METHOD FOR SCREENING FOR MODIFICATIONS IN THE INFECTIVITY RANGE OF BACTERIOPHAGES DUE TO EPIGENETIC IMPRINTING

TECHNICAL FIELD

The invention relates to methods for screening for modifications in the infectivity of bacteriophages that are due to epigenetic imprinting, compositions comprising bacteriophages obtained or obtainable by these methods and their use in therapy and for the selective killing of bacteria.

BACKGROUND OF THE INVENTION

Phage therapy is a practice used to treat bacterial infections. The principle of this treatment is based on the use of a bacteriophage. Phages are non-living biological entities which consist of RNA or DNA and which can selectively kill bacteria depending on their host specificity. While phage therapy has already existed for almost a century its usage for therapy was seen controversial. Nowadays, phage therapy has widely been reconsidered as an alternative to the use of antibiotics to treat bacterial infections. Antibiotic treatment is often linked to the occurrence of antibiotic resistance within the pathogenic phenotypes but also kills beneficial non-pathogenic bacteria due to its non-selective nature. In contrast to antibiotics, phages can be highly specific in killing bacteria and kill selectively even on strain level.

The bacteriophages capable of selectively killing bacteria can either be isolated from the bacterial strain that shall be targeted itself, or they can be modified by genetic modification to specifically target the desired strain. One method of genetically modifying phages is through traditional homologous recombination-based techniques, such as phage crosses. A more targeted approach is the homologous recombination between the plasmid and phage genome generating recombinant phages with gene replacements, deletions, or insertions. Bacteriophage Recombineering of Electroporated DNA (BRED), CRISPR-Cas-Based Phage Engineering and Rebooting Phages Using Assembled Phage Genomic DNA are further known methods of genetically engineering phages (Chen et. al, 2019). Phage engineering provides one strategy to generate phage variants with unique properties, which might accelerate the development of phage therapy.

Another way of modifying a bacteriophage is to alter its epigenetic imprint. It is generally known in the art that growing, or culturing, a phage in different strains of bacteria can alter the properties of the phage. This alteration may be epigenetic, meaning that it is not due to changes in the nucleic acid sequence encoded by the genome of the phage, but instead appears to be due to chemical changes modifying the bases in the DNA. Changes include, for example, changes in the methylation of the nucleic acid in the phage due to DNA methylases.

Bacteria employ restriction-modification (R-M) systems to destroy invading DNA, while keeping the self-DNA safe by methylation of specific sites (Samson et al., 2013; Seed, 2015). Furi et. al. (2019) have shown that in Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome and it was hypothesized that the ability of the R-M systems to switch between six target DNA specificities has a key role in preventing the spread of bacteriophages. However, phages can incorporate base modification systems to keep their genome resistant to the bacterial R-M systems (Samson et al., 2013). For example, T4 phage modifies the cytosines by two modifications, 5-hydroxymethylation and glucosylation, which make it highly resistant to virtually all the restriction endonucleases of E. coli (Bryson et al., 2015). MTases have been described in the prior art as a protective mechanism against bacteriophage infection that selectively transfer the methyl group from SAM to the nitrogen atoms at position 4 in cytosine and position 6 of adenine (m4C, m6A) or the fifth carbon of cytosine (m5C) within specific sequence motifs along the bacterial genome identified by the R-M system recognition domain (Vasu and Nagaraja (2013)). These methylated sequences are resistant to endonuclease digestion by the restriction enzyme and are recognized by the R-M system as a means of establishing self from nonself. Any phage DNA entering the host is assessed by the R-M system and digested by the R-M endonuclease if methylation is not detected by the corresponding recognition domain. To circumvent host restriction of phage DNA, bacteriophages often introduces their own MTases during infection.

The endless coevolution of phages and their host bacteria makes bacteria less resistant to phage therapy than for example the antibiotic treatment, especially when phage cocktails are used.

For example, WO2009087356A1 discloses that culturing phages in Staphylococcus aureus can be used to increase the host range of Staphylococcus aureus strains that can be infected by a phage. This reduces the number of different phages that need to be included in a panel of phages to ensure a broad coverage of different bacterial strains. This heterogenous panel of phages can then be used for example in disinfectant compositions for surfaces as it provides a mix of phages capable of killing many different bacteria.

Cutibacterium acnes (C. acnes), also known as Propionibacterium acnes (P. acnes) is the major skin commensal of the human skin microbiome but has been associated with the skin condition acne vulgaris. C. acnes harbors multiple lytic or temperate phages. Phages within the skin modulate skin microbiome composition by infecting either a narrow range of strains or broadly within a population. It has been shown that healthy skin harbors more phages than acne/unhealthy skin. C. acnes is resistant to multiple antibiotics either directly or by the formation of a stable biofilm which can protect bacteria from antibiotic activity. However, phage therapy can provide a suitable therapy because it has been shown that phages can dissolve biofilms. WO2019113066A1 discloses a bacteriophage treatment for acne where lytic bacteriophages are capable of lysing P. acnes thus preventing the formation of biofilms and acne.

However, due to the fact that C. acnes is at the same time the major component of healthy skin its concrete involvement in the disease is not fully understood. C. acnes contains various strains, some of which are beneficial to the skin microbiome, such as strain H1 (clade IB), whilst others, such as strain A1 (clade IA1), are associated with pathogenic states (Dreno B. et al. 2018). It would therefore be useful when treating skin to be able to distinguish between the beneficial and the pathogenic strains of C. acnes in order to provide for a healthy microbiome of the skin.

To date none of the available phage therapies provide a possibility to target only certain strains of C. acnes, while not affecting other beneficial probiotic strains. There is thus a need for a method for obtaining a selective and specific phage population that is targeted to the killing of preferably only one specific strain of C. acnes while not harming other probiotic strains.

Other applications where bacteriophage therapy is an interesting alternative to the use of antibiotics include targeting Methicillin-resistant Staplylococcus aureus (MRSA) that can cause systemic infections in sick, elderly or immune-compromised patients as well as other bacteria increasingly causing problems in hospitals, such as Clostridium difficile or Pseudomonas aeruginosa. Especially, since mutations confer antibiotic resistance, or genes encoding antibiotic resistance enzymes, such as penicillinases, are becoming increasingly common in pathogenic bacteria world-wide, there is a great need for a different approach in fighting bacteria.

With the large amount of possible applications of bacteriophages against all kinds of pathogenic bacteria, there is a need for a simple, rapid, economic and efficient method of screening for bacteriophages with a desired infectivity range allowing to identify and isolate the bacteriophages that have obtained the desired specific properties without the need of applying lengthy genetical modification to the phages as have been abundantly described in the prior art.

