Effect of erythromycin treatment delay on therapeutic outcome of experimental acute otitis media caused by Streptococcus pneumoniae

Carmina Martínez-Marín1, Lorena Huelves1, Gema del Prado1, José I. García-Cía1, Carlos Cenjor2, Carmen Ponte1, Juan J. Granizo3 and Francisco Soriano1,*

1 Unit for Research in Medical Microbiology and Antimicrobial Chemotherapy, 2 Otolaryngology, and 3 Epidemiology, Fundación Jiménez Díaz—UTE, Avenida de Reyes Católicos 2, 28040 Madrid, Spain


* Corresponding author. Tel: +34-91-544-73-87; Fax: +34-91-549-47-64; E-mail: fsoriano{at}fjd.es

Received 8 April 2005; returned 21 June 2005; revised 28 June 2005; accepted 15 August 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: To evaluate the effect of delayed administration of erythromycin in the course of acute otitis media caused by an erythromycin-susceptible Streptococcus pneumoniae strain in the gerbil model.

Methods: The bacterium was inoculated by transbullar challenge in the middle ear (ME) and antibiotic treatment at different doses was administered at various times thereafter.

Results: When 2.5 mg/kg of erythromycin was administered as a single dose 2, 5, 18 or 21 h post-inoculation (pi) the bacterial eradication rate was 55, 40, 0 and 0%, respectively. A higher dose (5 mg/kg) administered also as a single dose 2, 5, 18 and 21 h pi achieved bacterial eradication rates of 62.5, 43.8, 0 and 0%, respectively. Using a very high dose (50 mg/kg) repeated three times at 3 h intervals (total dose 150 mg/kg) and starting the treatment 21 h pi only achieved bacterial eradication in 25% of cases. The concentration of erythromycin achieved in the ME 90 min after administration of 5 mg/kg 5 or 21 h pi was very similar (0.74 and 0.79 mg/L) but the ME half-life was longer (98.2 min) with the early administration as compared with the delayed form (47.5 min), which could partially explain the different results. Further experiments showed that the failures observed with the delayed administration were not related to the time elapsed from antibiotic administration to ME sampling or selection of antibiotic-resistant mutants.

Conclusion: Bacteriological and clinical efficacies were significantly diminished if antibiotic administration was delayed.

Keywords: otitis , pneumococcal , therapy delay , gerbils


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Acute otitis media (AOM) is one of the most frequent illnesses of childhood, the predominant bacterium involved being Streptococcus pneumoniae.1 As many cases of AOM will resolve spontaneously, it has been suggested that treatment with antimicrobials should be delayed awaiting a possible clinical improvement.2,3 However, the effect of delayed antibiotic treatment has not been studied. We have previously shown that the efficacy of amoxicillin in the treatment of experimental AOM caused by S. pneumoniae decreased with delayed antibiotic treatment.4

The aim of this study was to evaluate the effect of delayed administration of erythromycin in the course of an experimental AOM caused by an erythromycin-susceptible S. pneumoniae strain.


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

A strain of S. pneumoniae (serotype 23F) (erythromycin MIC 0.12 mg/L) was used.

Antibiotic

Erythromycin (Sigma Chemicals Co., St Louis, MO, USA) and erythromycin lactobionate (Pantomicina; Abbott Laboratories, S.A., Madrid, Spain) were used for in vitro and in vivo studies.

In vitro studies

Erythromycin MICs were determined in 32 isolates collected from treated animals without complete bacterial eradication and in four from controls.

Animals

Mongolian gerbils were purchased from the Centre d'Élevage R. Janvier (Le Genest, St-Isle, France) and managed as previously described.5 The study was performed following current regulations regarding the use of laboratory animals in the European Community and was approved by our Ethics Committee.

Experimental otitis

Animals were inoculated bilaterally into the middle ear (ME) bulla with ~106 cfu of S. pneumoniae in 20 µL following published methods.4,5

Treatment regimens and efficacy studies

Erythromycin was given subcutaneously as a single dose (2.5 and 5 mg/kg) administered at 2, 5, 18 and 21 h post-inoculation (pi). Experiments using three infusions of 5 and 50 mg/kg (total dose of 15 and 150 mg/kg, respectively) at different times (2, 5 and 8, or 21, 24 and 27 h pi) were also carried out.

Animals in the control groups received pyrogen-free sterile distilled water. Groups of 8–10 animals per treatment and control groups were included. Efficacy was evaluated at 48 h pi for single and repeated doses. Other groups of animals were evaluated at 29 h pi after repeated doses administered at 2, 5 and 8 h pi.

