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Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42 Page 7 of 17

Table 2. qPCR primers for expression level analysis
qPCR primers
ATCC 6919 RecA forward 5’-GAC CGT TAA GAT CGC CGC TA-3’
ATCC 6919 RecA reverse 5’-CGT GCT CGG CGT CAA TAA AG-3’
Aquarius gp41 forward 5’-CTC CCT ACA AGC CGA ACA GG-3’
Aquarius gp41 reverse 5’-AGG TGT CTT TGT GAG CTC CG-3’

Table 3. C. acnes phage gene content similarity
Phage ATCC29399BC Lauchelly Bruce lethal Queen bey P100A P100D P104A P105
Aquarius 87.3 88.3 89.3 86 88.3 86.3 88.3 90.4
ATCC29399BC 94.5 95.6 92.2 96.7 92.5 94.5 92.3
Lauchelly 96.6 93.2 95.6 93.5 95.6 93.3
BruceLethal 94.3 96.6 94.6 96.6 94.4
QueenBey 93.2 91.3 93.2 91
P100A 93.5 95.6 93.3
P100D 95.7 91.4
P104A 93.3

surrounding the bacterial patches, indicating spontaneous phage release following lytic induction of
pseudolysogenized phage. PCR primers that anneal to the end of C. acnes phage genomes were used to
assess if circularized phage genomes were present in the bacterial samples [Supplementary Figure 1]. Gel
electrophoresis showed characteristic bands at roughly 735 base pairs (corresponding to the size of the
overlapping portion of the phage genome that was amplified) for all putative pseudolysogens. No bands
were produced on the uninfected ATCC 6919. These findings were consistent with previous experiments
demonstrating the capacity of C. acnes phages to undergo pseudolysogeny [11,24] .

Pseudolysogens are generally known to be less stable than full lysogens and, therefore, at greater
susceptibility of being diluted out over time . Given this, it was considered that the numerous lytic centers
[24]
scattered on the pseudolysogen lawns may be a manifestation of induction of the lytic life cycle in bacteria
after sequential passaging. After initial patch testing, sub-isolates of two pseudolysogens with either a
persistently sustained (“high”) or quickly lost (“low”) capacity for spontaneous lytic phage release were
assessed for pseudolysogen stability via serial patch testing for phage release over the course of several
months [Figure 4]. The sub-isolates of the “low stability” group lost the ability to lyse the surrounding lawn
after six total passages, which corresponded to about 20 days. This isolate was the first to lose its lysing
capacity after beginning serial patch testing, as demonstrated by the lack of an area of clearing surrounding
the bacterial patch on the bacterial lawn. The “high stability” group, however, sustained lytic capacity for
over six months of passages, corresponding to roughly 180 days. It was at this point that the study was
stopped.

Pseudolysogeny PCR
Pseudolysogeny PCR was performed on both the high- and low-stability groups as described above to assay
for maintenance of the circularized genome using the primers described by Liu et al. . All 27 high stability
[11]
group sub-isolates produced a 735 bp band indicative of the presence of the phage genome, while the two
representative samples for the low stability group did not, indicating true loss of the phage genome. A
Fisher’s exact test was conducted to assess independence between PCR result and patch test phenotype, and
the results indicated a strong association between them (P < 0.002) [Table 4]. Additionally, viral spot testing
on cultures of the high- and low-stability groups after reaching five months of passages revealed lysis of the