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Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42 Page 3 of 17

In the present study, C. acnes phage Aquarius was isolated from the facial microcomedones of a donor
without a history of acne. An early host range assay revealed the SIR phenotype in which bacterial
proliferation was observed in the centers of Aquarius plaque clearings on bacterial lawns of C. acnes ATCC
6919. This observation prompted a search for SIE characteristics, including verifying Aquarius’s ability to
undergo and maintain pseudolysogeny [11,23] . Sequencing and genomic annotation of the Aquarius genome
was performed, and bioinformatics were used to identify and attempt characterization of a putative SIE
gene. Evidence suggesting a phage-mediated SIE phenotype could begin to shed light on shortcomings of
past phage therapy experiments, as well as present a target for future studies aimed at fine-tuning phage
therapies for the purpose of controlling pathological C. acnes presence.

METHODS
C. acnes host strains and bacteriophages
C. acnes ATCC 6919, clinical isolates [strains 060PA1, 110PA3, and 020PA1, described in Fitz-Gibbon et al.
(2013)], and cultures of putative pseudolysogens were cultivated as described previously by Marinelli et al.
by incubation at 37 °C for three days under anaerobic conditions using the AnaeroPack System (Mitsubishi
Gas Chemical Company, Tokyo, Japan) . For all subsequent experiments, C. acnes plates were incubated
[1,3]
under these conditions, unless otherwise noted. C. acnes bacteriophage isolates were isolated and purified
from facial microcomedones using Bioré® pore strips (Kao USA Incorporated, Cincinnati, OH, USA)
[1,3]
applied to the nose as previously described . The microcomedone samples were scraped off of the strip
and re-suspended in 1 mL of liquid A Media agar (12 g casitone, 12 g yeast extract, 4 g D+ glucose, 4 g
KH PO , 1 g magnesium sulfate heptahydrate, ddH O up to 1.0 L). The inoculated A Media was then passed
2
4
2
through a 0.22-micron filter to isolate all particles smaller than 0.22 microns, including phages. The filtered
sterilized contents were then added to 500 μL C. acnes culture and plated on A Media hard agar via the soft
agar overlay technique using 0.5% A Media Top Agar (liquid A Media + 5 g agar). A control of C. acnes and
SM buffer (10 mL 1M Tris stock at pH 7.5, 10 mL MgSO stock, 4 g NaCl, 970 mL ddH O, 10 mL of
2
4
CaCl stock) was also plated. After obtaining plaques, samples were taken from eight plaques and plated
2
with C. acnes on A Media hard agar. The phage lysate used throughout the study was obtained by flooding
the web lysis plate from this isolation (using the sample that initially generated the most plaques) with
reinforced clostridial media (RCM) buffer and then collecting and filtering the lysate. A plaque assay was
performed to determine the titer of the lysate, and a dilution scheme was generated to yield complete lysis,
-3
-4
-1
-2
web lysis, and countable plaque plates using ten-fold serial dilutions of the phage at 10 , 10 , 10 , 10 , and
10 . This process was conducted twice using the highest titer dilution web lysis plate to generate a high-titer
-5
phage lysate. Bacteriophages used in this study are described in Table 1 [3,23,26-28] .
Electron microscopy
The phage sample was prepared by placing an aliquot of phage lysate on a carbon EM-grid and staining
with 1% uranyl acetate (0.1 g uranyl acetate, 10 mL ddH O). Images were taken with a Philips CM120
2
electron microscope (F.E.I. Company, Hillsboro, OR, USA).

Viral DNA purification and sequencing
Phage DNA was isolated by incubating a sample of phage lysate with 5 mg/mL DNase I and 10 mg/mL
RNase A for 30 min. The sample was then treated with Promega Wizard® DNA Clean-Up System (Madison,
Wisconsin) as described by the manufacturer’s protocol to isolate viral DNA. The concentration of the
isolated phage DNA was obtained using a NanoVue spectrophotometer (GE Healthcare, Chicago, Illinois).
The phage genomes were sequenced using an Illumina MiSeq (Illumina, San Diego, CA, USA) and
assembled using the software program Newbler (Roche, Branford, Connecticut, USA) at the Pittsburgh
Bacteriophage Institute in Pennsylvania as previously described .
[29]