SUMMARY OF THE INVENTION

The present invention therefore relates to an or in vitro method for screening for changes in the infectivity range of one or more bacteriophages due to epigenetic imprinting of the bacteriophage(s), the method comprising the following steps:

- (a) obtaining an isolate of the one or more bacteriophages after infection of at least a bacterium with said one or more bacteriophages, said bacterium preferably comprising a restriction methylation system; and
- (b) infecting a bacterium different from the bacterium used in step a), said bacterium preferably comprising a restriction methylation system, with the isolate of the one or more bacteriophages that previously infected the bacterium of a) in order to allow multiplication of said phage; and
- (c) obtaining an isolate of the one or more bacteriophages after infection of step b), and determining the changes in the infectivity range of the one or more bacteriophages due to the epigenetic imprinting of the bacteriophage(s) after the infection of the bacterium of step b); and optionally
- (d) selecting said isolate of the one or more bacteriophages if the sought-after specific infectivity range due to the change in the epigenetic imprinting is obtained and, optionally, formulating said bacteriophage(s) into a product.

In a further embodiment the method comprises characterizing the epigenetic imprinting of the isolates of step c) and/or d).

The present invention furthermore relates to an in vitro method for screening for one or more bacteriophages with a specific infectivity range due to the epigenetic imprinting of the bacteriophage(s), the method comprising the following steps:

- (a) obtaining an isolate of the one or more bacteriophages after infection of at least a bacterium with said one or more bacteriophages, said bacterium preferably comprising a restriction methylation system; and
- (b) infecting a bacterium different from a), said bacterium preferably comprising a restriction methylation system, with the one or more bacteriophages that previously infected the bacterium of a) in order to allow multiplication of said phage; and
- (c) obtaining an isolate of the one or more bacteriophages after infection of step b), and determining whether the one or more bacteriophages have the specific infectivity range due to the epigenetic imprinting of the bacteriophage(s) after the infection of the bacterium of step b), and
- (d) selecting said isolate of the one or more bacteriophage if the sought-after specific infectivity range due to the change in the epigenetic imprinting is obtained and, optionally, formulating said bacteriophage(s) into a product.

In a further embodiment of the method of present invention the bacterium of step b) comprises a restriction methylation system.

In a further embodiment of the method of present invention, the isolate of bacteriophages after infection of step b) comprises a substantially homogenous population of bacteriophages with a changed infectivity range due to the epigenetic imprinting.

In one embodiment the bacterium of a) and b) is selected from the group consisting of: C. acnes, E. coli, Salmonella species, such as Salmonella typhimurium, Shigella species, such as Shigella flexneri, Enterococcus species, such as Klebsiella, Proteobacteria species, such as Pseudomonas species, specifically Pseudomonas aeruginosa, Haemophilus species, Corynebacterium species and Firmicutes species, such as Bacillus species, Streptococcus and Staphylococcus, specifically Staphylococcus aureus. In a preferred embodiment the bacterium is C. acnes.

In one embodiment the bacterium of a) and b) of the methods described above are different strains of C. acnes, the bacterium of step b) being characterized by comprising an active restriction methylation system such as a type Ig, type IIa, type IIb, type IIg or type IIIb restriction methylation system.

In a further embodiment the bacterium of a) and b) is a different strain of C. acnes and each of said strains pertains to any of the following clades IA1, IA2, IB, IC, type II or type III.

In one embodiment the bacterium of a) pertains to the IA1, IA2 or IB clade and the bacterium of b) pertains to any of the following clades IA1 or IA2, IB, IC, type II or type III.

In another embodiment the bacterium of a) pertains to the IA1, IA2 or IB clade and the bacterium of b) pertains to any of the following clades IB, IC, type II or type III. In one embodiment the present invention relates to a composition comprising a phage population as identified and obtained or obtainable by the methods as described above.

In a further embodiment the present invention relates to the composition comprising a phage population as identified and obtained or obtainable by the methods as described above for use in therapy.

In one embodiment the present invention relates to the composition comprising a phage population as identified and obtained or obtainable by the methods as described above for use in the selective bacterial killing of one or more bacterial species or strains preferably selected from any species or strains from the group consisting of C. acnes, E. coli, Salmonella species, such as Salmonella typhimurium, Shigella species, such as Shigella flexneri, Enterococcus species, such as Klebsiella, Proteobacteria species, such as Pseudomonas species, specifically Pseudomonas aeruginosa. Haemophilus species, Corynebacterium species and Firmicutes species, such as Bacillus species, Streptococcus and Staphylococcus, specifically Staphylococcus aureus.

In a further embodiment the present invention relates to the composition comprising a phage obtained or obtainable by the methods as described above for use in the selective bacterial killing of the one or more bacterial species or strains to enhance engraftment of probiotic strains.

In a further embodiment the present invention relates to the use, preferably the in vitro use, of the epigenetic imprint which is changed during phage reinfection of a suitable bacterium with a restriction methylation system to select phages capable of lysing and/or killing one or more bacterial species or strains and to obtain a substantially homogenous population of phages with a specific infectivity range.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: C. acnes H2 (also referred to as KPA171202) has a methylation motif in the AGCAGY sequence. A) Fraction of methylated reads across the positions of the AGCAGY motif for the wild-type (in black) and the methyltransferase knock-out (KO) H2 (in grey) strains. B) Fraction of methylated reads in the methylated adenine at 4th position of the AGCAGY motif detected in the pMW535 plasmid after recombinant expression of the indicated methyltransferase originating from H1 or H2 C. acnes in a methylation deficient E. coli strain. Negative is the methylation deficient E. coli not being co-transformed with any methyltransferase of C. acnes. C) Fraction of methylated reads in the methylated adenine at 4th position of the AGCAGY motif in the PAD20 phage genome. Samples are named first after the infected strain where the phage was sequenced and second after the strain from which the phage was produced (i.e, H2-H1 means a phage produced in H1 was used to infect H2 and then extracted for sequencing from H2). Significance was tested using ANOVA analysis (***, p<0.001).

