Aztreonam, cefoperazone,
and gentamicin in the
treatment of experimental
Enterobacter aerogenes
endocarditis in rabbits.
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W D Kobasa and D Kaye
Antimicrob. Agents Chemother. 1983,
24(3):321. DOI: 10.1128/AAC.24.3.321.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1983, p. 321-324
0066-4804/83/090321-04$02.00/0
Copyright C 1983, American Society for Microbiology
Vol. 24, No. 3
Aztreonam, Cefoperazone, and Gentamicin in the Treatment
of Experimental Enterobacter aerogenes Endocarditis in
Rabbits
WILLIAM D. KOBASA AND DONALD KAYE*
Department of Medicine, The Medical College ofPennsylvania, Philadelphia, Pennsylvania 19129
Received 18 April 1983/Accepted 17 June 1983
Endocarditis caused by gram-negative bacilli
has been increasing in frequency especially in
intravenous drug abusers and in patients who
have had cardiac valves replaced (3). In a recent
review, Enterobacter spp. were reported to have
caused nine cases of endocarditis, of which only
two were cured (3). Of the older agents, carbenicillin, ticarcillin, and cefamandole, together with
aminoglycoside antibiotics, have been recommended in the therapy of endocarditis due to
Enterobacter spp. (3). The expanded-spectrum
cephalosporins, including cefoperazone, are extremely active against Enterobacter spp. (7). A
recent study from our laboratory demonstrated
that cefoperazone was more effective than cefamandole in the treatment of Enterobacter aerogenes endocarditis in rabbits (10). Aztreonam, a
synthetic monocyclic P-lactam antimicrobial
agent, has a spectrum of activity primarily
against aerobic, gram-negative bacilli, including
Pseudomonas aeruginosa (6, 12).
In this study, aztreonam was compared with
cefoperazine with and without gentamicin in the
treatment of left-sided E. aerogenes endocarditis in rabbits.
MATERUILS AND METHODS
Bacterium. The E. aerogenes strain used in this
study was a clinical isolate and has been previously
studied (10). The minimal inhibitory concentrations
and minimal bactericidal concentrations of aztreonam,
cefoperazone, and gentamicin for E. aerogenes were
determined by an antibiotic dilution method in
Mueller-Hinton broth (MHB). The antibiotics were
diluted in twofold steps in tubes containing 0.5 ml of
MHB. The bacterial inoculum was added to each tube
in 0.5 ml of MHB diluted from an 18-h MHB culture to
give a final concentration of 105 E. aerogenes organisms per ml. The minimal inhibitory concentration
was defined as the lowest concentration of antibiotic
that prevented turbidity for 24 h of incubation at 37°C.
The minimal bactericidal concentration was defined as
the lowest concentration of antibiotic that killed 99.9o
of the organisms within 24 h as determined by plating
portions of the minimal inhibitory concentration dilutions.
Stock cultures were made by incubating the organisms in MHB at 37°C for 24 h and storing 1-ml
321
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The effectiveness of aztreonam, cefoperazone, and gentamicin alone and in
combination was evaluated in Enterobacter aerogenes endocarditis in rabbits.
The minimal inhibitory concentration/minimal bactericidal concentration ratios
for E. aerogenes were as follows: aztreonam, 0.4/0.4 ,ug/ml; cefoperazone, 0.8/0.8
,ug/ml; and gentamicin, 3.1/3.1 ,ug/ml. With an inoculum of 109 organisms per ml,
aztreonam and cefoperazone were equivalent in reducing titers of E. aerogenes in
broth, and both drugs demonstrated an increased rate of reduction when gentamicin was added; gentamicin alone was least effective. E. aerogenes endocarditis in
rabbits was treated intramuscularly with aztreonam (60 mg/kg) every 6 h, with
cefoperazone (60 mg/kg) every 6 h, with gentamicin (1.7 mg/kg) every 8 h, and
with aztreonam plus gentamicin or cefoperazone plus gentamicin for 5 and 10
days, respectively. All of the therapeutic regimens were effective in reducing
vegetation titers as compared with untreated controls. Aztreonam plus gentamicin
was more effective than either aztreonam or gentamicin alone. Cefoperazone plus
gentamicin was more effective than cefoperazone alone but was not more effective
than gentamicin alone. Neither aztreonam and cefoperazone nor aztreonam and
gentamicin differed significantly, but gentamicin was significantly more effective
than cefoperazone. Aztreonam plus gentamicin did not differ significantly in
effectiveness from cefoperazone plus gentamicin. Aztreonam gave a peak level of
about 135 ,ug/ml and a half-life of 0.7 h. Cefoperazone gave a peak level of about
155 ,ug/ml and a half-life of 1.1 h. Gentamicin gave a peak level of 7.4 ,ug/ml and a
half-life of 1.3 h.
