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Antimicrobial Agents and Chemotherapy, April 2001, p. 1078-1085, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1078-1085.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Penicillin Pharmacodynamics in Four Experimental
Pneumococcal Infection Models
Helga
Erlendsdottir,1
Jenny Dahl
Knudsen,2
Inga
Odenholt,3
Otto
Cars,3
Frank
Espersen,2
Niels
Frimodt-Møller,2
Kurt
Fuursted,2
Karl G.
Kristinsson,1 and
Sigurdur
Gudmundsson4,*
Departments of
Microbiology1 and Internal
Medicine,4 Landspitalinn (University Hospital),
Reykjavík, Iceland; Department of Infectious Diseases,
University Hospital, Uppsala, Sweden3; and
Statens Serum Institut, Copenhagen, Denmark2
Received 27 January 2000/Returned for modification 14 May
2000/Accepted 23 December 2000
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ABSTRACT |
Clinical and animal studies indicate that with optimal dosing,
penicillin may still be effective against
penicillin-nonsusceptible pneumococci (PNSP). The present study
examined whether the same strains of penicillin-susceptible pneumococci
(PSP) and PNSP differed in their pharmacodynamic responses to
penicillin by using comparable penicillin dosing regimens in four
animal models: peritonitis, pneumonia, and thigh infection in mice and
tissue cage infection in rabbits. Two multidrug-resistant isolates of
Streptococcus pneumoniae type 6B were used, one for which
the penicillin MIC was 0.016 µg/ml and the other for which the
penicillin MIC was 1.0 µg/ml. Two additional strains of PNSP were
studied in the rabbit. The animals were treated with five different
penicillin regimens resulting in different maximum concentrations of
drugs in serum (Cmaxs) and times that the
concentrations were greater than the MIC
(T>MICs). The endpoints were bacterial
viability counts after 6 h of treatment in the mice and 24 h
of treatment in the rabbits. Similar pharmacodynamic effects were
observed in all models. In the mouse models bactericidal activity
depended on the T>MIC and to a lesser extent
on the Cmax/MIC and the generation time but not
on the area under the concentration-time curve (AUC)/MIC. Maximal
bactericidal activities were similar for both PSP and PNSP, being the
highest in the peritoneum and blood (~6 log10 CFU/ml),
followed by the thigh (~3 log10 CFU/thigh), and being the
lowest in the lung (~1 log10 CFU/lung). In the rabbit model the maximal effect was ~6 log10 CFU/ml after
24 h. In the mouse models bactericidal activity became marked when
T>MIC was 
65% of the experimental time and
Cmax was 15 times the MIC, and in the rabbit
model bactericidal activity became marked when T>MIC was 35%, Cmax
was 5 times the MIC, and the AUC at 24 h/MIC exceeded 25. By
optimization of the Cmax/MIC ratio and
T>MIC, the MIC of penicillin for pneumococci
can be used to guide therapy and maximize therapeutic efficacy in
nonmeningeal infections caused by PNSP.
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INTRODUCTION |
Streptococcus pneumoniae
remains one of the leading causes of community-acquired bacterial
infections, and severe S. pneumoniae infections
such as pneumonia and meningitis have significant morbidity and
mortality rates (22). Strains of
penicillin-nonsusceptible pneumococci were first reported in 1967 (16) and have spread throughout the world and become
prevalent in many countries. The high prevalence of
penicillin-nonsusceptible pneumococci has created problems in
antimicrobial chemotherapy and has resulted in renewed interest in the
management of pneumococcal infections (1, 25, 30).
Clinical and animal model studies indicate that by optimizing the
dosing of penicillin, it may still be effective against penicillin-nonsusceptible pneumococci (25; V. Magnusson, H. Erlendsdottir, K. G. Kristinsson, and S. Gudmundsson,
Program Abstr. 35th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. A89, p. 17, 1995). Studies in vitro (20;
Magnusson et al., 35th ICAAC) and with experimental animals have also
demonstrated that the most important pharmacokinetic parameter for
establishment of the efficacies of 
-lactam antibiotics against both
penicillin-susceptible and penicillin-nonsusceptible pneumococci is the
time that the antibiotic concentration remains above the MIC
(T>MIC) (8, 11, 14, 20, 26, 32; A. Thoroddsen, T. Asgeirsson, H. Erlendsdóttir, and S. Gudmundsson, Program Abstr. 37th Intersci. Conf. Antimicrob.
Agents Chemother., abstr. B-5b, p. 27, 1997). Most studies have
examined the correlation of pharmacokinetic and pharmacodynamic
parameters with efficacy by using experimental models and several
bacterial strains with different antimicrobial susceptibilities
(20, 32; Thoroddsen et al., 37th ICAAC). This raises the
question of the comparability of infections at different sites. Use of
the same bacterial strains in several animal models should
eliminate the effect of different strain characteristics and thus
provide a better understanding of the effects of pharmacokinetic and
pharmacodynamic parameters at the different sites of infection
(Thoroddsen et al., 37th ICAAC).
The purpose of the study described here was to examine whether
penicillin-susceptible and penicillin-nonsusceptible pneumococci exhibited different pharmacodynamic responses to penicillin at different sites of infection by using the same pneumococcal strains and
comparable penicillin dosing regimens in four animal models: peritonitis, pneumonia, and thigh infection in mice and tissue cage
infection in rabbits.
(This work was presented in part at the 38th Interscience Conference on
Antimicrobial Agents and Chemotherapy, San Diego, Calif., 24 to
27 September 1998 [H. Erlendsdóttir, J. D. Knudsen, I. Odenholt, N. Frimodt-Møller, K. Fuursted, F. Espersen, O. Cars, K. G. Kristinsson, and S. Gudmundsson, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-2, p. 1, 1998].)
