Extracellular and intracellular killing in neutrophil granulocytes of Staphylococcus aureus with rifampicin in combination with dicloxacillin or fusidic acid
S. L. Nielsen and
F. T. Black*
Department of Medicine and Infectious Diseases, Marselisborg Hospital, Aarhus University
Hospital, P. P. Ørums Gade 11, DK-8000 Aarhus C, Denmark
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Abstract
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The effect of rifampicin in combination with dicloxacillin or fusidic acid on the
extracellular and intracellular killing of Staphylococcus aureus in human neutrophil
granulocytes in the presence of serum was studied. At the extracellular level rifampicin
significantly reduced the bactericidal activity of dicloxacillin, but had an indifferent effect on the
activity of fusidic acid. The combination of rifampicin with dicloxacillin or fusidic acid led to
intracellular killing no different from that produced by rifampicin alone. However, owing to the
high intracellular activity of rifampicin, the intracellular killing by the drug combinations was
greater than that by dicloxacillin or fusidic acid alone.
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Introduction
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Survival of Staphylococcus aureus within phagocytes may result in recurrent infections
and failure of antibiotic treatment if the drugs penetrate cells poorly.
1 For treatment of S. aureus infections, a
ß-lactamase-stable penicillin is often used, sometimes in combination with fusidic acid.2 The ß-lactam antibiotics in general have poor
intracellular penetration, whereas fusidic acid penetrates well and accumulates in granulocytes.
3 However, we have recently shown that the degree of
killing of S. aureus within granulocytes by the combination of dicloxacillin and fusidic
acid was less than the killing by dicloxacillin alone.
4 As an alternative treatment of severe S. aureus
infections, rifampicin in combination with a ß-lactam antibiotic or with fusidic acid has been
recommended.2 As there is a high risk of emerging
resistance during therapy, rifampicin should never be used alone.
2 Rifampicin has high activity against intracellular
susceptible bacteria.5 We studied the effect of rifampicin in
combination with dicloxacillin or fusidic acid on the intracellular killing of S. aureus in
human neutrophil granulocytes, and the relationship between this and extracellular killing.
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Materials and methods
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Reagents
Rifampicin was from Ciba-Geigy (Basel, Switzerland), dicloxacillin from Bristol-Myers Squibb
(Sermoneta Latina, Italy) and sodium fusidate from Leo (Ballerup, Denmark). RPMI 1640
medium without L-glutamine was from Gibco-BRL, Life Technologies (Paisley,
UK) and Ficoll-Paque from Pharmacia (Uppsala, Sweden). Human serum samples obtained from
healthy volunteers were pooled and stored in aliquots at -70°C.
Bacteria
Three clinical isolates (strains A, B and D) of S. aureus were obtained from the
Departments of Clinical Microbiology at Aarhus University Hospital and Rigshospitalet,
Denmark. Testing by disc diffusion at the Statens Seruminstitut (Copenhagen, Denmark) showed
that the three strains were susceptible to rifampicin, methicillin and fusidic acid.
Human neutrophil granulocytes
Blood was obtained from healthy adult volunteers and the neutrophil granulocytes were isolated
by the Hypaque-Ficoll technique.4
MIC determination
The MIC was determined in RPMI with 40% serum (RPMI-40% serum)
using a standard macrodilution method.
4 The MIC was defined as the lowest concentration of drug
that inhibited visible growth of S. aureus.
Assay for extracellular killing
The assay for extracellular killing of S. aureus in RPMI-40% serum with
antibiotics
has previously been described.
4
Assay for intracellular killing
The effect of antibiotics on the intracellular killing of S. aureus in granulocytes was
measured independently of phagocytosis in RPMI-40% serum as previously described.
4 The intracellular survival index (SI
t), i.e. the percentage of viable intracellular bacteria at time t, was
calculated using the formula:
The intraexperimental coefficient of variation of the survival index was 5.1%.
Statistical analysis
Data on extracellular killing were compared using Student's unpaired t-test,
whereas data on intracellular killing were compared using the Wilcoxon signed ranks test for
paired data.
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Results and discussion
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In order to mimic the clinical situation, the extracellular and intracellular killing of S. aureus was measured in the presence of serum and fixed antibiotic concentrations were used that
were comparable to therapeutic serum concentrations in patients.
