Therapeutic effects of parenteral ß-lactam antibiotics on experimental otitis media caused by penicillin-resistant Streptococcus pneumoniae in guinea-pigs

Ritsuko Hori*, Harumi Araki, Minoru Yonezawa, Shinzaburo Minami and Yasuo Watanabe

Research Laboratories, Toyama Chemical Co. Ltd, 2-4-1 Shimookui, Toyama City, Toyama 930-8508, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Reference
 
The therapeutic effects of parenteral ß-lactam antibiotics were evaluated in experimental acute otitis media caused by penicillin-resistant Streptococcus pneumoniae (PRSP) in guinea-pigs. Cefotaxime, ceftriaxone and piperacillin significantly reduced viable cell counts of PRSP in the middle ear at a dose of 50 mg/kg bd for 3 days (P < 0.01 compared with control). The therapeutic effects of cefotaxime, ceftriaxone and piperacillin were superior to those of cefotiam and ceftazidime. These therapeutic effects reflected both in vitro activity and pharmacokinetic properties of the drugs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Reference
 
Streptococcus pneumoniae is a major cause of otitis media and is traditionally susceptible to penicillins and cephalosporins. Therefore, ß-lactams have been widely used in the clinical treatment of this disease.1–3 Since penicillin-intermediately resistant S. pneumoniae (PISP) was isolated in Australia in 1967,4 the emergence of PISP and penicillin-resistant S. pneumoniae (PRSP) has been reported in many countries and frequencies of PISP and PRSP have been increasing year by year.5–9 Furthermore, reports have indicated that isolates are commonly resistant not only to penicillins but also to cephalosporins, macrolides and tetracyclines, and others are resistant only to cephalosporins with an oxyimino group.10–12

High frequencies of both PISP and PRSP have been observed in juvenile and elderly patients. Therefore, it was felt necessary to study the optimum antibiotic and dosing route against experimental otitis media with PISP or PRSP; the comparative efficacy of several drugs was investigated in Mongolian gerbils and chinchilla.13–15 However, only one or two drugs were compared in these previous reports, and few studies have compared several drugs at the same time. In addition, few researchers have discussed the effect of in vitro activity and pharmacokinetics on the drug efficacy in models with otitis media at the same time.

We previously reported the dual requirements of an increased dosage of oral cephalosporin and prolonged treatment period to cure otitis media caused by PISP in guinea-pigs, and also showed that clinical efficacy reflected both the in vitro activity and pharmacokinetic properties of the antimicrobial.16 However, the appearance of PRSP has become important clinically. For example, Sato & Mimura reported that oral cephalosporins such as cefaclor were not effective and iv administration of antibiotics was required for treatment of otitis media with PRSP.17

In the present study, we evaluated efficacies of five parenteral ß-lactams with various degrees of antipneumococcal activity in experimental otitis media caused by PRSP in guinea-pigs. We also investigated the effects of in vitro activities and pharmacokinetics of the drugs on the efficacies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Reference
 
Bacterial isolate

The clinical isolate used in this study was penicillinresistant S. pneumoniae D-1051 (MIC of penicillin G, 1.56 mg/L).

Antibiotics

The antibiotics used in this study were cefotiam (Takeda Chemical Industries, Osaka, Japan), ceftazidime (Nippon Glaxo, Tokyo, Japan), cefotaxime (Hoechst Japan, Tokyo, Japan), ceftriaxone (Nippon Roche K.K., Tokyo, Japan) and piperacillin (Toyama Chemical Co., Tokyo, Japan).

Animals

Male Hartley guinea-pigs (bodyweight 250–300 g) were purchased from Japan SLC (Shizuoka, Japan). Animals were housed in regulation cages and were given free access to food and water.

In vitro studies

MICs were determined by the two-fold agar dilution method using Heart Infusion Agar (Eiken Chemical Co., Tokyo, Japan) supplemented with 5% defibrinized sheep blood. Bacteria stored in skim milk were spread on the blood agar and incubated at 37°C for 18 h. The bacteria grown on agar were suspended in Brain Heart Infusion Broth (Eiken Chemical Co., Tokyo, Japan), and then further incubated at 37°C for 6 h. The cells were inoculated on agar plates containing a two-fold dilution of antibiotics using a microplanter (Sakuma, Tokyo, Japan). The final inoculum was 104 cfu per spot. The MICs were determined after incubation at 37°C for 18 h.