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MATERIALS AND METHODS |
Bacteria.
For the mouse models, two clinical isolates
belonging to a Spanish-Icelandic clone were used (28). One
was an isolate (isolate A-2000) from the middle ear of a child with
otitis media, and the other was an isolate (isolate R-2151) from the
sputum of a patient with pneumonia. Both strains were
multidrug-resistant isolates of serotype 6B with similar susceptibility
profiles except for susceptibility to penicillin. They were resistant
to tetracycline, trimethoprim-sulfamethoxazole, and erythromycin.
Strain A-2000 was susceptible to penicillin (MIC, 0.016 µg/ml), and
strain R-2151 was not susceptible to penicillin (MIC, 1.0 µg/ml). For
the rabbit model, two clinical penicillin-nonsusceptible isolates were
used as well: strain 07126 of serotype 6B (MIC, 0.25 µg/ml) and
strain 40932 of serotype 9V (MIC, 2 µg/ml).
MICs.
The MICs were determined by the E test (AB Biodisk,
Solna, Sweden), according to the manufacturer's instructions.
Animals.
Female NMRI mice (age, approximately 6 to 8 weeks;
weight, 30 ± 2 g) were used for the mouse pneumonia and the
mouse thigh infection models. For the mouse peritonitis model, ssc:CF1
female mice (age, approximately 8 weeks; weight, 30 ± 2 g)
were used. Female New Zealand White rabbits (age, 3 to 4 months;
weight, 2.5 to 3.2 kg) were used in the tissue cage model.
Antibiotic and determination of antibiotic concentrations.
Benzylpenicillin was obtained from Astra AB, Södertälje,
Sweden, as a dry powder with known potency. Dilutions were made in
distilled water.
Penicillin concentrations were determined by the disk plate bioassay
method (19). For the mouse models the bioassay organism was Micrococcus luteus ATCC 9341 and the growth medium was
Mueller-Hinton agar (Difco Laboratories), and for the rabbit model the
bioassay organism was Bacillus stearothermophilus ATCC 3032 and the growth medium was tryptose-glucose agar (23).
Penicillin was dissolved in pooled normal mouse serum or in 50% rabbit
serum for preparation of the standard curves, and the concentrations
were derived from the standard curves. The samples were diluted in
either pooled normal mouse serum or 50% rabbit serum, so their
concentrations would be within the range of those on the standard
curve, and the samples were assayed in duplicate. Results were
expressed as micrograms per milliliter of fluid. The correlation
coefficients of the standard curves were at least 0.99, and the
coefficients of variation were below 5%. The lower limit of detection
was 0.1 µg/ml for the experiments with mice and 0.01 µg/ml for the
experiments with rabbits. At each dose given, the
T>MICs for the different strains were
calculated from the serum elimination regression line. The maximum
concentration in serum (Cmax) in mice was
measured 10 min after injection in all mice, and the
Cmax in rabbit serum was measured within 2 h after injection. The serum elimination half-life was estimated by the
expression 
log 2/, where is the slope of the serum elimination
regression line (log serum concentration versus time).
Protein binding.
The level of protein binding of penicillin
G in human serum, pooled mouse serum, and rabbit serum was determined
by the standard ultrafiltration method (7). The
concentrations used for human and mouse serum were 100, 50, 25, 12.5, and 6.25 µg/ml, and those used for rabbit serum were 150, 100, and 50 µg/ml. The results obtained with the different concentrations were averaged.
Animal models. (i) Mouse pneumonia and thigh infection
model.
Bacterial suspensions were prepared from fresh overnight
cultures (made from frozen stock cultures) on 5% blood agar plates. The bacteria were grown in heart infusion broth with 10% horse serum
for 6 h at 35°C to an inoculum of ~108 CFU/ml. The
culture was centrifuged (1,600 × g for 20 min) and resuspended in an equal volume of 0.9% saline. The size of the inoculum was determined by viability counting on blood agar plates.
The mice were anesthetized by with pentobarbital (50 mg/kg) by
intraperitoneal injection. Pulmonary inoculation was performed
by nasal
installation of 75 µl (~7.5 × 10
6 CFU) of the
bacterial suspension, which was dripped onto the
nares of each mouse
(Magnusson et al., 35th ICAAC). The mice readily
aspirated the solution
and were suspended on a string by the upper
incisors for 10 to 15 min.
Ten hours later the same animals were
infected in the thigh by
injecting 0.1 ml of ~10
6 CFU of the same organism in the
logarithmic phase per ml of heart
infusion broth with 10% horse serum
(
15). Antibiotic therapy
was initiated 12 h after
lung inoculation and 2 h after thigh
infection and was continued
for 6 h thereafter. The penicillin
was administered subcutaneously in
the neck region in a volume
of 0.2 ml per dose (see Tables
2 and
3).
Three mice were in
each treatment group, and the experiments were done
twice. Inoculated
untreated control mice were included in all
trials.
At the end of the experiment, the animals were killed by cervical
dislocation, and the lungs and thighs were removed and homogenized
(Omni tissue homogenizer; Omni, Gainesville, Va.) in cold saline
with
-lactamase (Penase; 100,000 U/ml) to neutralize residual
antibiotics. The total volume of lung homogenate was made to 3
ml, and
that of the thigh homogenate was made to 10 ml. The bacterial
densities
in the lungs and thighs were determined by plating serial
10-fold
dilutions on blood agar plates (Difco) containing 5 µg
of gentamicin
per ml (
17) to prevent growth of contaminating
bacteria
(the MICs of gentamicin for the test strains were 12
to 24 µg/ml).
