
clinical features of the patients carrying these organisms, are
summarized in Table 1. Positive cultures of CTX-M
enzyme producers were obtained from 8 patients after 72
h of hospitalization. CTX-M-48 producers were isolated from 2
patients in the Intensive Care Unit (ICU). Patient 3 had
accepted urethral catheterization. Patients 1, 5, and 8 had been
hospitalized for 3 to 6 months before clinical presentations
of their infections. Patient 4 had little history of
hospitalization before she underwent gastrectomy in hospital. Since it
was not known that they were infected with ESBL producers,
patient 6 was treated with piperacillin, cefotaxime, and
ceftriaxone, and patient 7 was treated with cefuroxime,
cefotaxime, ciprofloxacin, and levofloxacin.
Discussion
In recent reports, ESBL phenotypes were detected in 45%
of K. pneumoniae strains from Latin America, 23% from the
Western Pacific, 23% from Europe, 8% from the United States,
and 5% from Canada[21]. CTX-M enzymes were initially
isolated from strains in Europe and Argentina in the late 1980s
and early 1990s[22]. Since then, the plasmid-encoded CTX-M
enzyme, which is one of the most prevalent types in Asian
countries, has now been encountered on 5 continents
owing to the ease of global travel;
blaCTX-M may easily be spread worldwide as a means to adapt to environmental hazards
for bacterial growth and has a high survival capacity in the
environment. The inherent ability of K. pneumoniae
and E. coli may facilitate the development of resistance profiles
with the widespread use of antimicrobial agents through the
selection of strains with ever-accumulating antimicrobial
resistance profiles in hospital environments. Numerous
reports from eastern Europe, Asia, and South America have
identified CTX-M-type ESBL.
The wild-type isolates, transconjugants, and transformants
of our data exhibited a moderate or high resistance to
cefotaxime and ceftriaxone; however, most of them are
susceptible to ceftazidime and aztreonam, except for
E. coli 151, K. pneumoniae 247, and
K. pneumoniae 301. We consider that the reason is other ESBL involved in the resistant
patterns. K. pneumoniae 247, producing the SHV-12 enzyme,
obviously accounts for the resistance ability to ceftazidime.
E. coli 151 and K. pneumoniae 301, with broad-spectrum
β-lactamase (TEM-1), were simultaneously detected. This
could not be easily explained and questions still remain.
Therefore, we supposed that multicopies of plasmids or/and
the hyperproduction of TEM-1 in the wild-type strain may
account for the resistant property, which is resistant to
ceftazidime, and found that aztreonam declined in its
transconjugants[23]. In addition, the wild-type isolates and
the transconjugants displayed resistance to the 2
fluoro-quinolones (ciprofloxacin and levofloxacin). We can
clearly see the difference between the 2 groups of strains:
most of the wild-type strains were resistant to ciprofloxacin
and levofloxacin. However, all of the transconjugants were
susceptible to ciprofloxacin and levofloxacin. Therefore, we
infer that the resistance to ciprofloxacin and levofloxacin in
our strains is not plasmid-borne, but chromosomally
mediated.
Like CTX-M-9, CTX-M-14, has been described in China,
Korea, France, Japan, and Spain since
2002[24]. Enzymes of the CTX-M-9 group were defined as a pI ranged from 7.9 to
8.2, and had a higher catalytic activity against cefotaxime
than against ceftazidime and aztreonam. To our knowledge,
amino acid residues Asn104, Asn132, Phe160, Gly232,
Ser237, and Arg276 are thought to play an important role
in the catalytic properties in the CTX-M
β-lactamases[16, 25]. The substitution of Ser237, which is known to enhance
hydrolysis of cefotaxime, is observed in
CTX-M-14[16, 26]. The amino acid substitutions at positions 164, 179, 238, and
240[27], which are associated with the expansion of the spectrum of
activity towards oxyimino-cephalosporins and aztreonam in
TEM- and SHV-type ESBL [16, 28], were not observed in
CTX-M-46 to CTX-M-50, for which the MIC of aztreonam and
ceftazidime were low. In our report, the 3 substitution sites
(Lys31Asn, Gly46Arg, and Ala51Pro) do not belong to the
active sites or are not adjacent to the indirect active sites,
such as the Ω loop, which plays an important role in
influencing substrates, so we supposed that the substitutions
would not result in the resistance pattern changing, or a
neutral mutation. From our data, we also concluded that
cefotaxime was the specific substrate to the 5 CTX-M-14
derivatives as their parent enzymes.
In this study, the PFGE of XbaI-digested genomic DNA
and the electrophoresis of the PstI-digested
blaCTX-M-containing plasmid showed that 8 isolates and the resistant
plasmids had diverse patterns despite 1-3 amino acid
substitutions occurring intensively from the same parent enzyme
from the same hospital. The closely-related restriction
patterns only were found between strains of E. coli
269 and E coli 182, which were obtained from the First Affiliated
Hospital of Anhui Medical University and the First People's
hospital of Hefei, respectively. As both are teaching
hospitals, the patients, interchange between the 2 hospitals
was frequent. We excluded 2 isolates obtained from the
same patient (their addresses were indefinite). Therefore,
we conclude that CTX-M-14-derivatives could be the
multiplex genesis or 2 patients could come from the same area, but
the definite mechanisms that 8 CTX-M-14 derivatives
occurred remain unknown.
