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Antimicrobial Agents and Chemotherapy, April 1999, p. 963-965, Vol. 43, No. 4
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Trovafloxacin in Combination with Vancomycin
against Penicillin-Resistant Pneumococci in the Rabbit
Meningitis Model
Dina
Rodoni,1
Fränzi
Hänni,1
Cynthia M.
Gerber,2
Marianne
Cottagnoud,2
Klaus
Neftel,2
Martin G.
Täuber,3 and
Philippe
Cottagnoud1,*
Department of Internal Medicine,
Inselspital,1 Department of Internal
Medicine, Zieglerspital,2 and
Institute for Medical Microbiology, University of
Berne,3 Berne, Switzerland
Received 8 July 1998/Returned for modification 8 September
1998/Accepted 30 January 1999
 |
ABSTRACT |
Trovafloxacin, a new fluoroquinolone, produced bactericidal
activity (
0.33 ± 0.13 
log10 CFU/ml · h; intravenously [i.v.] administered dose, 15 mg/kg)
comparable to that of vancomycin (0.39 ± 0.18 log10 CFU/ml · h; i.v. administered dose, 20 mg/kg) in the treatment of experimental meningitis in rabbits due to a
pneumococcal strain highly resistant to penicillin (MIC of penicillin G, 4 µg/ml). The combination of both drugs significantly
increased (P < 0.05) the killing rate (0.60 ± 0.23 log10 CFU/ml · h) compared to that produced
by either monotherapy. These results were also confirmed in vitro.
| |
TEXT |
The worldwide spread of
penicillin-resistant pneumococci presents a major challenge for
clinicians. The new quinolone trovafloxacin {7-(3-azabicyclo[3.1.0]hexyl) quinolone} has
excellent antimicrobial activity against
penicillin-sensitive and penicillin-resistant pneumococci in
vitro and good penetration into the cerebrospinal fluid (CSF) due to
its lipophilic properties (2, 7, 15). In previous studies,
it has been demonstrated that trovafloxacin is very active
against penicillin-resistant pneumococci in the rabbit meningitis
model, provided that the antibiotic level in the CSF remains above the
MIC (12, 17). In the present study, we tested trovafloxacin
alone and in combination with vancomycin against a pneumococcus highly
resistant to penicillin in the rabbit meningitis model and in vitro.
Rabbit meningitis model.
The meningitis model originally
described by Dacey and Sande (4) was used in a slightly
modified way. In brief, young New Zealand White rabbits weighing 2 to
2.5 kg were anesthetized by intramuscular injections of ketamine (30 mg/kg) and xylazine (15 mg/kg) and were immobilized in stereotactic
frames for induction of meningitis and CSF samplings. An inoculum
containing 105 CFU of pneumococcus serotype 6 was directly
injected into the cisterna magna. The organism had originally been
isolated from a patient with pneumonia at the University Hospital of
Berne, Switzerland, and was highly resistant to penicillin (the MICs were as follows: penicillin G, 4 µg/ml; ceftriaxone, 0.5 µg/ml; vancomycin, 0.12 to 0.25 µg/ml; and trovafloxacin, 0.12 µg/ml). A
long-acting anesthetic (ethyl carbamate, or urethane; 3.5 g/rabbit) was injected subcutaneously. The animals were then returned to their
cages. Fourteen hours later the cisterna magna was punctured again for
periodic CSF sampling before and 0.75, 2.5, 4, 6, and 8 h after
initiation of therapy. Antibiotics were administered through a
peripheral ear vein as bolus injections at the following concentrations: alatrofloxacin (a prodrug of trovafloxacin with activity corresponding to 80.2% of the activity of trovafloxacin), 15 mg/kg; and vancomycin, 20 mg/kg. Alatrofloxacin was injected at hour 0, and vancomycin was injected at hours 0 and 4. During injection, alatrofloxacin was protected from light. Untreated controls
received the same volume of saline.