Efficacy experiment

Four or five guinea-pigs were used for each dosage group. Animals were anaesthetized with diethyl ether, and inoculated bilaterally with 50 µL of the bacterial suspension (5.4 x 108 cfu/mL) in the tympanic cavity through the tympanic membrane. Treatment was initiated 1 day after infection and antimicrobial agent at 50 mg/kg was administered iv bd for 3 days. Control animals were infected in the same manner but did not receive any antimicrobial agent. At 24 h after the final administration of antimicrobials, the animals were anaesthetized with diethyl ether and killed by bleeding from the abdominal artery. The middle ear bullae (containing the tympanic cavity and auditory tube) were removed, opened and suspended in saline. The suspension was diluted in saline and cultured on blood agar. Colonies were counted after incubation at 37°C for 18 h.

Pharmacokinetic experiment

Animals with otitis media caused by S. pneumoniae D-1051 were given an iv dose of cefotiam, ceftazidime, cefotaxime, ceftriaxone or piperacillin at 100 mg/kg, 1 day after infection. At 5, 15, 30, 60 and 120 min after antimicrobial administration, a serum sample was taken and middle ear mucosa (MEM) was removed from the guinea-pigs. Each group comprised three animals. MEM was homogenized in 0.066 M phosphate buffer (pH 7.0) and then centrifuged at 5000g for 10 min. Serum and MEM concentrations of cefotiam, ceftazidime, cefotaxime, ceftriaxone and piperacillin were measured by a bioassay method using Klebsiella pneumoniae ATCC10031, Proteus mirabilis ATCC21100, Micrococcus luteus ATCC9341, Escherichia coli NIHJ JC-2 and Micrococcus luteus ATCC9341, respectively. The detection limits were as follows: cefotiam, cefotaxime and piperacillin, 0.39 mg/L; ceftazidime, 0.1 mg/L; ceftriaxone, 0.78 mg/L.

Statistical analysis

Data were analysed for statistical significance by use of Tukey's test. A P value of <0.05 was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Reference
 
In vitro data

The MICs of cefotaxime, ceftriaxone and piperacillin for S. pneumoniae D-1051 were 1.56 mg/L, and superior to those of cefotiam (6.25 mg/L) and ceftazidime (12.5 mg/L).

Efficacy experiment

Figure 1Go shows the viable cell counts in the middle ear after the administration of the antimicrobial at 50 mg/kg bd for 3 days to guinea-pigs with otitis media caused by S. pneumoniae D-1051. The bacterial cell count in the untreated group was 6.49 log10 cfu/middle ear. Bacterial cell counts in the groups treated with cefotiam and ceftazidime were 6.14 and 3.84 log10 cfu/middle ear, respectively, and these decreases were not statistically significant in comparison with the value for the untreated group. On the other hand, the values in the groups treated with cefotaxime, ceftriaxone and piperacillin were <3.34, <2.59 and <2.68 log10 cfu/middle ear, respectively. These decreases were statistically significant in comparison with the value for the untreated group (P < 0.01).



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Figure 1. Therapeutic effects of ß-lactam antibiotics on experimental otitis media in guinea-pigs caused by S. pneumoniae D-1051 (PRSP). •, bacterial numbers per ear; {circ}, mean of bacterial numbers in one group. * P < 0.01 versus control by Tukey's test.

 
Pharmacokinetic experiment

Figure 2Go shows serum and MEM concentrations and the TableGo shows the pharmacokinetic variables of the parenteral ß-lactams after administration of 100 mg/kg to guinea-pigs with otitis media caused by S. pneumoniae D-1051. The highest concentration (Cmax) and the area under the concentration–time curve from time zero to infinity (AUC0–{infty}) in MEM were in the order ceftazidime > piperacillin > cefotiam > ceftriaxone > cefotaxime and ceftazidime > ceftriaxone > piperacillin > cefotaxime >= cefotiam, respectively. In addition, the times above MIC in MEM were in the order cefotaxime > ceftriaxone > piperacillin > ceftazidime > cefotiam.