Spreading between patients can be easy when a
bacterium with an antimicrobial resistance mechanism has been
established, especially if this mechanism is associated with
plasmids or other mobile genetic elements. We should alert
the medical community of the increase of these
β-lactamases in our area (especially isolates collected from patients with
underlying diseases in the hospital environment, such as
ICU). These hospitals are large-scale general hospitals, and
the interchange of patients who come from other
areas between them is frequent. Therefore, it is necessary to
strengthen the surveillance of antimicrobial resistance in
local areas and exchange data between different areas. There
is the extremely important epidemiology significance in this
work to prevent dissemination of resistant genes.
Five novel CTX-M enzymes are clinical variants found in
Anhui Province. The continuing evolution of genes
encoding ESBL which are caused by the misuse of antimicrobial
agents (especially the empirical administration of cefotaxime
in serious infections) are reflected in the increasingly large
number of derivative of β-lactamases, and their widespread
dissemination on resistant plasmids significantly limits
therapeutic choices. The rational use of other antimicrobial agents
may improve the situation. In addition, this research
emphasizes that improving the detection of ESBL with molecular
procedures and the implementation of appropriate infection
control procedures will provide a more accurate assessment
of their prevalence and lead to more rational uses of
antimicrobial agents in order to prevent clinical
complications[29], which in turn will reduce the selection and spread of
organisms producing these enzymes. We also should increase
efforts in surveillance and the study of risk factors (ie
hospitalized time and invasive operation) associated with the
acquisition of these isolates. This will guide future prevention
and control measures, which will continue to present
challenges for clinical microbiologists and clinicians
alike[30].
References
1 Pitout JD, Hossain A, Hanson ND. Phenotypic and molecular
detection of CTX-M-β-lactamases produced by Escherichia
coli and Klebsiella spp. J Clin Microbiol 2004; 42: 5715_21.
2 Bush K. New beta-lactamases in gram-negative bacteria:
diversity and impact on the selection of antimicrobial therapy. Clin
Infect Dis 2001; 32: 1085_9.
3 Bonnet R. Growing group of extended-spectrum beta-lactamases: the
CTX-M enzymes. Antimicrob Agents Chemother 2004; 48: 1_14.
4 Moland ES, Black JA, Hossain A, Hanson ND, Thomson KS,
Pottumarthy S. Discovery of CTX-M-like extended-spectrum
beta-lactamases in Escherichia coli isolates from five US states.
Antimicrob Agents Chemother 2003; 47: 2382_3.
5 Tzouvelekis LS, Tzelepi E, Tassios PT, Legakis NJ.
CTX-M-type beta-lactamases: an emerging group of extended-spectrum
enzymes. Int J Antimicrob Agents 2000; 14: 137_42.
6 Pagani L, Dell'Amico E, Migliavacca R, D'Andrea
MM, Giacobone E, Amicosante G, et al. Multiple CTX-M-type
extended-spectrum beta-lactamases in nosocomial isolates of
Enterobacteriaceae from a hospital in northern Italy. J Clin Microbiol 2003;
41: 4264_9.
7 Poirel L, Kämpfer P, Nordmann P. Chromosome-encoded Ambler
class A beta-lactamase of Kluyvera
georgiana, a probable progenitor of a subgroup of CTX-M extended-spectrum
beta-lactamases. Antimicrob Agents Chemother 2002; 46: 4038_40.
8 Decousser JW, Poirel L, Nordmann P. Characterization of a
chromosomally encoded extended-spectrum class A
beta-lactamase from Kluyvera
cryocrescens. Antimicrob Agents Chemother 2001; 45: 3595_8.
9 Humeniuk C, Arlet G, Gautier V, Grimont P, Labia R, Philippon
A. Beta-lactamases of Kluyvera ascorbata, probable progenitors
of some plasmid-encoded CTX-M types. Antimicrob Agents
Chemother 2002; 46: 3045_9.
10 Li H, Li JB. Detection of five novel CTX-M-type extended
spectrum beta-lactamases with one to three CTX-M-14 point
mutations in isolates from Hefei, Anhui Province, China. J Clin
Microbiol 2005; 43: 4301_2.
11 Liebana E, Gibbs M, Clouting C, Barker L, Clifton-Hadley FA,
Pleydell E, et al. Characterization of beta-lactamases responsible
for resistance to extended-spectrum cephalosporins in
Escherichia coli and Salmonella
enterica strains from food-producing animals in the United Kingdom. Microb Drug Resist 2004; 10:
1_9.