322
KOBASA AND KAYE
were mixed for 20 s, allowed to stand at room temperature for 10 min, and centrifuged at 1,400 x g for 10
min. Assayable supernatants were separated from
precipitates and kept on ice until analyzed. The chromatographic eluent used was a mixture of 2.4 ml of 1
M triethylamine in acetonitrile, 5.6 ml of 1 M acetic
acid, 240 ml of acetonitrile, and 752 ml of deionized
water.
Analysis was performed on a system composed of a
model M-45 solvent delivery system and model MU6K injector (Waters Associates, Inc., Milford,
Mass.). A C18 column was used for aztreonam and a
Radial-Pak ,-Bondapak C18 cartridge in a Z module
for cefoperazone. Samples were monitored by a
Lambda-Max model 480LC spectrophotometer set at
293 am for aztreonam and 254 nm for cefoperazone.
Results were collected and analyzed on an M730 data
module. Gentamicin levels in serun were analyzed by
an agar diffusion method with paper disks (14).
Statistical analysi. The half-lives of the antibiotics in
serum were calculated by the method of least squares
(5). Two-factor analysis of variance, followed by the
Newman-Keuls post hoc procedure with the harmonic
mean cell size, was used to determine significant
differences in vegetation titers where the independent
variables were therapeutic agents and duration of
therapy. The dependent variable was CFU per gram of
vegetation.
RESULTS
In vitro studis. The minimal inhibitory concentration/minimal bactericidal concentration
ratios for E. aerogenes were as follows: aztreonam, 0.4/0.4 >&g/ml; cefoperazone, 0.8/0.8
ig/ml; gentamicin, 3.1/3.1 ,g/ml, with an inoculum of 10' CFU/ml for each.
Figure 1 shows the rate of decrease in numbers of E. aerogenes organisms in broth containing 60 ,ug of aztreonam, 60 Fg of cefoperazone,
and 3 ,ug of gentamicin per ml alone and in
combinations. The inoculum was lop organisms
per ml. All antibiotics and combinations resulted
in a decrease in numbers of E. aerogenes organisms. Titers were most rapidly reduced by the
combinations of aztreonam plus gentamicin and
cefoperazone plus gentamicin. Gentamicin alone
was the least effective in reducing titers.
Animal experhnents. After therapy for 5 days,
the mean log10 (±- standard deviation) CFU per
gram of vegetation were as follows: aztreonam,
7.1 ± 1.2; aztreonam plus gentamicin, 3.8 ± 1.7;
cefoperazone, 8.4 ± 1.2; cefoperazone plus gentamicin, 4.4 ± 2.8; gentamicin, 5.9 ± 2.6; and,
untreated controls, 9.2 ± 0.7 (Table 1). Ten days
of therapy resulted in titers of 6.2 ± 3.0 with
aztreonam, 2.0 ± 0 with aztreonam plus gentamicin, 6.3 ± 2.1 with cefoperazone, 2.0 ± 0 with
cefoperazone plus gentamicin, 4.8 ± 1.3 with
gentamicin, and 9.5 ± 0.3 for untreated controls.
The two-factor analysis of variance revealed a
significant main effect of antibiotics (P < 0.001)
and a significant main effect of duration of
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samples at -20'C. For each experiment a sample was
subcultured into MHB and incubated at 37C for 18 h.
In vItro studies. The killing rate of E. aerogenes was
studied in flasks with MHB containing aztreonam,
gentamicin, aztreonam plus gentamicin, cefoperazone,
cefoperazone plus gentamicin, and MHB only. The
inoculum in MHB was added to each flask and incubated at 37°C. Samples were removed at 0, 3, 6, 24,
and 48 h and serially diluted in 10-fold steps in MHB,
and 0.1 ml of each dilution was plated on the surfaces
of tryptic soy agar plates with 5% sheep blood. After
24 h of incubation at 37°C, the numbers of colonies on
the plates were counted, and the numbers of CFU in
the flasks were calculated.
