Proteomic characterization of bacteriophage peptides from mastitis producer Staphylococcus aureus by LC-ESI-MS/MS and the bacteriophage phylogenomic analysis

foods Article Proteomic Characterization of Bacteriophage Peptides from the Mastitis Producer Staphylococcus aureus by LC-ESI-MS/MS and the Bacteriophage Phylogenomic Analysis Ana G. Abril 1, Mónica Carrera 2,* , Karola Böhme 3, Jorge Barros-Velázquez 4, Benito Cañas 5, José-Luis R. Rama 1, Tomás G. Villa 1and Pilar Calo-Mata 4,*   Citation: Abril, A.G.; Carrera, M.; Böhme, K.; Barros-Velázquez, J.; Cañas, B.; Rama, J.-L.R.; Villa, T.G.; Calo-Mata, P. Proteomic Characterization of Bacteriophage Peptides from the Mastitis Producer Staphylococcus aureus by LC-ESI-MS/MS and the Bacteriophage Phylogenomic Analysis. Foods 2021,10, 799. https://doi.org/10.3390/foods10040799 Received: 8 March 2021 Accepted: 6 April 2021 Published: 8 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, 15898 Santiago de Compostela, Spain; [email protected] (A.G.A.); [email protected] (J.-L.R.R.); [email protected] (T.G.V.) 2Department of Food Technology, Spanish National Research Council, Marine Research Institute, 36208 Vigo, Spain 3Agroalimentary Technological Center of Lugo, 27002 Lugo, Spain; [email protected] 4Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spain; jorge.barr[email protected] 5Department of Analytical Chemistry, Complutense University of Madrid, 28040 Madrid, Spain; [email protected] *Correspondence: [email protected] (M.C.); [email protected] (P.C.-M.) Abstract: The present work describes LC-ESI-MS/MS MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analyses of tryptic digestion peptides from phages that infect mastitis-causing Staphylococcus aureus isolated from dairy products. A total of 1933 nonredundant peptides belonging to 1282 proteins were identified and analyzed. Among them, 79 staphylococcal peptides from phages were confirmed. These peptides belong to proteins such as phage repressors, structural phage proteins, uncharacterized phage proteins and complement inhibitors. Moreover, eighteen of the phage origin peptides found were specific to S. aureus strains. These diagnostic peptides could be useful for the identification and characterization of S. aureus strains that cause mastitis. Furthermore, a study of bacteriophage phylogeny and the relationship among the identified phage peptides and the bacteria they infect was also performed. The results show the specific peptides that are present in closely related phages and the existing links between bacteriophage phylogeny and the respective Staphylococcus spp. infected. Keywords: pathogendetection;LC-ESI-MS/MS;proteomics; massspectrometry;phagepeptidebiomarker 1. Introduction The vast majority of mastitis cases are due to an intramammary infection caused by a microorganism belonging to either the Staphylococcus or Streptococcus genus [ 1 , 2 ]. Staphylococcus aureus is considered one of the major foodborne pathogens that can cause serious food intoxication in humans due to the production of endotoxins; this pathogen remains a major issue in the dairy industry due to its persistence in cows, its pathogenicity, its contagiousness and its ease of colonization of the skin and mucosal epithelia [3–5]. It is well-known that S. aureus bacteriophages encode genes for staphylococcal virulence factors, such as Panton-Valentine leucocidin, staphylokinase, enterotoxins, chemotaxisinhibitory proteins or exfoliative toxins [ 6 ]. These phages are usually integrated into bacterial chromosomes as prophages, wherein they encode new properties in the host, or vice versa, as transcriptions may hardly be affected by gene disruptions [ 7 ]. Phage-encoded recombinases, rather than the host recombinase, RecA, are involved in bacterial genome excisions and integrations [ 8 , 9 ]. These integrations