The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Abstract

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

Keywords: Burkholderia cepacia lineage IIIA; Peduovirinae; Temperate phage.

Fig. 1

Transmission electron micrograph of AP3. The sample was negatively stained with 2 % uranyl acetate and viewed at 80,000-fold magnification. The size bar is 100 nm

Fig. 2

Genome map of Burkholderia phage AP3. Structural protein genes (yellow), lysis cassette (green), DNA metabolism and replication (red), host interaction protein genes (blue), and hypothetical protein genes (gray). Both terminators and promoters are also noted on the map

Fig. 3

Pairwise comparison using TBLASTX of the AP3 and six selected BCC P2-like phages. Lines linking genomes represent high pairwise similarity between phages. Putative phage specific promoter sites (green lines), rho-independent terminator (red lines), and percentage of similarity between similar regions (shade scale on the bottom of the figure)

Fig. 4

The organization of the lysis cassette of Enterobacteria P2 and selected BCC P2-like viruses

Fig. 5

Antibacterial activity of AP3 phage (MOI 10) in the treatment of B. cenocepacia 7780-infected Galleria larvae. Statistical analysis was calculated for pairwise comparisons between infected larvae and phage-treated infected larvae using Mantel–Cox test (denoted P values <0.05).

Fig. 6

The scheme of AP3 phage integration site