Animal exper nts. Female New Zealand white
rabbits (Ace Animals Inc., Boyertown, Pa.) weighing 2
to 2.7 kg each were anesthetized, and the right carotid
artery of each was cannulated as previously described
(4). At 24 h after the placement of the catheter, each
rabbit was inoculated by ear vein with 1 ml, of MHB
containing 3 x 109 CFU of E. aerogenes. This inoculum produced endocarditis in all rabbits injected.
After 7 days of infections, the rabbits were randomly divided into the following six groups: those treated
with aztreonam, those treated with aztreonam plus
gentamicin, those treated with cefoperazone, those
treated with cefoperazone plus gentamicin, those
treated with gentamicin, and a group of untreated,
infected rabbits that served as controls. Antibiotics
were administered intramuscularly as 60 mg of aztreonam per kg every 6 h, 60 mg of cefoperazone per
kg every 6 h, and 1.7 mg of gentamicin per kg every 8
h.
After 5 and 10 days of therapy, the surviving rabbits
were sacrificed with an intravenous injection of sodium pentobarbital at 12 h after the last injection of
antibiotic. The chest was opened, and all aortic valve
vegetations from the rabbit were excised, pooled, and
weighed. The vegetations from each rabbit weighed a
total of 0.01 to 0.8 g. After a 1:10 suspension of each
vegetation pool in MHB was homogenized, the number of CFU was determined by serial dilution and
plating techniques as previously described (2). In
sterile vegetations, the number of CFU was calculated
as 2 log10 CFU, since the largest weight of vegetation
plated was 0.01 g. The catheter was left in place
throughout the experiment.
Drug ncatm In serum. Blood was taken from
the ear veins of rabbits at 0.25, 0.5, 1, 2, and 4 h after
the first injection of each antibiotic. These rabbits
were then discarded from the experiments. The serum
was separated and stored at -20°C until assay for
serum antibiotic levels.
Concentrations of aztreonam and cefoperazone in
serum were measured by high-pressure liquid chromatography (4a, 8). Assayable samples of aztreonam
were prepared by adding an equal volume of acetonitrile, followed by centrifuigation for 2 min at 1,400 x g.
The supernatant was then removed and kept on ice
until analyzed. The mobile phase was made up of 80%o
0.005 M tetrabutylammonium hydrogen sulfate and
0.005 M ammonium sulfate adjusted to pH 3.0 with 1.0
M K2HPO4 and 20% acetonitrile (vol/vol).
Assayable samples of cefoperazone were produced
by combining one part serum, one part 100% methanol, and one part 100%6 methanol containing the internal standard hydrochlorothiazide. These mixtures
ANTIMICROB. AGENTS CHEMOTHER.
TREATMENT OF E. AEROGENES ENDOCARDITIS
VOL. 24, 1983
323
Q
DISCUSSION
In these studies, we compared aztreonam, a
monocyclic 3-lactam antimicrobial agent, with
cefoperazone, a third-generation cephalosporin,
STERLE
in the treatment of E. aerogenes endocarditis in
0 3 6
24
48
rabbits. The two agents had equivalent in vitro
TIME (h)
activities for the E. aerogenes strain, with MICs
FIG. 1. Rate of decrease in numbers of E. aero- of 0.4 ,g/ml for aztreonam and 0.8 pug(ml for
cefoperazone.
genes organisms in broth containing 60 ,ug of aztreonam, 60 Fag of cefoperazone, and 3 pg of gentamiThe in vitro time-kill studies used inocula of
cin per ml alone and in combinations. *, Broth 109 E. aerogenes organisms per ml, which is
control;
gentamicin; 0, cefoperazone; 0, az- equivalent to titers in vegetations. Concentratreonam; A, aztreonam plus gentamicin; A, cefopera- tions of aztreonam, cefoperazone, and gentamizone plus gentamicin.
cin were used that were present in the sera of
rabbits between 60 and 120 min after injection.
