clavulanic acid and erythromycin in experimental otitis media caused by Streptococcus pneumoniae plus Haemophilus influenzae

International Journal of Antimicrobial Agents 23 (2004) 25–31

In vivo activity of amoxicillin/clavulanic acid and erythromycin in experimental otitis media caused by Streptococcus pneumoniae plus Haemophilus influenzae Araceli Parra a , Carmen Ponte a , Carlos Cenjor b , Marta Garc´ıa-Olmos a , Mar´ıa J. Giménez c , Lorenzo Aguilar c , Francisco Soriano a,∗ a

Department of Medical Microbiology, Fundación Jiménez D´ıaz, Avenida de Reyes Católicos 2, 28040 Madrid, Spain b Department of Otolaryngology, Fundación Jiménez D´ıaz, Avenida de Reyes Católicos 2, 28040 Madrid, Spain c Medical Department, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain Received 9 April 2003; accepted 14 May 2003

Abstract A gerbil model of acute otitis media induced by Streptococcus pneumoniae plus Haemophilus influenzae was used to assess the efficacy of amoxicillin/clavulanic acid (A/C) (1.5/0.3, 2.5/0.5 and 10/2 mg/kg) and erythromycin (2.5, 10, 20 and 50 mg/kg) with or without acetaminophen. The amoxicillin/clavulanic acid MIC was 1/0.5 mg/l for both organisms and the erythromycin MICs were 0.12 and 4 mg/l for S. pneumoniae and H. influenzae, respectively. The organisms were inoculated directly into the middle ear (ME) and antibiotic treatment started 2 h post-inoculation and continued at 8 h intervals for three doses. Acetaminophen was administered at 50 mg/kg. Samples for bacterial counting were obtained from the ME on day 2. Amoxicillin/clavulanic acid peri-MIC concentrations in ME were effective in eradicating both organisms. Despite the inflammation induced by S. pneumoniae, erythromycin did not eradicate H. influenzae at ME concentrations (2.4 mg/l for erythromycin 50 mg/kg) higher than those obtained in humans but lower than the MIC. © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: S. pneumoniae; H. influenzae; Acute otitis media; Animal model

1. Introduction Otitis media is the inflammation of the middle ear (ME), associated to the presence of fluid and/or otorrhoea. Streptococcus pneumoniae and Haemophilus influenzae are the bacterial pathogens most commonly involved, and both organisms can be isolated in mixed culture from 10–24% of patients [1–5]. We have shown in previous experimental models that S. pneumoniae induces acute otitis media (AOM) while H. influenzae induces otitis media with effusion (OME) [6]. Beta-lactam antibiotic concentrations in ME fluid were lower in OME than in AOM, probably due to the lower inflammatory reaction associated with OME [6]. Doses of amoxicillin/clavulanic acid (A/C) that achieve ME concentrations in OME [7] that are similar to those obtained in

∗

Corresponding author. Tel.: +34-91-5447387; fax: +34-91-5494764. E-mail address: [email protected] (F. Soriano).

humans after standard doses [8] are effective in eradicating H. influenzae (with an MIC of 1 mg/l, a value similar to the MIC50/MIC90 in Spain), but eradication did not occur with lower doses [7]. In contrast, these lower doses in a mixed (S. pneumoniae plus H. influenzae) AOM model were able to eradicate both organisms due to the higher antibiotic concentration in the ME fluid, probably due to a greater inflammatory reaction [6]. Amoxicillin/clavulanic acid and macrolides are commonly prescribed for the empirical treatment of otitis media often together with an anti-inflammatory or analgesic drug such as acetaminophen [9–12]. As erythromycin achieves higher concentrations in cerebrospinal fluid in the presence of meningeal inflammation [13], we speculated that, if this antibiotic is administered for the empirical treatment of AOM caused by S. pneumoniae plus H. influenzae, both organisms might be eradicated. The second objective was to know if acetaminophen, could prevent the development of OME and to study its possible interference with the ME antibiotic pharmacokinetics as well as therapeutic efficacy.

