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Antimicrobial Agents and Chemotherapy, March 2000, p. 654-657, Vol. 44, No. 3
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Efficacy of Linezolid in Experimental Otitis
Media
S. I.
Pelton,1,*
M.
Figueira,1
R.
Albut,1 and
D.
Stalker2
Section of Pediatric Infectious Diseases,
Maxwell Finland Laboratory for Infectious Diseases Boston Medical
Center/Boston University School of Medicine, Boston,
Massachusetts,1 and Clinical
Pharmacokinetics, Pharmacia & Upjohn, Kalamazoo,
Michigan2
Received 28 January 1999/Returned for modification 23 July
1999/Accepted 28 October 1999
 |
ABSTRACT |
Therapy for otitis media (OM) due to resistant Streptococcus
pneumoniae (MIC of penicillin, 
2.0 µg/ml) is challenging.
Linezolid, an oxazolidinone, represent a new class of antimicrobial
agents with excellent in vitro activity against penicillin-resistant S. pneumoniae; however, in vitro activity against
nontypeable Haemophilus influenzae (NTHI) is limited. We
evaluated its efficacy against experimental acute OM due to a
multidrug-resistant S. pneumoniae isolate and two isolates
of NTHI. The chinchilla model was utilized to evaluate the
efficacy of linezolid against experimental infection due to
S. pneumoniae or NTHI. Serum and middle ear antibiotic concentrations were determined, and sterilization of experimental OM was evaluated. Chinchillas were inoculated directly with S. pneumoniae into the superior bulla. Twenty-four hours after
inoculation, all animals had positive middle ear and nasopharyngeal
cultures. Animals were given linezolid at 25 mg/kg/dose twice a day
(b.i.d.) by orogastric feeding tube or amoxicillin at 40 mg/kg/dose
b.i.d. intramuscularly for 5 days. By day 5, all animals in the
linezolid group had sterile middle ear cultures and eradication of
S. pneumoniae from the nasopharynx. In the amoxicillin
group, all nine animals remained middle ear and nasopharynx positive
(P < 0.01). In animals inoculated with NTHI, 25 and
37.5 mg/kg b.i.d. failed to sterilize middle ear infection or eradicate
colonization. Mean levels in middle ear fluid measured during
experimental infection were 12.8 µg/ml at 2 to 6 h and 4.1 µg/ml at 16 to 17 h after orogastric dosing at 25 mg/kg.
Linezolid achieved a high concentration in the middle ear during
experimental OM. Linezolid eradicated multidrug-resistant S. pneumoniae from the middle ear and nasopharynx. Experimental infection and nasopharyngeal colonization due to NTHI persisted despite
achievement of concentrations in the middle ear that were above the MIC
(for NTHI).
| |
INTRODUCTION |
Acute otitis media remains the most
common bacterial infection in childhood. Marchant and colleagues
demonstrated that a clinical response was highly correlated with
eradication of middle ear pathogens (11). Dagan and
colleagues have recently reaffirmed this principle (6).
Prior to 1990, studies of currently used antimicrobial regimens for the
treatment of acute otitis media due to Streptococcus
pneumoniae demonstrated eradication from the middle ear in greater
than 90% of cases of acute otitis media (9). However, as
the prevalence of S. pneumoniae with reduced susceptibility
to penicillin, cephalosporins, and other antimicrobial agents has
increased, microbiologic and clinical failures in children with middle
ear infection due to nonsusceptible isolates have been reported
(5, 7, 10; E. Leibovitz, O. Abramson, D. Greenberg, P. Yagupsky, D. M. Fliss, A. Lieberman, and R. Dagan, Abstr. 36th
Intersci. Conf. Antimicrob. Agents Chemother., abstr. LM59, 1996).
New therapies for acute otitis media due to resistant pathogens will be
required if the prevalence of resistant isolates of S. pneumoniae (penicillin MIC of 2.0 µg/ml) continues to
increase. The chinchilla model for experimental otitis media has proven useful in evaluating the ability of new antimicrobial agents to eradicate middle ear infection due to S. pneumoniae or
nontypeable Haemophilus influenzae (NTHI) (1, 10, 13,
14, 15).
We studied the efficacy of linezolid, a novel antimicrobial agent, for
treatment of acute otitis media. In vitro studies have demonstrated
linezolid to have antibacterial activity similar to that of vancomycin,
with a MIC90 (the concentration of drug required to inhibit
the growth of 90% of strains) of between 0.25 and 1.0 µg/ml for
S. pneumoniae and limited activity against H. influenzae (MIC90 = 16 µg/ml) (G. V. Doern, Abstr. 8th Int. Congress Infect. Dis., abstr. 40.004, 1998; L. Verbist and J. Verhaegen, Abstr. 38th Intersci. Conf. Antimicrob.