FIG. 2: A) Variation of infection properties of PAD20 phage propagated on either SLST A1, H1, H2 or H2 KO. B) PAD20 phage infection properties on different C. acnes strains depending on propagation origin (SLST A1, H1, H1 or H2 KO). Infection of a selection of C. acnes strain belonging to different clades with PAD20 propagated on SLST A1, H1, H2 and H2 KO. Phages acquire different infection properties depending on their origin. C) Selected killing of whole microbiome samples infected with PAD20 propagated on SLST A1 after 3 different time points. Samples were taken after 24, 48, 120 h of on-going PAD20 infection. Samples were amplified using SLST methodology and subjected to Illumina sequencing. Similarity to SLST alleles is used to classify sequencing reads into clades IA, IB and II. Clade IA contains following SLST types: A1, A2, A3, A4, A7, A11, A13, A16, A17, A18, A21, A24, A27, A28, A29, A30, A31, A33, A34, A35, A38, A40, A42, A46, A47, A48, A49, A51, A52, C1, C5, F4, F6, F10, F15, F16, F17, F22, F24; Clade IB contains: H1, H2, H3, H4, H7, H8, H10, H11, H12; Clade II contains: K1, K2, K7, K9, K17, K19.

FIG. 3: Selective killing of a mixed population of SLST strain A1 and H1 by PAD20 Phage extracted from SLST A1. After 3 days serial dilutions were plated, and 10 representative colonies screened for their SLST type. All colonies showed to be of SLST H1.

FIG. 4: Transcriptional profile of restriction methylation locus and surroundings in C. acnes KPA171202 Wild Type (SLST H2 WT) and C. acnes restriction methylation knock-out (SLST H2 R-M KO). Expression levels are displayed by RNA-seq read counts per genomic position (mean of two replicates). R-M IIG corresponds to PPA 1614 and R-M IIIB to PPA1611.

FIG. 5: A) & B) Expression of the complete CRISPR/Cas type I-E locus in K1 and K2 shown by RNAseq analysis. C) Expression levels of genes belonging to the CRISPR system comparing threshold cycle (Ct) obtained by each candidate gene in both strains tested. D) RNA sequencing showed no expression of the IIIB and IIG locus in the KO strain.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention, and to the examples included therein.

The present invention relates to an in vitro method for screening for changes in the infectivity range of one or more bacteriophages due to epigenetic imprinting of the bacteriophage(s) as well as an in vitro method for screening for one or more bacteriophages with a specific infectivity range due to the epigenetic imprinting of the bacteriophages. The invention furthermore relates to a composition comprising bacteriophages identified and obtained or obtainable by these methods. The invention furthermore relates to such compositions for use in therapy. The invention also relates to such compositions for use in selective killing of bacteria.

The inventors herein provide for a method to obtain an isolate of phages with a modified epigenetic imprinting. The epigenetic profiles of the phages can be adapted to program killing to specific subsets of bacteria. In contrast to the genetic engineering techniques used and described in the prior art for genetically modifying phages, such as for example homologous recombination-based techniques, Bacteriophage Recombineering of Electroporated DNA (BRED), CRISPR-Cas-Based Phage Engineering and Rebooting Phages Using Assembled Phage Genomic DNA, the bacteriophages used in present invention do not need to be subjected to such techniques in order to provide them with the desired specificity. Instead, the bacteriophages are modified by altering their epigenetic imprinting.

Cutibacterium acnes (C. acnes) has been used as a model organism by the inventors to develop the methods of present invention. It is, however, to be understood that these methods are not limited to C. acnes but can be applied to any desired bacterial species. Particularly suitable are all bacteria comprising a restriction methylation system.

While C. acnes phages have an overall sequence identity of more than 85%, their ability of infecting certain strains is very diverse. Since the reason for this diversity was not well understood, the inventors investigated other factors involved in host selectivity. Restriction methylation (R-M) is a host mechanism that protects the bacterial host from foreign incoming DNA. This restriction methylation can be detected by nanopore sequencing. The present invention takes into account epigenetic changes which can occur on the phage genome when injecting its DNA into the host and change the scope of a single phage to infect one additional specific strain of C. acnes allowing to specifically erase pathogenic strains and sustain probiotic ones.

In particular, the C. acnes bacteriophage named PAD20 extracted from AD20 which was found in a deep tissue infection and belongs to type II C. acnes strains was investigated (Lood et. al 2008). Since all C. acnes phages are very similar in protein pattern, morphology and coding genes the phages are classified depending on their host range. PAD20 was classified into the PA I group. As shown in Example 2, different C. acnes strains belonging to different classes of C. acnes like type IA1, IA2, IB, type II and type III were infected with C. acnes bacteriophage PAD20, which was previously extracted from either a type IA1 or IB strain of C. acnes. It was observed that the lysis properties differed from strain to strain depending on which host the phage was first isolated from (see Example 2, FIG. 2A and FIG. 2B). While the phage extracted from a type IA strain like A1 was unable to infect type IB strains like H1 and H2, it was able to infect other type IA1 or IA2 strains and K1. On the other hand, PAD20 extracted from a type IB strain like H2 was able to kill IA1 and IA2 strains, H1 and K1 and also itself. PAD20 from H1 on the other hand could infect strains belonging to clade IA1 and IA2, K1 and itself but was unable to kill H2. K2 belonging to type II strains was unable to be lysed by any of the extracted PAD20 phages (FIG. 2A and FIG. 2B).

Then, the inventors investigated differences in the host and saw differences in active restriction methylation systems. One system in KPA171202 (also called “H2”) which belongs to type IIIB

R-M was found and its activity verified by nanopore sequencing (see FIG. 1A). To understand the involvement of R-M in host range specificity of C. acnes phage PAD20, a knockout (KO) mutant in KPA171202 (H2) was created as described in detail in Example 3. This strain did not have the corresponding R-M system anymore and was unable to methylate its own DNA in a specific pattern (see FIG. 1A). FIG. 4 and FIG. 5D verifies correct deletion of the R-M IIG and IIIB. RNA sequencing was performed on both the wild type and on the knockout strain and the expression of the R-M locus compared to each other.

After infection of this strain with PAD20 extracted from either H1, A1 and H2 infectivity of all three phages was observed confirming the hypothesis that epigenetic changes and specifically the R-M system is involved in phage host range specificity and that epigenetic profiles of the phages can be adapted to program killing to specific subsets of bacteria (FIGS. 2A and B). For this the inventors concluded that they the active R-M system could potentially protect the beneficial bacteria (type IB) while bacteria which do not have the R-M system will be killed by the bacteriophage.

To understand if C. acnes H2 is methylating phage DNA during infection the inventors extracted phages isolated from H2 and also infected H2 with H1 PAD20 phage. They verified methylation of phage DNA by nanopore sequencing and identified the specific methylation pattern (FIG. 1C).

To test specific killing of a population comprising two different C. acnes strains, the authors inoculated 1:1 a strain from subgroup IA1 (A1) with one of subgroup IB (H1) and infected those with a phage extracted from A1 host. After 3 days the inventors plated a dilution of bacteria on agar plates and they checked a subset for their classification by SLST sequencing. They verified that all tested colonies belonged to H1 C. acnes strain which meant a 100% host range specificity of PAD20 phage (see FIG. 3).

