541 Modern Skincare
investigated for its capacity to similarly diminish C. acnes biofilms. The biofilm impact
assessments followed standard protocol detailed under ASTM Standard E2799. As shown
in Figure 4, the triple cocktail of phages leveraged a dose-dependent decrease in C. acnes
biofilms 24 hours post-incubation. Cumulatively, the findings shown in Figures 3 and 4
validate the strong lytic activity of the constructed C. acnes triple phage cocktail against the
cognate bacteria at both planktonic and biofilm levels.
Figure 3. Phage cocktail consisting of phages targeted to C. acnes diminishes the bacterial concentration.
LEFT PANEL: qualitative bacteria reduction assay, where the ability to visualize lined paper set in the
background is used to assess diminished C. acnes levels, where (A) growth media alone, (B) C. acnes culture
post-exponential growth, (C) culture shown in (B) after 24 h incubation with the phage cocktail, and (D)
shows (A) and (C) side-by-side for comparison of relative translucency. RIGHT PANEL: quantitative bacteria
reduction assay, where the relative turbidity of C. acnes cultures pre- and post-24 h incubation with the phage
cocktail is quantified by spectrophotometry at 600 nm.
Figure 4. Phage cocktail consisting of phages targeted to C. acnes diminishes C. acnes biofilms. Following
ASTM Standard E2799, C. acnes biofilms were prepared and dilutions of the C. acnes targeted phage cocktail
were incubated with the biofilms for 24 h. The biofilms were rinsed in PBS, bacteria were sonicated into
suspension, and incubated in LB growth media for 48 h. Colony counts reflect the surviving viable bacteria
contained within the residual biofilm. These values were compared to untreated negative control biofilms.

542 JOURNAL OF COSMETIC SCIENCE
TRIPLE PHAGE COCKTAIL WAS NOT TOXIC AND REDUCED C. ACNES AND INTERLEUKIN-8 (IL-8)
IN A THREE-DIMENSIONAL MODEL OF BLEMISHED SKIN
Shortly after confirming the efficacy of the phage cocktail against C. acnes cultures and
biofilms, the three phages were applied to monolayers of normal human keratinocytes,
normal human dermal fibroblasts, and the HaCaT keratinocyte-derived cell line to
confirm their presumed non-cytotoxicity to skin cells as prokaryotic viruses should not
be able to infect eukaryotic cells. All of the concentrations applied to the cell monolayers
exhibited no adverse impact on cell viability as assessed by a resazurin viability assay
(data not shown). While this was an affirming result, a better evaluation of both the
efficacy and the non-toxicity of the phage cocktail would be on actual skin tissue. To cover
both objectives, a three-dimensional reconstituted human epidermal model of blemished
skin was employed, where the blemished skin tissue with elevated levels of C. acnes was
constructed. Topical application of a dilution series of the triple phage cocktail was non-
toxic to skin tissue at all the tested concentrations, which exhibited no morphological
disruptions upon histological examination (Figure 5). Even at the highest dose of the
phage cocktail, histological images of the treated tissue showed no toxic effect and
structurally looked identical to untreated control samples (Figure 5A). Toxicity was only
detected when 0.1% sodium dodecyl sulfate (SDS) was applied as a positive control, where
significant structural disruption was observed.
Furthermore, treatment with the phage cocktail showed a significant decrease in the
infused C. acnes population at all of the dilutions tested, extending the capacity of the
phage cocktail to diminish C. acnes from laboratory cultures to skin tissue (Figure 5B).
Finally, the blemished three-dimensional skin model was also examined for an impact
on the levels of inflammatory factors normally seen as elevated in blemished skin. One
such mediator, interleukin-8 (IL-8) was detected at significantly reduced levels relative to
untreated tissue (Figure 5C). Notably, treatment with the phage cocktail diminished IL-8
to concentrations normally observed in healthy, unblemished skin tissue. Collectively, these
findings confirmed that the phage cocktail was efficacious at decreasing C. acnes levels in
a three-dimensional model of blemished human skin coupled with a notable reduction
in IL-8 concentration. Also, importantly, no tissue toxicity was observed with any of the
treatment concentrations.
PILOT IN VIVO STUDY DEMONSTRATED THE EFFICACY OF A TOPICAL FORMULATION INFUSED
WITH THE TRIPLE BACTERIOPHAGE COCKTAIL TO REDUCE C. ACNES ON THE SKIN WITHOUT
COLLATERAL MICROBIAL IMPACTS AND TO AMELIORATE BLEMISHED CONDITIONS
Following the successes with laboratory cultures and a three-dimensional skin model, a
small pilot clinical study was commissioned to determine the impact of twice daily topical
application of a finished formulation containing the triple phage combination targeted to
C. acnes for a duration of 7 days. Participants in the study were evaluated for reductions
in sebum, coproporphyrin III fluorescence, and C. acnes genomes through 16S rRNA
sequencing.
As C. acnes is known to stimulate production of sebum in the skin,54–57 the administration of
any countermeasure (including phage) to the bacteria could potentially have a downstream
impact on the amount of sebum in the region of topical application. Sebum levels were
assessed at baseline, day 3, day 5, and day 7 via the tape-lifting technique. Relative to