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Antimicrobial Agents and Chemotherapy, April 2001, p. 1287-1291, Vol. 45, No. 4
Institute of Medical Microbiology, Semmelweis
University,1 Clinical Epidemiology Unit
of the National Institute of Traumatology,2 and
Department of Infectology, Central Military
Hospital,3 Budapest, Hungary
Received 7 April 2000/Returned for modification 16 November
2000/Accepted 17 January 2001
The in vitro and in vivo effectiveness of amikacin, cefepime, and
imipenem was studied using a high inoculum of an extended-spectrum Extended-spectrum Our aim was to compare the activities of amikacin, cefepime, amikacin
plus cefepime, and imipenem in septic mice infected by an SHV-5
ESBL-producing Klebsiella pneumoniae strain using a high
initial inoculum.
The SHV-5 ESBL-producing K. pneumoniae strain originated in
a premature intensive-care unit (26). Amikacin and
cefepime (Bristol-Myers Squibb), imipenem (Merck, Sharp & Dohme), and
cisplatin (Ebewe) were used according to the manufacturers'
instructions. The MICs and the minimal bactericidal concentrations
(MBCs) were determined by the microdilution method with inocula of
105 and 107 CFU/ml, respectively (20,
21). For the killing curve, the initial bacterial concentration
was 8 log10 CFU/ml. The concentrations of antibiotics
chosen were close to the in vivo mean levels in serum: amikacin, 4 µg/ml; cefepime, 40 µg/ml; amikacin plus cefepime in the same
concentrations listed above; and imipenem, 16 µg/ml. Synergy was
defined as a Randomly selected male CD-1 mice (30 to 35 g) were used for the
pharmacokinetic study, for the determination of blood bacterial counts,
and for survival analysis. Each group contained 15 mice. Cisplatin (18 mg/kg of body weight as determined in a pilot study) had been
administered by intraperitoneal (i.p.) injection 3 days before
infection in order to cause renal impairment (18). The mice were infected i.p. with 107 CFU/g; the uninfected
group received only cisplatin. Three groups of uninfected mice were
used for pharmacokinetic analysis, and they were followed up for
48 h (3, 6, 9, 10, 16, 19, 28). Single doses of
amikacin (7.5 mg/kg), cefepime (80 mg/kg), and imipenem (40 mg/kg) were
administered i.p. Blood samples were taken 15 and 30 min and 1, 2, and
3 h after drug administration. Antibiotic levels in sera were
determined by a paper disk method for imipenem and cefepime with
Bacillus subtilis ATCC 6633 and Escherichia coli
ATCC 25922, respectively, as indicator organisms on antibiotic medium 1 (Becton Dickinson) (1). The amikacin serum levels were
detected by the fluorescence polarization immunoassay (Abbott TDx
system) (30). The lower limits of detection were 0.05 µg/ml for amikacin, 8 µg/ml for cefepime, and 1 µg/ml for imipenem. The coefficient of variability was Four infected treated groups and one infected untreated group were used
for the determination of blood bacterial counts. The treatment started
3 h after infection and lasted for 24 h. Two doses of
amikacin (7.5 mg/kg every 10.5 h), three doses of cefepime (80 mg/kg every 7 h), the same doses of amikacin plus cefepime as in
monotherapy, or four doses of imipenem (40 mg/kg every 5.25 h)
were given i.p. Blood samples were taken 3 and 9 h after the beginning of antibiotic therapy. The Kruskal-Wallis test followed by
the Mann-Whitney U test were used for statistical analysis, taking a
P value of <0.05 as significant. Four infected treated groups and two control groups (uninfected treated and infected untreated) were used for the survival analysis after up to 48 h,
with death as the end point. Survival was assessed at 24 and 48 h
for calculating lethality, and 24-h survival was assessed by estimating
the cumulative probability using the Kaplan-Meier survival curve. The
relative risk between groups was estimated by the hazard ratio with the
appropriate 95% confidence interval comparing the observed deaths with
expected deaths. The log rank test was used for statistical analysis,
accepting a P value of <0.05 as significant.
The MIC and MBC of amikacin were 0.5 µg/ml at both 105
and 107 CFU/ml. The MIC and MBC of cefepime were 1 µg/ml
at 105 CFU/ml, and the MIC was >256 µg/ml at
107 CFU/ml. The MIC and MBC of imipenem were 0.125 µg/ml
at 105 CFU/ml, and the MIC and MBC were 0.5 and 1 µg/ml
at 107 CFU/ml, respectively. The killing curve study is
shown in Fig. 1A. Synergy was not
detected due to the limits of detection of bacterial counts.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1287-1291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vitro and In Vivo Activities of Amikacin,
Cefepime, Amikacin plus Cefepime, and Imipenem against an SHV-5
Extended-Spectrum
-Lactamase-Producing Klebsiella
pneumoniae Strain
ABSTRACT
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-lactamase-producing Klebsiella pneumoniae strain. An in
vitro susceptibility test at the standard inoculum predicted the in vivo outcome of amikacin or imipenem while it did not do so for cefepime due to the inoculum effect.
