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

543 Modern Skincare
baseline, there was an approximate 25% drop in sebum levels as early as day 3 in.the pilot
clinical study (Figure 6A).
As a part of their life cycle, C. acnes bacteria produce the molecule coproporphyrin III,
which will fluoresce a red-orange color under specific wavelengths of light.58 As such, it
has been utilized as a means of evaluating the population levels of C. acnes on the skin
of patients experiencing the effects of acne vulgaris.59 As shown in Figure 6B, there was
a visible decrease in C. acnes associated coproporphyrin III fluorescence from baseline to
day 7. The change in fluorescence intensity from baseline to day 7 was quantified using
ImageJ software (NIH), which indicated an approximate 20% reduction (data not shown).
To corroborate the fluorescence measurements from the coproporphyrin III examination,
16S rRNA sequencing was conducted on skin swabs collected from the study participants.
This sequencing method, first established by Dr. Carl Woese, allows for the differentiation
Figure 5. Three-dimensional blemished skin model. A) C. acnes targeted phage cocktail exhibits no tissue
toxicity when topically applied on a C. acnes-infused blemish-prone three-dimensional skin tissue model. The
phage cocktail was applied at three different dilution levels and allowed to incubate for 24 h. Histological
examination was conducted on those tissues as well as untreated tissue (negative control) and tissue exposed
to 0.1% SDS (cytotoxic positive control). B) The tissue described in (A) was further examined for a reduction
in C. acnes levels in response to the phage cocktail application. As shown, at all of the dilutions applied, the
phage cocktail diminished the C. acnes levels relative to tissue not exposed to the phage cocktail. C) The tissue
was also tested for changes in the levels of inflammatory factors normally associated with blemished skin.
The levels of IL-8 were significantly reduced in the blemished skin tissue model relative to untreated controls
(right side of graph, orange bars), and achieved levels as low as those observed in healthy skin tissue (left side
of graph, peach bars).