Colony counts were performed after 20 h of incubation
at 35°C in
5% CO
2-supplemented air. The lowest detection levels
for
the viability counts in the lungs and thighs were 30 and 100
CFU per
lung and thigh,
respectively.
(ii) Mouse peritonitis model.
Bacterial suspensions were
prepared from fresh overnight cultures (made from frozen stock
cultures) on 5% blood agar plates as described above. The inoculum for
the mouse peritonitis model was prepared immediately before use by
suspending colonies in sterile beef broth medium and was adjusted to an
optical density at 540 nm of 0.5, giving a density of approximately
108 CFU/ml. The size of the inoculum was determined by
viability counting on 5% blood agar.
Mucin (M-2378; Sigma Chemical Company, St. Louis, Mo.), an enzyme
extract of porcine stomach, was used as an adjuvant for
inoculation of
the peritoneum (
12,
20). Immediately before
inoculation, a
mucin stock solution (10% [wt/vol]) was diluted
1:1 with the
pneumococcal suspension in beef broth (final mucin
concentration, 5%
[wt/vol]), resulting in an inoculum of 5 × 10
6 to
1 × 10
7 CFU/ml. The mice were injected
intraperitoneally with 0.5 ml
of the pneumococcal suspension, resulting
in bacteremia within
1 h of inoculation (
12).
Antibiotic therapy was initiated 1
h after inoculation. Penicillin was
administered subcutaneously
in the neck region in a volume of 0.1 ml
per dose (see Tables
2 and
3). Three mice were in each treatment group.
Inoculated
untreated control mice were included in all
trials.
The effects of the various treatment regimens were determined after
6 h of treatment by evaluation of bacterial counts in
the blood
and peritoneal fluid. Blood samples were obtained by
periorbital cuts
after anesthetization of the mice with CO
2. After
the mice
were killed, peritoneal washes were performed by injecting
2 ml of
sterile saline intraperitoneally, followed by massage
of the abdomen
and then opening of the peritoneum to collect the
fluid. Blood and
peritoneal fluids were immediately diluted 10-fold
in saline, from
which 20 µl was plated onto 5% blood agar plates
in spots, with
subsequent counting of colonies after incubation
overnight at 35°C in
ambient air. The lowest detection levels
for bacterial counts in blood
and peritoneal fluid were 50 and
250 CFU/ml,
respectively.
(iii) Rabbit tissue cage model.
The animals were
anesthetized by intramuscular injection of 0.5 ml of
fentanyl-fluanisone (Hyponorm), followed by disinfection of the back of
the rabbits with 70% alcohol and the administration of local
anesthesia (lidocaine at 40 mg/ml). A 5-cm incision was then made in
the midline, and four well-separated pouches were bluntly dissected in
the subcutaneous layer. An autoclaved cylindrical steel-net cage with a
volume of 4 ml was implanted in each pouch (23). The
incision was closed with sutures. To reverse the anesthesia, the
rabbits were given 0.3 to 0.4 ml of naloxone hydrochloride (0.4 mg/ml;
Narcante) through an intravenous needle in the ear vein. Three to
4 weeks after the implantation, the tissue cages had sealed with a
thin layer of connective tissue and filled with clear, yellowish tissue
cage fluid (TCF). Earlier studies have shown that the TCF albumin/serum
albumin ratio corresponds very well to the skin albumin/plasma albumin
ratio of 0.45 found in humans (3, 23).
The bacterial suspensions used in the rabbit tissue cage model were
grown in Todd-Hewitt broth for 6 h at 37°C in 5%
CO
2-supplemented
air, resulting in an inoculum of
approximate 5 × 10
8 CFU/ml. The four test strains, for
which the MICs were different,
then in the logarithmic phase of growth,
were diluted 10
2 in phosphate-buffered saline (PBS), and
thereafter, 0.4 ml of
the dilutions was injected into the four
different cages, one
strain in each cage, giving a starting inoculum in
the cages of
approximately 5 × 10
5 CFU/ml.
Since the
Cmax of penicillin in the tissue cages
is reached approximately 1 h after injection, antibiotic therapy
was initiated
1 h before inoculation (
23). The
penicillin was administered
by intravenous injection in the ear vein of
one dose of 4, 7.5,
15, 75, or 150 mg of penicillin per kg of body
weight. Samples
from the cages were obtained by percutaneous aspiration
every
2nd h up to 12 h and, in addition, after 24 h. Two rabbits
were
used for each dosing regimen. To avoid antibiotic carryover, the
samples were treated with penicillinase (Penase; 100,000 U/ml)
and, if
necessary, were diluted in PBS before seeding onto blood
agar plates.
Only plates with 20 to 200 colonies were counted.
The lower limit of
detection was 20 CFU/ml.
Pharmacokinetic studies with mice.
Pharmacokinetic studies
were done with NMRI mice. Concentrations in sera were determined after
the administration of a single dose of 1, 10, 50, or 100 mg of
penicillin per kg. The drug was administered by subcutaneous injection
in the neck region in a volume of 0.2 ml per dose. At 10, 20, 40, 60, 90, 120, and 180 min following penicillin administration, blood samples
were obtained from the mice in groups of three by periorbital cuts
during ether anesthesia. Three to four groups of mice with three mice
per group were used, and two to three samples were obtained from each
mouse. After collection, the blood was centrifuged and the serum was stored at 80°C until it was analyzed.
Pharmacokinetic studies with rabbits.
Penicillin
concentrations in TCF were determined after the administration of
single doses of 4, 7.5, 15, 75, or 150 mg of penicillin per kg. The
drug was administered by intravenous injection in the ear vein. Samples
from the cages were obtained by percutanous aspiration every 2nd h up
to 14 h. TCF pooled from all four cages was used. Two rabbits were used
for each dosing regimen.