12 Nagano N, Nagano Y, Cordevant C, Shibata N, Arakawa Y.
Nosocomial transmission of CTX-M-2 beta-lactamase-producing
Acinetobacter baumannii in a neurosurgery ward. J Clin Microbiol
2004; 42: 3978_84.
13 Bou G, Martínez-Beltrán J. Cloning, nucleotide sequencing, and
analysis of the gene encoding an AmpC β-lactamase in
Acinetobacter baumannii. Antimicrob Agents Chemother 2000;
44: 428_32.
14 Ishii Y, Ohno A, Taguchi H, Imajo S, Ishiguro M, Matsuzawa H.
Cloning and sequence of the gene encoding a
cefotaxime-hydrolyzing class A beta-lactamase isolated
from Escherichia coli. Antimicrob Agents Chemother 1995; 39: 2269_75.
15 Clinical and Laboratory Standards Institute/NCCLS. Performance
standards for antimicrobial susceptibility testing; Fifteenth
informational supplement. CLSI/NCCLS document M100-S15
2005; 25: 1_167.
16 Bonnet R, Dutour C, Sampaio JL, Chanal C, Sirot D, Labia R,
et al. Novel cefotaximase (CTX-M-16) with increased catalytic
efficiency due to substitution
Asp-240¡úGly. Antimicrob Agents Chemother 2001; 45: 2269_75.
17 Pechère JC, Guay R, Dubois J, Letarte R. Hydrolysis of cefotaxime
by a beta-lactamase from Bacteroides
fragilis. Antimicrob Agents Chemother 1980; 17: 1001_3.
18 Stürenburg E, Kühn A, Mack D, Laufs R. A novel
extended-spectrum beta-lactamase CTX-M-23 with a P167T substitution in
the active-site omega loop associated with ceftazidime resistance.
J Antimicrob Chemother 2004; 54: 406_9.
19 Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE,
Persing DH, et al. Interpreting chromosomal DNA restriction
patterns produced by pulsed-field gel electrophoresis: criteria for
bacterial strain typing. J Clin Microbiol 1995; 33: 2233_9.
20 Machado E, Cantón R, Baquero F, Galán JC, Rollán A, Peixe
L, et al. Integron content of
extended-spectrum-β-lactamase-producing Escherichia coli
strains over 12 years in a single hospital in Madrid, Spain. Antimicrob Agents Chemother 2005; 49:
1823_9.
21 Winokur PL, Canton R, Casellas JM, Legakis N. Variations in the
prevalence of strains expressing an extended-spectrum
beta-lactamase phenotype and characterization of isolates from
Europe, the Americas, and the Western Pacific region. Clin
Infect Dis 2001; 32 (Suppl 2): S94_103.
22 Bauernfeind A, Grimm H, Schweighart S. A new plasmidic
cefotaximase in a clinical isolate of Escherichia
coli. Infection 1990; 18: 294_8.
23 Wu PJ, Shannon K, Phillips I. Effect of hyperproduction of
TEM-1 beta-lactamase on in vitro susceptibility of
Escherichia coli to beta-lactam
antibiotics. Antimicrob Agents Chemother 1994; 38:
494_8.
24 Quale JM, Landman D, Bradford PA, Visalli M, Ravishankar J,
Flores C, et al. Molecular epidemiology of a citywide outbreak of
extended-spectrum beta-lactamase-producing Klebsiella
pneumoniae infection. Clin Infect Dis 2002; 35: 834_41.
25 Ma L, Ishii Y, Chang FY, Yamaguchi K, Ho M, Siu LK.
CTX-M-14, a plasmid-mediated CTX-M type extended-spectrum
beta-lactamase isolated from Escherichia
coli. Antimicrob Agents Chemother 2002; 46: 1985_8.
26 Ambler RP, Coulson AF, Frere JM, Ghuysen JM, Joris B, Forsman
M, et al. A standard numbering scheme for the class A
beta-lactamases. Biochem J 1991; 276 (Pt 1): 269_70.
27 Barthélémy M, Péduzzi J, Bernard H, Tancrède C, Labia R. Close
amino acid sequence relationship between the new
plasmid-mediated extended-spectrum beta-lactamase MEN-1 and
chromosomally encoded enzymes of Klebsiella
oxytoca. Biochim Biophys Acta 1992; 1122: 15_22.
28 Knox JR. Extended-spectrum and inhibitor-resistant TEM-type
beta-lactamases: mutations, specificity, and three-dimensional structure.
Antimicrob Agents Chemother 1995; 39: 2593_601.
29 Rahal JJ, Urban C, Horn D, Freeman K, Segal-Maurer S, Maurer J,
et al. Class restriction of cephalosporin use to control total
cephalosporin resistance in nosocomial
Klebsiella. JAMA 1998; 280: 1233_7.
30 Pitout JD, Gregson DB, Church DL, Elsayed S, Laupland KB.
Community-wide outbreaks of clonally related CTX-M-14
beta-lactamase-producing Escherichia coli strains in the Calgary health
region. J Clin Microbiol 2005; 43: 2844_9.
|