may occur at specific bacterial genome sites that are identical to those present in the DNA of the phage, or, as in the case of phage Foods 2021,10, 799. https://doi.org/10.3390/foods10040799 https://www.mdpi.com/journal/foods Foods 2021,10, 799 2 of 21 Mu (as long as the given gene is not expressed), some phages can integrate randomly within the bacterial genome. In addition, bacteriophage and staphylococcal species interactions may substantially alter the variability of the bacterial population [10,11]. All known S. aureus phages are composed of an icosahedral capsid filled with doublestranded DNA and a thin, filamentous tail, and they belong to the order Caudovirales (tailed phages) [ 12 , 13 ]. Some Podoviridae family phages, such as the Staphylococcus viruses S13 0 and S24-1, have been reported, characterized and used in phage therapy against S. aureus infections [ 14 ]. There are some well-known Siphoviridae phages of S. aureus, such as the prophage ϕSaBov, which is integrated into a bovine mastitis-causing S. aureus strain [15]. The interaction between bacteria and bacteriophages leads to an exchange of genetic information, which enables bacteria to rapidly adapt to challenging environmental conditions and to be highly dynamic [ 11 , 16 ]. As closely related phages normally occupy the same genome location in different bacteria, a specific site in different bacterial strains can be occupied by completely different phages or can be empty. Conventional culture-based methods have been used for the detection of pathogenic bacteria [ 17 , 18 ] and their phages [ 19 , 20 ]; however, at this point, these procedures are timeconsuming and laborious. For this reason, new, rapid molecular microbial diagnostic methods based on genomics and proteomics tools have been developed to achieve faster and more efficient bacterial and bacteriophage identification [ 1 , 21 – 24 ]. Specifically, phage typing is a classic technique for such purposes [ 25 ]. Moreover, biosensors based on phage nucleic acids, receptor-binding proteins (RBPs), antibodies and phage display peptides (PDPs) have been used for pathogen detection [26–30]. Mass spectrometry techniques, such as MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and LC-ESI-MS/MS (liquid chromatographyelectrospray ionization-tandem mass spectrometry), have been used for the analysis and detection of specific diagnostic peptides in pathogenic bacterial strains [ 31 , 32 ]. In addition, LC-ESI-MS/MS methods have been employed for the identification and detection of bacteriophages [ 19 ]. In the case of bacteriophage detection and identification by a mass spectrometry analysis, the required production of viruses may be time-consuming. The detection of prophages based on protein biomarkers can be an alternative to genomic detection, and in this sense, proteomic techniques can be cheaper and faster and can ascertain different bacteriophage species by using a single analysis [ 33 ]. Based on the specificity of many bacteriophages with their hosts, bacteriophages are considered signal amplifiers; therefore, the detection of peptides from phages is suitable for pathogen identification. For example, Serafim et al. 2017 [ 33 ] identified bacteriophage lambda by a LC-ESI-MS/MS analysis. Moreover, the identification of peptides by means of LC-ESIMS/MS from bacteriophage-infected Streptococcus has been performed, which revealed new information on phage phylogenomics and their interactions with the bacteria they infect [ 19 ]. However, no study has been published on S. aureus phage detection and identification by LC-ESI-MS/MS or on S. aureus phage characterization without a previous phage purification step. Viral genomic detection and phage display are time-consuming methods. Here, we describe an easy, fast and accurate method for the detection of bacteriophages