The clinical P. acne strain Pacne11-13 was separated from facial abscesses in patients with severe acne vulgaris, identified by VITEK 2 Compact system (BioMérieux, France) (Dréno et al., 2018). The gene sequencing of Pacne11-13 was performed using PacBio Sequel and Illumina NovaSeq PE150 platforms. Software Diamond and the Antibiotic Resistance Genes Database (ARDB) (Liu and Pop, 2009) combined the sequenced genes with the antibiotic-resistance functional annotation information to obtain the results. The antibiotics susceptibility test of Pacne11-13 (tetracycline, aminoglycosides, cephalosporins, and clindamycin) was tested using the disk diffusion method (Rosco Neo-Sensitab) (Schuetz, 2014). The Pacne11-13 was grown in a BHI medium (Brain-Heart-Infusion, Oxoid, United Kingdom) and incubated at 37°C in a hypoxic incubator (HR900-IIB2, ESCO).

P. acne phage was screened and enriched as previously described (Huang et al., 2014; Yang et al., 2019b), with some alterations. Briefly, sewage water samples from the sewage management center at Xinqiao Hospital used as sources of phages were collected and then centrifuged (Ultracentrifuge AG22331, Eppendorf, German) at 10000 × g for 15 min at 4°C. The supernatant was filtered through 0.22-μm PES needle filters (Millipore, United States) to remove any remaining bacteria. Then P. acne was cultured to the exponential phase (OD600 = 0.6) and was taken 1 ml to mix with 10 ml sewage filter liquor. The growth curve of host bacteria is in Supplementary Figure S1. Then the mixture was added to a 100 ml BHI liquid medium and co-cultivated for 24 h on a hypoxic rocking platform at 37°C to enrich any P. acne phages in the sewage samples. The next day, this mixture was centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was passed through 0.22-μm PES needle filters. The phage was purified and separated by picking single-plaque with the double-layer agar method (Shi et al., 2021). And the resulting isolation was stored at 4°C.

Three 3-μL aliquots of the high-titer phage preparations (10¹⁰ ~ 10¹³ PFU /mL) were pipetted on carbon-coated copper grids as instructed (Shi et al., 2021). After allowing the phages to adsorb for 1 min, the grids were stained with 2% uranyl acetate (pH 4.2) for 30 s. Then the morphology of φPaP11-13 was observed and photographed under TEM (Hitachi HTT700, Japan). The heads and tails of 5 individual phage particles were measured by Image-Pro Plus6.0 image analysis software (Media Cybernetics 6.0, United States) to calculate the averages and standard errors for the dimensions (Yang et al., 2019a).

The multiplicity of infection (MOI) refers to the ratio of the plaque-forming unit (PFU) to the colony-forming unit (CFU). Firstly, the titer of P. acne was adjusted to 1 × 10⁸ CFU/ml and φPaP11-13 to 1 × 10⁸ PFU/mL. Then mixed the P. acne with the φPaP11-13 in proportion (10.000, 1.0000, 0.1000, 0.0100, 0.0010, and 0.0001). After 24 h, phage titers were measured by the spot test (Shi et al., 2020). The proportion with the highest phage titer was the optimal MOI.

The phages and bacteria were mixed at the optimal MOI tested in the above experiments. Placed it in a hypoxia incubator and incubated for 5, 10, and 15 min. It was the optimal adsorption time when the phage titer was the lowest. Those experiments were repeated three times, and three parallel averages were taken under per-scale titers. The measurement data conformed to the normal distribution and were expressed as mean ± standard errors.

A one-step growth curve, including incubation, lysis, and platform period, can demonstrate the phage life cycle. φPaP11-13 was mixed with P. acne at optimal MOI (0.0100) and adsorption at 37°C for optimal adsorption time (10 min). The mixture was centrifuged at 10000 × g for 60 s to clear unabsorbed phages from the supernatant. Then the residue was washed twice with BHI liquid medium on ice, resuspended by 5 ml BHI liquid medium, and cultured in a hypoxic incubator at 37°C. Samples were taken out at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h after injection, and the spot test measured the titer. According to previous reports, the burst size was calculated based on the one-step growth curve (Kropinski, 2018). This experiment was performed in triplicate.

To test the stability of φPaP11-13 under various conditions, we measured its titer at different temperatures, pH, UV, and Chloroform conditions, using our previously described method with some alterations (Shi et al., 2020). The φPaP11-13 solution was incubated in a water bath at different temperatures (−20, 4, 37, 50, 60, 70, and 80°C), and phage titers were measured after 1 h. Likewise, to measure the pH stability of φPaP11-13, samples were incubated at different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) at 37°C and samples were taken out for phage titer determination after 1 h. Then, to measure the UV stability of the phage. The phage sample was added to a 16-well plate, and the cover was removed and placed under UV light. After 0, 5, 10, 30, and 60 min of irradiation, the phage titers were measured. Finally, measured the stability of Chloroform. The spot test was used to measure the phage titer in the above experiments, and those experiments were repeated three times. The data were analyzed by GraphPad Prism version 9 for Windows (GraphPad Software, La Jolla, CA, United States).