4,6 The MICs in
RPMI-40% serum of dicloxacillin, fusidic acid and rifampicin respectively, were
1, 128 and 0.25 mg/L for strain A, 2, 128 and 0.125 mg/L for strain B, and 2, 1024 and 0.125
mg/L for strain D. The high MICs are explained by the presence of serum and the high protein
binding of the three antibiotics.4,6
Data on extracellular killing are shown in the Table. The bactericidal
effect of dicloxacillin was
significantly (P< 0.0003) reduced in all three strains when dicloxacillin was
combined with rifampicin. When rifampicin and fusidic acid were combined the effect was
indifferent, i.e. the extracellular killing by the combination was no different from that by
rifampicin alone.
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Table. Extracellular killing of S. aureus (mean (S.D.) log
cfu/mL) in RPMI- 40% serum after 3 h incubation with antibiotics alone or in combination
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The data on intracellular killing are shown in the Figure. Rifampicin and
dicloxacillin both
caused a significant (P < 0.05) increase in the intracellular killing of all three strains
compared with the controls without antibiotics. Fusidic acid only caused a significant (P
< .05) increase in the killing of strains B and D. For each strain the increase in the
intracellular killing with rifampicin was significantly (P < .05) higher than that of
dicloxacillin or fusidic acid alone. The high intracellular activity of rifampicin is in accordance
with previous studies on the killing of S. aureus in granulocytes.
5 Rifampicin accumulates rapidly within granulocytes,
probably by passive diffusion, as it is highly lipid-soluble.
3,7 High intracellular
concentrations of rifampicin seem to play a major role in its intracellular activity.
7 Because the activity of rifampicin is greatly enhanced at
low pH, it has been suggested that this activity is potentiated inside phagolysosomes.
8 The enhanced intracellular killing with rifampicin
probably results from a direct antibacterial effect.
9

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Figure. Intracellular killing of three strains of S.
aureus with antibiotics. (a) Rifampicin and dicloxacillin alone or in combination. (b)
Rifampicin and fusidic acid alone or in combination. Control is without antibiotics. The
concentrations tested were rifampicin 4 mg/L, dicloxacillin 8 mg/L and fusidic acid 64 mg/L.
The survival index after 3 h is shown. For each strain the data are the mean and S.E.M. of killing in neutrophil granulocytes from six or seven individuals.
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When rifampicin was combined with dicloxacillin or fusidic acid, the intracellular killing of S.
aureus by the drug combinations was the same as that by rifampicin alone (Figure). However, because of the effect of rifampicin, the combinations significantly
(P < .05) increased the intracellular killing compared with that of dicloxacillin or
fusidic acid alone. Thus the antagonism at the extracellular level between dicloxacillin and
rifampicin was not found at the intracellular level. In contrast, we previously found that fusidic
acid inhibited the effect of dicloxacillin at both the extracellular and intracellular levels.4 Studies on rifampicin in combination with a penicillin and
their effect on the intracellular killing of S. aureus in granulocytes have not previously
been published. The combined effect of rifampicin and fusidic acid on intracellular killing was
studied by Røder et al., 10 whose findings
agree with ours.
In conclusion, we found no synergy in the intracellular killing of S. aureus when
rifampicin was combined with dicloxacillin or fusidic acid. However, compared with the effect
of dicloxacillin or fusidic acid alone, the drug combinations showed significantly higher
intracellular killing, comparable to that of rifampicin alone. Since rifampicin should never be
used alone for treatment of S. aureus infections, our results support the use of either
dicloxacillin or fusidic acid in combination with rifampicin for the treatment of infections in
patients where intracellular survival of S. aureus is a problem, e.g. in patients with
chronic granulomatous disease.
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Acknowledgments
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We thank Else Madsen for excellent technical assistance. This work was supported by the SSAC
Foundation for Research and the Research Foundation, University of Aarhus, Denmark.
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Notes
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* Tel: +45-89-49-33-33; Fax: +45-89-49-28-20 
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Received 29 April 1998;
returned 29 June 1998; revised 8 December 1998;
accepted 7 October 1998