without the need for the pretreatment of bacterial lysis for bacteriophage replication. This method led to the identification of putative temperate and virulent phages present in the analyzed strains. A previously published work performed by our laboratory [ 3 ] studied the global proteome of several strains of S. aureus by shotgun proteomics. Important virulence protein factors and functional pathways were characterized by a protein network analysis. In this work, and for the first time, we aimed to use proteomics to characterize phage contents in different S. aureus strains to identify the relevant phage-specific peptides of several S. aureus strains and to identify both phages and bacterial strains by LC-ESI-MS/MS. Foods 2021,10, 799 3 of 21 2. Materials and Methods 2.1. Bacteria In this study, a total of 20 different S. aureus strains obtained from different sources were analyzed (Table S1 in Supplemental Data 2). These strains were previously characterized by MALDI-TOF mass spectrometry [ 1 ] after being obtained from the Institute of Science of Food Production of the National Research Council of Italy (Italy) and from the Spanish Type Culture Collection (Spain). The majority of the strains are from food origins, except for strain U17, which is a human clinical strain. Strains ATCC (American Type Culture Collection) 9144 and ATCC 29213 are classified as S. aureus subsp. aureus, while strain ATCC 35845 is categorized as S. aureus subsp. anaerobius. In previous works, the species identification of S. aureus and the presence of enterotoxins were evaluated by multiplex polymerase chain reactions (multiplex PCRs) [ 3 , 34 , 35 ]. The strains were reactivated in a brain–heart infusion medium (BHI, Oxoid Ltd., Hampshire, UK) and incubated at 31 ◦ C for 24 h. Bacterial cultures were then grown on plate count agar (PCA, Oxoid) at 31 ◦ C for 24 h [1,3,36]. Tubes of broth were inoculated under aerobic conditions. 2.2. Protein Extraction and Peptide Sample Preparation Protein extraction was prepared as described previously [ 37 ]. All analyses were performed in triplicate. Protein extracts were subjected to in-solution tryptic digestion [ 38 ]. 2.3. Shotgun LC-MS/MS Analysis Peptide digests were acidified with formic acid (FA), cleaned on a C18 MicroSpin ™ column (The Nest Group, South-borough, MA, USA) and analyzed by LC-ESI-MS/MS using a Proxeon EASY-nLC II Nanoflow system (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) [ 3 ]. Peptide separation (2 µ g) was performed on a reverse-phase (RP) column (EASY-Spray column, 50 cm × 75 µ m ID, PepMap C18, 2µ m particles, 100-Å pore size, Thermo Fisher Scientific, San Jose, CA, USA) with a 10-mm precolumn (Accucore XL C18, Thermo Fisher Scientific, San Jose, CA, USA) using a linear 120-min gradient from 5% to 35% solvent B (solvent A: 98% water, 2% ACN (Acetonitrile) and 0.1% FA and solvent B: 98% ACN, 2% water and 0.1% FA) at a flow rate of 300 nL/min. For ionization, a spray voltage of 1.95 kV and a capillary temperature of 230 ◦ C were used. Peptides were analyzed in the positive mode from 400 to 1600 amu (1 µ scan), which was followed by 10 data-dependent collision-induced dissociation (CID) MS/MS scans (1 µ scan) using an isolation width of 3 amu and a normalized collision energy of 35%. Fragmented masses were set in dynamic exclusion for 30 s after the second fragmentation event, and unassigned charged ions were excluded from the MS/MS analysis. 