The treatment of Haemophilus influenzae acute otitis media with amoxicillin protects against reinfection but not against structural changes

Eva Westmana and Åsa Melhusb,*

a Department of Oto-Rhino-Laryngology, Umeå University Hospital, Umeå; b Department of Medical Microbiology, Malmö University Hospital, S-205 02 Malmö, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Acute otitis media (AOM) is the most common reason for outpatient antimicrobial therapy today. With increasing problems with antibiotic resistance, a more restrictive use of antibiotics has been advocated for both single and recurrent episodes. The arguments advanced have been immunological as well as ecological. To study the effects of a 5 day course of amoxicillin on recurrent AOM caused by non-typeable Haemophilus influenzae, Sprague–Dawley rats were used. Amoxicillin was introduced at the clinical peak of the first infection. One month later the animals were rechallenged. Local and systemic changes were monitored by otomicroscopy, bacterial cultures, and analyses of systemic IgG responses and histological changes. Antibiotic treatment accelerated the resolution of the primary infection. After resolution, only minor morphological changes were observed. The protective rate at rechallenge was 100% in the treatment group, compared with 80% in the untreated control group. Although the production of serum IgG antibodies was initially slightly impeded by the treatment, it was significantly higher in treated animals after rechallenge. Major structural changes could not be avoided at the second infection, however, and a significantly higher frequency of myringosclerosis was observed in treated animals. These experimental findings constitute support for further studies of antimicrobial drugs and recurrent AOM.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Acute otitis media (AOM) is a common clinical problem. Before the age of 2 years about 40% of all children will have experienced an episode of AOM, and 4% will have recurrent episodes.1 A major aetiological agent of both AOM and recurrent AOM is non-typeable Haemophilus influenzae, an organism also associated with therapeutic failure and secretory otitis media.2–4

Owing to its Gram-negative cell wall composition, non-typeable H. influenzae induces an intense inflammatory host response in the middle ear cavity.5–7 After a resolved infection persistent structural changes develop.8–10 In experimental models, the use of antimicrobial agents alters the gene expression of cytokines significantly and minimizes histopathological changes induced by bacteria.11,12 In spite of obvious histological differences between treated and untreated experimental AOM, few studies have explored the long-term consequences of these differences. Structural changes have been suggested to be of importance for subsequent AOM episodes in children.13,14

Currently, AOM is the most common reason for outpatient antimicrobial therapy in the USA.15 In Europe, with the exception of The Netherlands,15 74–97% of the children with AOM receive treatment.1,16,17 The antibiotic load in younger age groups is consequently high, and a high selective pressure increases the risk of antibiotic resistance.18,19 With the emergence of penicillin-resistant pneumococci, there has been massive propaganda against antibiotic misuse and overuse in Sweden. It has led to a clear decline in the prescription rate of these drugs,20 but also to questions about the benefits of antibiotic treatment. The arguments for non-treatment of AOM have been not only ecological but also immunological. In the latter case the arguments have been founded on the assumption that an untreated middle ear infection is better than a treated one in promoting protective host responses, e.g. antibody production, but data supporting this assumption are scarce,21,22 especially in children with recurrent disease.

The aim of the present study was to investigate whether antibiotic treatment of experimental AOM induced by non-typeable H. influenzae influences the ability of the host to develop protection against reinfection or not. The treatment consisted of a 5 day course of amoxicillin initiated at the clinical peak of the infection. Morphological changes of the middle ear mucosa and the humoral response were also analysed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals and surgical procedures

Healthy male Sprague–Dawley rats weighing 350–400 g were used, and the study protocol was approved by the ethics committee of Lund University. A ventral midline incision was made in the neck during anaesthesia induced by intraperitoneally administered chloralhydrate (Apoteksbolaget, Malmö, Sweden). After exposing the bulla, the bone structure surrounding the middle ear of the rat, the right middle ear was inoculated with c. 0.05 mL of the bacterial suspension through the bony wall without perforating the tympanic membrane.23

Bacteria and media

Non-typeable H. influenzae strain 3655 (biotype II, MIC of ampicillin 0.5 mg/L, ß-lactamase negative; kindly provided by Robert S. Munson, Jr, Ohio State University, Columbus, OH, USA) was used throughout the study. The bacteria were grown on chocolate agar or in brain–heart infusion broth (BHI; Difco Laboratories, Detroit, MI, USA) supplemented with NAD and haemin (Sigma, St Louis, MO, USA), each at 10 mg/L.

