Efficacy of ß-lactam antibiotics combined with gentamicin against penicillin-resistant pneumococcal pneumonia in CBA/J mice

Kazuhiro Tateda*, Tetsuya Matsumoto, Shuichi Miyazaki and Keizo Yamaguchi

Department of Microbiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohta-ku, Tokyo 143, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We examined the efficacy of gentamicin combined with ß-lactam antibiotics against penicillin-resistant Streptococcus pneumoniae (PRSP) in a noncompromised mouse model of pneumonia. In the presence of 8 mg/L (0.25 x MIC) and 16 mg/L (0.5 x MIC) of gentamicin, MICs of penicillin G against 23 strains of PRSP decreased from 1–4 mg/L to 0.03 mg/L in 14 (61%) and 23 strains (100%), respectively. A short-time killing study using strain 741 showed that 8 mg/L of gentamicin (0.25 x MIC) increased the killing activity of penicillin G, cefotaxime and imipenem (at 0.25, 1 and 4 x MIC) during a 6 h incubation period. Survival studies showed that the combined treatment of penicillin-G (160 mg/kg) and gentamicin (10 mg/kg), which commenced 2 days after infection (twice a day for 5 days), provided complete protection, while no animal survived when either antibiotic was used alone. A significant improvement in mortality was observed when a small dose of imipenem (2.5 and 10 mg/kg) was used with gentamicin. Our results suggest that gentamicin, when combined with ß-lactam antibiotics, especially imipenem, may be potentially useful against PRSP pneumonia in noncompromised individuals.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is still the leading cause of bacterial pneumonia, which is associated with significant morbidity and mortality. 1,2 Epidemiological surveys in the past several years have demonstrated a dramatic increase in the prevalence of penicillin-resistant S. pneumoniae (PRSP) throughout the world. 3,4,5 Moreover, the appearance of multiresistant strains, which also exhibit resistance to extended-spectrum cephalosporins, such as cefotaxime and ceftriaxone, limits the therapeutic choice in clinical practice. 6,7,8 Experience with clinical treatment failures in infections caused by PRSP have prompted investigators to re-examine the in-vitro and in-vivo therapeutic efficacies of antibiotics. 9,10,11 Combination therapy is an alternative form of therapy available for the treatment of PRSP infections. Several investigators have reported synergy between ß- lactams and aminoglycosides against PRSP in killing curves and chequerboard studies. 11,12,13 In addition to these in-vitro studies, Darras-Joly et al. 14 have clearly demonstrated the usefulness of amoxycillin combined with gentamicin in a leukopenic mouse model of pneumonia. Although this combination has generally been accepted as a potential alternative therapy, some points regarding the effectiveness of this strategy still need confirming. These include the exact type of ß-lactam that forms the best partner with gentamicin in vivo and whether the combined use of gentamicin is also effective in immunocompetent individuals, since the majority of PRSP pneumonia occurs in otherwise healthy individuals as community-acquired pneumonia. 15,16

We have recently described a noncompromised CBA/J mouse model of PRSP pneumonia. 17,18 This model resembles human PRSP community-acquired pneumonia, particularly with regard to progression of the disease and pathological findings. In the present study, we investigated the efficacy of gentamicin combined with several ß-lactam antibiotics, such as penicillin G, cefotaxime and imipenem, using this newly developed PRSP pneumonia model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S. pneumoniae strains

Twenty-three strains of S. pneumoniae isolated at Toho University School of Medicine were used in the present study, including strain 741, reported previously. 18 These strains were isolated from different patients between 1993 to 1995, with no two representing the same outbreak. All strains were frozen at -80°C in skimmed milk until used.

Animals

Five-week-old male CBA/J mice (body weight range, 16–20 g) were purchased from Charles River Japan, Kanagawa, Japan. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Toho University School of Medicine.

Antimicrobial agents

Penicillin G, cefotaxime, imipenem and gentamicin were obtained from Meiji Seika (Tokyo, Japan), Hoechst Japan (Tokyo, Japan), Banyu Pharmaceutical Co. (Tokyo, Japan) and Schering-Plough Co. (Osaka, Japan), respectively. Imipenem was mixed with cilastatin (Banyu) at a ratio of 1:1, and was used in an in-vivo pneumonia model.

Antimicrobial susceptibility test

The MICs of penicillin G in the presence or absence of gentamicin were determined by a broth dilution method using Mueller- Hinton broth (Difco, Detroit, MI, USA) supplemented with 5% lysed horse blood. Microtitre plates, containing 5.0 x 10 4 cfu/well were incubated at 35°C for 18 h. The MIC was defined as the lowest concentration of antimicrobial agent that prevented visible growth of S. pneumoniae. In preliminary experiments, MICs of penicillin G and gentamicin for 23 strains were examined. These results showed that MIC of penicillin G ranged between 1 and 4 mg/L while the MIC of gentamicin for all strains was 32 mg/L. MICs of penicillin G, cefotaxime and imipenem for strain 741 were 1, 0.5 and 0.25 mg/L, respectively.