Agents Chemother., abstr. 122-E, 1998). Our studies demonstrate
sufficient middle ear penetration to eradicate resistant S. pneumoniae (penicillin MIC = 8.0 µg/ml) but persistence of
infection due to NTHI.
| |
MATERIALS AND METHODS |
Isolates of S. pneumoniae and H. influenzae.
S. pneumoniae 645, a serotype 9 pneumococcus, was obtained
from Michael Jacobs (Case Western Reserve, Cleveland, Ohio). The isolate was cultured from the respiratory tract of a pediatric patient
and is characterized by its resistance to amoxicillin and erythromycin
(MIC = 4.0 and 8.0 µg/ml, respectively) and susceptibility to
linezolid (0.5 and 1.0 µg/ml) (Table
1). The MIC and minimal bactericidal
concentration (MBC) were determined using an inoculum of
105 CFU incubated overnight in brain heart infusion plus
NADH plus lysed horse blood with appropriate concentrations of
antibiotic. MBCs were determined by streaking a standard loop from each
tube following overnight incubation.
Two isolates of NTHI cultured from the respiratory tracts of pediatric
patients were used in the experimental studies. The
amoxicillin and
linezolid MICs for isolate BCH3 are 0.5 and 16
µg/ml, respectively,
and those for isolate no. 2 are 0.125 and
8 µg/ml, respectively
(Table
1).
Experimental infection.
Prior to inoculation, the middle
ears of adult chinchillas (8 to 48 months of age) were confirmed as
normal using otomicroscopy and tympanometry. Isolates of S. pneumoniae and NTHI were grown for 16 h at 31 and 37°C,
respectively, and cultures were diluted appropriately to a
concentration of 100 to 1,000 CFU/ml in Gey's balanced salt solution.
Ten to 100 CFU was inoculated directly into the middle ear with a
25-gauge needle and tuberculin syringe in a final volume of 100 µl.
Otomicroscopic and tympanometric examinations were performed at 24 to
48 h after inoculation. Middle ear contents were cultured through
a 3-mm hole in the superior bulla by using a Calgi swab. Calgi swabs
were streaked directly onto blood or chocolate agar plates.
Nasopharyngeal cultures were performed by cannulating the nares with a
24-gauge angiocatheter, instilling 0.5 to 1.0 ml of Gey's balanced
salt solution, and collecting the effluent (approximately 100 ml) from
the contralateral nares directly onto blood or chocolate agar plates.
Animals were randomly assigned to either the linezolid or amoxicillin
group, and therapy was initiated 24 to 48 h after inoculation
(after culture of middle ear fluid to document infection). Linezolid
was administered at 25 mg/kg/dose twice daily (b.i.d.) by orogastric
feeding tube (p.o.) at 9:00 a.m. and 6:00 p.m. for 5 days. Amoxicillin
was administered at 40 mg/kg/dose by intramuscular (i.m.) injection
on
the same schedule. Animals were reexamined at 2, 4 or 5, and
8 to 10 days after initiation of therapy. Otomicroscopic examination
of the
tympanic membrane and examination of the middle ear through
a small
hole in the superior bullae for fluid were done at each
time, and
middle ear culture was performed. Nasopharyngeal cultures
were
performed on day 4 or 5 and day 8 or 10. Comparisons of antibiotic
regimes were made using Fisher's exact
method.
Serum and/or middle ear fluid was obtained for measurement of linezolid
concentration by cardiac puncture (serum) or aspiration
of middle ear
contents through a 20-gauge angiocatheter inserted
through the hole
created in the bullar bone from animals infected
with NTHI. Middle ear
and serum specimens were frozen at
70°C.
Determination of linezolid in serum and middle ear fluid.
Linezolid concentrations in serum were determined by Dennis Stalker at
Pharmacia & Upjohn using a sensitive and selective high-performance
liquid chromatographic (HPLC) system (12). The method was
validated and run for Pharmacia & Upjohn at AVTech Laboratories, Inc.,
Kalamazoo, Mich. Briefly, serum specimens (0.1 ml) were spiked with an
internal standard (IS) and extracted using a solid phase cartridge.
Samples were eluted with methanol and injected onto the reversed-phase
analytical column (C8; 4.6 by 150 mm, 5 µm). The mobile
phase was acetonitrile and water (20:80, vol/vol), and a Beckman Gold
HPLC system was used for monitoring the samples. Peak height ratios,
based on UV detection at 251 nm, were used for quantitative analysis. A
nine-point calibration standard curve was linear over the range of 0.02 to 100 µg/ml, using a weighted (1/concentration) linear least-squares
regression. Coefficients of variation for quality control samples were
<8.3%, and the mean accuracy was from 99 to 105%.