Treatment regimens.
The designs of the treatment regimens
for the mouse models were based upon the results from the
pharmacokinetic studies (see Results) (Table
1). Dosages not included in Table 1 were
derived by interpolation. The mice were treated and monitored for
6 h. The treatment regimens and dosages for the animals infected
with penicillin-susceptible and penicillin-nonsusceptible organisms are
shown in Table 2. The regimens were
selected to provide a range of Cmaxs and
T>MICs. The range of
Cmaxs in serum was ~4 to 100 times the MIC,
and the range of T>MICs was 45 to 360 min;
i.e., the T>MIC lasted for 12.5 to 100% of the
6-h experiment (see Table 3).
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TABLE 1.
Half-lives, Cmaxs, and
T>MICs for the two strains used in the
mouse models achieved after administration of a single subcutaneous
dose of 1, 10, 50, or 100 mg of penicillin per kg
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TABLE 2.
Treatment regimens and dosages with time schedules
for the penicillin-susceptible (A-2000) and
penicillin-nonsusceptible (R-2151) pneumococci
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The rabbits were treated with one dose of either 4, 7.5, 15, 75, or 150 mg/kg to provide different
T>MICs (0 to 100%
of
the time during the 24 h after the administration of each dose)
for the four different strains used (see the Results and Table
4).
Data analysis and presentation.
The pharmacokinetic
parameters Cmax, half-lives, area under the
concentration-time curve (AUC)/MIC, T>MIC, and
the time that the antibiotic concentration after administration of the first dose (f.d.) of antibiotic remains above the MIC
(T>MIC f.d.) were determined from the
concentration curves. Total drug concentrations were used in all calculations.
The bactericidal efficacies of the treatment regimens in the mouse
models were calculated by subtracting the results for each
treated
mouse from the mean results for control mice at the end
of therapy (6 h). In the rabbit model the bactericidal efficacy
was determined as the
difference between the starting inoculum
and the colony count at the
end of the experiment at 24 h for
all strains. Correlation between
pharamacokinetic variables and
bactericidal activity was examined by
linear regression (Pearson's).
The generation time was defined as the time for the viability counts
for the untreated organisms in the different mouse models
to
double.
A
P value of <0.05 was considered significant. All
statistical comparisons were two-tailed.
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RESULTS |
Pharmacokinetic studies with mice.
Table 1 shows the different
half-lives and Cmaxs achieved after the
administration of various doses of penicillin and the times that the
serum drug levels exceed the MICs for the two strains used in the mouse
models. Table 3 demonstrates the
Cmax/MICs and T>MICs for
both pneumococcal strains achieved with all five regimens. By
univariate analysis, the Cmaxs and
T>MICs correlated by a coefficient
(r2) of 0.14% (P was not
significant).
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TABLE 3.
Cmax/MIC,
T>MIC, and T>MIC f.d.
in mouse serum for both pneumococcal strains achieved with all five
regimensa used in the mouse models
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Pharmacokinetic studies with rabbits.
Tables
4 and 5
similarly show the different half-lives,
T>MICs, and Cmaxs
achieved after the administration of five different single-dose
regimens. For all the regimens the Cmax was
reached within 2 h.
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TABLE 4.
Half-lives and T>MIC in the TCF
for the four strains used in the rabbit model achieved after the
administration of a single intravenous dose of 4, 7.5, 15, 75, or 150 mg of penicillin per kg
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TABLE 5.
Cmax and
Cmax/MIC in TCF for the four pneumococcal
strains achieved with all five regimens in the rabbit model
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Protein binding.
The mean protein binding of penicillin G in
human serum was 29.5% (range, 25 to 46%), that in mouse serum was
8.5% (range, 0 to 17%), and that in rabbit serum was 24% (range, 13 to 36%).
Bactericidal activity in mouse lungs and thighs.
The
bactericidal activities of the different treatment regimens in the
lungs and thighs are shown in Fig. 1A and 1B. The
pharmacodynamic parameters were similar for both the
susceptible (Fig. 1A) and the
nonsusceptible (Fig. 1B) strains, with maximal bactericidal activities in the thighs of 3.5 and 2.9 log10 CFU,
respectively, and maximal bactericidal activities in the lungs of 1.1 and 0.9 log10 CFU, respectively. In the thigh model,
bactericidal activity became pronounced only when
T>MIC was 65% of the experimental time
or Cmax was at least 15 times the MIC, or both.
Similarly, even though the bactericidal activity was very limited in
the lungs, it was observed only when T>MIC was
at least 65% of the experimental time. The correlation of
Cmax, T>MIC,
T>MIC f.d., and AUC/MIC with bactericial
activity by univariate analysis is shown in Table
6, and the association with AUC/MIC is
demonstrated further in Fig. 2.
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FIG. 1.
Bactericidal activities of five treatment regimens in
mouse lungs and thighs. (A) Susceptible strain A-2000; (B)
nonsusceptible strain R-2151. Results are given for each regimen as
log10 CFU reduction between control mice and treated mice
at 6 h. The bars show the means and standard deviations for six
mice. Below the x axis the multiples of the MIC
(Cmax/MIC) and the T>MIC
as a percentage of the 6-h experiment for each regimen are provided.
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TABLE 6.
Correlation (by univariate analysis) of bactericidal
activity with Cmax,
T>MIC, T>MIC f.d., and
AUC/MIC in the mouse infection models for two type 6B
pneumococcal isolates, A-2000 and R-2151a
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FIG. 2.