2.4. LC-MS/MS Mass Spectrometry Data Processing LC-ESI-MS/MS spectra were searched using SEQUEST-HT (Proteome Discoverer 2.4, Thermo Fisher Scientific, San Jose, CA, USA) against the S. aureus UniProt/TrEMBL database (208,158 protein sequence entries in July 2020). The following parameters were used: semi-tryptic cleavage with up to two missed cleavage sites and tolerance windows set at 10 ppm for the precursor ions and 0.06 Da for the MS/MS fragment ions. These additional identified semi-tryptic peptides increased the sequence coverage and confidence in protein assignments. The variable modifications that were allowed were as follows: (M*) methionine oxidation (+15.99 Da), (C*) carbamidomethylation of Cys (+57.02 Da) and acetylation of the N-terminus of the protein (+42.0106 Da). To validate the peptide assignments, the results were subjected to a statistical analysis with the Percolator algorithm [ 39 ]. The false discovery rate (FDR) was kept below 1%. The mass spectrometric data were deposited into the public database PRIDE (Proteomics Identification Database), with the dataset identifier PXD023530. Foods 2021,10, 799 4 of 21 2.5. Selection of Potential Peptide Biomarkers For each peptide identified by LC-ESI-MS/MS, we used the BLASTp program to determine the homologies and exclusiveness with protein sequences registered in the NCBI (National Center for Biotechnology Information) database [ 40 ]. For the BLASTp search, the Staphylococcus taxon was included and excluded with the aim of finding the peptides that belonged to the Staphylococcus phages, Staphylococcus spp. and only to S. aureus. 2.6. Phage Genome Comparison and Relatedness Genomes of all studied Staphylococcus spp. phages were downloaded from the GenBank database, analyzed and compared using the Web server VICTOR (Virus Classification and Tree Building Online Resource, http://ggdc.dsmz.de/victor.php, accessed on 27 November 2020) for the calculation of the intergenomic distances and the construction of the phylogenomic tree [41]. 3. Results 3.1. S. aureus Proteome Repository Protein mixtures from each of the 20 different S. aureus strains (Table S1 in Supplemental Data 2) were digested with trypsin and analyzed by LC-ESI-MS/MS. A total of 1933 nonredundant peptides corresponding to 1282 nonredundant annotated proteins were identified for all S. aureus strains (see the Excel dataset in Supplemental Data 1 ). Among them, 79 phage peptides were identified. These peptides belong to proteins such as phage repressors, structural phage proteins, uncharacterized phage proteins and complement inhibitors. Figure 1shows a comparative representation of the different types of phage proteins identified in this study. These phage peptides were selected and analyzed using the BLASTp algorithm. For the BLASTp search, Staphylococcus was included and excluded with the aim of finding peptides belonging to Staphylococcus bacteriophages. Foods 2021, 10, x FOR PEER REVIEW 12 of 23 S3 Uncharacterized phage protein EFRNKLNELGADK Staphylococcus aureus, Streptococcus pneumoniae, Terrabacteria group Staphylococcus phage phi7401PVL, Staphylococcus phage tp310-2, Staphylococcus phage vB_SauS_phi2, Staphylococcus virus IPLA35 , Staphylococcus phage phiSa2wa_st30, Staphylococcus virus 47, Staphylococcus virus 3a S3 Phage repressor, Cro/CI family HLEEVDIR Staphylococcus aureus, Paxillus involutus ATCC 200175, Brassica cretica, Staphylococcus epidermidis, Staphylococcus spp., Enterobacter hormaechei S4 YhgE/Pip; Phage infection protein APQSTSVKK Staphylococcus aureus, Staphylococcus schweitzeri, Staphylococcus sp. S4 YhgE/Pip Phage infection protein ALNFAADDVPAQFPK S. aureus, Staphylococcus sp. HMSC36A10, Staphylococcus sp. HMSC34H10, Pseudomonas aeruginosa, E. coli Figure 1. Comparative representation of different types of phage proteins identified in this study for the different strains (represented by different colors). The number of each type of protein is shown in parentheses. All staphylococcal phage peptides with 100% homology were found to belong to the Siphoviridae family: 52 staphylococcal phages belong to the Phietavirus genus, 37 belong to the Biseptimavirus genus, 30 are Triavirus, two are phieta-like viruses and one is a SPbetalike virus, and the others