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Introduction
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.35 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.68 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.911 Combination therapy is an alternative form
of therapy available for the treatment of PRSP infections.
Several investigators have reported synergy between lactams and aminoglycosides against PRSP in killing curves
and chequerboard studies.1113 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 effective-
367
1999 The British Society for Antimicrobial Chemotherapy
K. Tateda et al.
to 1995, with no two representing the same outbreak. All
strains were frozen at 280C in skimmed milk until used.
Animals
Five-week-old male CBA/J mice (body weight range,
1620 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.
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 1214 days
after infection.
Statistical analysis
The x2 test was used to compare the survival rates, and P
values of <0.05 were considered statistically significant.
Results
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 subinhibitory concentrations of gentamicin dramatically
reduced the MICs of penicillin G (Figure 1). When 8 mg/L
of gentamicin (0.25 3 MIC) was used, 14 strains (60.9%)
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
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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 3 MIC (circles), MIC (triangles) and 4 3 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.
Figure 3. Effects of gentamicin combined with penicillin G on survival of mice infected with strain 741. Penicillin G at 40 mg/kg (m),
160 mg/kg (M) or saline (V) 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.
369
K. Tateda et al.
Figure 4. Effects of gentamicin combined with imipenem on survival rates of mice infected with strain 741. Imipenem at 2.5 mg/kg
(m), 10 mg/kg (M) or saline (V) 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
The major finding of the present study was the high efficacy
of gentamicin combined with -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 3 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
-lactams (cefotaxime and imipenem) and gentamicin
when 0.25 3 MIC (8 mg/L) of the latter and 0.5 3 MICs
of -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 3
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
Acknowledgements
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.
370
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