treatment, i.e., 5 versus 10 days (P 0.001). The These studies demonstrated rapid bactericidal
two-factor interaction was not significant (P = activity of both aztreonam and cefoperazone,
0.529). The Newman-Keuls post hoc procedure with an increased rate of bactericidal activity
for the main effect of antibiotic revealed the when gentamicin was added. There was no
following. All of the therapeutic regimens were difference between aztreonam and cefoperazone
effective in reducing vegetation titers as com- alone or between aztreonam plus gentamicin and
pared with the untreated controls (P < 0.01 for cefoperazone plus gentamicin.
all comparisons, except for cefoperazone, where
The levels of aztreonam, cefoperazone, and
P < 0.05). Aztreonam plus gentamicin was more gentamicin achieved in the sera of rabbits were
effective than aztreonam alone (P < 0.01) or within the ranges found in humans (1, 9, 11).
gentamicin alone (P < 0.01). Cefoperazone plus Aztreonam and cefoperazone were equivalent in
gentamicin was more effective than cefopera- reducing titers in vegetations in rabbits. Howevzone alone (P < 0.01) but not more effective than er, addition of gentamicin to either agent ingentamicin alone (P > 0.05). Neither aztreonam creased the rate of titers reduction. In a previous
and cefoperazone nor aztreonam and gentamicin study of endocarditis in rabbits with the same
differed significantly, but gentamicin was signifi- strain of E. aerogenes, 40 mg of cefoperazone
cantly more effective than cefoperazone (P < per kg every 6 h given intramuscularly was
0.05). Aztreonam plus gentamicin did not differ found to be superior to 40 mg of cefamandole per
significantly in effectiveness from cefoperazone kg every 6 h given intramuscularly (10).
plus gentamicin.
In the present study, we compared two new
U,
<
Duration of
tepy
(deapys)
5
10
TABLE 1. Titers of E. aerogenes in vegetations
Mean ± SD log1o CFU/g of vegetation (no. sterile/total)
Aztreo|am
Gentamicin
en .mcn +
Azttreonamgentamte icinm + Cefoperazone | Cefoperazone
Controls
(untreated)
7.1 ± 1.2 (0/9) 3.8 ± 1.7 (3/8) 8.4 ± 1.2 (0/10) 4.4 ± 2.8 (3/12) 5.9 ± 2.6 (1/11) 9.2 ± 0.7 (0/12)
6.2 ± 3.0 (1/7) 2 ± 0 (6/6) 6.3 ± 2.1 (1/7)
2 ± 0 (4/4)
4.8 ± 1.3 (0/7) 9.5 ± 0.3 (0/3)
Downloaded from http://aac.asm.org/ on September 9, 2014 by guest
Drug concentrations in serum. Cefoperazone
achieved the highest levels in serum (peak,
about 155 ,ug/ml) and had a half-life in serum of
1.1 h (Table 2). Peak levels of aztreonam were
slightly lower (about 135 ,ug/ml), and the half-life
was shorter (0.7 h). The peak concentration of
gentamicin in serum was 7.4 ,ug/ml, and the halflife was 1.3 h.
To evaluate the possibility of nephrotoxicity
from gentamicin, serum creatinine levels were
measured after 10 days of therapy in rabbits
receiving gentamicin alone or in combination
with aztreonam or cefoperazone. The serum
creatinine levels were 0.6 to 1.3 mg/dl, which is
normal for rabbits (13).
324
KOBASA AND KAYE
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Concentrations of antibiotics in serum
Drug (dose [mg/kg])'
Aztreonam (60)
Cefoperazone (60)
15
Mean ± SD concn (gml of serum) at following time (min):
30
60
120
135.8 ± 44.1
154.6 ± 63.6
134.9 ± 34.8
153.2 ± 33.0
240
87.8 ± 21.8 24.8 ± 11.0
3.5 ± 1.7
100.1 ± 14.4 57.9 ± 5.0
14.6 ± 4.8
Gentamicin (1.7)
7.4 ± 0.9
6.0 ± 0.2
5.4 ± 0.3
2.6 ± 0.4
1.0 ± 0.2
a There were four rabbits in each group. All doses were administered intramuscularly.
ACKNOWLEDGMENT
This study was supported in part by a grant from E. R.
Squibb & Sons, Inc.
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agents, aztreonam and cefoperazone, each belonging to a different class of antibiotic. In
combination with gentamicin, they were both
shown to be effective in treatment of E. aerogenes endocarditis in rabbits.
Half-life