are nonclassified Siphoviridae viruses (Table S2 in Supplemental Data 2). Siphoviridae genomes are usually organized into functional modules, such as lysogeny, DNA replication, packaging, morphogenesis and lysis modules [6,42]. Figure 1. Comparative representation of different types of phage proteins identified in this study for the different strains (represented by different colors). The number of each type of protein is shown in parentheses. Foods 2021,10, 799 5 of 21 The obtained staphylococcal phage-specific peptides shared homology with the Staphylococcus phages and Staphylococcus spp. in the NCBI database. Among them, all shared homology with S. aureus; however, eighteen peptides were specific to S. aureus (IRLPYYDVK, LYVGVFNPEATK, SIINGKLDSQWTVPNEHK, M*NDSNQGLQANPQYTIHYLSQEITR, PCPALM*NKRNSIATIHR, SQDSNLTPELSTKAPK, ESINANTYINQNLEK, VAVLSTPLVTSFESK, KDGEILFDAIDIYLRNK, MPVYKDGNTGKWYFSI, KTTSEALKEVLSDT, EPKPVDATGADDPLKPDDRM*ITNFHANLVDQKVSY, MSHNALTTGIGIGAGAG, VQHPGKLVNKVM*SGLNINFGGGANATAK, QM*MEGLSGVMDLAAVSGEDLGAVSDIVTDGLTA FGLKAKDSG, KSNVEAFSNAVK,GMVASMQMQVVQVNVLTM*ELAQQNAMLTQQLTELK and DIITVYC*PENGTATDEY). Figure S1 shows the MS/MS spectra for these S. aureusspecific peptide biomarkers. Table 1summarizes the list of 79 specific staphylococcal bacteriophage peptides, bacterial peptides with putative phage origins and bacteria and phages with 100% homology with respect to the NCBI protein database. All staphylococcal phage peptides with 100% homology were found to belong to the Siphoviridae family: 52 staphylococcal phages belong to the Phietavirus genus, 37 belong to the Biseptimavirus genus, 30 are Triavirus, two are phieta-like viruses and one is a SPbeta-like virus, and the others are nonclassified Siphoviridae viruses (Table S2 in Supplemental Data 2 ). Siphoviridae genomes are usually organized into functional modules, such as lysogeny, DNA replication, packaging, morphogenesis and lysis modules [6,42]. Table 1. Phage origin peptides identified in Staphylococcus aureus strains. NCBI (National Center for Biotechnology Information). Strain Protein Peptide Bacteria with 100% Homology Based on the NCBI Protein Database Phages with 100% Homology Based on the NCBI Protein Database S4 Uncharacterized phage protein IRLPYYDVK Staphylococcus aureus Staphylococcus phage StauST398-2 S4 Uncharacterized phage protein AVAELLKEINR Staphylococcus argenteus Staphylococcus simiae Staphylococcus aureus Staphylococcus virus 71 Staphylococcus virus 55 Staphylococcus virus 88 S4 Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus aureus Staphylococcus xylosus Staphylococcus muscae Staphylococcus haemolyticus Staphylococcus argenteus Streptococcus pneumoniae Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3a Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL S4 Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus aureus 12S01399 Staphylococcus aureus Staphylococcus aureus A9299 Staphylococcus aureus A9765 Staphylococcus argenteus Staphylococcus aureus A6300 Staphylococcus sp. Terrabacteria group Escherichia coli Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 S7 Major tail protein LYVGVFNPEATK Staphylococcus aureus Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 S8 Uncharacterized phage protein M*NDSNQGLQANPQYTIHYLSQEITR Staphylococcus aureus Staphylococcus phage phiN315 Foods 2021,10, 799 6 of 21 Table 1. Cont. Strain Protein Peptide Bacteria with 100% Homology Based on the NCBI Protein Database Phages with 100% Homology Based on the NCBI Protein Database S8 Major tail protein AYINITGLGFAK Staphylococcus aureus Staphylococcus argenteus Pararheinheimera mesophila Staphylococcus phage phiNM3 Staphylococcus phage P282 Staphylococcus phage StauST398-4 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 S9 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus aureus Staphylococcus