Association of bactericidal activity of five different
regimens with AUC/MIC at four different infection sites in mice. (A)
Results for susceptible strain A-2000. (B) Results for nonsusceptible
strain R-2151.
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Bactericidal activity in mouse blood and peritoneum.
The
bactericidal activity of penicillin in blood and peritoneum is shown in
Fig. 3A and B. Pronounced bactericidal
efficacy was seen against both blood and peritoneal infections. The
pharmacodynamic parameters were similar for both the
susceptible (Fig. 3A) and the nonsusceptible (Fig. 3B) strains, with
maximal activities in blood of 5.4 and 6.0 log10 CFU/ml,
respectively, and maximal activities in the
peritoneum of 5.8 and 6.3 log10 CFU/ml, respectively. In
these infections, bactericidal activity became significant only
when T>MIC was 65% of the experimental time
or Cmax was at least 15 times the MIC, or both.
The correlation of Cmax,
T>MIC, T>MIC f.d., and
AUC/MIC with bactericial activity by univariate analysis is shown in
Table 6, and the association with AUC/MIC is demonstrated further in
Fig. 2.
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FIG. 3.
Bactericidal activities of five different treatment
regimens in mouse peritoneum and blood. (A) Results for susceptible
strain A-2000. (B) Results for nonsusceptible strain R-2151. Results
are given for each regimen as log10 CFU reduction between
control mice and treated mice at 6 h. The bars show the means and
standard deviations for three mice. Below the x axis the
multiples of the MIC (Cmax/MIC) and the
T>MIC as a percentage of the 6-h experiment for
each regimen are provided.
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Bactericidal activity in rabbit TCF.
As shown in Table 4, the
concentration of penicillin in the cages after the administration of a
dose of 15 mg/kg never exceeds the MICs for strain 40932 (MIC, 2.0 µg/ml). As an example, Fig. 4 shows the
different bactericidal activity curves for the four test strains
following the administration of a penicillin dose of 15 mg/kg. For the
strain for which the MIC was 1.0 µg/ml, the T>MIC after this dose was 3.2 h (13% of
the 24-h experiment), but the Cmax/MIC was only
1.6 and there was no bactericidal activity against the two
nonsusceptible strains. For the intermediate (MIC, 0.25 µg/ml), the
15-mg/kg dose provided a T>MIC of 13 h (54%) and the Cmax/MIC was 6.3. For the sensitive
strain, the T>MIC was 32 h (100%) and the
Cmax/MIC was 99. Significant bactericidal activity was achieved only against these two strains (MICs, 0.016 and
0.25 µg/ml, respectively).
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FIG. 4.
Killing and growth curves for the four
pneumococcal strains (MICs, 0.016, 0.25, 1, and 2 µg/ml,
respectively) for 24 h in the rabbit tissue cages following the
intravenous administration of 15 mg of penicillin per kg. The curves
show the mean number of CFU per milliliter for two rabbits at each time
point.
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The bactericidal activities of all five regimens against the four test
strains are shown in Fig.
5 as a
correlate of the respective
T>MICs,
Cmax/MICs, and AUC/MICs 24 h after
dosing. Maximal
bactericidal activity was obtained when the
T>MIC was at least
35% of the experimental
time,
Cmax exceeded 5 times the MIC, and
AUC/MIC
exceeded 25.
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FIG. 5.
Bactericidal activities of all five regimens (4, 7.5, 15, 75, and 100 mg/kg) against the four test strains in the rabbit
tissue cages as a function of the respective
T>MICs (A) Cmax/MIC (B),
and AUC/MIC (C) 24 h after dosing.
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Generation times.
Maximum bactericidal activity was associated
with the generation times of the untreated control organisms in the
different mouse models, as demonstrated in Fig.
6 (r2 = 0.94; P < 0.01). The generation time in the lungs
was long, approaching infinity, and strain R-2151 (MIC, 1.0 µg/ml)
had a 1.3- to 3-fold longer generation time than strain A-2000 (MIC, 0.016 µg/ml).
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FIG. 6.
Correlation between generation time of the untreated
control organisms in the different mouse models and maximum
bactericidal activity. The generation time in the lungs approached
infinity, but the duration was arbitrarily chosen as 1,000 min.
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DISCUSSION |
The major aim of the present study was to evaluate the
bactericidal activity of penicillin against penicillin-susceptible and
penicillin-nonsusceptible pneumococcal strains in four different animal
models. Previous studies with experimental animals (2, 13, 29;
Magnusson et al., 35th ICAAC) and limited studies with humans
(25) have shown that penicillin-nonsusceptible pneumococci can be treated with penicillin. The experimental studies were performed
with single animal models. The current study is the first that has
examined these issues concurrently in different animal models at
different sites of infection but with the same organisms and the same
antimicrobial agent at comparable dosages. Furthermore, the end points
in this study were not survival but bactericidal activity as determined
by the viable counts in all models. Since the half-life in the rabbit
model was 2.9 to 4.4 h but was only 12.1 to 16.1 min in mice, the
end point of therapy in the tissue cages was chosen to be 24 h
after dosing of the drug, but in the mouse models the end point was
chosen to be 6 h after the initiation of therapy.
The main findings of this study are as follows. (i) Similar
pharmacodynamic patterns were observed in all models, although bactericidal activity was lowest in the lung. (ii) The bactericidal effect of penicillin in all models against both the
penicillin-susceptible and penicillin-nonsusceptible pneumococci was
dependent on both T>MIC and, to a lesser
extent, Cmax, but it was not dependent on
AUC/MIC. (iii) The bactericidal activity in the mouse models correlated
with the in vivo generation time.
The virulence of pneumococci in mice varies with capsular type.
