Antimicrobial treatment of an experimental otitis media caused by a ß-lactamase positive isolate of Haemophilus influenzae

Carmen Pontea, Carlos Cenjorb, Araceli Parraa, Eva Nietoa, Gloria García-Calvoa, María José Giménezc, Lorenzo Aguilarc and Francisco Sorianoa,*

a Department of Medical Microbiology; b Department of Otolaryngology, Fundación Jiménez Díaz, Avenida de Reyes Católicos 2, 28040 Madrid; c Medical Department, SmithKline Beecham Pharmaceuticals, Madrid, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A gerbil model of otitis media induced by a ß-lactamase producing and non-serotypeable isolate of Haemophilus influenzae was used to assess the in-vivo efficacy of co-amoxiclav and cefuroxime at low (5 mg/kg) and high (20 mg/kg) doses. The MIC of the antibiotics tested against the pathogen was 1 mg/L (1/0.5 mg/L for co-amoxiclav). The organism was inoculated (±10 6 cfu) by transbullar challenge directly in the middle ear and antibiotic treatment was commenced 2 h post-inoculation and continued at 8 h intervals for three doses. Only high dose co-amoxiclav significantly reduced the number of culture-positive specimens as compared with untreated animals or with other treatment groups (91.7% as compared with 36.7% for high dose cefuroxime). The results obtained in any treatment group were related to middle ear antibiotic level/MIC. Antibiotic concentrations in the middle ear 90 min after administration were about 10% of serum levels at 15 min, probably related to a slight inflammatory response. Only after high dose co-amoxiclav did the concentration in the middle ear exceed the MIC by a factor of four. In otitis media with effusion, if indicated, antibiotics active in vitro should be administered in high doses and, to avoid side effects, probably in short courses.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Otitis media is one of the most frequently diagnosed infectious diseases of childhood, Streptococcus pneumoniae and Haemophilus influenzae being the predominant organisms involved. 1,2,3 The increasing rate of antibiotic resistance of these pathogens emphasizes the importance of demonstrating that they can be eradicated from the middle ear (ME) by the commonly used antibiotics. 4,5 Obtaining ME fluid before antibiotic administration and during the course of treatment has been considered as a useful method to evaluate antimicrobial efficacy. 5 However, such a method requires repeated tympanocentesis, which carries with it the risk of morbidity and super-infection, and could be argued as unethical. 4

Animal models of otitis media, although not without disadvantages, may also be useful in evaluating antibiotic efficacy, because it is unnecessary to take the first ME sample before administering the antibiotic, repeated samples can be taken from anaesthetized animals and an untreated control group can be included. The information gathered through such models may also be useful in explaining some of the results observed in clinical practice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

A clinical isolate of ß-lactamase producing biotype II and non-serotypeable H. influenzae obtained from a patient with otitis media was used.

Antibiotics

Lithium clavulanate and amoxycillin trihydrate (SmithKline Beecham Pharmaceuticals, Worthing, UK), and cefuroxime sodium (Sigma Chemical Co., St Louis, MO, USA) were used in the in-vitro studies.

For in-vivo use, co-amoxiclav 10:1 (Augmentin; SmithKline Beecham Pharmaceuticals, Toledo, Spain) and cefuroxime (Curoxima; Glaxo, Madrid, Spain), were reconstituted in apyrogen sterile distilled water to the desired concentrations.

In-vitro studies

MICs and MBCs were determined in Haemophilus Test Medium by the microdilution broth method following previously described procedures. 6 ,7 The median of five separate determinations was used to calculate the MIC and MBC of each antibiotic.

Animals

Eight- to nine-week-old adult female Mongolian gerbils ( Meriones unguiculatus)weighing 44-54 g each were purchased from the Centre d'Élevage R. Janvier (Le Genest St-Isle, France) and were managed as previously described.8

Experimental otitis

To prepare the inoculum, an overnight culture of H. influenzae was grown in brain heart infusion broth (Oxoid; Unipath Ltd, Basingstoke, UK ) enriched with 2% Fildes extract (Oxoid) (BHIF medium), incubated for 20 h at 35°C in a 5% CO 2 atmosphere, and frozen in aliquots at -70°C. The day before the experiment, a freshly thawed aliquot of 400 µL was added to 9.6 mL of BHIF, incubated at 35°C in 5% CO 2 for 20 h. The number of viable bacteria in this culture was determined by colony counting method after 10-fold serial dilutions in brain heart infusion broth. Animals were inoculated bilaterally with 20 µL of broth diluted to obtain approximately 10 6 cfu, introduced directly into the ME bulla. The tympanic membrane was left intact and swelled without rupture during the inoculation. A normal tympanic aspect and correct inoculation were verified with an operating microscope.