The inocula for middle ear challenge and rechallenge were prepared by growing the bacteria at 37°38;C to an optical density of 1 at 620 nm on a New Brunswick Environmental Incubator Shaker. The bacterial suspensions were thereafter diluted with supplemented fresh medium to an inoculum concentration of 107 cfu/mL after dose finding experiments. Viable counts of the bacterial suspensions were performed at the time of the inoculations. Middle ear cultures were carried out with a previously described and highly reproducible swab technique,11 in which no attention has to be paid to clot formation or to changes in the viscosity of the effusion during the AOM course.

Experimental design

A total of forty-eight animals were challenged with non-typeable H. influenzae. Antibiotic therapy with amoxicillin (Imacillin; AstraZeneca, Södertälje, Sweden) was introduced on day 3 via the drinking water (250 mg/500 mL, the recommended dose for rats by veterinary standards). Twenty animals were treated, and the remaining 28 animals served as controls. (for details of the experimental design, see Table 1Go). Water consumption was measured on a daily basis until day 8, when antibiotic treatment was discontinued. Water consumption was followed more closely in two animals, in whom the serum concentrations of amoxicillin were measured during two periods of 12 h in each animal. A rechallenge was performed 1 month after the initial inoculation. At this rechallenge, five unchallenged animals were added as controls to ensure established infection.


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Table 1. Experimental design
 
The animals were inspected under an otomicroscope on days 3, 4, 8 and 28 after the initial middle ear challenge, and on days 4, 8 and 28 after the rechallenge. The status of the tympanic membrane and the quantity and quality of the effusion behind the membrane were evaluated, and the inspections were carried out by a team member blinded to the identity of each animal. Direct visualization of opaque fluid behind the tympanic membrane was required for diagnosis of AOM.

Three individuals from each animal group were randomly selected for middle ear culture on days 3, 4 and 8 post-inoculation and on day 8 after the second inoculation. When the middle ear samples had been obtained, these animals were excluded from the study. Blood samples were collected for serological studies on days 0, 4, 8, 28 and 36 (day 8 after the rechallenge). The samples were stored at °32;20°38;C until analysed with an enzyme-linked immunosorbent assay (ELISA). Finally, three treated and three untreated animals were killed on day 28, and another five treated animals on day 56 (day 28 after the second inoculation), for histological studies.

Morphological examination

The bullae were removed, and fixation was performed in 4% paraformaldehyde for 24 h. The decalcification process was initiated by transferring the middle ears to vials containing 8% EDTA in 0.1 M Tris buffer (pH 7.4) with 4% paraformaldehyde. The process was completed in a microwave oven (Polaron, H2500; Bio-Rad, Sundbyberg, Sweden) set at 75% of 630 W. During this last stage, which lasted 57 h, the ears were placed in vials with a 10% EDTA solution in 0.1 M Tris buffer (pH 7.3).24 After dehydration in ethanol, the specimens were embedded in paraffin. Serial sections (5 µm) of the bullae were mounted on glass slides for routine haematoxylin and van Gieson staining. Sections in which both the pars tensa and the cochlea were present were examined under a light microscope at an object magnification of 40x. The examination was performed by a team member who was unaware of the identity of each animal. The epithelial lining in the middle ear cavity was studied and the degree of inflammation and the occurrence of metaplastic changes were registered. One unchallenged middle ear was added as a negative control.

