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Page 2 of 17 Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42

pseudolysogeny was a concomitantly present phenomenon. Phage genomic analysis identified the presence of a
conserved gene (gp41) with a product containing Ltp family-like protein signatures which may contribute to phage-
mediated bacterial superinfection resistance (SIR) in a pseudolysogeny-dependent manner. qPCR was performed
to analyze and roughly quantify gp41 activity, and mRNA expression was high during infection, implicating a role
for the protein during the phage life cycle.

Conclusions: This study confirms that C. acnes bacteria are capable of harboring phage pseudolysogens and
suggests that this phenomenon plays a role in bacterial SIR. This mechanism may be conferred by the expression of
phage proteins while the phage persists within the host in the pseudolysogenic state. This parameter must be
considered in future endeavors for efficacious application of C. acnes phage-based therapeutics.
Keywords: Cutibacterium acnes, Propionibacterium acnes, pseudolysogeny, superinfection resistance, bacteriophage,
antibiotic resistance, phage therapy, superinfection exclusion

INTRODUCTION
Cutibacterium acnes, formerly Propionibacterium acnes, is a gram-positive bacterium of the human
epidermal microbiome. It has been documented widely within human preclinical lesions, also called
microcomedones, regardless of skin microflora variability . Certain strains of C. acnes have been
[1-3]
[2,3]
implicated as key contributors to acne vulgaris (i.e., acne) . Though generally considered a mild affliction,
many individuals affected by acne may suffer from psychosocial problems and physical pain at affected
sites . Additionally, C. acnes may contribute to severe health complications such as post-operative
[2]
prosthetic hardware contamination, sarcoidosis, spondylodiscitis, prostate pathologies, and even
[4,5]
Parkinson’s Disease . Extensive time and effort have been dedicated to the study of C. acnes and C. acnes
bacteriophages, the viruses that infect C. acnes, to better understand and characterize the predominant
strains in humans, particularly those correlated with pathogenicity . Some studies such as those
[1,3]
investigating biofilm dynamics have evaluated the efficacy of phage therapy against C. acnes and have
demonstrated bacterial resistance to infection in certain cases . The leading proposal for this phenomenon
[6-9]
in C. acnes was that clustered regularly interspaced short palindromic repeat (CRISPR) elements conferred
resistance in a Cas protein-dependent manner [3,10] . Since then, experimental results aiming to induce
CRISPR-mediated resistance in clinical strains of C. acnes have suggested inconsistency with the notion of
CRISPR as an exclusive immunity mechanism, indicating other mechanisms are also at play [10,11] .

An alternative cause of bacteriophage (phage) resistance is the mechanism of superinfection exclusion (SIE).
SIE is a property conferred to the host bacterium via expression of certain temperate phage proteins which
give rise to superinfection resistance (SIR) [12-14] . SIE conferring SIR has been documented among both
viruses and phages such as those that infect Pseudomonas aeruginosa, Escherichia coli, and
Salmonella typhimurium [15-20] . Several other phages that infect Streptococcus thermophilus and Lactococcus
lactis including TP-J34 and TP-778L have also been found to display SIE conferring SIR, and proteins
involved in the mechanism have been identified and explored [13,20-23] . The SIR phenotype has been observed
in C. acnes by a failure of new plaque formation when phages that were previously able to cause lysis were
re-spotted onto bacterial lawns composed of bacteria that had proliferated within prior plaque centers .
[11]
The ability of phages to persist within a host cell supports the possibility of an SIE mechanism dependent on
the expression of SIE gene(s) from a latent phage genome. While C. acnes phages do not undergo a classic
lysogenic state as they do not encode integrases, they are known to exhibit pseudolysogeny in a state either
characterized by the phage genome remaining as an episome, or in a state of inefficient lysis [6,11,23-25] . Despite
documentation of C. acnes phage capacity to both undergo pseudolysogeny and to confer SIR, an SIE
mechanism has not been characterized and the mechanism conferring SIR has not been described.