0924-8579/$ – see front matter © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2003.05.016

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2. Materials and methods 2.1. Bacteria Two clinical isolates were used: S. pneumoniae serotype 23F (MIC benzylpenicillin, 2 mg/l) isolated from a patient with bacteraemia and a biotype II ␤-lactamase producing, non-serotypable H. influenzae (MIC amoxicillin, 8 mg/l) isolated from a patient with otitis media. 2.2. Pharmacological compounds Amoxicillin trihydrate and lithium clavulanate (SmithKline Beecham Pharmaceuticals, Worthing, UK), and erythromycin (Sigma, St. Louis, MO, USA) were used in the in vitro studies. For in vivo (therapeutic) use, propacetamol chlorhydrate, a pro-drug of acetaminophen, was used (Pro-efferalgan; Upsa Laboratories, Agent-Lot et Garonne, France), amoxicillin/clavulanic acid 5:1 (Augmentin; SmithKline Beecham S.A., Toledo, Spain) and erythromycin lactobionate (Pantomicina; Abbott Laboratories, S.A., Madrid, Spain) were reconstituted in pyrogen free sterile distilled water to the desired concentrations. 2.3. In vitro studies MICs and MBCs were determined by a microdilution broth method following previously described procedures [14,15]. Modal values of three separate determinations were recorded. 2.4. Animals Eight to 9-week-old adult female Mongolian gerbils (Meriones unguiculatus) weighing 49 ± 5 g each were purchased from the Centre d’Élevage R. Janvier (Le Genest St. Isle, France) and were managed as previously described [6]. The study was performed in accordance with prevailing regulations regarding the care and use of laboratory animals in the European Community [16]. 2.5. Experimental otitis Overnight cultures of the organisms were kept in aliquots at −70 ◦ C. The day before the experiment, a freshly thawed aliquot of H. influenzae was added to brain heart infusion broth (BHI) (Oxoid Ltd., Basingstoke, UK) enriched with 2% Fildes extract (Oxoid), incubated at 35 ◦ C in 5% CO2 for 20 h. On the day of the experiment, a freshly thawed aliquot of S. pneumoniae was incubated for 4 h at 37 ◦ C in a 5% CO2 atmosphere in BHI enriched with 5% horse serum (bio-Merieux, Marcy-l’Étoile, France). The number of viable bacteria in the different inocula was determined by colony counting. Animals were inoculated bilaterally with a mixture of both broths diluted in BHI to obtain approx-

imately 105 cfu of H. influenzae and 106 cfu of S. pneumoniae per 20 ␮l, introduced directly in 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. AOM was defined as otorrhoea through a perforation in the tympanic membrane and/or inflammatory signs with changes in the membrane’s normal yellowish-pink appearance to a grey, dark brownish-yellow, or whitish opaque area, with a very rough surface texture. OME was defined as no inflammatory signs of tympanic membrane with air fluid levels and ME fluid with or without signs of negative ME pressure. Three animals (six ears) were inoculated with 20 ␮l of BHI/ear to investigate the possible role of the broth as inducer of otitis media. Blood cultures were performed in four animals 20 h after bacterial inoculation to check for the presence of bacteraemia in infected animals. 2.6. Treatment regimen and efficacy studies Amoxicillin/clavulanic acid (1.5/0.3, 2.5/0.5 and 10/2 mg/ kg) and erythromycin (2.5, 10, 20 and 50 mg/kg) were administered subcutaneously (sc) in 500 ␮l at 2, 10, and 18 h post-inoculation (pi). The experiments were carried out in parallel administering the antibiotics alone or combined with 50 mg/kg of propacetamol sc administered 30 min before each antibiotic dose. Animals in the control groups received pyrogen free sterile distilled water instead of antibiotic with and without propacetamol. Groups of 6–8 animals were used per treatment and control groups. Treated and control animals were studied longitudinally for otorrhoea, weight, and behaviour. Otoscopic aspect and ME samples obtained by washing the ME fossa with 20 ␮l of saline solution injected and withdrawn via the epitympanic membrane with a 0.33 mm needle to determine bacterial counts in ME washing fluid, were obtained from both ears on day 2 pi. Aliquots of serial 10-fold dilutions in saline were plated on sheep blood agar and chocolate agar and incubated for 24 h at 35 ◦ C in a 5% CO2 atmosphere. Bacterial counts are expressed as log10 cfu/20 ␮l; the lowest detectable bacterial count was 4 cfu/20 ␮l (0.60 log10 cfu/20 ␮l). To evaluate the presence of polymorphonuclear (PMN) leukocytes, 3 ␮l of ME samples were spread over a 6 cm2 slide surface to be Gram stained, and observed under a high-power (1000×) microscope. The number of cells in 10 fields was calculated and expressed as mean [S.D.] per field. 2.7. Pharmacokinetic studies Serum levels of acetaminophen were determined in healthy animals 15 and 30 min after sc administration of 50 mg/kg of propacetamol, by fluorescence polarization immunoassay (FPIA) in an Axsym autoanalyser (Abbott Cient´ıfica, S.A., Spain). Groups of six animals per sample

