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Antimicrobial Agents and Chemotherapy, December 1998, p. 3304-3308, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Efficacies of Cefepime, Ceftazidime, and Imipenem Alone or in
Combination with Amikacin in Rats with Experimental Pneumonia Due
to Ceftazidime-Susceptible or -Resistant Enterobacter
cloacae Strains
Olivier
Mimoz,1,*
Anne
Jacolot,2
Sophie
Leotard,3
Nadia
Hidri,3
Kamran
Samii,1
Patrice
Nordmann,3 and
Olivier
Petitjean2
Service
d'Anesthésie-Réanimation, Hôpital Paul Brousse,
Assistance Publique-Hôpitaux de Paris, 94804 Villejuif
Cedex,1
Crépit 93, Centre de
Recherche en Pathologie Infectieuse et Tropicale, Hôpital
Avicenne, Assistance Publique-Hôpitaux de Paris, 93009 Bobigny Cedex,2 and
Service de
Bactériologie-Virologie, Hôpital de Bicêtre,
Assistance Publique-Hôpitaux de Paris, 94275 Le
Kremlin-Bicêtre Cedex,3 France
Received 8 May 1998/Returned for modification 5 August
1998/Accepted 12 September 1998
 |
ABSTRACT |
The antibacterial activities of human regimens of cefepime,
ceftazidime, and imipenem alone or in combination with amikacin against an isogenic pair of Enterobacter cloacae strains
(wild type and its corresponding derepressed cephalosporinase mutant) were compared by using our nonlethal model of pneumonia with 180 immunocompetent rats. Compared with untreated animals, all
-lactam-treated rats, except those inoculated with the mutant
isolate and receiving ceftazidime, had significantly lower bacterial
counts in their lungs 60 h after the onset of therapy. Although
the combination of a -lactam and amikacin was more bactericidal than
each corresponding antimicrobial agent alone, true synergy was
noted only with cefepime and imipenem against the
constitutive derepressed strain.
| |
TEXT |
Stably derepressed mutant strains of
Enterobacter spp. resist most available -lactams,
including ceftazidime, except for the carbapenems (15).
Cefepime, a newly extended-spectrum cephalosporin, retains in vitro
efficacy against these mutants, but few in vivo data have confirmed
these results. Comparing the bactericidal activities of cefepime and
amikacin against an isogenic pair of Enterobacter cloacae
strains (wild type and its ceftazidime-resistant mutant) in induced
experimental pneumonia with rats, we previously reported that each
antibiotic alone failed to decrease bacterial counts in the lungs,
while combined therapy significantly reduced the number of viable
microorganisms (12). The short duration of therapy in these
experiments (24 h) as well as the absence of groups treated with a
carbapenem or an expanded-spectrum cephalosporin does not permit the
definitive proposal of cefepime as the first choice for therapy against
stably derepressed cephalosporinase-producing enterobacterial
strains. Therefore, the purpose of the present study was to compare the
bactericidal activities of 60-h human regimens of cefepime,
ceftazidime, and imipenem-cilastatin alone or in combination with
amikacin with the same animal model.
Tested organisms.
An isogenic pair of E. cloacae
strains (wild type [474S] and its ceftazidime-resistant mutant
[474R]) identical to those inoculated into rats in our previous
experiments (12) was used for these studies. Each strain was
stored at 
70°C in Mueller-Hinton broth (bioMérieux,
Marçy-l'Etoile, France) supplemented with 10% glycerol. Fresh inocula were prepared for each experiment from cultures grown for
24 h in 10 ml of Trypticase soy broth (bioMérieux) and then
rinsed twice and suspended in normal saline prior to use.
Antimicrobial agents.
Cefepime and amikacin were from
Bristol-Myers Squibb (Paris, France), ceftazidime was from Glaxo
Wellcome (Evreux, France), and imipenem-cilastatin was from Merck
Sharp and Dohme-Chibret (Paris, France). Antibiotic powders were
freshly diluted with saline before each experiment according to the
manufacturer's instructions.
-Lactamase assay.