Penicillin-nonsusceptible pneumococci are dominated by capsular types
with low levels of virulence for mice, i.e., types 6B, 14, 19, and 23 (4). In the present study pneumococcal strains of serotype
6B were used in all models, and in addition, one strain of serotype 9V
was used in the tissue cage model. The serotype 6B strains were
multidrug resistant and exhibited similar levels of resistance to most
antibiotics tested except penicillin, which had MICs from 0.016 to 1.0 µg/ml. The studies were done with immunocompetent mice. Previous data
(21; H. Erlendsdóttir, S. Ómarsdóttir, V. Magnússon, and S. Gudmundsson,
Program Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother.,
abstr. B-5c, p. 27; 1997) have indicated that pharmacokinetic
parameters in infected and uninfected mice are comparable.
It has previously been shown with experimental animal models that the
most important pharmacokinetic parameter for prediction of the effects
of -lactam antibiotics in vivo against penicillin-susceptible pneumococci is T>MIC (8, 11, 20,
32). An earlier study examined the efficacy of penicillin in 100 different penicillin dosing regimens against a penicillin-sensitive
pneumococcus (MIC, 0.008 µg/ml) in the mouse thigh model
(32). Therapeutic efficacy was achieved when levels in
serum were constantly maintained above the MIC. In the present study,
the pharmacokinetic datum points did not allow the complete separation
of the interdependence between T>MIC and
Cmax, but it was not the primary goal.
Nevertheless, the correlation between the two parameters was poor by
univariate analysis (r2 = 14%).
Furthermore, the correlations of the bactericidal activity with
T>MIC were higher
(r2 = 54 to 94%) in all
mouse models for both isolates than the correlations with
Cmax (r2 =
0.2 to 49%) and AUC/MIC (r2 = 14 to 75%) except for those for the nonsusceptible
isolate in the lung (for T>MIC,
r2 = 37%; for
Cmax, r2 = 79%; for AUC/MIC, r2 = 97%). Thus, the results in the present study achieved with the
same isolates at several different infection sites are consistent with
those of previous single-infection-site studies (8, 11, 20,
31). In the present study the T>MIC
after administration of the first dose of antibiotic did not correlate
with the bactericidal activity at the end of treatment except for that
against the nonsusceptible isolate in the lung (P = 0.019). It should be noted, however, that a certain
maximum-effect threshold for Cmax,
T>MIC, and T>MIC f.d.
can be deducted from the results from studies with mice (Fig. 1 and 3).
Only a few studies have investigated whether the same
pharmacodynamic parameters apply to penicillin-susceptible and
penicillin-nonsusceptible pneumococci, but in the present studies
there have been indications that penicillin at the appropriate dosages
can be used to treat infections caused by penicillin-nonsusceptible
pneumococci. Azoulay-Dupuis et al. (2) showed in a
leukopenic mouse pneumonia model that the dose of amoxicillin (50 mg/kg) required to protect mice and to eradicate
penicillin-nonsusceptible strains was 10 times higher than the dose
which was effective against penicillin-susceptible strains. Tateda et
al. (29) showed that the ratio of the penicillin MICs for
the two pneumococcal strains tested (1.0/0.015 µg/ml) was the same as
the ratio of the two penicillin dosages (40/0.6 mg/kg) required to
clear the pneumonia from immunocompetent mice. The doses were given six
times at 1-h intervals. These results are consistent with those of
Knudsen et al. (20), who showed a highly significant
correlation between the log MIC and the log 50% effective dose in a
mouse peritonitis model. Moreover, earlier Magnusson et al. (35th
ICAAC) showed that high penicillin dosages of up to 200 mg/kg/24
h, with doses given every 1 or every 3 h, were efficacious
in the treatment of pneumonia due to penicillin-nonsusceptible pneumococci in mice. That study also indicated that a certain threshold
penicillin Cmax of approximately 6 µg/ml was
necessary for in vivo efficacy. However, beyond this, an increased
dosage did not improve the bactericidal activity further. This was seen with both the penicillin-susceptible and the penicillin-nonsusceptible strains tested and also in the in vitro experiments with the same strains.
Few studies with humans have obtained results consistent with these
findings. Pallares et al. (25) reported that penicillin resistance is not associated with an increased rate of mortality in
patients with bacteremic pneumococcal infections that were treated with
penicillin. Friedland (10) showed that penicillin nonsusceptibility was not associated with increased mortality in a
series of 208 children with pneumococcal bacteremia without infection
in the central nervous system. Einarsson et al. (9) found
in a recent case control study that patients with pneumonia due to
penicillin-nonsusceptible strains presented with a milder illness than
patients infected with susceptible strains but had prolonged hospital
stays and required more expensive antibiotics. These results might be
explained by the fact that penicillin resistance is more common among
serotypes with lower levels of virulence (18).
In the present study the bactericidal activity in lungs was limited,
the reason for which is not entirely clear, but the long generation
time in the lungs is probably the most important factor. Several
investigators have previously reported a significant association between the generation time in untreated organisms and the bactericidal activities of -lactams, for example, for Escherichia coli
(31) and group A beta-hemolytic streptococci
(24). In a previous study (Thoroddsen et al., 37th ICAAC)
of pneumococcal serotype 6B infection in the lungs and thighs in
healthy and neutropenic mice, the bactericidal activity of penicillin
was monitored at regular intervals for 24 h after the
administration of one 100-mg/kg dose of penicillin. The maximal
bactericidal effect was reached 4 to 12 hours later in the lungs than
in the thighs. The antibiotic concentration at the infection site does
not explain this difference, because values for the most important
parameter for establishment of the effect of -lactams,
T>MIC, were similar at both sites (Erlendsdóttir et al., 37th ICAAC). Thus, the bactericidal
activity in the lungs in the present study may have been more
pronounced if it had been monitored for a longer period, but the
studies were done with immunocompetent mice, which limited the
experimental time.