A. Parra et al. / International Journal of Antimicrobial Agents 23 (2004) 25–31

time were killed with CO2 and were exsanguined by intracardiac puncture. The limit of detection was 1 ␮g/ml. Serum levels of amoxicillin, clavulanic acid and erythromycin were determined in healthy animals after a single sc injection of the same doses used for the treatment. Groups of six animals per antibiotic dose and collection time (15, 30, 60 and 120 min after drug administration) were killed as described for acetaminophen. Antibiotic concentrations in ME fluid without washing were also determined in groups of 10 animals bilaterally inoculated under the same conditions as previously described in the experimental otitis model. Amoxicillin/clavulanic acid (1.5/0.3 mg/kg) and erythromycin (50 mg/kg) were administered alone or combined with propacetamol, 46 h after bacterial inoculation. ME samples were obtained 90 min later, via the epitympanic membrane, with a 0.33 mm needle. Aliquots of ME samples having ≥2 ␮l of exudate were pooled and frozen at −70 ◦ C for later determination of antibiotic levels. This procedure permits collection of ME samples at a time when effusion is consistently abundant with high bacterial density and without otorrhoea. Antibiotic concentrations were determined by microbiological assay using Micrococcus luteus ATCC 9341 for amoxicillin and erythromycin and Klebsiella pneumoniae NCTC 11228 for clavulanic acid. Standard curves for determination of antibiotic concentrations were derived from standard solutions prepared in pooled gerbil sera for serum levels and in 0.1 M phosphate buffer pH 6.0, for ME fluid. Assay variability for individual samples was <10%. Antibiotic concentration–time curves for each antimicrobial were analysed by a non-compartmental approach using the Win-Nonlin program (Pharsight, Mountainview, CA). Time over MIC was calculated graphically from the semilogarithmic representation of the concentration–time curve and the regression line representing the apparent elimination rate constant. The relationship between the maximum antibiotic serum concentration obtained (concentration at 15 min after antibiotic administration: C15 ) and the MIC of the infecting microorganism (serum inhibitory quotient: C15 /MIC) and the relationship between the ME antibiotic concentration after 90 min of drug administration and the MIC of the infecting microorganism (ME inhibitory quotient: ME level/MIC) were calculated. 2.8. Statistical analysis The number of ears with a positive count divided by the total number of ears was calculated to give the percentage of positive ears in each group of animals. To detect differences in eradication rates in each group, the Fisher’s exact test were used. Otorrhoea at day 1 was analysed using the chi-square test. Bacterial counts were expressed in untreated and treated animals as arithmetic means in log10 cfu per 20 ␮l of ME sample, culture negative samples being included in the calculation of means assuming a value at the detection limit. Analysis of covariance (ANCOVA) was

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used to compare the reduction of log10 cfu and loss of body weight at day 2 in each group controlled for the log10 cfu or weight basal values and for the differences by groups. The data on number of PMN cells in the ME sample were analysed by the ANOVA. When the ANCOVA or ANOVA P-value was significant (P < 0.05), contrast between groups was made by use of the Tukey–Kramer test to adjust the type I experiment-wise error.