Treatment regimen and efficacy studies

Each antibiotic was tested at two doses (5 and 20 mg/kg), administered subcutaneously in 500 µL at 2, 10 and 18 h post-inoculation (pi). Animals in the control group received apyrogen sterile distilled water in the same way.

Treated and control animals were studied longitudinally for otorrhea, weight and behaviour. Otoscopic aspect and ME samples to determine bacterial counts in ME effusion were obtained on day 2 pi by washing the ME fossa with 20 µL of saline injected and withdrawn via the epi-tympanic membrane with a 0.33 mm needle. Shortly after sampling, aliquots of serial 10-fold dilutions in saline were plated on chocolate agar. Plates were incubated for 24 h at 35°C in a 5% CO 2 atmosphere. Bacterial counts are expressed as log 10 cfu per 20 µL of ear washing fluid; the lowest detectable bacterial count was 2 cfu per 20 µL (0.30 log 10 cfu per 20 µL). Cell contents were evaluated as previously described. 8

Cerebrospinal fluid (CSF) was obtained on day 2 by percutaneous intracysternal puncture to detect possible meningeal involvement.

Pharmacokinetic studies

Serum levels of co-amoxiclav and cefuroxime were determined in healthy animals after subcutaneous injection of 5 and 20 mg/kg. Groups of six animals per dose and collection time were killed with CO 2 and exsanguinated by intracardiac puncture at 0.25, 0.5, 1 and 2 h after drug administration. Blood samples were centrifuged to obtain serum.

Antibiotic concentrations were also determined in ME exudate in groups of 10 animals per dose bilaterally inoculated with the same organism and conditions as previously indicated in the experimental otitis model. Co-amoxiclav and cefuroxime at dosages of 5 and 20 mg/kg were administered by the subcutaneous route 46 h after bacterial inoculation. ME samples were obtained, 90 min later, via the epitympanic membrane with a 0.33 mm needle. After sampling, aliquots of each ME having >=10 µL of exudate were pooled and frozen at -70°C to determine antibiotic levels. This standard procedure (see ref. 20, below) permits collection of ME samples without blood contamination at a time when effusion is consistently abundant and the bacterial density is about 5.0 log 10 cfu per 20 µL washing fluid.

Antibiotic concentrations were determined by microbiological assay using Micrococcus luteus ATCC 9341 for amoxycillin, Bacillus subtilis 1904E for cefuroxime, and Klebsiella pneumoniae NCTC 11228 for clavulanic acid. The antibiotic concentrations in the samples were derived from standard solutions prepared in pooled gerbil sera for blood levels and in 0.1 M phosphate buffer pH 6.0 for the ME exudates. Assay variability for individual samples was <10%. Pharmacokinetic analysis was performed by standard graphical methods. 9

Statistical analysis

The number of ears with a positive count over the total number of ears was calculated as a percentage in each group of animals. The percentages of treated animals were compared with that of the untreated control group and with each other by Fisher's exact {chi} 2 corrected by the Bonferroni method ({alpha} = 0.005). Bacterial counts were expressed as the arithmetic mean log 10 cfu per 20 µL of washing fluid; culture-negative samples were included in the calculation of means assuming a value at the detection limit, and the results were analysed by Kruskal- Wallis and Mann-Whitney tests, plus the Bonferroni correction ({alpha} = 0.005). These tests were also used to analyse the differences in volume of fluid recovered from ME, the relative weight loss and inflammatory response. The study was performed in accordance with prevailing regulation regarding the care and use of laboratory animals in the European Community. 10


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vitro studies

The five MIC determinations for the test isolate gave the same values: 8, 1 and 1 mg/L for amoxycillin, co-amoxiclav (1/0.5) and cefuroxime, respectively. MBC values were equal to those of MICs.

