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Macrophage interactions with biofilm-producing Staphylococcus epidermidis

Abstract number: P1802

Spiliopoulou A., Kolonitsiou F., Krevvata M., Spiliopoulou I., Dimitracopoulos G., Anastassiou E.D.

To compare immune responses of biofilm-producing S. epidermidis existing either in planktonic or in biofilm phase in terms of IL-12p40 production, bacterial adhesion on macrophages and resistance to phagocytosis process.

ATCC35983 and two clinical biofilm-positive, ica-positive S. epidermidis stains were used. Planktonic phase bacteria were obtained after 2 h incubation in Tryptic Soy Broth (TSB); whereas biofilm phase bacteria after 24 h incubation of bacterial suspensions and homogenisation of bacterial cells embedded in biofilm attached to the bottom of the tube. Monocytes were separated from human peripheral blood mononuclear cells by plastic adherence and differentiated in macrophages. Phagocytosis experiments were performed by co-incubation of cells with bacteria at 1:10 ratio for 20, 60, 90 and 120 min; removal of extracellular bacteria and further incubation in antibiotic supplemented medium. Intracellular bacteria were counted by serial dilutions on blood agar plates. For measuring bacterial adhesion to macrophages, a modified ELISA was used where macrophages cultured on 96-well plates were incubated with biotinylated bacterial suspensions. For cytokine determination, macrophages cultured on 24-well plates were stimulated with bacteria at 1:10 and 1:25 ratio for 45 min, extracellular bacteria were removed and macrophages were further incubated for 12 h. IL-12p40 concentrations were measured in supernatants by commercial ELISA kit.

Biofilm phase bacteria showed increased adhesion on macrophages compared to planktonic phase bacteria (7.6±0.02×106 for biofilm phase bacteria vs 3±0.06×106 CFU/macrophage monolayer for planktonic phase bacteria). Biofilm phase bacteria were internalised in greater proportion (10-fold) than planktonic phase bacteria and showed higher degree of intracellular survival (Table 1). Planktonic phase bacteria elicited higher amounts of IL-12p40 than biofilm phase bacteria (planktonic phase 645±95 and 1,029±48 pg/ml vs biofilm phase 231±72 and 396±26 pg/ml for 1:10 and 1:25 ratio respectively) (p < 0.05).

Biofilm phase bacteria are efficiently internalised but seem to be more resistant to killing by macrophages than their planktonic counterparts. Internalisation of biofilm phase bacteria does not promote IL-12 production and macrophages can not manage an efficient Th1 response. These findings could contribute to insight mechanism of resistance of biofilm-associated infections to immune system responses.

Phase% intracellular survival
 Compared to initial inoculumCompared to 2 h intracellular survival
 20 min60 min90 min120 min4 h12 h24 h48 h3 days5 days
Biofilm393127156640.2241.50.80.05
Planktonic3.642.31.30.411.42.051.80.40.110

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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