3. Results 3.1. In vitro studies For H. influenzae, median MICs/MBCs were 1/1 and 4/4 mg/l, for amoxicillin/clavulanic acid and erythromycin, respectively. For S. pneumoniae, median MICs/MBCs were 1/1 and 0.12/0.12 mg/l for amoxicillin/clavulanic acid and erythromycin, respectively. For amoxicillin/clavulanic acid (2/1) data are referred as the amoxicillin concentration. 3.2. Experimental otitis and therapeutic efficacy Since no significant differences could be found between the administration or not of acetaminophen in the different groups, results have been grouped according to the antibiotic regimen administered and in one untreated control group. After inoculation of S. pneumoniae (6.24±0.07 log10 cfu) and H. influenzae (4.78 ± 0.15 log10 cfu) bilateral AOM was observed in all ears from animals which did not receive antibiotics with 100% of culture positive ME samples. S. pneumoniae was isolated in 93.3% and H. influenzae in 96.7% of the samples with bacterial counts (log10 cfu) of 3.10 ± 1.10 and 5.40 ± 1.18, respectively. In these samples a mean polymorphonuclear cells of 8.3±7.8 per field with intra- and extracellular organisms was found. Animals showed lethargy, with an important weight loss (11.22 ± 3.82%) and 76.7% of ears showing otorrhoea. One of the four animals from which blood culture was taken was positive for both organisms inoculated. Table 1 presents the comparative therapeutic results. Any dose of amoxicillin/clavulanic acid showed a significant (P < 0.001) reduction in the number of culture-positive ME specimens and in the colony counts of both organisms compared with control animals. Erythromycin, at any dose, did not significantly reduce the number of culture-positive ME specimens due to its failure to eradicate H. influenzae. Erythromycin significantly (P < 0.001) reduced S. pneumoniae colony counts but H. influenzae counts remained at levels similar to the control group regardless of the dose administered (from 2.5 to 50 mg/kg). Table 2 shows otoscopic results and presence of otorrhoea. All ears from the control group showed AOM with a high percentage of ears with otorrhoea while more than 90% of those treated with any dose of any antibiotic showed OME, the animals having a very low percentage

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A. Parra et al. / International Journal of Antimicrobial Agents 23 (2004) 25–31

Table 1 Outcome (day 2) of experimental otitis media caused by S. pneumoniae plus H. influenzae treated with amoxicillin/clavulanic acid (A/C) and erythromycin Group

Control A/C A/C A/C Erythromycin Erythromycin Erythromycin Erythromycin

Dose (mg/kg)

No. of ears

30 24 24 24 24 24 24 24

1.5c 2.5c 10c 2.5 10 20 50

No. of MEa culture-positive samples (%)

Bacterial counts log10 cfub mean (S.D.)

S. pneumoniae

H. influenzae

Total

S. pneumoniae

H. influenzae

28 2 0 0 0 0 0 0

29 1 1 0 22 21 24 22

30 2 1 0 22 21 24 22

3.10 (1.10) 0.69 (0.40)d <0.60d <0.60d <0.60d <0.60d <0.60d <0.60d

5.40 (1.18) <0.60d,e 0.60 (0.06)d,e <0.60d,e 5.03 (1.78) 5.04 (1.80) 5.17 (1.60) 4.95 (1.92)

(93.3) (8.3)d (0.0)d (0.0)d (0.0)d (0.0)d (0.0)d (0.0)d

(96.7) (4.2)d,e (4.2)d,e (0.0)d,e (91.7) (87.5) (100) (91.7)

(100) (8.3)d,e (4.2)d,e (0.0)d,e (91.7) (87.5) (100) (91.7)

a

Middle ear washing fluid. Values are the mean ± S.D. log10 cfu in 20 ␮l of washing fluid. Data for culture-negative samples were included in the calculation of means by assuming a value at the detection limit (0.60 log10 cfu). c Referred to amoxicillin. d Differences statistically significant vs. controls. e Differences statistically significant vs. treated groups with erythromycin. b

Table 2 Relationship between otoscopic examination, otorrhoea and relative body weight loss in animals with experimental otitis media caused by S. pneumoniae plus H. influenzaea Group

Control A/C A/C A/C Erythromycin Erythromycin Erythromycin Erythromycin a b c d e f

Dose (mg/kg)

1.5d 2.5d 10d 2.5 10 20 50

No. of ears

30 24 24 24 24 24 24 24

Otoscopic examination (%) AOMb

OMEc

30 2 0 0 2 0 1 0

0 22 24 24 22 24 23 24

(100) (8.3)e (0.0)e (0.0)e (8.3)e (0.0)e (4.2)e (0.0)e

(0.0) (91.7)e (100)e (100)e (91.7)e (100)e (95.8)e (100)e

Otorrhoea (%)

% Body weight loss (S.D.)

23 2 1 0 5 1 0 0

11.2 5.3 2.1 2.8 2.7 2.7 5.4 5.6

(76.7) (8.3)e (4.2)e (0.0)e (20.8)e (4.2)e (0.0)e (0.0)e

(3.8) (3.6)e (3.1)e,f (2.8)e,f (2.1)e,f (2.1)e,f (3.0)e (2.8)e

All parameters are referred at day 2 except otorrhoea (day 1). Acute otitis media. Otitis media with effusion. Referred to amoxicillin. Differences statistically significant vs. controls. Differences statistically significant vs. amoxicillin/clavulanic 1.5 mg/kg, erythromycin 20 and 50 mg/kg.

