Harnessing the diversity of Burkholderia spp. prophages for therapeutic potential

Abstract

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested prophages were tested for lytic activity against the same 32 isolates. Lytic phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes, and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that B. cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Keywords: Burkholderia; antibiotic resistance; phage therapy; prophage.

Figure 1.. Approach for prophage induction and testing.

To induce prophage release, stationary phase liquid culture of 32 Burkholderia spp. clinical isolates were individually inoculated into LB media containing mitomycin C. Cultures were grown overnight at 37°C. The next day, bacterial cells were pelleted and liquid lysates were filtered through a 0.22μm filter. Filtered lysates were then spotted onto soft agar lawns containing each of the same 32 Burkholderia spp. clinical isolates. Plates were examined for growth inhibition, then inhibitory lysates were retested to confirm phage activity.

Figure 2.. Phage activity against genetically diverse Burkholderia spp. clinical isolates.

Bacterial isolates are ordered according to their core genome phylogeny and are grouped by species. Grey boxes indicate isolates collected from the same patient. Infectivity is shown as the log₁₀ titer (PFU/mL) of each phage against each isolate. Bolded values indicate the Burkholderia spp. target isolate that each phage was isolated and propagated on. Asterisks mark the source Burkholderia spp. isolate for each phage. Green shading corresponds to phage activity titer, with darker shading indicating a higher titer. Empty cells indicate no phage activity.

Figure 3.. Electron micrograph of prophage-derived phage BCC02.

Transmission electron micrograph showing Burkholderia phage BCC02. Image was taken at 200,000X magnification. Icosahedral head, tail, and contractile tail sheath are visible for 6 virions.

Figure 4.. Prophage abundance and phage susceptibility of diverse Burkholderia spp. clinical isolates.

(a) Core genome phylogeny of 35 Burkholderia spp. clinical isolates showing the number of prophages and phage susceptibility of each isolate. For phage susceptibility, BCC02/BCC03/BCC04 and BCC05/BCC06 were each combined into a single count. Darker red shading corresponds with higher values. (b) Prophage abundance among B. multivorans and B. cenocepacia genomes. (c) Phage susceptibility among B. multivorans and B. cenocepacia isolates. P-values are from unpaired two-tailed t-tests. * P < 0.05; *** P < 0.001.

Figure 5.. Clusters of prophages encoded by Burkholderia spp. clinical isolate genomes.

Bacterial isolate names and prophage number are listed inside the nodes of each cluster, and lines connect prophages that share >90% sequence coverage and >90% sequence identity. Yellow nodes indicate prophages in B. multivorans isolate genomes and purple nodes indicate prophages in B. cenocepacia isolate genomes. Isolates from the same patient (two separate patients) are boxed.