To verify whether the isolated phages target a certain subpopulation only, the inventors have used a whole microbiome sample and infected it with PAD20 originating from A1. The complex microbiome sample contains many different strains, and it was analyzed over time (24h, 48h and 120h) to see the dynamics of phage infection. While a decrease in SLST types belonging to IA1 and IA2 strains could be observed an increase in IB strains was seen. In the case of type II strains, a small decrease was observed which verifies again the existence and protection by CRISPR Cas systems in some of the sub types. (see FIG. 2C). This difference might indicate functional or unfunctional CRISPR type I-E system in the K strains. This experiment showed that Clade IA1 and IA2 bacteria (SLST types A, F, C) were targeted by using a phage which is not methylated and probiotic strains such as H1 and H2 were preserved. The abundance of IA1 and IA2 reduced after time while IB strains increased. To identify functional CRISPR Cas type I-E systems in K strains (type II), the authors studied the expression of the proteins involved in the CRISPR cascade. They verified transcription of the full cascade by qPCR and RNAseq in K1 and K2 SLST representatives (FIG. 5A, B, C). Additionally, the authors identified the origin of 6 spacers of K2, which were targeted against PAD20 phage genome. This verifies that K2 CRISPR Cas system actively protects K2 strain against phage infection.

From the results it can be deducted that this phage can be targeted to the killing of only one species of bacteria and contribute to a healthy microbiome without the use of antibiotics. Some C. acnes strains, such as H1, are beneficial, while others are more associated with pathogenic states, such as A1. The inventors have shown in the past that it is possible to engraft probiotic strains, such as H1 (Paetzold et al. (2019)). In this sense, it is possible to enhance the engraftment of probiotic strains, such as H1, by clearing the ecological niche using phages as prepared by the present invention. In other words, applying the probiotic strain H1 together with phages programmed to lyse/kill the more pathogenic strain A1, which at the same time is inactive against H1, engraftment can be significantly enhanced.

Therefore, the inventors have shown the use of phages for the selective bacterial killing based on epigenetic imprints and the use of phages to enhance engraftment of probiotic strains, wherein such engraftment is enhanced by the selective bacterial killing based on epigenetic imprints.

The present invention therefore provides for a method to use epigenetic imprinting which occurs on the phage genome when injecting its DNA into the host to target the scope of a single phage to infect a specific C. acnes strain providing a possibility to erase pathogenic strains and sustain probiotic ones.

The C. acnes strains suitable for the use in the methods or compositions of present invention can be selected from the following SLST types:

- H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, K14, K15, K16, K17, K18, K19, K₂O, K21, K22, K23, K24, K25, K26, K27, K28, K29, K30, K31, K32, K33, K34, K35, L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, M1 and M2, or a combination thereof.

The C. acnes strains suitable for the use in the methods or compositions of present invention can be selected from the following:

- C.acnes subsp. defendens, C.acnes subsp. elongatum, C.acnes subsp. acnes, C.acnes Asn10, C.acnes 5/1/3, C.acnes ADL-409, C.acnes P15, C.acnes P10, C.acnes P09, C.acnes P07B, C.acnes P06B, C.acnes PO6A, C.acnes PO3, C.acnes JCM 18920, C.acnes JCM 18918, C.acnes JCM 18916, C.acnes JCM 18909, C.acnes P6, C.acnes PA2, C.acnes FZ1/2/0, C.acnes hdn-1, C.acnes HL202PA1, C.acnes HL201PA1, C.acnes 25G, C.acnes 21G, C.acnes PA20B, C.acnes 19B2, C.acnes 19B1, C.acnes C1, C.acnes HL042PA3, C.acnes P07A, C.acnes P05, C.acnes 18B, C.acnes 17B, C.acnes Asn12, C.acnes PRP-38, C.acnes HL096PA1, C.acnes DSM 1897, C.acnes TypeIA2 P.acn31, C.acnes TypeIA2 P.acn17, C.acnes TypelA2 P.acn33, C.acnes subsp. defendens ATCC 11828, C.acnes 6609, C.acnes 266, C.acnes ACS-068-V-Col6, C.acnes HL037PA1, C.acnes HL082PA2, C.acnes HL082PA1, C.acnes HL056PA1, C.acnes HL007PA1, C.acnes HL002PA3, C.acnes HL002PA2, C.acnes HL002PA1, C.acnes HL063PA2, C.acnes HL063PA1, C.acnes HL027PA2, C.acnes HL027PA1, C.acnes HL067PA1, C.acnes HL036PA3, C.acnes HL036PA2, C.acnes HL036PA1, C.acnes HL001PA1, C.acnes HL030PA2, C.acnes HL030PA1, C.acnes HL050PA3, C.acnes HL050PA2, C.acnes HL050PA1, C.acnes HL005PA4, C.acnes HL005PA3, C.acnes HL005PA2, C.acnes HL005PA1, C.acnes HL046PA2, C.acnes HL046PA1, C.acnes HL086PA1, C.acnes HL059PA2, C.acnes HL059PA1, C.acnes HL025PA2, C.acnes HL025PA1, C.acnes HL083PA2, C.acnes HL083PA1, C.acnes HL092PA1, C.acnes HL038PA1, C.acnes HL060PA1, C.acnes HL087PA3, C.acnes HL087PA2, C.acnes HL087PA1, C.acnes HL110PA4, C.acnes HL110PA3, C.acnes HL110PA2, C.acnes HL110PA1, C.acnes HL074PA1, C.acnes HL072PA2, C.acnes HL072PA1, C.acnes HL043PA2, C.acnes HL043PA1, C.acnes HL078PA1, C.acnes HL013PA2, C.acnes HL013PA1, C.acnes HL045PA1, C.acnes HL053PA2, C.acnes HL053PA1, C.acnes HL020PA1, C.acnes HL097PA1, C.acnes HL099PA1, C.acnes HL103PA1, C.acnes HL096PA2, C.acnes HL096PA3, C.acnes SK187, C.acnes J165, C.acnes J139, C.acnes SK182, C.acnes SK137 and C.acnes KPA171202, or a combination thereof.