argenteus Staphylococcus virus 85 Staphylococcus phage SP5 Staphylococcus virus phiETA2 Staphylococcus phage phiNM2 Staphylococcus virus SAP26 Staphylococcus phage SA12 Staphylococcus virus Baq Sau1 S11 and S20 Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus aureus Staphylococcus argenteus Staphylococcus sciuri Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 S11 and S20 Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus aureus Staphylococcus sciuri Staphylococcus pseudintermedius Staphylococcus devriesei Staphylococcus warneri Staphylococcus capitis Staphylococcus argenteus Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Staphylococcus virus IPLA35 S12 S10 and S14 Complement inhibitor IYNEIDEALKSK Staphylococcus aureus, Enterobacter sp. IF2SW-B1 Klebsiella pneumoniae Staphylococcus phage 13 Staphylococcus phage phiNM3 Staphylococcus phage StauST398-1 S20 Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus aureus Staphylococcus haemolyticus Staphylococcus saprophyticus Staphylococcus warneri Staphylococcus argenteus Streptococcus pneumoniae Staphylococcus sciuri Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL S20 Phage protein (DUF2479 domain) SIINGKLDSQWTVPNEHK Staphylococcus aureus Staphylococcus phage DW2 Staphylococcus virus IPLA88 S18 N-acetylmuramoyl-Lalanine amidase KEAGNYTVANVK Bacilli, Staphylococcus argenteus Staphylococcus aureus Staphylococcus sp. HMSC34H10 Staphylococcus phage tp310-1 Staphylococcus phage tp310-2 Staphylococcus phage phi2958PVL Staphylococcus phage PVL Staphylococcus phage SA137ruMSSAST121PVL Staphylococcus virus IPLA35 S4 Phage protein NrdI VETFLENETNQNNLIAVM* SSGNRNWGTNFAIAGDTISK Staphylococcus haemolyticus Staphylococcus hominis Staphyloccus aureus Staphylococcus aureus subsp. aureus Z172 S12 Complement inhibitor IYNEIDEALK Staphylococcus. Aureus Klebsiella pneumoniae Enterobacter sp. IF2SW-B1 Staphylococcus phage StauST398-1 Staphylococcus virus 13 S10 Complement inhibitor IYNEIDEALKSKY Staphylococcus. aureus Klebsiella pneumoniae Enterobacter sp. IF2SW-B2 Staphylococcus phage StauST398-1 Staphylococcus virus 13 S10 DDE-type integrase/transposase/ recombinase PC*PALM*NKRNSIATIHR Staphylococcus aureus S9 DNA primase phage-associated LLHHFYNPENTTALSF NDLNDKFKPANLQGKLVNIAD Staphylococcus aureus, Staphylococcus haemolyticus Staphylococcus capiti, Staphylococcus epidermidis Staphylococcus warneri Staphylococcus sp. HMSC077D08 Corynebacterium propinquum, Staphylococcus sp. U Staphylococcus lugdunensis Staphylococcus sp. HMSC077B09 Uncultured Caudovirales Phage Foods 2021,10, 799 7 of 21 Table 1. Cont. Strain Protein Peptide Bacteria with 100% Homology Based on the NCBI Protein Database Phages with 100% Homology Based on the NCBI Protein Database S2 Phage repressor, Cro/CI family AAHLEGELTDDEWQR Staphylococcus haemolyticus Staphylococcus warneri Staphylococcus agnetis, Staphylococcus chromogenes Staphylococcus haemolyticus Staphylococcus sp. 58-22 Staphylococcus capitis Staphylococcus pasteuri Bacillales Staphylococcus chromogenes Staphylococcus agnetis Escherichia coli, Staphylococcus aureus 08-02906 Staphylococcus aureus VET0383R, Staphylococcus aureus VET0098R Staphylococcus aureus M1487 Staphylococcus aureus, Staphylococcus aureus A6300 Staphylococcus aureus subsp. aureus str. Newman Staphylococcus aureus subsp. aureus WBG10049, Staphylococcus aureus A9635, Staphylococcus aureus subsp. aureus MN8 Staphylococcus virus 71 Staphylococcus phage phiSa2wa_st1 Staphylococcus phage phiSa2wa_st5 Staphylococcus phage Henu2 Staphylococcus phage ROSA Staphylococcus phage phi7401PVL S2 Phage repressor, Cro/CI family VLDYADYIR Staphylococcus aureus Staphylococcus epidermidis Staphylococcus warneri Staphylococcus agnetis Staphylococcus warneri Staphylococcus chromogenes, staphylococcus spp. Staphylococcus schleiferi Staphylococcus simulans Staphylococcus