Therapeutic efficacy in experimental otitis

After inoculation, uni- or bilateral otitis media was obtained in all untreated animals at day 2, with 96.7% of ME specimens being culture-positive. The mean ± S.D. ME volume recovered and bacterial concentration were 34.3 ± 11.9 µL and 5.0 ± 1.3 log 10 cfu per 20 µL, respectively. All ME specimens contained polymorphonuclear cells (mostly in moderate amounts) with intra- and extracellular bacteria detected by Gram staining. Most of the untreated animals remained active, with no significant weight loss, without otorrhea and showing by otoscopic examination, at day 2, presence of air fluid levels, ME effusion, and negative ME pressure.

From all treated animals ME washing fluid was recovered (mean range 28.3-36.2 µL) with no statistically significant differences between groups in the volume of the fluid obtained. A statistically significant (P = 0.0015) higher amount of polymorphonuclear cells in ME fluid was detected in the control group.

Table I presents the comparative therapeutic results in control and treated animals. Only animals treated with the high dose (20 mg/kg) of either co-amoxiclav or cefuroxime showed a significant reduction in the number of culture-positive ME specimens as compared with untreated controls (P <= 0.0013). The reduction in the number of positive ME samples with the high dose of both antibiotics was greater than that of the low dose of the same antibiotic, the differences being significant only for co-amoxiclav (P = 0.00001). When the results obtained with the different antibiotics and doses were compared, only the high dose of co-amoxiclav significantly (P = 0.00004) reduced the number of culture-positive ME samples as compared with any of the cefuroxime doses. As far as the mean concentration of bacteria in the ME samples is concerned, there were significant differences only between results obtained in animals treated with the high dose of co-amoxiclav and any other group.


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Table I. Outcome (day 2) of experimental otitis media caused by ß-lactamase producing H. influenzae treated with co-amoxiclav and cefuroxime
 
Table II shows otoscopic results at day 2 determined by the otolaryngologist. Otitis media with effusion (OME) was the predominant (70-90%) picture in both treated and untreated animals.


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Table II. Otoscopic results (as percentage) at day 2 in animals with experimental otitis media caused by H. influenzae
 
As far as body weight is concerned only animals treated with the high dose of co-amoxiclav showed a significant body weight loss (mean 3.97 ± 2.78%) at day 2 as compared with that of any other group (P < 0.05).

No animals, untreated or treated, had positive culture for CSF as studied at day 2.

Pharmacokinetic and pharmacodynamic data

Table III shows the pharmacokinetic and pharmacodynamic analysis of both serum and ME exudate levels in relation to the organism inoculated. The most favourable pharmacokinetic and pharmacodynamic data were obtained with the high dose of co-amoxiclav mainly in relation to inhibitory quotient (C 15 min /MIC), and area under the curve (AUC) (mg·h/L)/MIC (mg/L). As far as ME antibiotic concentrations are concerned, higher concentration of co-amoxiclav than cefuroxime was found at any administered dose.


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Table III. Pharmacokinetic and pharmacodynamic data of co-amoxiclav and cefuroxime in relation with the H. influenzae susceptibility
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Otitis media is usually a self-limiting infection with a favourable outcome even in the absence of antimicrobial treatment. 11,12 The superiority of one regimen over another is therefore difficult to demonstrate and so clinical criteria must be accompanied by bacteriological controls. 13,14 On the other hand, clinical and bacteriological failures have been recently described with some antibiotics commonly used to treat such infections. 3,15,16,17,18,19 Animal models of otitis media can explain some of the differences found, as well as provide both clinical and bacteriological data to compare efficacy between antibiotics.8,20,21

We previously carried out a gerbil model of otitis media induced by a penicillin-resistant isolate of S. pneumoniae where the activity of amoxycillin and cefuroxime was evaluated. 8 In this study we selected, for a similar model, a ß-lactamase producing isolate of H. influenzae to compare the efficacy of co-amoxiclav and cefuroxime, antibiotics which are usually active in vitro against this organism.

In this model of otitis media caused by H. influenzae, animals showed fewer alterations of the ME compared with those shown in our model of otitis media by S. pneumoniae. Furthermore, animals with otitis caused by H. influenzae did not show otorrhoea, changes in behaviour or body weight loss as compared with animals with otitis caused by S. pneumoniae, a clinical picture closer to the so-called OME than to an acute otitis media (AOM). Also in humans, AOM is related to infection by organisms with high pathogenic potential such as S. pneumoniae, 1,2,22 whereas OME seems to be related to infection by less pathogenic organisms such as non-serotypeable isolates of H. influenzae, mainly from biotype II. 19,22

Serum levels after 5 mg/kg of any antibiotic were similar to those usually attained in humans after oral administration of such antibiotics. 23 This dose of co-amoxiclav and both doses (low and high) of cefuroxime were unable to achieve a bacteriological efficacy >36.7% at day 2, even though very favourable serum pharmacokinetic and pharmacodynamic values were reached, probably because the ME antibiotic concentrations were rather low.