-Lactamase activity was assayed by UV
spectrometry as previously reported (17), with cephalothin
as the substrate, and expressed as units of specific activity. One unit
of specific activity was defined as the amount of enzyme that
hydrolyzed 1 nmol of cephalothin per min/mg of protein.
In vitro studies.
MICs were determined by an agar dilution
technique with Mueller-Hinton agar (bioMérieux) and an inoculum
of 4 log10 CFU per spot.
Pharmacokinetics.
Preliminary drug-dosing studies were run in
uninfected rats as described elsewhere (11) to determine if
the subcutaneous dose of 1 mg of uranyl nitrate (Merck, Darmstadt,
Germany) per kg of body weight that was previously recommended
(11, 12) was optimal to impair their renal function so as to
simulate the pharmacokinetics of cefepime, ceftazidime, imipenem,
and amikacin in healthy humans. Briefly, 4 days after the
uranyl nitrate injection, each rat received a single 1-ml
intraperitoneal injection of each antimicrobial agent studied. Multiple
blood samples were collected via a femoral catheter during the 8 h
following antibiotic administration and immediately centrifuged to
separate the plasma. Plasma samples were stored at 70°C and
assayed within 7 days. Individual antibiotic pharmacokinetic
parameters were determined with the Siphar software package (Simed,
Créteil, France).
Antibiotic assay.
Imipenem, ceftazidime, and cefepime
concentrations in plasma were determined by using modified versions of
high-pressure liquid chromatography assays described elsewhere
(1, 6, 7). The amikacin concentration was determined by an
immunoenzyme assay (Emit; Syva, Dardilly, France). The lower
detection limits of the assays were 0.5, 5, 1, and 1 µg/ml for
imipenem, ceftazidime, cefepime, and amikacin, respectively.
Pneumonia model.
The animal model used was previously
developed in our laboratory (11, 12). Briefly, male Wistar
rats weighting 280 to 300 g were rendered renally insufficient by
the subcutaneous administration of 1 mg of uranyl nitrate per kg and
intraperitoneally anesthetized 93 h later with phenobarbital (60 mg/kg). Each rat trachea was exposed by a vertical midline incision. A
0.5-ml portion of a bacterial suspension containing 8.9 ± 0.1 log10 CFU (mean ± standard deviation) of E. cloacae was injected intratracheally. Following inoculation,
animals were gently shaken for 15 s to help distribute the
inoculum in the lungs. Previous studies had shown that, 3 h after
bacterial inoculation, all animals had developed bilateral pneumonia
with bacterial densities (>7.5 log10 CFU/g of tissue) in
both lungs and an intense inflammatory reaction.
Treatment regimens.
Each strain used to induce pneumonia was
studied separately. Among the 180 animals utilized in this study, 64 of
the 80 wild-type and 90 of the 100 ceftazidime-resistant mutant strain
recipients were still alive 3 h after bacterial inoculation; at
this time, 8 rats from each study group were killed to document the
existence of pneumonia. The remaining rats were randomly assigned
to one control group (i.e., no antibiotic) and seven treatment groups. Treatment groups received intraperitoneal injections of either imipenem-cilastatin (30 mg/kg/8 h), ceftazidime (60 mg/kg/8 h), cefepime (60 mg/kg/12 h), or amikacin (25 mg/kg once a day)
or a combination of each -lactam with amikacin given at the same dosages. These dosages were retained so that plasma concentrations could be obtained close to those observed in adult humans receiving 1 g of imipenem three times a day, 2 g of ceftazidime three
times a day, 2 g of cefepime twice a day, or 25 mg of amikacin per
kg once a day. Therapy was started 3 h after bacterial inoculation and continued for 2.5 days.
Evaluation of antibiotic treatment.