Spot test and double-layer agar plate were used to determine the host range of φPaP11-13. Sixteen strains of P. acnes isolated from facial abscesses in patients were used for the host range experiment. 300 μl host bacteria with logarithmic phase (OD600 = 0.6) of each strain was mixed with 5 ml semi-solid agar and then poured into the bottom agar plate. 5 μl φPaP11-13 solution (10¹⁰ PFU/ml) was dropped onto the upper agar with more than three spots per plate. After the phage suspension was adsorbed, the plate was incubated in a hypoxic incubator at 37°C for 24 h.

Phage DNA was extracted using the standard Protease-K/SDS DNA extraction method (Shi et al., 2020). The nucleic acid concentration was measured using a NanoDrop spectrophotometer (ND-1000, Wilmington, DE, United States). This genome sequencing adopted the Whole Genome Shotgun (WGS) strategy to construct libraries of different insertions and utilized Next-Generation Sequencing (Dréno et al.) technology based on the Illumina NovaSeq Sequencing platform. Paired-end (PE) sequencing was performed on these libraries.

Phageterm (Garneau et al., 2017) was used to predict the genome ends of φPaP11-13, and GeneMarkS (Brettin et al., 2015), RAST (Aziz et al., 2008), and PHASTER (Arndt et al., 2016) were used as genome prediction software to predict ORFs of the assembled φPaP11-13 genome sequence. Then the predicted ORF was proofread using the Basic Local Alignment Search Tool (BLAST) of NCBI.¹ The annotated nucleotide sequence of the φPaP11-13 genome was submitted to the NCBI database under the accession number: ON557706, and the CGView Server BETA² (Stothard et al., 2019) was used to visualize the φPaP11-13 genome. To analyze the evolution trend and relation of φPaP11-13 with other known phages, Mauve 20,150,226 was used for collinearity analysis (Darling et al., 2004), and Mega-X 10. 0. 2 was used for phylogenetic tree analysis of the major terminal large subunit sequences (Kumar et al., 2018).

Liquid nitrogen was added to the phage particle, and then it was ground to powder. Then the phage particle was lysed by ultrasound, and a BCA kit measured its protein concentration. 5 μl of 4 × loading buffer and 2% SDS were added to the sample and then electrophoretic. The silver dyeing method was transferred into the chromogenic solution for about 10 min at room temperature, and clear bands were finally visible. Then the cut protein bands were tested by LC–MS/MS system (UPLC, EASY-nLC 1,200 system, PTM BioLab, Hangzhou) for phage major protein analysis. Then cross-checking underwent between LC–MS/MS results and whole genome annotation.

The whole genome of Pacne11-13 underwent sequencing. And AMR genes were annotated and listed in Table 1. The bacteria carried 17 AMR genes, including drug efflux-related, antibiotic target protection, antibiotic inactivation, and antibiotic target replacement genes, resulting in resistance against tetracycline, aminoglycosides, fluoroquinolones, macrolides, rifamycin, peptide antibiotics and cephalosporin, and other antibiotics. In addition, the AST results indicated that Pacne11-13 was multi-drug resistant to tetracycline, aminoglycosides, cephalosporins, and clindamycin (Table 2).

A P. acne phage was isolated from the sewage management center at Xinqiao Hospital and named “φPaP11-13.” This phage can form transparent circular plaques (diameter ranges from 1.0 to 5.0 mm, Figure 1). TEM showed that φPaP11-13 had a polyhedron head (diameter 60 ± 4.5 nm) and an untraceable flexible tail (length 170 ± 6.4 nm, and width 14 ± 2.4 nm, Figure 2). These morphological features suggested that φPaP11-13 was a member of the Siphoviridae family.

Three groups of parallel experiments were conducted for MOI tests, and the optimal MOI of φPaP11-13 was 0.0100 (Figure 3). After 6 h of incubation, the titers of φPaP11-13 were 2.7 ± 0.6, 3.3 ± 0.9, 3.3 ± 0.4, 5.7 ± 0.9, 2.3 ± 1.1, 0.63 ± 0.3 × 10⁷ PFU/mL when the MOI was 10.0000, 1.0000, 0.1000, 0.0100, 0.0010, 0.0001. When incubated with host bacteria for 5, 10, and 15 min, the titers of φPaP11-13 respectively, were 20.0 ± 1.0, 1.56 ± 3.1, and 3.4 ± 0.9 × 10⁷ PFU/mL, suggesting an optimal adsorption time of φPaP11-13 at 10 min.