Only the high dose of co-amoxiclav significantly reduced, in this gerbil model, the number of ME culture-positive samples, as compared with untreated animals or with those in other treatment groups, achieving 91.7% of negative cultures, after just three doses of antibiotic. However, these animals showed a significant loss of body weight. The efficacy of this dose of co-amoxiclav was probably related to the higher antibiotic ME concentration as compared with that obtained with any other antibiotic or dose. For technical reasons, such antibiotic concentrations could only be determined after 46 h post-inoculation, to obtain enough blood-free sample. Only after the high dose of co-amoxiclav was the antibiotic ME concentration greater than three times the MIC for the pathogen. This agrees with previously published papers showing that a ME antibiotic concentration/MIC ratio of between 3.2 and 6.3 correlates with 80-85% bacterial eradication in otitis media in humans. 23

Although bacteriological efficacy in otitis media is said to be achieved when antibiotic concentrations in serum exceed the MIC for 30-40% of the dosing interval, 23 in this H. influenzae model we did not observe such a correlation because similar time serum levels above the MIC achieved very different bacteriological results and only the high dose of co-amoxiclav obtained >90% of bacteriological efficacy, in spite of serum levels exceeding the MIC for just 20% of the dosing interval. This could be explained by the pharmacokinetics of antimicrobials in ME resulting in lower peak and higher trough values than in serum 23 and/or longer antibiotic half-life in the ME. 20

In this animal model of otitis media caused by H. influenzae, the ME antibiotic concentration was about 10% of serum levels at 15 min. In the otitis media model caused by our isolate of S. pneumoniae, three times higher ME amoxycillin concentrations were found (unpublished data), probably because of the higher ME inflammation in the pneumococcal model. This finding agrees with a report showing ME concentrations of about 30% of serum levels in otitis media induced by S. pneumoniae in the gerbil model. 20 This may explain why lower doses of amoxycillin (or co-amoxiclav for H. influenzae) may be more efficacious in clearing pneumococci than haemophili, both having equal MIC values, from the ME in gerbils. 8

Published results on clinical and bacteriological efficacy of antibiotics in the treatment of otitis media in humans are varied and depend not only on patients' characteristics but also on the organisms involved and, possibly, on the time when ME samples are taken. Nevertheless, in most published series, aminopenicillins and cefuroxime have shown efficacy in cases of otitis media by S. pneumoniae 4,16,17,19 probably because most isolates involved were penicillin-sensitive and sufficient ME concentrations were obtained, although failure of cefuroxime has been recently described for pneumococcal isolates with cefuroxime MIC >= 0.5 mg/L. 13,24,25 On the other hand, the efficacy of antibiotics in cases of otitis media caused by H. influenzae is less encouraging. 16,17,19 These results observed in children could be explained, at least in part, by the MICs of ß-lactams for S. pneumoniae being lower than for H. influenzae 26,27 and also by the fact that the latter organism is mainly involved in OME 19,28 where the antibiotic seems to penetrate poorly into the ME. The results obtained in these gerbil models of otitis media corroborate the pharmacodynamic explicative basis.

The most suitable treatment for otitis media in children has not yet been established, as it depends not only on the host characteristics, inflammatory response, pathogen involved, and its antimicrobial susceptibility, but also on the antibiotic ME penetration, which could be diminished in cases where H. influenzae is involved. If an antibiotic is indicated, in cases of OME, it should probably be administered at high doses and in short courses.


    Acknowledgments
 
E.N. and G.G.-C. were aided with scholarships from the Fundación Conchita Rábago (Madrid, Spain). This study was supported by a grant from SmithKline Beecham S.A., Madrid, Spain.


    Notes
 
* Tel: +33-1-45593219; Fax: +33-1-45593834; E-mail: olivier.mimoz{at}pbr.ap-hop-paris.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bluestone, C. D., Stephenson, J. S. & Martin, L. M. (1992).Ten-year review of otitis media pathogens. Pediatric Infectious Disease Journal 11, Suppl.,7 –11

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Received 24 November 1998; returned 13 January 1999; revised 2 February 1999; accepted 25 February 1999