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Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42 Page 9 of 17

Figure 3. Genome-BLAST Distance Phylogeny tree for C. acnes and S. thermophilus phages. The numbers above the branches are GBDP
pseudo-bootstrap support values from 100 replications. The branch lengths of the resulting trees are scaled in terms of the
recommended VICTOR formula (D0) [40] . GBDP: Genome-BLAST Distance Phylogeny.

Figure 4. High Stability/Low Stability Patch Test Samples, derived from passage 4, on ATCC 6919. The above picture illustrates the
capacity of the passaged lysogenic bacteria taken from the fourth pass of the patch test to produce an area of clearing surrounding the
lysogen streak (high stability group, on the right). Also shown is a prior putative pseudolysogen that possessed the ability to cause lysis
but lost its lysing capacity after the fourth passage (low stability group, on the left). Positive and negative controls were also prepared
(lawn of pure ATCC 6919, and putative pseudolysogens with no bacterial lawn, respectively), and demonstrated successful bacterial
growth (not shown).

Evidence for SIR
When phage Aquarius was spotted onto lawns of C. acnes ATCC 6919, bacterial proliferation was observed
in the center of areas of clearing. To test for SIR, bacteria were isolated from plaque centers, cultured, and
used to create bacterial lawns for spot tests with ten-fold dilutions of the Aquarius phage lysate. The spot
tests revealed no evidence of lysis on the lawn of previously infected bacteria, indicating SIR. Additionally,
putative Aquarius lysogens of three C. acnes clinical isolates demonstrated SIR by phage Aquarius
[Table 1]. To assess the range of cross-immunity that the SIR mechanism may confer, eight additional
representative C. acnes phages that were previously isolated and characterized were spot tested for their
ability to infect putative Aquarius lysogens, and no lysis was observed on the lawns for any of the C. acnes
phages tested [Table 1]. To further test the range of this SIR mechanism, putative ATCC 6919 lysogens for
seven different C. acnes phages were isolated and cross-tested for SIR for a variety of phages [Table 1]. No
lysis was observed for any of the tested combinations of pseudolysogens and re-infecting phages, suggesting
a mechanism for broadly preventing superinfection by C. acnes phages.