Treated and control animals were evaluated for otorrhoea, weight and otoscopic appearance. Otoscopic features were evaluated as previously defined4 and ME washings were obtained from both ears at 29 and 48 h pi and plated for colony counting.

Pharmacokinetic studies

Serum levels of erythromycin were determined in healthy animals after a single subcutaneous injection of 5 mg/kg of the antibiotic. Groups of six animals per dose had blood samples obtained at 15, 30, 60 and 120 min after drug administration. Antibiotic concentrations in ME fluid (MEF) without washing were determined in groups of 10 animals bilaterally inoculated with the organism. A single 5 mg/kg dose of erythromycin was subcutaneously administered 5 or 21 h pi and MEF samples were obtained 60, 90 and 120 min thereafter. Aliquots of MEF samples were pooled for determination of antibiotic levels.

Antibiotic concentrations were determined by microbiological assay. Assay variability for individual samples was <10%.

Statistical analysis

For qualitative variables the Fisher's exact test was used. The Mann–Whitney test was used to compare the reduction of log10 cfu and loss of body weight. To compare the otoscopic appearance the {chi}2 test for trends was used.


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

The erythromycin MIC values for all isolates from control and treated animals remained at 0.12 mg/L.

Experimental otitis and therapeutic efficacy

After inoculation of 6.22 ± 0.36 log10 cfu (mean ± SD of all experiments) bilateral AOM and otorrhoea was observed in all untreated animals that showed lethargy and significant weight loss. Table 1 presents the bacteriological and clinical efficacy of erythromycin administered as a single dose at different intervals. A significant reduction in otorrhoea only occurred in those animals treated with any dose 2 h after bacterial challenge as compared with untreated controls. A significant reduction in the AOM was observed only in those animals treated with 5 mg/kg of erythromycin administered 2 h pi as compared with any other groups. Animals treated with any dose between 2 and 5 h pi showed significant reduction in the number of culture-positive ears as compared with any other group within the same dose and untreated control. A significant reduction in the number of organisms recovered from the ME, as compared with untreated controls, was observed after administration of 2.5 mg/kg 2 h pi, and 5 mg/kg administered 2 and 5 h pi. The reduction in body weight was significantly lower as compared with untreated controls in those animals receiving any dose of erythromycin but only when administered 2 and 5 h pi. Increasing the erythromycin dose from 2.5 to 5 mg/kg only significantly improved the otoscopic appearance when the antibiotic was administered 2 h pi.


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Table 1. Bacteriological and clinical efficaciesa of erythromycin administered in two doses in a single shot at different intervals after bacterial inoculation

 
Table 2 presents the bacteriological and clinical results after administering three shots of 5 and 50 mg/kg (total dose of 15 and 150 mg/kg, respectively) starting 2 and 21 h pi for the first dose and 21 h pi for the latter. The most effective regimen for all evaluated parameters was the administration of 5 mg/kg of erythromycin 2 h pi, the delayed treatment being less efficacious even after using a very high dose. The only parameter that improved with the highest dose, as compared with the 5 mg/kg both administered 21 h pi, was the reduction in bacterial colony counts.


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Table 2. Bacteriological and clinical efficaciesa of erythromycin administered in doses of 5 and 50 mg/kg in three shots (total dose of 15 and 150 mg/kg, respectively) at early (2, 5 and 8 h) or delayed (21, 24 and 27 h) starting times after bacterial inoculation

 
When the results obtained after the administration of 5 mg/kg of erythromycin as a single dose are compared with those obtained with the dose of 5 mg/kg in three doses (Tables 1 and 2) the antibiotic efficacy increased only when administered 2 h pi (P < 0.05).

Because the antibiotic efficacy was determined 48 h pi, while the treatment was started 2 and 21 h pi, the results could have been influenced by the difference in the time elapsed from antibiotic administration to ME sampling. To rule out this possibility a group of animals received 5 mg/kg in three doses starting the treatment 2 h pi and making the bacteriological evaluation 27 h after treatment initiation (29 h pi). At this latter period of evaluation the antibiotic had already achieved complete bacterial eradication while all untreated animals were ME culture-positive with a mean (±SD) bacterial count of 4.29 ± 0.75 log10 cfu in 20 µL.

