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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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
 
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.

 
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.


    Acknowledgments
 
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.


    Notes
 
* Tel: +45-89-49-33-33; Fax: +45-89-49-28-20 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Yancey, R. J., Sanchez, M. S. & Ford, C. W. (1991). Activity of antibiotics against Staphylococcus aureus within polymorphonuclear neutrophils. European Journal of Clinical Microbiology and Infectious Diseases 10, 107-13.[ISI][Medline]

2 . Turnidge, J. & Grayson, M. L. (1993). Optimum treatment of staphylococcal infections. Drugs 45, 353-66.[ISI][Medline]

3 . Forsgren, A. & Bellahsène, A. (1985). Antibiotic accumulation in human polymorphonuclear leucocytes and lymphocytes. Scandinavian Journal of Infectious Diseases, 44, Suppl., 16-23.

4 . Nielsen, S. L. & Black, F. T. (1998). The effect of dicloxacillin and fusidic acid on the extracellular and intracellular killing of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 42, 221-6.[Abstract]

5 . van den Broek, P. J. (1989). Antimicrobial drugs, microorganisms, and phagocytes. Reviews of Infectious Diseases 11, 213-45.[ISI][Medline]

6 . Lambert, H. P. & O'Grady, F. W. (1992). Rifamycins. In Antibiotic and Chemotherapy , 6th edn (Lambert, H. P. & O'Grady, F. W., Eds), pp.263-267. Churchill ivingstone, Edinburgh.

7 . Höger, P. H., Vosbeck, K., Seger, R. & Hitzig, W. H. (1985). Uptake, intracellular activity, and influence of rifampin on normal function of polymorphonuclear leukocytes. Antimicrobial Agents and Chemotherapy 28, 667-74.[ISI][Medline]

8 . Sanchez, M. S., Ford, C. W. & Yancey, R. J. (1988). Evaluation of antibiotic effectiveness against Staphylococcus aureus surviving within the bovine mammary gland macrophage. Journal of Antimicrobial Chemotherapy 21, 773-86.[Abstract]

9 . Jacobs, R. F. & Wilson, C. B. (1983). Intracellular penetration and antimicrobial activity of antibiotics. Journal of Antimicrobial Chemotherapy 12, Suppl. C , 13-20.[ISI][Medline]

10 . Røder, B. L., Forsgren, A. & Gutschik, E. (1991). The effect of antistaphylococcal agents used alone and in combinations on the survival of Staphylococcus aureus ingested by human polymorphonuclear leukocytes. APMIS 99, 521-29.[ISI][Medline]

Received 29 April 1998; returned 29 June 1998; revised 8 December 1998; accepted 7 October 1998





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