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Figure 2. Serum and middle ear concentration of ß-lactam antibiotics after iv injection of 100 mg/kg in guinea-pigs with acute otitis media caused by S. pneumoniae D-1051 (PRSP). {circ}, serum concentration; •, middle ear concentration. Parentheses indicate the tentative mean, in which the detectable limit was tentatively used for the calculation if the value was under the detectable limit.

 

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Table. Pharmacokinetic variables of ß-lactam antibiotics after iv injection of 100 mg/kg in guinea-pigs with acute otitis media caused by S. pneumoniae D-1051 (PRSP)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Reference
 
S. pneumoniae is a major pathogen of acute otitis media. Recently, the increase of PISP and PRSP has become a clinical problem. Until now, it has been reported that an increased dosage of penicillins is effective in the clinical treatment of otitis media caused by PISP and PRSP. However, the reports indicated that iv administration of ß-lactams was needed to treat such infections because oral administration was not completely effective.17–19 In the present study, we evaluated the efficacies of some parenteral ß-lactams on an experimental model of otitis media caused by PRSP and also investigated the effects of in vitro antipneumococcal activity and pharmacokinetic profiles of the antimicrobials on the therapeutic efficacies. The following antimicrobials were used: cefotiam, ceftazidime, cefotaxime, ceftriaxone and piperacillin; these were chosen because of their different in vitro antipneumococcal activities and pharmacokinetic profiles.

Previously, we used the guinea-pig model of otitis media by inoculation of penicillin-susceptible S. pneumoniae (PSSP) or PISP into the middle ear through the tympanic membrane to evaluate the efficacies of oral cephalosporins.16 This model with S. pneumoniae D-1051 (PRSP) could be also used to evaluate the efficacy on otitis media because 106–107 cells/ear persisted for at least 1 week (data not shown).

In the otitis media model, cefotaxime, ceftriaxone and piperacillin reduced the viable cell counts in the middle ear close to the detection limit, following iv doses of 50 mg/kg bd for 3 days. On the other hand, administration of cefotiam and ceftazidime did not lead to a significant decrease in the viable cell counts at the same dosage. All the drugs were expected to give concentrations over MICs in MEM after iv administration. However, cefotiam had only a short time above MIC, because of both its inferior antipneumococcal activity and its short t1/2, resulting in no therapeutic effect. Ceftazidime had a longer time above MIC than cefotiam because of its superior distribution to MEM despite having the lowest antipneumococcal activity of the five antimicrobials. This longer time above MIC seemed to contribute to the reduction in viable cell counts in some cases. Cefotaxime, ceftriaxone and piperacillin had a long time above the MIC owing to their low MICs, resulting in good efficacy. These results indicate that the therapeutic effects of parenteral ß-lactams on otitis media caused by PRSP reflect both the in vitro antipneumococcal activity and the distribution to MEM.

In conclusion, cefotaxime, ceftriaxone and piperacillin, on account of having a longer time above their MICs in the MEM, showed greater efficacy than cefotiam and ceftazidime against the otitis media model with PRSP.


    Acknowledgments
 
We would like to express our thanks to Ms Helen Lavin for reading our manuscripts.


    Notes
 
* Corresponding author. Tel: +81-76-431-8268; Fax: +81-76-431-8208. Back


    Reference
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Reference
 
1 . Bluestone, C. D., Stephenson, J. S. & Martin, L. M. (1992). Ten-year review of otitis media pathogens. Pediatric Infectious Disease Journal 11, S7–11.[ISI][Medline]

2 . Klein, J. O. (1994). Otitis media. Clinical Infectious Diseases 19, 823–33.[ISI][Medline]

3 . Pichichero, M. E. & Pichichero, C. L. (1995). Persistent acute otitis media: I. Causative pathogens. Pediatric Infectious Disease Journal 14, 178–83.[ISI][Medline]

4 . Hansman, D. & Bullen, M. M. (1967). A resistant pneumococcus. Lancet 2, 264–5.[ISI]