Further bacteria suitable for the use in the methods or compositions of present invention can be selected from any species or strains from the group consisting of Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp., Streptococcus spp., Staphylococcus spp., Bacteroides spp., Prevotella spp., Clostridium spp., Bifidobacterium spp., Clostridium spp., Brevibacterium spp., Lactococcus spp., Leuconostoc spp., Actinobacillus spp., Selnomonas spp., Shigella spp., Zymonas spp., Mycoplasma spp., Treponema spp., Leuconostoc spp., Corynebacterium spp., Enterococcus spp., Enterobacter spp., Pyrococcus spp., Serratia spp., Morganella spp., Parvimonas spp., Fusobacterium spp., Actinomyces spp., Porphyromonas spp., Micrococcus spp., Bartonella spp., Borrelia spp., Brucella spp., Campylobacter spp., Chlamydophilia spp., Ehrlichia spp., Haemophilus spp., Leptospira spp., Listeria spp., Mycoplasma spp., Nocardia spp., Rickettsia spp., Ureaplasma spp., Cutibacterium (formerly Propionibacterium) spp., and Lactobacillus spp., or a combination thereof.

Specifically, the bacteria can be selected from the group consisting of Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Actinobacillus actinobycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphilococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Corynebacterium glutamicum, Corynebacterium jeikeium, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Enterococcus faecalis, Bacillus coagulans, Bacillus cereus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Streptomyces phaechromogenes, Streptomyces ghanaenis, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Morganella morganii, Citrobacter freundii, Pseudomonas aerigunosa, Parvimonas micra, Prevotella intermedia, Fusobacterium nucleatum, Prevotella nigrescens, Actinomyces israelii, Porphyromonas endodontalis, Porphyromonas gingivalis Micrococcus luteus, Bacillus megaterium, Aeromonas hydrophila, Aeromonas caviae, Bacillus anthracis, Bartonella henselae, Bartonella Quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Cutibacterium acnes (formerly Propionibacterium acnes), Cutibacterium granulosum, Ehrlichia canis, Ehrlichia chaff eensis, Enterococcus faecium, Francisella tularensis, Haemophilus influenza, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Nocardia asteroids, Rickettsia rickettsia, Salmonella enteritidis, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Shigella flexnerii, Shigella dysenteriae, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholera, Vibrio parahaemolyticus, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Actinobacter baumanii, Pseudomonas aerigunosa, or a combination thereof.

The step of characterizing the epigenetic imprinting of the isolates of step c) and/or d) as described in the methods of present invention can be performed using any method commonly known in the art for methylation detection. Such methods include, but are not limited to plaque assays, nanopore (ONT) sequencing, SMRT (Single-Molecule Real-Time sequencing), Dot blots, Methylation sensitive restriction digest coupled with sequencing, BS-seq (bisulfite sequencing) (Beaulaurier et al. (2019), Kurdyukov and Bullock (2016), Zhang et al. (2012)).

A plaque assay allows differentiation of bacterial lysis and whilst it does not provide for the exact sequence of the methylation pattern it gives a hint that the R-M system might be the cause for the differentiation.

Nanopore (ONT) sequencing as used in the Examples allows the detection of base-modifications (6 mA, 4mC, 5mC, 5hmC) by detecting changes in the ionic current induced by the passage of different nucleotides. Covalently modified bases can be differentiated from canonical bases which allows to identify chemical DNA modifications additionally to acquiring sequencing data.

SMRT (Single-Molecule Real-Time sequencing) allows detection of base-modifications and reports sequence and all three types of methylation (6 mA, 4mC, 5mC whereas confidence is high, moderate, low). DNA modifications are detected by identification of changes in kinetics of the Polymerase and comparison to control inter-pulse duration values.

Dot blots use an antibody against specific methylation which detect modified DNA by binding to it.

Methylation sensitive restriction digest coupled with sequencing use digestion of DNA by restriction enzymes followed by next generation sequencing. This method can detect all three methylation types (6 mA, 4mC, 5mC) but is limited by the restriction enzyme availability with known target sequence.

BS-seq (bisulfite sequencing) uses treatment of DNA by bisulfite followed by next generation Sequencing and can detect methylated cytosines in genomic DNA. The treatment with bisulfite of DNA mediates the deamination of cytosine into uracil which will be read a thymine and therefore modified cytosines can be differentiated from canonical ones. This technique can be used for 5mC.

It is finally contemplated that any features described herein can optionally be combined with any of the embodiments of any method, medical uses and compositions of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word “a” or “an” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one”. The use of the term “another” may also refer to one or more. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprises” also encompasses and expressly discloses the terms “consists of” and “consists essentially of”. As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.

As used herein, words of approximation such as, without limitation, “about”, “around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by +1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Accordingly, the term “about” may mean the indicated value±5% of its value, preferably the indicated value±2% of its value, most preferably the term “about” means exactly the indicated value (+0%).

As used herein, the term “bacteriophage” or “phage” refers to a functional phage particle comprising a nucleic acid genome packaged in a proteinaceous envelope or capsid. The term also refers to portions of the bacteriophage, including, e.g., a head portion, or an assembly of phage components, which provide substantially the same functional activity. Further phages that can be used in the methods and compositions according to present invention are summarized in the table here below:

Phage Name
Species
Strain
Sequenced
Cluster

Anatole

P. freudenreichii

TL 110
Yes
BV

Aquarius

P. acnes

ATCC 6919
Yes
BU

ATCC29399BC

P. acnes

ATCC 29399
Yes
BU

ATCC29399BT

P. acnes

ATCC 29399
Yes
BU

Attacne

P. acnes

ATCC 6919
Yes
BU

B22

P. freudenreichii

TL 110
Yes
BW

B3

P. freudenreichii

TL 110
Yes
BV

Boba

P. acnes

ATCC 6919
No
None

BruceLethal

P. acnes

ATCC 6919
Yes
BU

Cota

P. acnes

ATCC 6919
Yes
BU

Doucette

P. freudenreichii

TL 110
Yes
BW

DrParker

P. acnes

ATCC 6919
Yes
BU

E1

P. freudenreichii

TL 110
Yes
BV

E6

P. freudenreichii

LS2500
Yes
BW

Enoki

P. acnes

ATCC 6919
Yes
BU

G4

P. freudenreichii

Z435
Yes
BW

Keiki

P. acnes

ATCC 6919
Yes
BU

Kubed

P. acnes

ATCC 6919
Yes
BU

Lauchelly

P. acnes

ATCC 6919
Yes
BU

Leviosa

P. acnes

ATCC 6919
Yes
BU

LilBandit

P. acnes

ATCC 6919
Yes
BU

MEAK

P. acnes

ATCC 6919
Yes
BU

Moyashi

P. acnes

ATCC 6919
Yes
BU

MrAK

P. acnes

ATCC 6919
Yes
BU

Ouroboros

P. acnes

ATCC 6919
Yes
BU

P1.1

P. acnes

ATCC 6919
Yes
BU

P100.1

P. acnes

B100.4
Yes
BU

P100A

P. acnes

ATCC 6919
Yes
BU

P100D

P. acnes

ATCC 6919
Yes
BU

P101A

P. acnes

ATCC 6919
Yes
BU

P104A

P. acnes

ATCC 6919
Yes
BU

P104B

P. acnes

ATCC 6919
Yes
BU

P105

P. acnes

ATCC 29399
Yes
BU

P106A

P. acnes

ATCC 6919
Yes
BU

P106C

P. acnes

ATCC 6919
Yes
BU

P106I

P. acnes

ATCC 29399
Yes
BU

P106L

P. acnes

ATCC 6919
Yes
BU

P106M

P. acnes

ATCC 6919
Yes
BU

P107A

P. acnes

ATCC 29399
Yes
BU

P107C

P. acnes

ATCC 6919
Yes
BU

P108C

P. acnes

ATCC 6919
Yes
BU

P14.4

P. acnes

ATCC 6919
Yes
BU

P9.1

P. acnes

ATCC 6919
Yes
BU

PA6

P. acnes

AT1
Yes
BU

PAD20

P. acnes

AD20
Yes
BU

PAS50

P. acnes

AS50
Yes
BU

PFR1

P. freudenreichii

FD DVS PS-1
Yes
BX

PFR2

P. freudenreichii

str. (Australian Swiss)
Yes
BX

Pirate

P. acnes

ATCC 6919
Yes
BU

Procrass1

P. acnes

ATCC 6919
Yes
BU

QueenBey

P. acnes

ATCC 6919
Yes
BU

SKKY

P. acnes

ATCC 6919
Yes
BU

Solid

P. acnes

ATCC 6919
Yes
BU

Stormborn

P. acnes

ATCC 6919
Yes
BU

Supernova

P. acnes

ATCC 6919
Yes
BU

Wizzo

P. acnes

ATCC 6919
Yes
BU

The term “lysis” as used in the invention refers to the lytic activity of a bacteriophage causing the breaking down of the bacterial cell compromising its integrity. The term “killing” of the bacteria is equally used herein and can mean that the bacterium is killed due to the lytic activity of the bacteriophage. The lytic activity of a bacteriophage can be tested on for example C. acnes according to techniques known per se in the art (see also experimental section).

The term “specific” or “specificity” in relation to a bacteriophage refers to the type of host that said bacteriophage is able to infect, lyse and/or kill. A bacteriophage “specific” for a C. acnes strain refers to a bacteriophage which can infect, lyse and/or kill one specific C. acnes strain. A bacteriophage “specific” to C. acnes is a phage that is able to infect, lyse and/or kill only C. acnes and which essentially does not infect bacteria other than C. acnes under physiological conditions.

In the context of the present specification, the term “isolated bacteriophage” or “isolate of the one or more bacteriophages” should be considered to mean one or more bacteriophage(s) that is/are removed from its/their natural environment and/or separated from a component of its natural environment, such as for example its host bacterium.

The term “substantially homogenous population of phages” or “substantially homogenous population of bacteriophages” refers to a bacteriophage population with a high number of phages having the same infectivity patterns due to their epigenetic imprints as produced by the method of present invention, the number being high enough to confer the desired specificity for a certain strain. In a preferred embodiment of the invention the substantially homogenous population comprises at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of phages having the same infectivity patterns due to their epigenetic imprints as produced by the method of present invention.

EXAMPLES
Example 1: A) Homology-Based Search of Potential Restriction Methylation Systems Using REBASE, b) Genomic DNA Extraction c) Bacterial Lysates Preparation and d) C. Acnes Lysate Assay with Plasmid DNA

a) Homology-based search of potential restriction methylation systems using REBASE In order to identify the potential R-M systems present in C. acnes, all the predicted ORFs corresponding to either methyltransferases or restriction enzymes from those strains for which complete genome assembly was available were retrieved from the REBASE database. For the strains with no information available, their respective genomes from the NCBI Genome database and performed protein-protein BLAST with the annotated coding sequences to identify the potential R-M homologs were retrieved. To further assess the difference between systems across strains, pairwise comparisons between ORFs belonging to the same putative system type by sequence alignment using the Clustal Omega algorithm available at EMBL-EBI portal were performed (Madeira et al. 2019). A complete list of strains and accession numbers used in this study can be found in Table 1 below:

TABLE 1

List of strains and accession numbers used in this study

Strain
SLST
GeneBank

designation
type
Accession number
Notes

6609
H1
NC_017535.1

KCOM1315
H1
NZ_CP031442.1

KPA171202
H2
NC_006085.1
used in this study

PA_21_1_L1
H1
NZ_CP012351.1

PA_30_2_L1
D1
NZ_CP012350.1

09-9
K1
LKVB00000000.1
used in this study

ATCC11828
K9
NC_017550.1

KCOM1861
K2
NZ_CP012647.1

P.acnes17
F5
NC_016512.1

P.acnes31
F4
NC_016511.1

P.acnes33
F1
NC_016516.1

P.acnes 266
A1
NC_017534.1

ATCC6919
A1
NZ_CP023676.1

FDAARGOS 503
A1
NZ_CP033842.1

FDAARGOS 577
C1
NZ_CP033718.1

Hdn-1
A1
NZ_CP006032.1

HL096PA1
C1
NC_021085.1

PA_12.1.L1
A1
NZ_CP012354.1

PA_12.1.R1
A1
NZ_CP012353.1

PA_15.1.R1
C1
NZ_CP012355.1

PA_15.2.L1
A1
NZ_CP012352.1

SK137
C1
NC_014039.1

TP-CU 389
F1
NZ_AP019664.1

Laboratory Strain H1
H1

used in this study

Laboratory Strain A1
A1

used in this study

Laboratory Strain K2
K2

used in this study

Laboratory Strain A1
A1

used in this study

Laboratory Strain C1
C1

used in this study

b) Genomic DNA Extraction

C. acnes SLST types A1, C1, H1, H2, K1, K2 were grown on Brucella agar plates for 3 days. Bacteria were collected with a sterile cotton swab and inoculated in 1 mL BHI media, spun down 3 min 8000×g and resuspended in 1 mL lysis buffer (9,4 parts TE buffer pH 8,0 and 0,6 parts 10% SDS). All content was transferred to tubes prefilled with 0.1 mm silica beads and lysed in a mechanical homogenizer (Savant Bio 101 FastPrep FP120 Cell disruption system) for 25 sec at 6,5 speed. Samples were placed on ice for 1 min and the cycle was repeated once more. Next, 6 ul Proteinase K (20 mg/mL) and 5 ul RNAse (1 mg/mL) were added and incubated 1 h at 37° C. water bath. Equal volume Phenol/Chloroform was added, mixed and spun down at max speed for 5 min. Then, the aqueous phase was collected and repeated the previous step. Equal volume of chloroform was added, mixed and spun down at max speed for 5 min. The aqueous phase was collected and 2,5 volumes 100% ice-cold EtOH was added to precipitate DNA. Samples were kept for at least 1 h at −20° C. Then, they were spun down at max speed for 15 min at 4° C. Supernatant was discarded and the pellet was washed with 70° C. EtOH. After, the sample was spun down for 2 min at max speed. Once dried, the pellet was resuspended in 50 ul H2O and DNA concentration was measured using Qubit high sensitivity DNA kit (Thermo Fisher).