In-vitro short-time killing study

The in-vitro bactericidal effect of ß-lactams (penicillin, cefotaxime and imipenem) was examined in the presence of gentamicin. Strain 741 was inoculated at a final concentration of 10 7 cfu/mL into Mueller–Hinton broth supplemented with 5% lysed horse blood, in which a ß-lactam antibiotic with or without gentamicin was added. The suspensions were incubated at 35°C, and then a count of the number of bacteria was performed after 1.5, 3 and 6 h by plating serial ten-fold dilutions of the samples on to 5% blood agar. Data were expressed as the average of two experiments.

Pneumonia model

The noncompromised mouse model of penicillin-resistant pneumococcal pneumonia was used as reported previously. 18 For this purpose, strain 741 was inoculated into Mueller- Hinton broth supplemented with 5% lysed horse blood and incubated at 35°C until the culture appeared turbid to the naked eye. This exponential-growth culture was suspended in 0.9% saline to the desired concentration (confirmed by plating serial ten-fold dilutions on to 5% blood agar). Mice were anaesthetized lightly by intramuscular injection of a mixture of 60 mg/kg ketamine (Sankyo Pharmaceutical, Tokyo, Japan) and 10 mg/kg xylazine (Bayer Japan, Tokyo, Japan), after which each mouse was challenged intranasally with approximately 10 5 logarithmic-phase organisms.

Survival studies

To investigate the effects of gentamicin combined with penicillin G or imipenem on survival, the indicated doses of antibiotics were administered subcutaneously to a group of ten animals twice a day for 5 days, commencing 2 days after infection. Survival rates were recorded daily for 12–14 days after infection.

Statistical analysis

The {chi}2 test was used to compare the survival rates, and P values of <=0.05 were considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of gentamicin combination on MIC of penicillin G

In the absence of gentamicin, the MICs of penicillin G for the 23 strains were 1, 2 and 4 mg/L for six, 16 and one strains, respectively. Simultaneous presence of sub-inhibitory concentrations of gentamicin dramatically reduced the MICs of penicillin G (Figure 1). When 8 mg/L of gentamicin (0.25 x MIC) was used, 14 strains (60.9%) became highly sensitive to penicillin G. Moreover, the addition of 16 mg/L of gentamicin (0.5 x MIC) decreased MICs of penicillin G for all strains tested to <=0.03 mg/L. These results indicated that a combination of sub-inhibitory concentrations of gentamicin generally sensitize clinical isolates of PRSP to penicillin G in a gentamicin concentration-dependent manner.



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Figure 1. Effects of gentamicin on MICs of penicillin G against PRSP. MICs of penicillin G for 23 strains of PRSP were examined in the presence or absence of various concentrations of gentamicin.

 
Effects of gentamicin combination on short-time killing of ß-lactams

Short-time killing (1.5, 3 and 6 h) by ß-lactam antibiotics was examined in strain 741 in the presence or absence of gentamicin (Figure 2). Without gentamicin, the best killing activity was noted with imipenem at all tested concentrations, followed by cefotaxime and penicillin G. In the presence of 8 mg/L of gentamicin (0.25 x MIC), these activities were generally enhanced, although the reduction of bacterial numbers by gentamicin alone was <1 log during the 6 h observation period. For example, in the presence of 0.25 x MIC of imipenem at 3 h, a gentamicin combination reduced the bacterial count from 6.7 to 3.3 log cfu/mL.



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Figure 2. Effects of gentamicin combination on short-time killing of penicillin G, cefotaxime and imipenem against PRSP strain 741. Short-time killing activities of penicillin G (a), cefotaxime (b) and imipenem (c) in 0.25 x MIC (circles), MIC (triangles) and 4 x MIC (squares) were examined in the absence (open symbols) or presence (solid symbols) of 8 mg/L of gentamicin. Horizontal dotted line represents the detection limit of bacterial counts.

 
Effects of gentamicin combination on survival (Figures 3 and 4)

Untreated control mice died between 6 and 10 days after infection. Penicillin G (160 mg/kg) alone or gentamicin (10 mg/kg) alone did not improve the survival rate at 10 days after infection although gentamicin-treated mice demonstrated slightly higher survival rates between 7 and 9 days. In contrast, a dramatic improvement of survival was observed when mice were treated simultaneously with penicillin G (160 mg/kg) and gentamicin (100% survival at the end of observation) A significant decrease in mortality was observed using a considerably smaller doses of imipenem (2.5 and 10 mg/kg), relative to penicillin G, when combined with gentamicin.