5 . Appelbaum, P. C., Bhamjee, A., Scragg, J. N., Hallett, A. F., Bowen, A. J. & Cooper, R. C. (1977). Streptococcus pneumoniae resistant to penicillin and chloramphenicol. Lancet 2, 995–7.[ISI][Medline]

6 . Fenoll, A., Bourgon, C. M., Munoz, R., Vicioso, D. & Casal, J. (1991). Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing systemic infections in Spain, 1979–1989. Reviews of Infectious Diseases 13, 56–60.[ISI][Medline]

7 . Appelbaum, P. C. (1992). Antimicrobial resistance in Streptococcus pneumoniae: an overview. Clinical Infectious Diseases 15, 77–83.[ISI][Medline]

8 . Geeslin, P., Buu-Hoi, A., Fremaux, A. & Acar, J. F. (1992). Antimicrobial resistance in Streptococcus pneumoniae: an epidemiological survey in France, 1970–1990. Clinical Infectious Diseases 15, 95–8.[ISI][Medline]

9 . Konno, M., Yoshida, S., Inoue, M., Shimizu, Y., Akizawa, K., Uehara, N. et al. (1994). An epidemiological study of penicillinresistant Streptococcus pneumoniae in Japan. Journal of the Japanese Association for Infectious Diseases 68, 1338–51.[Medline]

10 . Kitayama, R., Minami, S., Horii, T., Maehana, J., Yasuda, T., Watanabe, Y. et al. (1994). Sensitivity of Streptococcus pneumoniae isolated from clinical materials to oral antimicrobial agents. Chemotherapy (Tokyo) 42, 592–8.

11 . Yoshida, R., Kaku, M., Kohno, S., Ishida, K., Mizukane, R., Takemura, H. et al. (1995). Trends in antimicrobial resistance of Streptococcus pneumoniae in Japan. Antimicrobial Agents and Chemotherapy 39, 96–8.

12 . McDougal, L. K., Rasheed, J. K., Biddle, J. W. & Tenover, F. C. (1995). Identification of multiple clones of extended-spectrum cephalosporin-resistant Streptococcus pneumoniae isolates in the United States. Antimicrobial Agents and Chemotherapy 39, 2282–8.[Abstract]

13 . Barry, B., Muffat-Joly, M., Gehanno, P. & Pocidalo, J.-J. (1993). Effect of increased dosages of amoxicillin in treatment of experimental middle ear otitis due to penicillin-resistant Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 37, 1599– 603.[Abstract]

14 . Barry, B., Muffat-Joly, M., Bauchet, J., Faurisson, F., Gehannno, P., Pocidalo, J.-J. et al. (1996). Efficacy of single dose ceftriaxone in experimental otitis media induced by penicillin- and cephalosporin-resistant Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 1977–82.[Abstract]

15 . Alper, C. M., Doyle, W. J., Seroky, J. T. & Bluestone, C. D. (1996). Efficacy of clarithromycin treatment of acute otitis media caused by infection with penicillin-susceptible, -intermediate, and -resistant Streptococcus pneumoniae in the chinchilla. Antimicrobial Agents and Chemotherapy 40, 1889–92.[Abstract]

16 . Hori, R., Yamamoto, S., Kitayama, R., Sanzen, T., Araki, H., Minami, S. et al. (1996). Therapeutic effect of oral cephem antibiotics on experimental otitis media caused by Streptococcus pneumoniae in guinea pigs. Japanese Journal of Chemotherapy 44, 746–52.

17 . Sato, K. & Mimura, M. (1988). Penicillin insensitive and resistant Streptococcus pneumoniae infection in children. Journal of the Japanese Association for Infectious Disease 63, 189–93.

18 . Friedland, I. R., Med, M. & McCracken, G. H., Jr (1994). Management of infections caused by antibiotic-resistant Streptococcus pneumoniae. New England Journal of Medicine 331, 377–82.[Free Full Text]

19 . Barnett, E. D. & Klein, J. O. (1995). The problem of resistant bacteria for the management of acute otitis media. Pediatric Clinics of North America 42, 509–17.[ISI][Medline]

Received 3 March 1999; returned 25 June 1999; revised 30 July 1999; accepted 2 November 1999





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