Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models

Abstract

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.

Fig 1. Treatment of mature biofilms formed in 96-well MTPs.

Number of CFU per biofilm after treating mature biofilms of B. cenocepacia LMG 16656 for 24 h with 100 μL PS (untreated control, CTRL), 1, 10, 100 μM econazole (ECO) or 1, 10, 100 μM miconazole (MICO), in combination with 512 μg/mL tobramycin (TOB), 512 and 2560 μg/mL gentamicin (GEN), 320 and 3200 μg/mL meropenem (MEM), or 250 μg/mL ciprofloxacin (CIP). Asterisks indicate a significantly different number in log CFU/BF compared to treatment with the respective antibiotic alone (P value < 0.05). (Data shown are average; n ≥ 3; error bars indicate SEM).

Fig 2. Infection of 3D lung epithelial cells.

Left image showing an uninfected control where the microcarrier bead scaffolds are covered with A549 human lung epithelial cells, right image shows the 3D lung epithelial cells 17 h p.i., green dots (white arrows) indicate intracellular growth of B. cenocepacia K56-2, while the large green structure (blue arrow) suggests that biofilm-like structures are formed. Magnification is 300x. Scale bar is 400 μm.

Fig 3. Effect of treatment in the 3D model.

Effect of treatment with 10 μM econazole (ECO), 10 μM miconazole (MICO), 156 μg/mL tobramycin (TOB) or the combination on inhibition of biofilm formation of B. cenocepacia K56-2 with a plastic surface (left, number of biofilm cells per well) or the effect on the treatment on the number of bacterial cells associated with human 3D lung epithelial cells (right, number of host associated cells per well). Asterisks indicate a significantly different number in CFU/well compared to treatment with tobramycin (P value < 0.05). (Data shown are average; n ≥ 3; error bars indicate SEM).

Fig 4. G. mellonella survival assay.

Percentage survival of G. mellonella in uninfected control groups (NI) and infected groups (I) 24, 48, and 72 h after administration of the different treatments (tobramycin (TOB, 512 μg/ml), econazole (ECO, 50 μM), miconazole (MICO, 50 μM) or the combinations) and/or infection. (Data shown are average, n = 6, error bars indicate SEM).

Fig 5. Effect of the combination therapy in a mouse lung infection model.

Colony counts in lung (a), spleen (b), and liver (c) in mice treated with: (i) vehicle, (ii) 22.4 mg/kg bw miconazole (MICO), (iii) 5.6 mg/kg bw miconazole, (iv) 120 mg/kg bw tobramycin (TOB) and (v) 120 mg/kg bw tobramycin combined with 5.6 mg/kg bw miconazole (TOB + MICO). Error bars indicate SEM. Three independent repeats of mice treated with vehicle (n ≤ 3), two independent repeats of mice treated with MICO 22.4 mg/kg (n ≤ 3), one independent repeat of mice treated with MICO 5.6 mg/kg (n = 2), one independent repeat of mice treated with TOB 120 mg/kg (n = 6) and one independent repeat of mice treated with TOB + MICO (n = 6).