Efficacy of rifampicin combination therapy against MRSA prosthetic vascular graft infections in a rat model

Abstract

Staphylococcus aureus is a major cause of prosthetic vascular graft or endograft infections (VGEIs) and the optimal choice of antibiotics is unclear. We investigated various antibiotic choices as either monotherapy or combination therapy with rifampicin against MRSA in vitro and in vivo. Fosfomycin, daptomycin and vancomycin alone or in combination with rifampicin was used against MRSA USA300 FPR3757. Each antibiotic was tested for synergism or antagonism with rifampicin in vitro, and all antibiotic regimens were tested against actively growing bacteria in media and non-growing bacteria in buffer, both as planktonic cells and in biofilms. A rat model of VGEI was used to quantify the therapeutic efficacy of antibiotics in vivo by measuring bacterial load on grafts and in spleen, liver and kidneys. In vitro, rifampicin combinations did not reveal any synergism or antagonism in relation to growth inhibition. However, quantification of bactericidal activity revealed a strong antagonistic effect, both on biofilms and planktonic cells. This effect was only observed when treating active bacteria, as all antibiotics had little or no effect on inactive cells. Only daptomycin showed some biocidal activity against inactive cells. In vivo evaluation of therapy against VGEI contrasted the in vitro results. Rifampicin significantly increased the efficacy of both daptomycin and vancomycin. The combination of daptomycin and rifampicin was by far the most effective, curing 8 of 13 infected animals. Our study demonstrates that daptomycin in combination with rifampicin shows promising potential against VGEI caused by MRSA. Furthermore, we show how in vitro evaluation of antibiotic combinations in laboratory media does not predict their therapeutic effect against VGEI in vivo, presumably due to a difference in the metabolic state of the bacteria.

Keywords: Biofilm; Infections; MRSA; Persister cells; Prosthesis; Rat model; Rifampicin; Staphylococcus aureus; VGEI; Vascular graft or endograft infections.

Fig. 1

Active and inactive populations of MRSA (planktonic and biofilms) treated with antibiotic monotherapy or in combination with rifampicin. 24 h treatment of planktonic cells (A, B, C, D, E, F) or biofilms (G, H), treated with daptomycin, fosfomycin, or vancomycin as mono-therapy or in combination with rifampicin (10 mg/l). For planktonic cultures, the change in CFU during the incubation is displayed. For biofilms, the absolute values of surviving bacteria is shown. Symbols with an “ × ” denote that this sample had no visible survival and the value is under the limit of detection (LOD). Statistical tests compare treated samples with untreated controls, and mono-vs. combination therapy. n = 3–4 (one-way ANOVA, ns: p > 0.05, *p = 0.0217–0.0435, **p = 0.003–0.0079, ***p = 0.0005, ****p < 0.0001) (Mann-Whitney test, ^$p = 0.0286).

Fig. 2

Bacterial load on vascular grafts following 7 days of antibiotic treatment. Each data point represents the median log 10 CFU/ml of triplicates from one graft. The asterisks represent pairwise comparison of each monotherapy with combination therapy with rifampicin (Fisher’s exact test ns: p > 0.05, **p ≤ 0.005). NaCl = Control, DAP = Daptomycin, RIF = Rifampicin, FOS = Fosfomycin, VAN = Vancomycin.

Fig. 3

Bacterial load in organs following 7 days of treatment. Each data point represents median log 10 CFU/ml with interquartile range from triplicates of homogenate of (A) liver (B) left and right kidney (C) spleen. The asterisks represent pairwise comparison of each monotherapy with combination therapy with rifampicin (Mann-Whitney test *p ≤ 0.05). NaCl=Control, DAP = Daptomycin, RIF = Rifampicin, FOS = Fosfomycin, VAN = Vancomycin.

Fig. 4

Cumulated cure rate of graft and organs. Each column represents percent of animals with no growth of bacteria from graft and organs (liver, kidney and spleen). NaCl = Control, DAP = Daptomycin, RIF = Rifampicin, FOS = Fosfomycin, VAN = Vancomycin. Asterisks represent pairwise comparison of monotherapy with combination therapy with rifampicin (Fisher’s exact test *p ≤ 0.05).