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Figure 3. Effects of gentamicin combined with penicillin G on survival of mice infected with strain 741. Penicillin G at 40 mg/kg ({triangleup}), 160 mg/kg ({square}) or saline ({circ}) was subcutaneously administered without (a) or with (b) 10 mg/kg of gentamicin from 2 days after infection twice a day for 5 days. *P < 0.05, compared with the corresponding control group.

 


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Figure 4. Effects of gentamicin combined with imipenem on survival rates of mice infected with strain 741. Imipenem at 2.5 mg/kg ({triangleup}), 10 mg/kg ({square}) or saline ({circ}) was subcutaneously administered without (a) or with (b) 10 mg/kg of gentamicin from 2 days after infection twice a day for 5 days, * P < 0.05, compared with the corresponding control group.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The major finding of the present study was the high efficacy of gentamicin combined with ß-lactam antibiotics against PRSP pneumonia in noncompromised mice. Our data were consistent with previous results in neutropenic mouse model, 14 and suggest the clinical usefulness of such combination therapy in community-acquired PRSP pneumonia.

The use of gentamicin concentrations of 1/16 of the MIC (2 mg/L) had little or no effect on MICs of penicillin G while the use of a higher dose of gentamicin (0.25 x MIC, 8 mg/L) dramatically sensitized PRSP to penicillin G in a gentamicin concentration-dependent manner. In this regard, Schlegel et al., 13 have reported synergy between ß-lactams (cefotaxime and imipenem) and gentamicin when 0.25 x MIC (8 mg/L) of the latter and 0.5 x MICs of ß-lactams were used. In contrast, using a lower concentration of gentamicin (1 mg/L), Gross et al. 19 were unable to demonstrate a significant enhancement of the killing activity of penicillin and cefotaxime. Our data, together with those of previous reports suggest that 8 mg/L (0.25 x MICs) of gentamicin may be the critical concentration necessary to express its synergic activity in vitro.

In the pneumonia model of leukopenic mice, a combination of gentamicin 8 mg/kg with amoxycillin 100 mg/kg yielded almost complete protection and survival of all animals, which occurred at a maximum gentamicin serum concentration of 12 mg/L. 14 The results of our pulmonary clearance and survival studies indicated the effectiveness of 10 mg/kg dose of gentamicin, although serum gentamicin concentrations were not determined. Pharmacokinetic studies of gentamicin in humans, conducted by Chung and co-workers, 20 reported peak serum concentrations of gentamicin of 5.76 and 11.0 mg/L following intramuscular administration of 1.0 and 2.5 mg/kg, respectively.

Of the combinations examined in the present study, the combination of imipenem and gentamicin exhibited potent bactericidal activity in the lungs, which was associated with higher survival rates than other combinations. In our previous study using the CBA/J mice model, we reported that in a single antibiotic regimen, imipenem was the most active among antibiotics tested in clearing penicillin-resistant pneumococci from the lungs. 18 Our results are also consistent with other previous reports showing excellent bactericidal activity of imipenem plus gentamicin against PRSP in an in-vitro killing study 12 and in a rabbit model of penicillin-resistant S. sanguis endocarditis. 21 These data suggest that imipenem may be one of the most active antimicrobials when used in combination with gentamicin for the treatment of PRSP infection.

Although there are pharmacokinetic differences between species, our mouse survival studies employed the usual twice-a-day human dosage regimen. Whether this is the most appropriate regimen, or whether different results might be obtained with approaches such as frequent dosing or constant infusion that address specific features of mouse pharmacokinetics, certainly requires further study. Although caution must be exercised in applying the results of the present animal studies to the management of patients, we believe that the combination of ß-lactams and gentamicin should be considered in those patients in whom infections caused by strains showing high-level penicillin resistance is suspected, or in systemic infections in critically-ill patients.


    Acknowledgments
 
We thank Professor Shogo Kuwahara for his critical reading of the manuscript and helpful suggestions. We also thank Dr F. G. Issa for his expert editorial assistance.


    Notes
 
* Corresponding author. Tel:+81-3-3762-4151; Fax:+81-3-5493-5415; E-mail: kazu{at}med.tobo-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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11 . Friedland, I. R., Paris, M., Shelton, S. & McCracken, G. H. (1994). Time- kill studies of antibiotic combinations against penicillin-resistant and -susceptible Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 34, 231–7.[Abstract]

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18 . Tateda, K., Takashima, K., Miyazaki, H., Matsumoto, T., Hatori, T. & Yamaguchi, K. (1996). Noncompromised penicillin-resistant pneumococcal pneumonia CBA/J mouse model and comparative efficacies of antibiotics in this model. Antimicrobial Agents and Chemotherapy 40, 1520–5.[Abstract]

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Received 11 May 1998; returned 20 June 1998; revised 28 July 1998; accepted 17 October 1998





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