Animals were sacrificed
60 h after the onset of therapy. Blood was obtained by aortic
puncture, put in a tube containing EDTA, and centrifuged. The
plasma was stored in two or three aliquots at 70°C for
determination of antibiotic concentrations and creatinine levels within
7 days after sampling. The plasma containing imipenem was
immediately mixed after sampling 1:1 with a stabilizing buffer containing equal volumes of 1 M morphilino-ethane sulfonate and ethylene glycol before freezing. Creatinine levels in plasma were determined to document the fact that renal impairment was well established. The lungs were aseptically removed, gently blotted with sterile absorbent paper to remove blood, weighed, placed in 25 ml
of ice-cold saline, and homogenized (Ultraturax, Staufen, Germany). The
homogenate was quantitatively cultured after serial dilution on
Drigalski agar (bioMérieux) with a Spiral Système plater (Interscience, Saint-Nom-La-Bretèche, France). After
overnight incubation at 37°C, viable bacteria were counted and
expressed as log10 CFU/g of lung. When no bacterial growth
was noted, the value of the detection limit for the specific animal
was entered into the statistical analysis.
Determination of emergence of antibiotic resistance during
therapy.
Emergence of resistance during -lactam therapy was
examined at the end of each experiment by plating 2 × 200 µl of
the lung homogenates onto agar containing imipenem (1 µg/ml),
cefepime (1 µg/ml), or ceftazidime (8 µg/ml) for the wild-type
strain group and imipenem (2 µg/ml) or cefepime (4 µg/ml) for the
ceftazidime-resistant mutant strain group. After incubation in air for
48 h at 37°C, emergence of a resistant strain(s) was defined as
growth of at least one colony of E. cloacae.
Statistical analysis.
Results are expressed as medians and
their ranges. Bacterial counts in the lungs of the control and
treatment groups were compared by one-way nonparametric analysis of
variance (Kruskal-Wallis test); when the value of this test was
statistically significant, each treatment group was compared to the
control group and to each of the other treatment groups by using the
Mann-Whitney U test without modifications. For all tests, a
P value of <0.05 was considered significant.
The susceptibilities of both organisms to the antimicrobial agents
studied are presented in Table 1. The
isolates remained susceptible to the four antibiotics tested, except
for the stably derepressed cephalosporinase-producing mutant, which was
highly resistant to ceftazidime. The cephalosporinase level in the
mutant strain was 16,900 U of specific activity, i.e., 110-fold higher than the level determined in the wild-type strain. Such results clearly
indicated that overproduction of cephalosporinase was the molecular
mechanism explaining resistance to ceftazidime.
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TABLE 1.
In vitro susceptibility of the isogenic pair of
E. cloacae strains (wild type and stably-derepressed
cephalosporinase-producing mutant) to the antibiotics studied
|
|
The pharmacokinetic parameters of the antibiotics tested in
renally insufficient rats simulated those reported in healthy humans given high doses of the same antibiotics (Table
2). Creatinine levels in plasma
measured at sacrifice were not statistically different between
the study groups, indicating that renal impairment was identical
regardless of the treatment received (Table
3). Antibiotic concentrations in plasma
observed 60 h after the beginning of therapy did not differ
significantly when the antibiotics were administered to animals
infected with the two strains. Results for each antibiotic for each
strain were then pooled to simplify presentation, and the data are
shown in Table 3. Antibiotic levels measured in the plasma of rats were
broadly similar to those usually reported in adult humans given high
doses of the same antibiotics.
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TABLE 2.
Pharmacokinetics for antibiotics given intraperitoneally
to noninfected rats with uranyl nitrate-induced renal impairment
|
|
The eight animals from each study group killed at the beginning of
therapy presented with bilateral pneumonia, with median
E. cloacae counts of log
10 8.3 (range, 7.8 to 8.5) and
log
10 8.1
(range, 8.0 to 8.5) CFU/g of lung for the
wild-type strain and
its ceftazidime-resistant mutant, respectively.
Nine animals died
during the antibiotic treatment period (seven rats
inoculated
with the wild-type strain and two with the mutant strain).
At
sacrifice, all untreated animals showed a spontaneous decrease
in
the number of bacteria 60 h after therapy was begun in the
treated
animals (Fig.
