Phage-antibiotic synergy reduces Burkholderia cenocepacia population

Abstract

Background: Burkholderia cenocepacia is an opportunistic pathogen that can cause acute and chronic infections in patients with weakened immune systems and in patients with cystic fibrosis. B. cenocepacia is resistant to many antibiotics making treatment challenging. Consequently, there is a critical need for alternative strategies to treat B. cenocepacia infections such as using bacteriophages and/or bacteriophages with subinhibitory doses of antibiotic called phage-antibiotic synergy.

Results: We isolated a bacteriophage, KP1, from raw sewage that infects B. cenocepacia. Its morphological characteristics indicate it belongs in the family Siphoviridae, it has a 52 Kb ds DNA genome, and it has a narrow host range. We determined it rescued infections in Lemna minor (duckweed) and moderately reduced bacterial populations in our artificial sputum medium model.

Conclusion: These results suggest that KP1 phage alone in the duckweed model or in combination with antibiotics in the ASMDM model improves the efficacy of reducing B. cenocepacia populations.

Keywords: ASMDM model; Bacteriophage; Burkholderia; Phage-antibiotic therapy; cenocepacia.

Fig. 1

Plaque morphology of KP1 phage at 37°C. Plaques were visualized with a Zeiss Stemi 2000-C stereo microscope at 16X magnification. Scale bar represents 1 mm

Fig. 2

Transmission electron micrograph of phosphotungstic acid-stained KP1 phage at 120,000X magnification. Scale bar represents 100 nm. Arrow is pointing to tail fibers. Images were collected by The Ohio State University Campus Microscopy and Imaging Faci

Fig. 3

A one-step growth curve of KP1. The phage was adsorbed to log phase B. cenocepacia K56-2 cells at low MOI (0.073). Virion production was measured from samples taken at 10-minute intervals for 90 min to quantify intracellular (adsorbed) and free virion (unadsorbed) PFU. Unadsorbed samples are represented by squares. Adsorbed samples are represented by circles. The latent period is at 50 min. The eclipse period occurred from time 0 until 25 min when the first virion was detected in the unadsorbed samples. This is a representative of two independent experiments

Fig. 4

Bacteriophages rescued B. cenocepacia K56-2 infected duckweed plants. One plant per well containing SHS medium was treated with bacteria (20 µl of 100xLD₅₀) at 30°C for four hours to establish an infection. KP-1 bacteriophages (20 µl at MOI 1 per well) were added to the wells and incubated at 30°C. Plant survival was examined after 96 h. Each experimental group had 14–18 plants and each control had eight plants per experiment. Mean ± standard error is from three independent experiments. Letters show statistically pairwise differences (ANOVA, P < .05; post-hoc Tukey Kramer HSD)

Fig. 5

Effect of PAS on B. cenocepacia K56-2 growth in ASMDM over time. Bacterial cells suspended in 60% ASMDM were treated with PAS, phage, or trimethoprim at two doses. A) Treatment with high dose of trimethoprim (0.88 mg/well). N = 4. B) Treatment with low dose of trimethoprim (0.0088 mg/well). N = 3. B. cenocepacia K56-2 no treatment (circle), PAS treatment (diamond), trimethoprim (square), KP1 phage (triangle). MOI = 17. Values are mean ± standard deviation. Statistical analysis was performed with MANOVA, P < .05. The PAS treatments are significantly different from the untreated K56-2 and K56-2 treated with phage or trimethoprim

Fig. 6

Effect of ASMDM, trimethoprim, and B. cenocepacia on KP1 viability. Phage viability was measured in the wells that contained phage. At 0 and every 24 h for 72 h, a plaque assay was performed to determine PFU/ml. A) PAS treatment with high dose of trimethoprim (0.88 mg/well). N = 4. B) Treatment with low dose of trimethoprim (0.0088 mg/well). N = 3. Values are mean ± standard deviation. MOI = 17. PAS treatment (circle), KP1 phage alone (square). Statistical analysis was performed with MANOVA, P < .05. There were no significant differences

Fig. 7

B. cenocepacia pellicle formation when grown in ASMDM for 72 h at 37°C, 5% CO₂. A) Untreated K56-2, B) KP1 phage treatment, C) Low dose trimethoprim, D) High dose trimethoprim, E) PAS with low dose trimethoprim, and F) PAS with high dose trimethoprim. Asterisks in well C denote holes in the pellicle. Images collected with the assistance of Chris Faur, Denison University

Fig. 8

Colonial morphology of untreated B. cenocepacia (A) and treated with PAS with low dose trimethoprim (B). Images collected using a Zeiss Stemi 2000-C stereo microscope at 16X magnification