The antibiotic concentration at the infection site is also important.
In rabbits it has been shown that the concentrations of -lactams in
muscle interstitial fluid closely approximate those observed in serum,
with only a short lag time (5, 27). Similarly, comparison
of the pharmacokinetics of penicillin in mouse serum and lungs
(Erlendsdóttir et al., 37th ICAAC) has shown good penetration of
penicillin into murine lung tissue. However,
Cmax was much higher in serum than in lungs,
with a ratio of 2/1 to 3/1. In contrast, the half-lives were
considerably longer in lungs than in serum (20%), resulting in
comparable T>MICs. However, in the tissue cage,
due to a large volume-to-surface area ratio, an even antibiotic
concentration at the infection site is achieved during the induction
phase (23). The -lactams accumulated slowly in TCF, and
the Cmax in TCF was lower than that in serum,
but the half-life was much longer. For instance, a single intravenous
injection of ampicillin (20 mg/kg) to rabbits resulted in a half-life
of 28 min in serum and one of approximately 2.5 h in TCF
(6). In the present study the half-life of penicillin in
TCF ranged from 2.9 to 4.9 h. Therefore, levels in serum were used
as surrogate markers for the concentration at the infection sites in
the mouse models, while the concentrations in TCF were used as
surrogate markers in the rabbit model. The question arises as to
whether infection alters the pharmacokinetic parameters. It has been
shown that the pharmacokinetic parameters for penicillin 18 to 22 h
after infection were the same in both serum and lungs in infected and
uninfected mice (Erlendsdóttir et al., 37th ICAAC). By the same
token, peritonitis does not alter the parameters at the start of the
infection (21).
In summary, the present study showed that the pharmacokinetic
parameters associated with the efficacy of penicillin against pneumococci with different susceptibilities were remarkably similar in
the four animal models and nearly identical at the different sites of
infection in the mice. Thus, in conclusion, the MIC of penicillin for
pneumococci can be used to adjust the dosage required to achieve an
optimum outcome with penicillin treatment regimens when one is treating
nonmeningeal infections.
| |
ACKNOWLEDGMENT |
This work was supported by a Scandinavian Society of Chemotherapy
research grant for research in antimicrobial chemotherapy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Directorate of
Health, Laugavegur 116, IS-101 Reykjavik, Iceland. Phone: 354 510 1900. Fax: 354 510 1919. E-mail: sigurdur{at}landlaeknir.is.
| |
REFERENCES |
| 1.
|
Appelbaum, P. C.
1992.
Antimicrobial resistance in Streptococcus pneumoniae: an overview.
Clin. Infect. Dis.
15:77-83[Medline].
|
| 2.
|
Azoulay-Dupuis, E.,
P. Moine,
J. P. Bedos,
V. Rieux, and E. Vallee.
1996.
Amoxicillin dose-effect relationship with Streptococcus pneumoniae in a mouse pneumonia model and roles of in vitro penicillin susceptibilities, autolysis, and tolerance properties of the strains.
Antimicrob. Agents Chemother.
40:941-946[Abstract].
|
| 3.
|
Bergholm, A. M.,
C. Henning, and S. E. Holm.
1984.
Static and dynamic properties of tissue cage fluids in rabbits.
Eur. J. Clin. Microbiol.
3:126-130[CrossRef][Medline].
|
| 4.
|
Briles, D. E.,
M. J. Crain,
B. M. Gray,
C. Forman, and J. Yother.
1992.
Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae.
Infect. Immun.
60:111-116[Abstract/Free Full Text].
|
| 5.
|
Cars, O.
1981.
Tissue distribution of ampicillin: assay in muscle tissue and subcutaneous tissue cage fluid from normal and nephrectomized rabbits.
Scand. J. Infect. Dis.
13:283-289[Medline].
|
| 6.
|
Cars, O.,
C. Henning, and S. E. Holm.
1981.
Penetration of ampicillin and dicloxacillin into tissue cage fluid in rabbits: relation to serum and tissue protein binding.
Scand. J. Infect. Dis.
13:69-74[Medline].
|
| 7.
|
Craig, W. A., and B. Suh.
1991.
Protein binding and antimicrobial effects: methods for determination of protein binding, p. 367-393.
In
V. Lorian (ed.), Antibiotics in labaratory medicine, 3rd ed. The Williams & Wilkins Co., Baltimore, Md.
|
| 8.
|
Drusano, G. L., and F. W. Goldstein.
1996.
Relevance of the Alexander Project: pharmacodynamic considerations.
J. Antimicrob. Chemother.
38(Suppl. A):141-154.
|
| 9.
|
Einarsson, S.,
M. Kristjansson,
K. G. Kristinsson,
G. Kjartansson, and S. Jonsson.
1998.
Pneumonia caused by penicillin non-susceptible and penicillin susceptible pneumococci in adults: a case-control study.
Scand. J. Infect. Dis.
30:253-256[CrossRef][Medline].
|
| 10.
|
Friedland, I. R.
1995.
Comparison of the response to antimicrobial therapy of penicillin-resistant and penicillin-susceptible pneumococcal disease.
Pediatr. Infect. Dis. J.
14:885-890[Medline].
|
| 11.
|
Frimodt-Møller, N.,
M. W. Bentzon, and V. F. Thomsen.
1986.
Experimental pneumococcus infection in mice: comparative in vitro activity and pharmacokinetic parameters with in vivo effect for 14 cephalosporins.
