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Page 6 of 17 Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42

Figure 1. Phage Aquarius Characterization. (A) Plaque morphology of Aquarius was of variable size with clear to turbid plaques. The
plate image was enhanced by 30% in brightness and contrast to show detail; (B) Transmission electron microscopy at 52000X
magnification. The length of the phage tail and the diameter of the phage head were measured using the software program ImageJ [52]
and were found to be 150.3 and 58.8 nm, respectively. The presence of the long non-contractile tail and the icosahedral head are
characteristic of Caudoviricetes phages; (C) SIR phenotype characterized by the growth of bacteria in the center of an area of clearing
following spot inoculation of phage Aquarius on cultures of C. acnes ATCC 6919 (second row streak) and two C. acnes clinical isolates
(third and fourth row streaks) during an initial host range assay. No bacterial lysis was observed for the negative control spot inoculation
on E. coli (first row streak). SIR: superinfection resistance.

iSCRIPT cDNA kit protocol using a Bio-Rad thermocycler (Bio-Rad Laboratories, Inc.; Hercules, CA,
USA). After obtaining cDNA, qPCR was performed with primers for Aquarius gp41 and ATCC 6919 RecA
reference gene [Table 2] on undiluted cDNA using the Roche KAPA SYBR FAST qPCR kit (Roche, Basel,
Switzerland). A one-way ANOVA with post hoc Tukey HSD was used to assess statistical significance.

RESULTS
Phage Aquarius characterization
Aquarius, which was isolated from the microcomedones of a donor individual, displayed variable plaque
sizes with clear to turbid morphologies on C. acnes ATCC 6919 [Figure 1A]. Aquarius virions were
visualized using transmission electron microscopy and displayed a morphology similar to other previously
[52]
isolated C. acnes phages [Figure 1B] . After initial isolation of the phage, an early host range experiment
additionally demonstrated the SIR phenotype plaque morphology as has been described previously by Liu
[11]
et al. (2011) [Figure 1C] .

Genomic analysis demonstrated a genome of 30112 bp with 54.5% GC content and 48 putative ORFs were
identified. The genome ends have 11 base 3’ sticky overhangs (TCGTACGGCTT), suggesting that the
genome is capable of the circularization necessary for pseudolysogeny [3,11,24] . Comparative genomics of the
representative C. acnes phage genomes used in this study [Table 1] indicated 86%-96.7% GCS with a high
degree of synteny and nucleotide conservation [Table 3 and Figure 2] . These genome characteristics were
[34]
demonstrative of homology with other previously isolated C. acnes phage genomes, which are known to
[11]
have very limited diversity . This was further supported by the GBDP that were calculated for all of the
phages in Table 1, which included Aquarius and eight representative C. acnes phages, along with two
Streptococcus thermophilus phages to root the tree [Figure 3]. All the C. acnes phages exhibited a high degree
of genomic homology, as evidenced by the short branch lengths and low bootstrap values.

Evidence for pseudolysogeny and stability
The identification of 3’ sticky overhangs suggested the genome’s capacity to undergo circularization and
exist as a pseudolysogen. To further assess for pseudolysogenic characteristics, putative Aquarius
pseudolysogens were streaked (patched) onto an uninfected bacterial lawn. Areas of clearing were observed