Table 3 Pharmacokinetic data of amoxicillin/clavulanic acid (A/C) and erythromycin Antibiotic

A/C A/C A/C A/C Erythromycin Erythromycin Erythromycin Erythromycin Erythromycin a b c

Dose (mg/kg)

1.5/0.3 1.5/0.3 + ACEc 50 2.5/0.5 10/2 2.5 10 20 50 50 + ACEc 50

Serum C15 (mg/l)b

2.58 ± 0.33/0.52 ± 0.05 – 5.49 ± 0.48/0.86 ± 0.16 23.92 ± 2.76/2.98 ± 1.39 0.81 ± 0.14 1.77 ± 0.43 4.87 ± 1.72 5.66 ± 0.74 –

MEFa Concentration (mg/l)

Penetration (%)

1.17/0.14 1.21/0.12 – – – – – 2.30 2.48

45.3/26.9 46.9/23.1 – – – – – 40.6 43.8

Middle ear fluid. Values are means ± S.D. for six animals measured 15 min after administration for each antibiotic and dose. Acetaminophen.

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Table 4 Pharmacodynamic data of amoxicillin/clavulanic acid (A/C) and erythromycin in serum and ME fluid in relation with the S. pneumoniae and H. influenzae susceptibility Antibiotic

Dose (mg/kg)

Serum C15 /MIC

1.5b

S. pneumoniae

H. influenzae

54 (11.2) – 72 (15.0) 114 (23.7)

54 (11.2) – 72 (15.0) 114 (23.7)

1.17 1.21 – –

1.17 1.21 – –

– – 30 (6.2) 48 (10.0) –

– – – 19.17 20.67

– – – 0.57 0.62

2.58 – 5.49 23.92

2.58 – 5.49 23.92

Erythromycin Erythromycin Erythromycin Erythromycin Erythromycin

2.5 10 20 50 50 + ACEc 50

6.75 14.75 40.58 47.17 –

0.20 0.44 1.22 1.41 –

c

S. pneumoniae

H. influenzae

1.5b + ACEc 50 2.5b 10b

b

MEa concentration/MIC

S. pneumoniae A/C A/C A/C A/C

a

Time > MIC (min) (% dose)

96 120 264 276 –

(20.0) (25.0) (55.0) (57.5)

H. influenzae

Middle ear fluid. Referred to amoxicillin. Acetaminophen.

of otorrhoea (0–20.8%). Non-significant lower numbers of polymorphonuclear cells were found in the ME of animals treated with antibiotics compared with untreated controls. All antibiotic-treated groups had lost less weight by day 2 compared with the control group (P < 0.001) (Table 2). 3.3. Pharmacokinetic and pharmacodynamic data Serum levels of acetaminophen after administration of 50 mg/kg were 39.6 ± 8.2, and 24.7 ± 4.4 ␮g/ml at 15 and 30 min, respectively. Table 3 shows serum and ME pharmacokinetic data. The ME antibiotic penetration (expressed as percentage in relation to serum concentration at 15 min) was similar for both antibiotics. The concomitant administration of acetaminophen did not influence the antibiotic concentration in ME fluid. Table 4 shows the pharmacodynamic analysis in relation to the organisms inoculated. The favourable therapeutic and bacteriological results achieved with amoxicillin/clavulanic acid were related to serum T > MIC higher than 11% of the dosing interval and ME inhibitory quotients ≥1.17 for both pathogens. Erythromycin changed the initial AOM pattern to OME by eradicating S. pneumoniae, with serum T > MIC equal or higher than 20% of the dosing interval. The failure of erythromycin in eradicating H. influenzae was due to serum T > MIC equal or lower than 10 and ME sub-MIC concentrations (inhibitory quotient ≤0.57).