Linezolid in middle ear fluid was quantitated using a selective HPLC
system coupled with a triple-quadrupole mass spectrometer
(LC/MS/MS)
(PE Sciex-API 300) developed and validated for Pharmacia
& Upjohn for
quantitation of linezolid in human plasma (proprietary
method). Because
of the low calibration range of this method,
the chinchilla middle ear
fluid (0.1 ml) was diluted 1:200 with
blank human plasma before it was
spiked with a deuterated IS and
extracted using liquid-liquid
techniques. Mean recoveries for
linezolid and the IS were 106 and 101%
respectively. Calibration
standard curves were created from eight
calibration points and
were linear over the range of 1.0 to 250 ng/ml,
using a weighted
(1/concentration
2) linear least-squares
regression. Coefficients of variation for
the quality control samples
were <11.5%, with a mean accuracy
of 98.7 to 101%.
| |
RESULTS |
Concentration of linezolid in serum and middle ear during
experimental otitis media.
Serum specimens collected 2 1/2 to
6 h after the third dose had a mean linezolid concentration of
17.5 µg/ml (range, 13.0 to 23.6 µg/ml). Middle ear specimens
obtained simultaneously had a mean concentration of 12.8 µg/ml
(range, 7.3 to 19.9 µg/ml) (Fig. 1).
Middle ear specimens obtained 16 to 17 h after the third dose had
a mean concentration of 4.1 µg/ml (range, 0.96 to 13.6 µg/ml). The
mean ratio of the linezolid concentration in middle ear fluid to that
in serum was 81% (range, 48 to 120%).
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FIG. 1.
Concentration of linezolid in the middle ear in
experimental otitis media. Samples were collected 2 to 6 or 16 to
17 h after the third dose of linezolid (25 mg/kg/dose per
gavage).
|
|
Treatment of experimental otitis media due to Streptococcus
pneumoniae: linezolid (25 mg/kg b.i.d., p.o.) compared with
amoxicillin (40 mg/kg b.i.d., i.m.).
Amoxicillin failed to
eradicate middle ear infection or nasopharyngeal carriage due to
S. pneumoniae 645, while linezolid sterilized 100% of
middle ear cultures by day 2 and 100% of nasopharyngeal cultures by
day 4 (Table 2). Four of the
linezolid-treated animals had 1 to 5 CFU reappear in the middle ear
culture performed 72 h after completion of therapy. The
proportions of animals with middle ear fluid on the final day of
therapy (10 of 15 treated with linezolid versus 9 of 9 treated with
amoxicillin) and 3 days after completing therapy (6 of 12 versus 5 of
6) suggested more rapid resolution in the linezolid-treated group, but
the difference was not statistically significant. Nasopharyngeal
carriage of S. pneumoniae was rapidly cleared in the
linezolid-treated cohort but persisted in the cohort treated with
amoxicillin.
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TABLE 2.
Treatment of experimental otitis media due to
S. pneumoniae: linezolid (25 mg/kg b.i.d., p.o.)
compared with amoxicillin (40 mg/kg b.i.d., i.m.)
|
|
Treatment of experimental otitis media due to NTHI isolate BCH3
(linezolid MIC = 16 µg/ml): linezolid (25 µg/kg/dose b.i.d.)
versus amoxicillin (40 mg/kg/dose b.i.d.).
Linezolid failed to
eradicate middle ear infection due to BCH3. Infection persisted through
the entire course of therapy. Amoxicillin rapidly eradicated infection,
and by day 5 middle ear cultures had been sterilized in all
amoxicillin-treated animals (Table 3).
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TABLE 3.
Treatment of experimental otitis media due to NTHI BCH3:
linezolid (25 mg/kg b.i.d., p.o.) versus amoxicillin (40 mg/kg
b.i.d., i.m.)
|
|
Treatments of experimental otitis media due to NTHI isolate no. 2 (linezolid MIC = 8.0 µg/ml): high-dose (37.5 mg/kg) versus
low-dose (25 mg/kg) linezolid.
We selected a second isolate of
NTHI and evaluated dosing at both 25 mg/kg b.i.d. and 37.5 mg/kg b.i.d.
in a small pilot experiment to determine if an isolate for which the
MIC is lower or treatment with a higher dose would successfully
eradicate in middle ear infection or nasopharyngeal carriage with NTHI.
