Vascular Graft Impregnation with Antibiotics: The Influence of High Concentrations of Rifampin, Vancomycin, Daptomycin, and Bacteriophage Endolysin HY-133 on Viability of Vascular Cells

Abstract

BACKGROUND Rifampin-soaked synthetic prosthetic grafts have been widely used for prevention or treatment of vascular graft infections (VGIs). This in vitro study investigated the effect of the antibiotics daptomycin and vancomycin and the new recombinant bacteriophage endolysin HY-133 on vascular cells, as potential alternatives compared to rifampin. MATERIAL AND METHODS Primary human ECs, vascular smooth muscle cells (vSMC), and fibroblasts were cultivated in 96-well plates and incubated with rifampin, daptomycin, vancomycin, and endolysin HY-133 for 24 h. Subsequently, after washing, cell viability was determined by measuring mitochondrial ATP concentration. Antibiotics were used in their corresponding minimum and maximum serum concentrations, in decimal multiples and in maximum soaking concentration. The experiments were performed in triplicate. RESULTS The 10-fold max serum concentrations of rifampin, daptomycin, and vancomycin did not influence viability of EC and vSMC (100 µg/ml, p>0.170). Higher concentrations of rifampin (>1 mg/ml) significantly (p<0.001) reduced cell viability of all cell types. For the other antibiotics, high concentrations (close to maximum soaking concentration) were most cytotoxic for EC and vSMC and fibroblasts (p<0.001). Endolysin did not display any cytotoxicity towards vascular cells. CONCLUSIONS Results of this in vitro study show the high cytotoxicity of rifampin against vascular cells, and may re-initiate the discussion about the benefit of prophylactic pre-soaking in high concentrations of rifampin. Further studies are necessary to determine the influence of rifampin on the restoration of vessel functionality versus its prophylactic effect against VGIs. Future use of recombinant phage endolysins for alternative prophylactic strategies needs further investigations.

Figure 1

Influence of rifampin on vascular cells: EC (endothelial cells), vSMC (vascular smooth muscle cells), and fibroblasts. Cell viability of ECs and fibroblasts started to decrease at max serum concentrations of rifampin, while for SMC it occurred at 10-fold higher rifampin values. No cell survival of SMCs and fibroblasts was detected with 1000 μg/ml rifampin; the threshold was 10-fold higher for ECs. MIC₉₀ – minimum inhibitory concentration, Min – minimum serum concentration, Max – maximum serum concentration. For each concentration and cell type, at least n= 4 wells were chosen. The experiment was repeated 3 times. Mean intra-assay coefficient of variability (CV) was 2.00±0.89.

Figure 2

Influence of vancomycin on vascular cells. Cell viability of ECs, fibroblasts, and SMCs was not affected up to a vancomycin concentration of 100 μg/ml. Compared to the negative control and to the other cell types, ECs displayed higher viability in the presence of vancomycin. We found no survival of the 3 cell types at 100 000 μg/ml vancomycin. MIC₉₀ – minimum inhibitory concentration, Min – minimum serum concentration, Max – maximum serum concentration. For each concentration and cell type, at least n=4 wells were chosen. The experiment was repeated 3 times. Mean intra-assay coefficient of variability (CV) was 1.31±0.76.

Figure 3

Influence of daptomycin on vascular cells. Cell viability of ECs already decreased with daptomycin concentration at MIC₉₀. Viability of fibroblast and SMC was not affected up to 100 μg/ml daptomycin and decreased with higher concentrations. No survival of the 3 cell types could be detected at 100 000 μg/ml daptomycin. MIC₉₀ – minimum inhibitory concentration, Min – minimum serum concentration, Max – maximum serum concentration. For each concentration and cell type, at least n=4 wells were chosen. The experiment was repeated 3 times. Mean intra-assay coefficient of variability (CV) was 2.01±0.65.

Figure 4

Influence of endolysin on vascular cells. Cell viability of all 3 cell types was not affected up to 100×MIC₉₀ (50 μg/ml) endolysin. MIC₉₀ – minimum inhibitory concentration. For each concentration and cell type, at least n=4 wells were chosen. The experiment was repeated 3 times. Mean intra-assay coefficient of variability (CV) was 1.79±0.51.