ELISA

The IgG response to untreated and treated non-typeable H. influenzae AOM was measured with a previously described ELISA25 with modifications. In brief, microtitre plates were coated overnight with whole strain 3655 cells suspended in NaHCO3 buffer (pH 9.5). The antigen concentration was predetermined to yield optimal readings. The plates were blocked and after each step washed. Test sera diluted with blocking buffer were allowed to react. A 1:4000 dilution of peroxidase-conjugated goat anti-rat IgG (Dakopatts A/S, Glostrup, Denmark) was added. After incubation the process was halted with 1 M H2SO4. Optical densities were read at 405 nm. Experiments were performed in triplicate. Two internal controls were included on each plate. The titres were averages of the three optical density readings.

Statistics

Fisher's exact test was used for statistical analysis of the otomicroscopy results and Student's t-test for the serological findings. A value of P <= 0.05 was selected as the minimal level of significance for both tests.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Clinical findings and otomicroscopy

All animals developed AOM after the first bacterial challenge. Apart from the induced middle ear infection, the animals appeared clinically healthy throughout the study. Five deaths were recorded after the rechallenge, caused by respiratory insufficiency during anaesthesia (for group distribution, see Table 1Go).

The amoxicillin treatment was well tolerated. No side effects were observed except slight diarrhoea. The mean dose of amoxicillin was c. 53 ± 6 mg/kg body weight/day deduced from the water consumption, yielding peak serum concentrations of 4 ± 3 mg/L. Once the amoxicillin had been introduced, there was no progress of the AOM otomicroscopically.

The course of the untreated and treated AOM after the first and second challenge is shown in Figure 1Go. Treatment with amoxicillin accelerated the resolution of the AOM significantly. On day 8, eight of the treated animals had recovered fully, whereas the corresponding figure for the untreated controls was zero (P = 0.03). The bacteriological recovery was faster than the clinical recovery. On day 4, the middle ear cultures showed sparse (two out of three cultures) or no growth (one out of three cultures) of H. influenzae in treated animals, whereas all cultures from untreated animals yielded abundant growth. After 5 days of treatment three out of three cultures were negative. In the untreated animal group there was sparse growth in the middle ear in one out of three cultures.



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Figure 1. Otomicroscopic changes in rat middle ears recorded after the first and second challenge with non-typeable H. influenzae in untreated animals and animals treated with amoxicillin during the first AOM episode. Animals challenged only once are not shown. Key: {blacksquare}, opaque effusion; , turbid effusion; , clear effusion; {square}, no effusion.

 
On day 28, the otomicroscopic status was normal in all but two animals, one treated and one untreated, which both exhibited myringosclerosis for the first time. After rechallenge, AOM was only observed in two animals. These animals belonged to the untreated group. The difference in protection between the animal groups was not significant, and none of the rechallenged animals was culture-positive on day 36 (day 8 after the second inoculation). Four additional cases of myringosclerosis were observed on day 4 (n = 2) and day 8 (n = 2) after the rechallenge. All four animals had received antibiotic treatment (P = 0.04).

Structural observations

Three different categories of morphological changes were identified (summarized in Table 2Go). Representative light micrographs of categories 1 and 3 are shown in Figure 2Go.


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Table 2. Summary of the structural observations found in the middle ear after challenge and rechallenge in treated and untreated animals
 


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Figure 2. Light micrographs (400x) representing sulcus occipitalis in (a) an amoxicillin-treated animal on day 28 (no changes), (b) an untreated control animal on day 28 (major changes), and (c) an initially amoxicillin-treated and thereafter rechallenged animal on day 56 (major changes). For definitions of no or major changes, see Table 2Go. Bar = 25 µm.

 
Humoral immune response

Low levels of IgG antibodies to non-typeable H. influenzae cells (ELISA value <0.4) were detected in the pre-challenge sera. As shown in Figure 3Go, the antibody levels increased in both untreated and treated animals during the AOM. The ELISA values had a tendency to be slightly higher in the untreated animal group during the treatment period, but not significantly so, and all animals mounted IgG antibodies after the middle ear challenge. Twenty-eight days after the initial inoculation, the mean ELISA values for the two groups were similar. On day 36, i.e. 8 days after the rechallenge, the ELISA values were significantly higher in the animal group that had received amoxicillin during the first middle ear infection (P = 0.003). There was no difference in antibody levels between animals that were protected against reinfection and those that were not.