Neither dose was successful, as middle ear infection and nasopharyngeal
carriage persisted throughout the course of therapy (Table
4).
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|
TABLE 4.
Treatment of experimental otitis media due to NTHI
isolate no. 2 (linezolid MIC = 8.0 µg/ml): high-dose (37.5 µg/kg/b.i.d., p.o.) versus low-dose (25 mg/kg b.i.d.,
p.o.) linezolid
|
|
| |
DISCUSSION |
The chinchilla model of experimental otitis media has proven
valuable for assessing the potential efficacy of antimicrobial agents.
Studies demonstrating the efficacy of ampicillin, broad-spectrum cephalosporins, and macrolides (1, 10, 15) for isolates of
S. pneumoniae susceptible to penicillin and of ceftibuten
and ampicillin-sulbactam (13, 14) for isolates of NTHI all
predated human studies of acute otitis media by using microbiologic
endpoints to affirm the efficacy of these agents. Studies of macrolides for acute otitis media due to NTHI in chinchillas failed to demonstrate rapid sterilization of middle ear infection (2) and
correlate with observations of persistent infection at day 3 to 5 in
children treated with macrolides, as reported by Howie (8)
and more recently by Dagan et al. (R. Dagan, E. Leibovitz, M. Jacobs,
D. Fliss, A. Leiberman, and P. Yagupsky, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. K-102, 1997).
The present study suggests that linezolid will be a valuable antibiotic
in the treatment of respiratory infection due to S. pneumoniae, including those isolates resistant to high levels of
amoxicillin. The dose utilized in these studies, 25 mg/kg b.i.d., gives
peak levels in serum similar to those reported for adults administered
625 mg p.o. b.i.d. Concentrations in the middle ear were 80% of those
in serum and exceeded the MIC for S. pneumoniae for the
entire 12-h dosing interval. These pharmacokinetic parameters should be
achievable in children, and therefore the results from this
experimental model should be applicable to pediatric patients.
The ability to eradicate nasopharyngeal carriage may be an important
benefit not currently used in the selection of an antimicrobial agent
for therapy. It appears likely that currently used antibiotic regimens
(in children) selectively decrease colonization with susceptible
S. pneumoniae but frequently fail to eradicate
nasopharyngeal colonization with resistant isolates (4).
This favors the spread of resistant isolates to close contacts. The
eradication of resistant isolates at both the site of infection and the
nasopharynx is likely to be associated with clinical improvement as
well as to diminish the increasing spread of resistant isolates within
the community.
The failure to sterilize middle ear infection due to NTHI correlates
with the in vitro susceptibility data. Even with peak levels of
linezolid in the middle ear of greater than 8 µg/ml, sterilization of
an NTHI isolate for which the MIC was 8.0 µg/ml was not possible.
Craig has suggested that the middle ear antimicrobial levels need to
exceed the MIC for greater than 40 to 50% of the dosing interval
(3). Although the limited pharmacokinetic studies performed
suggest that we achieved that goal for at least one isolate of NTHI,
infection persisted. Potential explanations include the high MBC for
NTHI with linezolid. Craig's model may fit better for 
-lactamase
antibiotics which are bactericidal. Antimicrobial agents that are
bacterostatic, such as linezolid against NTHI, may need to achieve
different parameters for successful bacterial eradication. Second,
failure to eradicate nasopharyngeal colonization may result in
continuous reinfection within the middle ear. Finally, the biologic
activity of linezolid may be less than that determined by chemical
detection methods. Further studies will be necessary to precisely
define the contributions these factors make to antibiotic failure in
this model.
Our results support the need for clinical studies of linezolid for the
treatment of acute otitis media due to S. pneumoniae, including highly resistant isolates. This is especially relevant given
the recent report of increased numbers of isolates of S. pneumoniae for which the MICs are 4.0 µg/ml (7a).
If studies with children confirm our observations, linezolid may
provide an effective method for treating infection and reducing the
risk of spread of resistant S. pneumoniae within the
community. However, since linezolid is unlikely to be effective in
disease due to NTHI, its use should be limited to patients with
documented or high suspicion of infection with S. pneumoniae, especially resistant isolates.
| |
ACKNOWLEDGMENT |
This project was supported by a research grant from Pharmacia Upjohn.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Maxwell Finland
Laboratory for Infectious Diseases, Boston Medical Center, 774 Albany St., Boston, MA 02118. Phone: (617) 414-7407. Fax: (617) 414-5806. E-mail: spelton{at}bu.edu.
| |
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Antimicrobial Agents and Chemotherapy, March 2000, p. 654-657, Vol. 44, No. 3
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
