Table 1.
PAC1 to PAC10 genomic features and nucleotide similarity to P101A phage.
Fig 1.
PAC1 to PAC10 phage are able to circularise.
PCR was performed across a region incorporating the 11 bp overhang at the 3’ end of the genome. A band of approximately 735 bp was seen in all the phage and was only possible if the genomes were circular. Lanes 1 to 10 represent DNA from PAC phages and lane 11 is Propionibacterium freudenreichii prophage DNA as negative control.
Fig 2.
The coloured arrows indicate predicted ORFs and direction of translation. Modular structure is indicated in blocks of colour and the putative gene function is noted below.
Fig 3.
Alignment of genomes PAC1 to PAC10 using Mauve alignment program.
Mauve colour indicates regions of conserved sequence between all phage, ‘gaps’ indicate insertions in a given phage genome and the regions of colour indicate sequence similarity across some phage and not others.
Fig 4.
Phylogenetic tree of the 72 known sequenced P. acnes phages.
When bootstrap values <80 were collapsed, 53 distinct strains emerged, with the PAC phage effectively adding another ten strains to the 43 reported previously. Of these, nine highly homologous groups, with members differing by as little as 11–14 base pairs, emerged as distinct clonal strains (strains I-IX), as reported recently (33). Insert shows phylogenetic relationships between PAC1 to PAC10.
Fig 5.
DNA was extracted from the phage resistant subcultured cells derived from the regrowth area within plaques on P. acnes (ATCC 6919).
PCR across the 3’ phage genome overhang displayed a band of approximately 735 bp, potentially indicating the presence of circularised phage episomal DNA. Lanes 1–10 represent DNA extracted from resistant P. acnes (ATCC 6919) strains arising from exposure to PAC1-PAC10. Lane 11 represents control DNA extracted from Propionibacterium freudenreichii.
Fig 6.
Lytic capacity of the P. acnes phage cream formulation.
All PAC1 to PAC10 phage displayed similar behaviour when formulated in cetomacrogol cream aqueous and placed onto P. acnes lawns. Fig 6 shows results of experiments of cream formulated with PAC1 phage placed onto one of the P. acnes cutaneous strains isolated in this study. Legend: A. Cetomacrogol cream aqueous; B. Cetomacrogol cream aqueous with PBS; C. Phage cream, PAC1 at a concentration of 5.0x103 PFU per gram of cream D. Phage cream, PAC1 at a concentration of 2.5x108 PFU per gram of cream.
Fig 7.
Quantitative assessment of the phage lytic activity and stability following storage of the creams at various temperatures and light exposures: 0.05 g samples of the phage cream were taken at weekly intervals, mixed into PBS to a volume of 1 mL, vortexed and centrifuged.
An aliquot of a dilution series of the supernatant was then placed onto a lawn of P. acnes so as to ascertain the numbers of viable phage. Storage at 4°C in a light protected bottle resulted in optimal stability of the cream and efficacy of the phage activity. ● indicates storage at 4°C in a light protected bottle. ○ indicates storage at 20–25°C in a light protected bottle. ▼ indicates storage at 45°C in a light protected bottle. Δ indicates storage in constant light at 20–25°C.