4. Discussion Amoxicillin/clavulanic acid and erythromycin were chosen because both drugs are usually recommended for the empirical treatment of otitis media [9–12]. The strains tested were representative of the susceptibility patterns of the Spanish isolates: MIC values for S. pneumoniae were similar to MIC90 and MIC50 values of amoxicillin/clavulanic acid

and erythromycin, respectively, while MIC values for H. influenzae were similar to MIC50 values of both antibiotics [17,18]. From the clinical perspective, antibiotic treatment (regardless the antibiotic or the dose administered) changed the initial AOM pattern to a post-treatment OME pattern, significantly lowering the percentage of otorrhoea versus controls. This change occurred as a result of the bacteriological efficacy obtained by both drugs at any dose against S. pneumoniae, since as previously reported with ␤-lactams, efficacious antibiotic treatment of AOM is followed by a sterile OME [19]. This is also the natural history of appropriately treated AOM in children with approximately 90% of cases of post-treatment OME resolved within 3 months [20]. From the bacteriological perspective, amoxicillin/ clavulanic acid was able to eradicate both organisms in the mixed infection but the macrolide did not eradicate H. influenzae, even at high doses. It has been speculated that ␤-lactams, due to their mechanism of action, could induce greater inflammatory response than macrolides [21,22]. This inflammatory response could be responsible for the higher ME concentrations (achieving H. influenzae peri-MIC concentrations) that produces bacterial eradication. In the previously reported model [6], the increase in amoxicillin/clavulanic acid ME concentrations by the increased inflammation induced by S. pneumoniae allowed the ␤-lactam to eradicate H. influenzae even at low doses. In this AOM model, amoxicillin/clavulanic acid administered at 1.5 mg/kg (referred as amoxicillin) achieved ME concentrations higher than those obtained after the administration of doses three times higher (5 mg/kg) in an OME model (1.17 ␮g/ml versus 0.95 ␮g/ml with 45% versus 10% penetration in relation to serum concentrations) [7]. This was not the case for the macrolide, since the penetration of erythromycin into the ME in this AOM (mixed) model was not higher than the one achieved in an OME model caused by H. influenzae alone (40.6% versus 43.6%)

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(unpublished data). In this model the systemic translation of local (ME) therapeutic failure can be generally seen as loss of body weight, being higher in untreated animals versus erythromycin-treated animals and in the latter versus amoxicillin/clavulanic acid-treated animals (with the highest bacterial eradication rates). Considering both perspectives, in this study, two types of post-treatment OME can be distinguished: (a) an sterile post-inflammatory condition after the H. influenzae and S. pneumoniae eradication produced by amoxicillin/clavulanic acid administration; and (b) a non-sterile post-inflammatory condition with persistence of H. influenzae not eradicated by erythromycin sub-MIC concentrations in ME fluid. None of these conditions were modified by the concomitant administration of an analgesic drug with weak anti-inflammatory activity such as acetaminophen, although some pharmacokinetic interactions between antibiotics and non-steroidal anti-inflammatory drugs have been described in experimental models and exceptionally in humans [23–26]. The results of this study suggest that bacterial eradication in otitis is dependent on two facts: the antibiotic concentration in ME and the MIC of the infecting organisms. With respect to antibiotic concentrations in ME fluid, the penetration of the ␤-lactam is dependent on the inflammatory response that the infecting organism induces, and this is the critical factor for amoxicillin/clavulanic acid, necessitating an increase the dose in infections caused by H. influenzae that induces OME. With respect to the MIC for the infecting pathogen, in Spain, MIC90 of amoxicillin/clavulanic acid for both organisms are within the susceptibility range (≤2 mg/l) while for erythromycin values are ≥64 and 8 mg/l for S. pneumoniae and H. influenzae, respectively [17,18]. Therefore, although erythromycin has the advantage of a high penetration in ME regardless of the inflammatory situation, in Spain, MIC90 values of erythromycin compromise its therapeutic efficacy in otitis media; susceptibility and not antibiotic penetration being the critical factor for efficacy. For this reason it is important to know local patterns of resistance when choosing an antibiotic for empirical treatment of otitis media. Acetaminophen did not prevent the appearance of OME suggesting that no clear benefit, nor harm, may be expected from combining antibiotics with acetaminophen. Nonetheless, since acetaminophen may alleviate ear pain and decrease body temperature [12,27], its use, alone or combined with an antibiotic, may be advocated.

Acknowledgements This study was funded by Fondo de Investigaciones Sanitarias (FIS 99/111) and SmithKline Beecham S.A., Madrid, Spain. A.P. and M.G.-O received scholarships from the Fundación Conchita Rábago, Madrid, Spain. We thank A. Carcas (Clinical Pharmacology, Universidad Autónoma, Madrid, Spain) for determining serum levels of acetaminophen.

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