Bacterial concentrations were determined as described by Kim et
al. (12). In order to determine a possible carryover
effect, samples were plated undiluted and at dilutions of 1:10
and 1:100 and titers were compared. No obvious carryover
effect was noted. The antimicrobial activities of the regimens during
the 8-h treatment were calculated by linear regression analysis and
expressed as the decrease of log10 CFU per
milliliter per hour (log10 CFU/ml · h),
as previously described (12). The results were
expressed as means ± standard deviations (SDs). Statistical
significance was determined by the Newman-Keuls test.
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TABLE 1.
Bactericidal activities of alatrofloxacin (prodrug of
trovafloxacin) and vancomycin alone and in combination in
experimental meningitis due to Streptococcus pneumoniae
highly resistant to penicillin
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|
Measurement of antibiotic concentrations in the CSF.
Trovafloxacin and vancomycin concentrations in the CSF of every
treated rabbit were determined by an agar diffusion method with
antibiotic medium 11 for trovafloxacin and antibiotic medium 1 for
vancomycin (Difco Laboratories, Detroit, Mich.). Standard curves were
generated by measuring levels in saline with 5% rabbit serum,
approximating the CSF protein concentration during meningitis (12,
15). Bacillus subtilis (ATCC 6633) was used as a test strain (18). Inter- and intraday assay variabilities
were both less than 10%. The limits of detection were 0.16 to 0.20 µg/ml for trovafloxacin and 0.5 µg/ml for vancomycin and thus below the lowest concentration measured in the CSF.
In vitro assays.
The pneumococcal strain was grown in C+Y
medium (13) to an optical density at 590 nm of 0.3 and then
diluted 40-fold to 106 CFU/ml, corresponding to the CSF
bacterial titer in rabbits before initiation of therapy. Antibiotics
were added at concentrations approximating levels achievable in the
CSF, as follows: vancomycin, 0.06 to 2.5 µg/ml (MIC, 0.25 µg/ml);
and trovafloxacin mesylate, 0.06 to 0.6 µg/ml (MIC, 0.12 µg/ml).
Combinations of trovafloxacin (0.12 µg/ml) with vancomycin (0.12 µg/ml) were also tested. Bacterial titers were determined at 0, 2, 4, and 6 h by serial dilution of samples, and the samples were plated
on agar plates containing 5% sheep blood and incubated at 37°C for
24 hours. Experiments were performed in triplicate, and results were
expressed in means ± SDs.
Results and discussion.
The results summarized in Table 1
confirm previous studies that demonstrated good activity of
trovafloxacin against penicillin-sensitive and -resistant pneumococci
and good CSF penetration in the rabbit meningitis model
(17). To date, the optimal dosage for treatment of
bacterial meningitis in humans has not been established. On account
of this fact, we used a dose (15 mg/kg) that led to concentrations in
CSF closely comparable to those found in healthy human volunteers following intravenous infusion of 300 mg of alatrofloxacin
(5) (Fig. 1). Due to its long
half-life, a single 300-mg dose of alatrofloxacin is usually
recommended for severe infections (10). The CSF
vancomycin concentration (Fig. 2)
corresponded to the level measured in the CSF in humans (1,
11). Although the trovafloxacin level after a single dose (15 mg/kg) of alatrofloxacin remained far above the MIC during the
treatment period (the lowest level in the CSF after 8 h was 0.6 µg/ml [MIC, 0.12 µg/ml]), trovafloxacin had only moderate
bactericidal activity (log10 CFU/ml · h,
0.33 ± 0.13 [Table 1]). The minimal CSF trovafloxacin
concentration/MIC ratio was 5. The bactericidal activity of
vancomycin (log10 CFU/ml · h, 0.39 ± 0.18) was similar to that of trovafloxacin (it showed no statistical
difference compared to trovafloxacin), confirming the findings of
previous studies (9). Combination of trovafloxacin with vancomycin improved the killing rate significantly
(log10 CFU/ml · h, 0.60 ± 0.23) compared
to that of either compound alone (P < 0.05). The
regimens tested were bactericidal during therapy, sterilizing the CSF
in four of nine animals treated with vancomycin, none of seven animals
in the alatrofloxacin group, and four of seven animals treated
with the combination.