TEXT
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References
-lactamase
(ESBL) production is one of the main mechanisms of resistance to
-lactam antibiotics among the strains of the family
Enterobacteriaceae (14). The therapeutic choices in infections caused by such strains remain limited because of
cross-resistance (4). Attempts have been made to compare the activities of -lactams with -lactamase inhibitors, showing inconsistent results (5, 20, 24, 27). Conflicting reports have been published concerning the activities of the broad-spectrum and
"fourth generation" cephalosporins with an explanation of the
inoculum effect (5, 15, 27).
2-log10 decrease in the number of CFU per
milliliter between the combination and its most active constituent after 24 h (17).
5% for each antibiotic assay. The pharmacokinetic values half-life
(T1/2), peak concentration (Cmax), first occurrence of
Cmax (Tmax), area under
the concentration-time curve extrapolated to infinity (AUC), and area
under the concentration-time curve from 0 to 24 h
(AUC0-24) were calculated by a noncompartmental method
(2).
View larger version (18K):
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FIG. 1.
(A) Killing curves of ESBL-producing K. pneumoniae incubated without antibiotic, with cefepime, with
amikacin, with imipenem, and with amikacin plus cefepime. (B) Mean
bacterial counts observed in untreated mice and in those receiving
amikacin, amikacin plus cefepime, cefepime, and imipenem after
infection with ESBL-producing K. pneumoniae. Error bars
indicate standard deviations. The difference was significant when all
groups were compared (P < 0.0001) and between the
infected untreated group and all treated groups (P < 0.01 in each pair compared). The cefepime-treated group differed
statistically from the other treated groups (P < 0.05
in each pair compared). There was no difference between groups
receiving amikacin, amikacin plus cefepime, and imipenem (P > 0.37 in each pair compared).
Data from the pharmacokinetic analysis are given in Table
1, except for amikacin plus cefepime
because they do not affect the elimination of each other
(2).
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The blood bacterial count increased persistently in the untreated group; cefepime initially decreased it, but an increase occurred after 6 h, while it decreased persistently in the other treated groups (Fig. 1B).
There were no deaths in the uninfected group. After 24 h,
the lethality was 14 of 15 in the infected untreated and
cefepime-treated groups, 6 of 15 in the amikacin- and
amikacin-plus-cefepime-treated groups, and 7 of 15 in the
imipenem-treated group (Fig. 2).
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The in vitro susceptibilities of ESBL-producing strains to cefepime have been found to be 52 or 90% (15, 25). Cefepime was recommended for the treatment based on this in vitro susceptibility (12). Others did not recommend it, despite its in vivo effectiveness, arguing on the basis of the inoculum effect, dose dependence, and other factors (15, 27). In our study, we saw an inoculum effect for cefepime, and it was ineffective in vitro and in vivo with a high inoculum.
Carbapenems have been reported to be stable against ESBL enzymes and are considered to be the treatment of choice (14, 24). In our study, imipenem showed a slight inoculum dependence, but the organisms remained susceptible. It persistently decreased the in vitro and in vivo bacterial counts, and it was biologically effective in the survival analysis.
Amikacin has good in vitro activity against ESBL-producing K. pneumoniae strains (15, 22). In our study, it did not
show an inoculum effect, which can be an advantage compared to
-lactams. Synergy between amikacin and cefepime was not detected in
vitro, and the combination was not more effective in vivo than amikacin alone.
Overall, the observed biological differences could not be explained by the pharmacokinetics because they were appropriate for all antibiotics, taking into account the susceptibility at the standard inoculum (7, 8, 11, 13, 23, 29). The in vitro susceptibility test and the percentage of T greater than the MIC calculated with the standard inoculum were not predictive for the biological effectiveness of cefepime due to a possible in vivo inoculum effect.
Based on our results, imipenem or amikacin can be the therapy of choice against ESBL-producing K. pneumoniae strains showing susceptibility at the standard inoculum, while cefepime cannot be recommended even though the organisms show susceptibility.
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ACKNOWLEDGMENTS |
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We thank Mónika Kapás, Györgyné Sümeghy, and Ágoston Ghidán for their valuable technical help and David Stevens for proofreading.
This study was supported by the Hungarian National Scientific Research Fund, grant no. OTKA T021251 and OTKA T032473.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Medical Microbiology, Semmelweis University Budapest, P.O. Box 370, H-1445 Budapest, Hungary. Phone and fax: 36-1210-29-59. E-mail: szabdor{at}net.sote.hu.
This study was part of the 10th accredited Ph.D. program at
Semmelweis University, Budapest, Hungary.
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Antimicrobial Agents and Chemotherapy, April 2001, p. 1287-1291, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1287-1291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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