The one-step growth curve revealed the latency time and burst size of φPaP11-13. As shown in Figure 4, the titer decreased slightly in the first 10 min but then remained stable for the first 5 h but increased to a large degree in the next 5 h, indicating that phage was released by the infected cells. Thus, the change in phage titer showed that φPaP11-13 had a long incubation period of approximately 5 h, followed by a burst period, and then we can calculate the burst size. The phage titer reached a plateau about 10–12 h after co-culture. The burst size of φPaP11-13 is 26 PFU/cell.

The stability of φPaP11-13 was tested under various temperatures, pH, and UV irradiation. Temperature stability results showed that the phage titer remained still from −20 to 50°C. However, a temperature over 50°C would decrease phage titer rapidly to 0 (Figure 5A). In the acid–base stability test, the pH tolerance of φPaP11-13 was stable from 2.0 to 12.0 (Figure 5B). Moreover, the titer of φPaP11-13 decreased rapidly under UV irradiation and was utterly inactivated when the irradiation time reached 60 min (Figure 5C). Chloroform had no significant effect on phage activity (Figure 5D).

A total of 16 strains of P. acne were involved in the analysis of the lytic range of φPaP11-13. The results showed that various-sized transparent plaques could be seen on most double-layer agar plates. The lysed rate of φPaP11-13 was 100% (16/16), demonstrating the phage has a broad host range (Table 3).

The linear dsDNA genome of φPaP11-13 consisted of 29,648 bp, with a G/C content of 54.08%. Forty five ORFs were predicted, accounting for 83.83% of the genome length, and the average length of each ORF was 606.22 bp. Among the 45 predicted ORFs and 20 ORFs were largely matched to genes with annotation functions in the GenBank database. No non-coding RNA, antibiotic resistance, or virulence factors were found in the phage genome (Table 4).

The predicted ORFs could be divided into four groups based on different functions: structural protein group, metabolism-related group, bacteria lysis group, and other functional groups. The structural protein contained minor tail protein (ORF34), H-type lectin domain-containing protein (ORF28), tail family protein (ORF30), tape measure protein (ORF31), major tail protein (ORF27), capsid protein (ORF39), scaffold protein (ORF40), MuF-like minor capsid protein (ORF42), and portal protein (ORF43). The metabolic-related group included HNH endonuclease (ORF1), P D-(D/E) X.K. nuclease family protein (ORF10), DNA Primase (ORF15), exonuclease (ORF19), helix-turn-helix DNA binding domain protein (ORF20), terminase large subunits (ORF44), and terminase small subunits (ORF45). The cleavage proteome included Amidase (ORF25) and Holin (ORF24). Other function groups include collagen triple helix repeat protein (ORF26) and sigma factor (ORF22). The annotation information of φPaP11-13 was uploaded to GenBank with the accession number: ON557706. The whole genome is visualized in Figure 6.

SDS-PAGE was applied to verify the major proteins of φPaP11-13. The results showed clear protein bands and normal distribution (Figure 7A), indicating an undegraded phage protein preparation. Then all visible bands were cut and analyzed by LC–MS/MS system. 17 phage proteins with high intensity were identified (Table 5). As shown in Figure 7B, the bands were identified as the lysis-related proteins Holin (12.528 kDa, ORF24), Amidase (31.354 kDa, ORF25), and the tape measure protein (93.79 kDa, ORF31). Furthermore, the molecular weight between 20 and 30 kDa included scaffold protein (ORF40), major tail protein (ORF34), MuF-like minor capsid protein (ORF42), and H-type lectin domain-containing protein (ORF28); the molecular weight of 30–40 kDa included capsid protein (ORF39) and tail family protein (ORF30); molecular weight of 40–50 kDa included portal protein (ORF43).

By comparing with the whole genome sequence in the NCBI database, the similarity between φPaP11-13 and known Sipoviridae family phages ranged from 41 to 98% (Supplementary Table S1). The two phages with the highest similarity to φPaP11-13 were φPA6 (dq4431235.1, 98%) and φct4Al2 (BK053742.1, 98%). However, the collinearity analysis revealed multiple local collinearity regions (LCB) between φPaP11-13 and these known phages. And these LCBs were rearranged and inverted. Even some genes within each LCB were distinct, with variants, rearrangements, and insertions (Figure 8). Collinearity analysis indicated that although φPaP11-13 was similar to these phages, there were significant internal differences (Orthologs and common conserved region sequence are listed in Supplementary Tables S2, S3). Phylogenetic trees were constructed based on the terminal large subunit protein because its amino acid sequence was highly conserved. As shown in Figure 9, φPaP11-13 and φPHL030N00 (KJ578760.1), a P. acne phage belonging to the Sipoviridae family, were on the same branch, with 100% reliability and short genetic distance, revealing the homology between φPaP11-13 and φPHL030N00. Collinearity and phylogenetic analysis confirmed P. acne phage φPaP11-13 as a member of the Sipoviridae family.