c) Bacterial Lysates Preparation

C. acnes strains were pre-cultured in 20 ml BHI broth with 180 rpm shaking at 37° C. during 24 hours under anaerobic conditions. Then, they were re-inoculated in 1 L of fresh BHI with 1200 rpm of stirring, maintaining the same conditions. After 72h, cells were harvested by centrifugation at 5000×g for 20 minutes and bacterial pellet was washed twice with 20 ml of cold K2PO4 50 mM, pH 7.4. Then, 0.33 ml of S30 buffer (10 mM Tris(CH3COO) (pH 8.2), 14 mM Mg (CH3COO) 2, 10 mM K (CH3COO) and 4 mM DTT) were added per gram of wet cell mass and the suspension was transferred into 2 ml microtubes prefilled with 0.1 mm glass beads. Using a mechanical homogenizer (Savant Bio 101 FastPrep FP120 Cell disruption system), cells were lysed following 2 cycles at 6000 rpm for 30 seconds. Samples were then filtered by centrifugation at 1000×g for 5 minutes at 4° C. using a 0.22 um filter unit. Then, the follow-through was centrifuged at 12.000 ×g for 12 minutes at 4° C. Finally, clear supernatants were collected and stored at −80° C. until they were used.

d) C. Acnes Lysate Assay with Plasmid DNA

C. acnes lysates were incubated with 0.5 ug plasmid DNA together with different buffers (NEB) supplemented with or without ATP or SAM. Cutsmart (NEB) buffer was chosen for further experiments. 8 ul of C. acnes concentrated lysate was mixed with 2 ul of Cutsmart buffer (1×final) and 0.5 ug of plasmid DNA was added. Final volume was adjusted to 20 ul and samples were incubated for 4 h at 37° C. Afterwards, DNA was observed on a 1% agarose gel.

Example 2: Infection of C. Acnes Strains Type IA1, IA2, IB, Type II and Type III with C. Acnes Bacteriophage PAD20

C. acnes SLST types A1, H1, H2 (KPA171202) and restriction-methylation deficient H2 (H2 KO) were infected with a high titer of C. acnes PAD20 phage extracted from either A1 or H1. Brucella top agar plates (0,6%) were prepared by mixing 4.5 mL Brucella agar with 0.5 ml Bacterial culture. After being solidified, phages were added in 10 ul drops and plates were incubated at 37° C. anaerobically for 3-5 days. After the first infection cycle, phages were extracted by adding 10 mL SM buffer (100 mM NaCl, 8 mM MgSO₄, 50 mM Tris-HCl PH 7.5, 0.01% gelatin) to the plates and incubating 30 min at RT carefully rotating. SM buffer was collected and NaCl was added to a final concentration of 0.5 M to release phages bound to bacterial debris. Samples were incubated for 30 min at RT and centrifuged at 8000 g for 20 min. Then, samples were filtered, and 10% (w/v) PEG 6000 was added. After 10 minutes of incubation, the phages were collected by centrifugation (10,000 ×g, 30 min). Finally, the pellet was resuspended in 300 ul of SM buffer. Phage concentration was determined by serial dilutions followed by plaque counting. EOP assay was performed by using an initial phage concentration of 5×10{circumflex over ( )}3 PFU/mL to re-infect C. acnes A1, A5, C1, C3, F4, H1, H2 (KPA171202), K1 (09-9) and K2 in serial dilutions. Plaquing efficiency of phages PAD20 A1, PAD20 H1, PAD20 H2 and PAD20 H2 KO was measured in PFU/mL and compared to each other.

As can be seen in FIG. 2, different lysis properties depending on which host the phage strain was extracted from could be observed. While the phage extracted from a type IA1 strain like A1 was unable to infect KPA (short for “KPA171202” also called “H2”), which belongs to type IB, it was able to infect other type IA1 or IA2 strains and H1. PAD20 extracted from KPA was able to kill A1, H1 and also itself. PAD20 from H1 on the other hand could infect A1 and itself but could not lyse H2. Strain K2, belonging to type II strains was unable to be lysed by any of the extracted PAD20 phages. This could be due to the availability of functional CRISPR Cas type I-E systems which includes spacers against PAD20 phages.

Example 3: Activity of the Restriction Methylation System

Differences in the activity of the type III restriction methylation system of the host KPA171202 were investigated by nanopore sequencing.

First, a methylation deficient KPA171202 mutant (KO mutant) was created following the method previously described by Sörensen et al. 2010. Shortly, 500 bp upstream (primers 468/469) and downstream (primers 470/471) of the locus to be replaced were amplified and cloned into the pGEM-T-easy vector. In a second step the erythromycin cassette was cloned between the homology arms using Acc65 I restriction sites and positive clones selected to be transformed into dam-GM2199 cells. C. acnes competent cells were prepared as previously described (Sörensen et al. 2010).

Correct knockouts were verified by junction PCR and WGS followed by a functional characterization using nanopore sequencing, RNAseq and phage infection assay. Nanopore sequencing verified the loss of methylated AGCAGY motifs in comparison to the wildtype genome and RNAseq showed loss of expression of the IIIB methylase.