1). Compared with
untreated animals, all
-lactam-treated rats, except those
inoculated with the mutant
isolate and receiving ceftazidime, had
significantly (
P 
0.02)
lower bacterial counts in
their lungs 60 h after the onset of
therapy. Significantly
decreased bacterial titers were also observed
in the lungs of animals
inoculated with the mutant strain and
receiving antibiotic therapy
combined with amikacin and imipenem
or cefepime compared to those in
the lungs of animals receiving
each corresponding antimicrobial agent
alone (Fig.
1). No
E. cloacae isolate resistant to
either of the
-lactam agents tested was
detected in any of the
antibiotic-treated animals.
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FIG. 1.
Lung CFU/gram of the isogenic pair of E. cloacae (top panel, wild type; bottom panel, stably-derepressed
cephalosporinase-producing mutant) in rats treated with either amikacin
(AMK), ceftazidime (CAZ), cefepime (FEP), or imipenem (IMP) or with
each -lactam in combination with amikacin. Each mark represents a
single animal. The horizontal bar indicates the median for each
group.
|
|
Imipenem has been reported to be stable in the presence of
chromosomally mediated cephalosporinase and is considered the therapy
of choice for infections due to enterobacterial strains which
constitutively overproduce
-lactamase (
5,
10,
14,
15).
Although they were initially regarded as stable, most
broad-spectrum
cephalosporins such as ceftazidime are hydrolyzed to
some extent
by high levels of enzyme, rendering them useless against
derepressed
strains (
5,
8,
9). Cefepime retains in vitro
activity
toward these strains, possibly because of a combination
of factors,
including faster penetration of the outer membrane of
gram-negative
bacteria, poor affinity for most
-lactamases, and
increased resistance
to hydrolysis (
2,
8,
9,
16).
Using an in vitro infection
model to compare the bactericidal
activities of human regimens
of various
-lactams against a
ceftazidime-resistant
Enterobacter strain, Palmer and
colleagues reported a reduction in bacterial
titer over the first
6 h that was similar for all regimens, but
significant regrowth
occurred with ceftazidime, cefotaxime, and
ceftriaxone, whereas no
regrowth was observed with cefepime during
the 48 h of therapy
(
13). In this study, performed in immunocompetent
animals,
the antimicrobial activities of human regimens of cefepime
and imipenem
were broadly similar against the wild-type
E. cloacae strain as well as its corresponding stably derepressed mutant,
and
their bactericidal effects were reduced against the latter.
These
encouraging results were recently supported by a clinical
study in
which 15 of 17 infections due to
Enterobacter spp. with
low
susceptibility or resistance to ceftazidime but with susceptibility
to
cefepime were successfully treated with cefepime. In particular,
cefepime was successfully used to manage chronic infections that
had
responded poorly to repeated therapy with imipenem, aminoglycosides,
or
ciprofloxacin (
19). However, more clinical experience is
required before cefepime can be systematically used in this
setting.
Antibiotic combinations including a
-lactam and an
aminoglycoside have frequently produced an increased
bactericidal effect
in vivo in experimental models of aerobic
gram-negative bacillary
infections that has generally paralleled an
increased rate of
killing observed in vitro (
3). It has
been suggested that such
combinations are necessary in order to
prevent the emergence of
resistance during therapy; however,
recent clinical experience
with older cephalosporins, such as
cefotaxime, indicates that
the addition of an aminoglycoside does not
always prevent resistance
(
4). Selection of resistance was
not observed with any antimicrobial
regimen in our model. It is
possible that our inoculum was too
low to detect resistant
subpopulations, as these mutants are reported
to occur in 1 of
10
5 to 10
8 wild-type strains possessing
inducible cephalosporinase (
18).
In addition, the duration
of our experiments (60 h) may not have
been sufficient for the
selection of such resistant
clones.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a grant-in-aid from
Bristol-Myers Squibb, Paris, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Service
d'Anesthésie-Réanimation, Hôpital Paul Brousse,
Assistance Publique-Hôpitaux de Paris, 94804 Villejuif Cedex,
France. Phone: 33 1 45 59 32 19. Fax: 33 1 45 59 38 34. E-mail:
olivier.mimoz{at}pbr.ap-hop-paris.fr.
| |
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Antimicrobial Agents and Chemotherapy, December 1998, p. 3304-3308, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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