J. Infect. Dis.
154:511-517[Medline].
|
| 12.
|
Frimodt-Møller, N.,
J. D. Knudsen, and F. Espersen.
1999.
The mouse peritonitis/sepsis model, p. 127-136.
In
O. Zak, et al. (ed.), Handbook of animal models of infection. Academic Press, Inc., New York, N.Y.
|
| 13.
|
Gavalda, J.,
J. A. Capdevila,
B. Almirante,
J. Otero,
I. Ruiz,
M. Laguarda,
H. Allende,
E. Crespo,
C. Pigrau, and A. Pahissa.
1997.
Treatment of experimental pneumonia due to penicillin-resistant Streptococcus pneumoniae in immunocompetent rats.
Antimicrob. Agents Chemother.
41:795-801[Abstract].
|
| 14.
|
Gerber, A. U.
1990.
Impact of the antibiotic dosages schedule on efficacy in experimental soft tissue infections.
Scand. J. Infect. Dis. Suppl.
74:147-154[Medline].
|
| 15.
|
Gudmundsson, S., and H. Erlendsdottir.
1999.
Murine thigh infection model, p. 137-144.
In
O. Zak, et al. (ed.), Handbook of animal models of infection. Academic Press, Inc., New York, N.Y.
|
| 16.
|
Hansman, D., and M. M. Bullen.
1967.
A resistant pneumococcus.
Lancet
ii:264-265.
|
| 17.
|
Hendley, J. O.
1975.
Spread of Streptococcus pneumoniae in families. I. Carriage rates and distribution of types.
J. Infect. Dis.
132:55-61[Medline].
|
| 18.
|
Kaplan, S. L., and E. O. Mason.
1998.
Management of infections due to antibiotic-resistant Streptococcus pneumoniae.
Clin. Microbiol. Rev.
11:628-644[Abstract/Free Full Text].
|
| 19.
|
Klassen, M., and S. C. Edberg.
1996.
Measurement of antibiotics in human body fluids: techniques and significance, p. 233-243.
In
V. Lorian (ed.), Antibiotics in laboratory medicine, 4th ed. The Williams & Wilkins Co., Baltimore, Md.
|
| 20.
|
Knudsen, J. D.,
N. Frimodt-Møller, and F. Espersen.
1995.
Experimental Streptococcus pneumoniae infection in mice for studying correlation of in vitro and in vivo activities of penicillin against pneumococci with various susceptibilities to penicillin.
Antimicrob. Agents Chemother.
39:1253-1258[Abstract].
|
| 21.
|
Knudsen, J. D.,
N. Frimodt-Møller, and F. Espersen.
1998.
Pharmacodynamics of penicillin are unaffected by bacterial growth phases of Streptococcus pneumoniae in the mouse peritonitis model.
J. Antimicrob. Chemother.
41:451-459[Abstract/Free Full Text].
|
| 22.
|
Mufson, M. A.
1990.
Streptococcus pneumoniae, p. 1539-1551.
In
G. L. Mandell, R. G. Douglas, and J. E. Bennet (ed.), Principles and practice of infectious diseases, 3rd ed. Churchill Livingstone Inc., New York, N.Y.
|
| 23.
|
Odenholt, I.,
S. E. Holm, and O. Cars.
1988.
An in vivo model for evaluation of the postantibiotic effect.
Scand. J. Infect. Dis.
20:97-103[Medline].
|
| 24.
|
Odenholt, I.
1989.
Pharmacodynamics of -lactam antibiotics, p. 29. Ph.D. thesis.
Akademitryk, Uppsala, Sweden.
|
| 25.
|
Pallares, R.,
J. Linares,
M. Vadillo,
C. Cabellos,
F. Manresa,
P. F. Viladrich,
R. Martin, and F. Gudiol.
1995.
Resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in Barcelona, Spain.
N. Engl. J. Med.
333:474-480[Abstract/Free Full Text].
|
| 26.
|
Roosendaal, R., and I. A. J. M Bakker-Woudenberg.
1990.
Impact of the antibiotic dosage schedule on efficacy in experimental lung infections.
Scand. J. Infect. Dis. Suppl.
74:147-154.
|
| 27.
|
Ryan, M., and O. Cars.
1980.
Antibiotic assays in muscle: are conventional tissue levels misleading as indicator of the antibacterial activity?
Scand. J. Infect. Dis.
12:307-309[Medline].
|
| 28.
|
Soares, S.,
K. G. Kristinsson,
J. M. Muser, and A. Tomasz.
1992.
Evidence for introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980's.
J. Infect. Dis.
168:158-163.
|
| 29.
|
Tateda, K.,
K. Takashima,
H. Miyazaki,
T. Matsumoto,
T. Hatori, and K. Yamaguchi.
1996.
Noncompromised penicillin-resistant pneumococcal pneumonia CBA/J mouse model and comparative efficacies of antibiotics in this model.
Antimicrob. Agents Chemother.
40:1520-1525[Abstract].
|
| 30.
|
Tomasz, A.
1995.
Pneumococcus at the gates.
N. Engl. J. Med.
333:514[Free Full Text].
|
| 31.
|
Tuomanen, E.,
R. Cozens,
W. Tosch,
O. Zak, and A. Tomasz.
1986.
The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth.
J. Gen. Microbiol.
132:1297-1304[Abstract/Free Full Text].
|
| 32.
|
Vogelman, B.,
S. Gudmundsson,
J. Leggett,
J. Turnidge,
S. Ebert, and W. A. Craig.
1988.
Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model.
J. Infect. Dis.
158:831-847[Medline].
|
Antimicrobial Agents and Chemotherapy, April 2001, p. 1078-1085, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1078-1085.2001
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Top
Abstract
Introduction