Pharmacokinetic and pharmacodynamic data

The serum concentration of erythromycin 15 min after administration of 5 mg/kg was 1.64 ± 0.21 mg/L with a half-life of 31 min and an AUC of 98.6 mg·min/L. This dose administered 5 or 21 h pi produced, 90 min thereafter, a MEF concentration of 0.74 or 0.79 mg/L. The half-lives and AUC/MIC ratios were 98.2 or 47.5 min, and 1236 or 732, which correlated with bacterial eradication of 43.8% and 0%, respectively.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most experiments were carried out using 2.5 or 5 mg/kg of erythromycin because such doses achieve serum and ME concentrations in gerbils similar to those obtained in children receiving the standard dose for the treatment of AOM.69 The best therapeutic regimen after once-only administration of the antibiotic was related to both time from challenge to treatment and erythromycin dose. When the antibiotic was administered 18 or 21 h pi neither a dose of 2.5 nor 5 mg/kg improved bacteriological and clinical results as compared with untreated controls.

The poorer efficacy observed with delayed administration of the antibiotic was not due to selection of subpopulations with higher MIC values. Furthermore, such results cannot be explained by the bacterial burden present at time of treatment initiation because the antibiotic efficacy was greater at 5 h than at 21 h, the bacterial burden being lower at later times in untreated controls.4 The delayed administration of three doses of either 5 or 50 mg/kg improves efficacy only slightly. The most significant pharmacokinetic/pharmacodynamic differences were the lower half-life and AUC values obtained when the antibiotic was delayed compared with early administration. Such differences could be due to the presence of otorrhoea at the time of delayed administration.

The better pharmacodynamic properties when 5 mg/kg of the antibiotic was administered early as a single dose compared with delayed administration could be responsible, in part, for the higher efficacy (bacterial eradication of 43.8 versus 0%). However when a higher dose was administered (5 mg/kg in three doses; total 15 mg/kg) in delayed form the eradication rate was not significantly improved (6.2%). Even administration of a very high dose (150 mg/kg) in three doses in delayed form increased the efficacy only modestly (25% eradication).

The clinical relevance of our results is unclear. The use of antibiotics in AOM is controversial but our results suggest that even if they are given in some cases, they should be administered as early as possible, as proposed by Hendley.10


    Acknowledgements
 
This study was funded by the Fondo de Investigaciones Sanitarias (PI030208) Ministerio de Sanidad y Consumo, and presented in part at the 15th European Congress of Clinical Microbiology and Infectious Diseases in Copenhagen, Denmark, 2–5 April 2005. CM-M and LH were aided by scholarships from the Fundación Conchita Rábago, and GdP by a scholarship from the FIS, Madrid, Spain.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 Bluestone CD, Stephenson JS, Martín, L.M. Ten-year review of otitis media pathogens. Pediatr Infect Dis J 1992; 11 Suppl 8: 7–11.

2 Little P, Gould C, Williamson I et al. Pragmatic randomised controlled trial of two prescribing strategies for childhood acute otitis media. Br Med J 2001; 322: 336–42.[Abstract/Free Full Text]

3 Froom J, Culpepper L, Jacobs M et al. Antimicrobials for acute otitis media? A review from the International Primary Care Network. Br Med J 1997; 315: 98–102.[Free Full Text]

4 Parra A, Ponte C, Cenjor C et al. Effect of antibiotic treatment delay on the therapeutic outcome of experimental acute otitis media caused by Streptococcus pneumoniae strains with different susceptibilities to amoxicillin. Antimicrob Agents Chemother 2004; 48: 860–6.[Abstract/Free Full Text]

5 Soriano F, Parra A, Cenjor C et al. Role of Streptococcus pneumoniae and Haemophilus influenzae in the development of acute otitis media with effusion in a gerbil model. J Infect Dis 2000; 181: 646–52.[CrossRef][ISI][Medline]

6 Craig WA, Andes D. Pharmacokinetics and pharmacodynamics of antibiotics in otitis media. Pediatr Infect Dis J 1996; 15: 255–9.[CrossRef][ISI][Medline]

7 Nelson CT, Mason EO JR, Kaplan SL. Activity of oral antibiotics in middle ear and sinus infections caused by penicillin-resistant Streptococcus pneumoniae: implications for treatment. Pediatr Infect Dis J 1994; 13: 585–9.[ISI][Medline]

8 Ginsburg CM, McCracken GH Jr, Nelson JD. Pharmacology of oral antibiotics used for treatment of otitis media and tonsillopharyngitis in infants and children. Ann Otol Rhinol Laryngol 1981; 90 Suppl: 37–43.[ISI]

9 Ponte C, Parra A, Cenjor C et al. Does acetaminophen interfere in the antibiotic treatment of acute otitis media caused by a penicillin- resistant pneumococcus strain? A gerbil model. Pediatr Res 2003; 54: 913–18.[Abstract/Free Full Text]

10 Hendley JO. Otitis media. N Engl J Med 2002; 347: 1169–74.[Free Full Text]





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