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Figure 3. Mean ELISA values (± S.D.) of serum IgG antibodies to whole non-typeable H. influenzae cells for untreated ({square}) and amoxicillin-treated ({circ}) animals shown in Figure 1Go on day 0 to day 36 (day 8 after rechallenge).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, the effects of an earlier amoxicillin course on local protection were explored in a rat model of recurrent AOM. As shown in an earlier study,12 antibiotic treatment accelerated the resolution of the middle ear infection and diminished the persistent morphological changes normally observed after an untreated AOM induced by non-typeable H. influenzae. No treated animal developed AOM at rechallenge, whereas two animals in the untreated group did. The assumption that antimicrobial chemotherapy leads to a less efficient protection against recurrent AOM could therefore not be confirmed.

Production of IgG antibodies in serum in a primary infection seemed initially to be slightly impeded by the amoxicillin therapy. Surprisingly, the situation was the opposite after the rechallenge, with significantly higher IgG antibody levels in the treatment group and an almost absent booster effect in the untreated group. Enhancement of antibody responses by ampicillin has also been observed after vaccination with live attenuated mucosal bacterial vaccines.26,27 Only modest immunomodulatory activities have been demonstrated for oral ß-lactams,22,28 but the observed phenomenon can be explained by the current knowledge of the effects of bacterial killing on the polarization of immune responses.29

In this study the therapy was initiated at the clinical peak of the infection, a stage at which it is not unlikely that the patient might consult a doctor. It is, however, possible that the serum IgG antibody profile would have been different if the treatment had been introduced at a different time point. Before determining the time at which to initiate antibiotic therapy for an optimal antibody response, the role systemically produced antibodies play in preventing AOM needs to be elucidated. Serum antibodies against non-typeable H. influenzae have been shown to protect against experimental AOM,30 but viable H. influenzae can persist in the middle ear cavity despite increasing antibody levels.31

Despite a protective rate of 100% and an inflammatory reaction of low intensity and very short duration at the renewed contact with H. influenzae, the mechanisms involved in middle ear protection could not prevent tissue damage or structural alterations in the middle ear cavities of the previously amoxicillin-treated animals. After the second inoculation, treated animals seemed to have a higher risk than untreated animals of developing myringosclerosis, a not uncommon finding in children treated with ventilation tubes for recurrent and secretory otitis media.32,33 As early as day 4 after the rechallenge, sclerotic lesions in the tympanic membrane were observed in the treatment group. This is at least 10 days earlier than previously recorded during an untreated non-typeable H. influenzae-induced AOM.7 It is not unlikely that the naturally occurring histopathological changes of the tympanic membrane during and after an infection diminish the vulnerability to new bacterial attacks by decreasing the mobility of the membrane under a critical healing period.8,34 The antibiotic therapy could therefore have contributed indirectly to the increased frequency of myringosclerosis. However, animal studies always call for circumspection, and their clinical application or relevance can be limited, especially if the route of infection used in a model, as in this rat otitis model, is not the natural one.

To summarize, under the given conditions, no proof of a better protection against recurrent disease could be established by abstaining from antibiotic therapy. The level of protection was at least as high in amoxicillin-treated animals as in untreated animals. The protection did not extend to the prevention of tissue damage and structural alterations, although the serum IgG levels were significantly higher in the amoxicillin-treated animals during the later phases of the study. These experimental findings constitute support for further studies of antimicrobial drugs and AOM, and of antibiotic treatment of recurrent AOM in particular.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The study was supported by grants from the Swedish Society of Medicine and Forssman's foundation.


    Notes
 
* Corresponding author. Tel: +46-40-331350; Fax: +46-40-336234; E-mail: asa.melhus{at}mikrobiol.mas.lu.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 4 April 2001; returned 26 July 2001; revised 31 August 2001; accepted 10 October 2001





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