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FIG. 1.
Mean trovafloxacin concentrations in CSF at 0.75, 2.5, 4, 6, and 8 h after intravenous injection of 15 mg of
alatrofloxacin per kg. The concentration of trovafloxacin remained
above the MIC (0.12 µg/ml) during the entire treatment period.
Vertical bars show ± SD.
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FIG. 2.
Mean vancomycin concentration in the CSF at 0.75, 2.5, 4, 6, and 8 h after intravenous injection of vancomycin (20 mg/kg). Vancomycin was injected intravenously at 4 h at the same
dose. The MIC for vancomycin was 0.25 µg/ml, and the concentrations
in CSF remained above the MIC for the entire treatment period. Vertical
bars show ± SD.
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|
In vitro, trovafloxacin showed good bactericidal activity provided that
the antibiotic concentrations were above the MIC (Fig.
3). Vancomycin at concentrations ranging
from 0.06 to 2.5 µg/ml
produced bactericidal rates comparable to
those of trovafloxacin
(Fig.
4). The
combination enhanced the killing rates by nearly
2 log
10
CFU/ml after 2, 4, and 6 h of incubation (
P < 0.05) (Fig.
5).
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FIG. 3.
Killing rates of trovafloxacin ( ) in vitro at
concentrations ranging from 0.06 µg/ml (1/2 of the MIC) to 0.6 µg/ml (5× the MIC).  , data for untreated controls (C).
Experiments were performed in triplicate, and killing rates are
expressed as means ± SDs.
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FIG. 4.
Bactericidal activity of vancomycin in vitro.
Concentrations of vancomycin ranging from 0.06 µg/ml (1/4 of the MIC)
to 2.5 µg/ml (10× the MIC) were tested. , data for untreated
controls (C). Experiments were performed in triplicate, and results are
expressed as means ± SDs.
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FIG. 5.
Killing rates of trovafloxacin (T) (0.12 µg/ml; 1×
the MIC), vancomycin (V) (0.12 µg/ml; 1/2 of the MIC), and the
combination of the two substances (V+T) in vitro. The experiments were
performed in triplicate, and results are expressed as means ± SDs. *, P < 0.05 versus either single-drug
therapy.
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|
In summary, addition of vancomycin improved the bactericidal
activity of trovafloxacin in vitro and in vivo. Synergistic effects
of
quinolones with other antibiotics, e.g., rifampin, have been
documented, and their therapeutic benefit in certain clinical
situations is suggested (
6,
13). To our knowledge, an
increased
bactericidal efficacy against pneumococci of the combination
of
trovafloxacin and vancomycin compared to single drugs in vivo
has
not been described. Recently, Nicolau et al. (
16)
demonstrated
a synergy between trovafloxacin and vancomycin in vitro by
calculating
fractional inhibitory concentration (FIC) indices, as
described
by Eliopoulos and Moellering (
8). However, synergy
was observed
in only 6% of all resistant isolates tested
(
16). The FIC indices
of the combination against the
resistant pneumococcal isolate
we studied showed a neutral effect (FIC
indices were 1.0). Time-killing
curves constructed by using data
measured in vitro, as used in
our study, may correlate more closely
with the situation in the
CSF in vivo. The findings of the present
study might be of relevance
to pneumococcal diseases like meningitis,
where the penetration
of antibiotics is limited and bactericidal
activity of therapeutic
regimens is an imperative
requirement.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from Pfizer Corporation and the
Wander Foundation, Berne, Switzerland.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Internal Medicine, Inselspital, Freiburgstrasse, 3010 Berne,
Switzerland. Phone: 41316322111. Fax: 41316323847. E-mail:
pcottagn{at}insel.ch.
| |
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Antimicrobial Agents and Chemotherapy, April 1999, p. 963-965, Vol. 43, No. 4
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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