The Nanopore libraries were prepared as follows: Genomic libraries corresponding to C. acnes A1, H1, H2, C1, K1 and K2 strains, PAD20 phage DNA and E. coli plasmids were prepared using the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore) according to the manufacturer's instructions. Native and WGA samples were barcoded using the Native Barcoding Kit (EXP-NBD104) and PCR Barcoding kit (EXP-PBC001), respectively. All samples were sequenced on the MinION platform either using a FLO-MIN106D (R9) flow cell or a FLO-FLG001 Flongle (R9.4.1) flow cell. Basecalling and demultiplexing were performed using Guppy v4.0.11. Nanopore sequencing data for the mentioned strain were analyzed using Tombo v1.5 software (Stoiber et al., n.d.) for detection of modified bases. In brief, current signals were compared against the KPA171202 reference genome using the “resquiggle” command. Then, methylation peaks were detected comparing the native sample and the whole genome amplification (WGA) sample for each strain using the “detect_modifications model_sample_compare” command. For the detection of methylated motifs, the 50 bp context of 1000 positions with the highest modified fraction (i.e., the fraction of reads identified as potentially methylated over all reads mapping to the given position in the genome) were extracted using the “text_output signif_sequence_context” command. This set of sequences was processed using MEME (Bailey et al. 2015) with ‘zoops’ mode to identify common sequences. Statistically significant modified fraction values for each genomic position were exported from tombo using tombo's “text_output browser_files” and were further analysed using a custom R script (see code availability). A similar procedure was used to analyse the Phage DNA and plasmid samples. For phage DNA, the genome reference was assembled de novo using Fyle (Kolmogorov et al. 2019) with the default parameters. Fine typing of the methylation motif was performed using the multi-label classification algorithm present in the Nanodisco (Tourancheau et al. 2021) toolkit according to its documentation.

As shown in FIG. 1A) the KO mutants (methylation deficient KPA 171202) did not have the corresponding restriction methylation system anymore and were therefore unable to methylate its own DNA in a specific pattern.

To verify the functionality of the methylase itself the inventors have expressed the methylase of H1 and H2 recombinantly by shuttling plasmid DNA through this dam-dcm-E. coli strain expressing the corresponding C. acnes methylase (FIG. 1B). Nanopore sequencing showed a functional H2 methylase by methylating plasmid DNA while H1 methylase was unable to do SO.

The KO mutant strain was then infected with PAD20 extracted from either H1, A1 or KPA and the activity of the Restriction methylation system measured. The KO mutant strain became susceptible to all phages regardless of where is was previously extracted from. This confirmed the hypothesis that epigenetic changes could be involved in phage host range specificity.

To understand if C. acnes H2 (KPA171202) is methylating phage DNA during infection, bacteria was infected with phages from H1 and H2 and respectively isolated after. Extracted phage DNA was subjected to nanopore sequencing. Methylation of phage DNA by nanopore sequencing was measured and the specific methylation pattern identified (FIG. 1C).

Example 4: Specific Killing of a Population Comprising Two Different C. Acnes Strains

A strain from subgroup IA1 (A1) was inoculated 1:1 with one of subgroup IB (H1) and this mix was infected with a phage extracted from A1 host. After 3 days a subset of bacteria was plated on agar plates and a subset was checked for classification by SLST sequencing. All tested colonies belonged to H1 C. acnes strain which meant a 100% host range specificity of PAD20 phage. These findings confirm strong selectivity of the tested phage to its host.

Example 5: Specific Killing on a Whole Microbiome Culture Extracted from Human Skin

A diverse bacterial population was inoculated with PAD20 C. acnes phage extracted from A1 and samples were taken at 3 different time points to observe specific killing of certain strains in the growing population. Changes depending on strain level but also Clade level were compared. A decrease in Clade IA1, especially A1 strain, was observed, while an increase could be observed in Clade IB. Type II strains had a slight decrease in its SLST types but stayed quite stable over time. This could be linked to potential CRISPR Cas systems in a subset of type II strains in the culture (FIG. 2C).

LIST OF REFERENCES

1. Chen, Y. et al. Genetic Engineering of Bacteriophages Against Infectious Diseases. Front. Microbiol. 10, 954 (2019).

2. Samson, J. E., Magadán, A. H., Sabri, M. & Moineau, S. Revenge of the phages: defeating bacterial defences. Nat. Rev. Microbiol. 11, 675-687 (2013).

3. Seed, K. D. Battling Phages: How Bacteria Defend against Viral Attack. PLOS Pathog. 11, e1004847 (2015).

4. Furi, L. et al. Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host. J. Bacteriol. 201, (2019).

5. Bryson, A. L. et al. Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9. MBio 6, e00648 (2015).

6. Dréno, B. et al. Cutibacterium acnes (Propionibacterium acnes) and acne vulgaris: a brief look at the latest updates. Journal of the European Academy of Dermatology and Venereology vol. 32 5-14 (2018).

7. Lood, R., Morgelin, M., Holmberg, A., Rasmussen, M. & Collin, M. Inducible Siphoviruses in superficial and deep tissue isolates of Propionibacterium acnes. BMC Microbiol. 8, 139 (2008).

8. Paetzold, B. et al. Skin microbiome modulation induced by probiotic solutions. Microbiome 7, 95 (2019).

9. Madeira, F. et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47, W636-W641 (2019).

10. Brüggemann, H. et al. The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Science 305, 671-673 (2004).

11. Sorensen, M. et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants. J. Microbiol. Methods 83, 211-216 (2010).

12. Stoiber, M. et al. De novoldentification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing. doi: 10.1101/094672.

13. Bailey, T. L., Johnson, J., Grant, C. E. & Noble, W. S. The MEME Suite. Nucleic Acids Res. 43, W39-49 (2015).

14. Kolmogorov, M., Yuan, J., Lin, Y. & Pevzner, P. A. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37, 540-546 (2019).

15. Tourancheau, A., Mead, E. A., Zhang, X.-S. & Fang, G. Discovering multiple types of DNA methylation from bacteria and microbiome using nanopore sequencing. Nat.

Methods 18, 491-498 (2021).

16. Vasu K, Nagaraja V. Diverse functions of restriction-modification systems in addition to cellular defense. Microbiol Mol Biol Rev. 77:53-72 (2013).

17. Beaulaurier J, Schadt E E, Fang G. Deciphering bacterial epigenomes using modern sequencing technologies. Nat Rev Genet. 2019; 20 (3): 157-172. doi: 10.1038/s41576-018-0081-3

18. Kurdyukov S, Bullock M. DNA Methylation Analysis: Choosing the Right Method. Biology (Basel). 2016; 5 (1): 3. Published 2016 Jan. 6. doi: 10.3390/biology5010003

19. Zhang G, Wang W, Deng A, Sun Z, Zhang Y, et al. (2012) A Mimicking-of-DNA Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria. PLOS Genetics 8 (9): e1002987. https://doi.org/10.1371/journal.pgen. 1002987

METHOD FOR SCREENING FOR MODIFICATIONS IN THE INFECTIVITY RANGE OF BACTERIOPHAGES DUE TO EPIGENETIC IMPRINTING

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