haemolyticus, Staphylococcus pettenkoferi Staphylococcus lugdunensis Escherichia coli Staphylococcus virus 71 Staphylococcus phage phiSa2wa_st1 Staphylococcus phage phiSa2wa_st5 Staphylococcus phage Henu2 Staphylococcus phage ROSA Staphylococcus phage phi7401PVL S9 DNA-binding protein SLDNM*SLK Striga asiática Staphylococcus aureus subsp. aureus 112808A Staphylococcus aureus A8819 Staphylococcus argenteus Staphylococcus spp. Pseudomonas aeruginosa Flectobacillus sp. BAB-3569 Eoetvoesia caeni Arabidopsis thaliana,Coxiellaceae bacterium,Clostridia bacterium Staphylococcus phage vB_SauS_phi2 S19 DUF2479, Phage tail fiber, BppU family phage baseplate upper protein HAGYVRC*KLF Staphylococcus aureus,Staphylococcus sp. HMSC055H07 Staphylococcus argenteus, Staphylococcus sp. KY49P Staphylococcus sp. HMSC035F11 Pseudomonas aeruginosa Escherichia coli Staphylococcus phage SA97 Staphylococcus virus 55 uncultured Caudovirales phage Staphylococcus virus 85 Staphylococcus virus 80 Staphylococcus virus phiETA3 Staphylococcus virus phiETA2 Staphylococcus phage 55-2 Staphylococcus phage B166 Staphylococcus phage B236 Staphylococcus virus SAP26 Staphylococcus virus 88 Staphylococcus virus phiETA Staphylococcus virus 11 Staphylococcus phage SP5 Staphylococcus virus 69 Staphylococcus phage ROSA Staphylococcus phage TEM123 Staphylococcus virus 92 Staphylococcus phage StauST398-1 Staphylococcus virus phiNM2 Staphylococcus virus phiNM1 Staphylococcus virus 29 Staphylococcus phage vB_SauS-SAP27 Staphylococcus virus 80alpha Staphylococcus phage HSA84 Staphylococcus virus phiMR11 Staphylococcus phage SAP33 Staphylococcus phage 3MRA S12 Phage protein (DUF4393 domain) NSPIDLNSTEISLNNLER Staphylococcus aureus Staphylococcus spp. Staphylococcus argenteus Staphylococcus phage StauST398-1 S12 Phage protein (DUF669 domain) MNFNLNLQGAQELGN Staphylococcus capitis Staphylococcus epidermidis Staphylococcus caprae Staphylococcus devriesei Staphylococcus warneri Staphylococcus virus phiMR11 S10 GNAT family N-acetyltransferase IINYARQNNYESLLTSIVSNNIGAK Staphylococcus aureus Staphylococcus aureus subsp. anaerobius Staphylococcus aureus subsp. aureus Mu50 Staphylococcus hominis Escherichia coli Foods 2021,10, 799 8 of 21 Table 1. Cont. Strain Protein Peptide Bacteria with 100% Homology Based on the NCBI Protein Database Phages with 100% Homology Based on the NCBI Protein Database S5 Holin, phage phi LC3 family SQDSNLTPELSTKAPK Staphylococcus aureus Staphylococcus phage HSA84 Staphylococcus phage SP5 S6 ImmA/IrrE family metallo-endopeptidase EKAKIFGDFDMNDSGVY DEENSTIIYNPLDSITR Staphylococcus aureus subsp. aureus H19 Staphylococcus aureus Staphylococcus aureus subsp. aureus Staphylococcus aureus subsp. aureus 21204 S16 Involved in the expression of fibrinogen-binding protein phage-associated ESINANTYINQNLEK Staphylococcus aureus S16 Involved in the expression of fibrinogen-binding protein phage-associated VAVLSTPLVTSFESK Staphylococcus aureus S17 N-6 DNA methylase; N6_Mtase domain-containing protein KDGEILFDAIDIYLRNK Staphylococcus aureus Staphylococcus phage phi-42 S4 Phage DNA-binding protein GDM*FVVITIM*MQQIK Staphylococcus aureus Staphylococcus warneri S9 Phage terminase KLYIIEEYVKQGM Staphylococcus aureus Staphylococcus argenteus Staphylococcus sp. HMSC58E11 Allobacillus sp. SKP4-8 Staphylococcus virus Baq_Sau1 Staphylococcus virus phiETA2 Staphylococcus virus 69 Staphylococcus virus 11 Staphylococcus virus 80alpha S14 Integrase M*PVYKDGNTGKWYFSI Staphylococcus aureus Staphylococcus phage B166 Staphylococcus virus phiMR25 Staphylococcus virus 88 S4 Phage repressor ISKVQQLADYFNVPK Staphylococcus aureus, Staphylococcus chromogenes Staphylococcus hyicus Staphylococcus virus 80 S13 Toxin Phage protein; Pathogenicity island protein NLDGVWLGDLILIKRGLSDR Staphylococcus aureus, Staphylococcus sp. HMSC58E11, Staphylococcus argenteus, Escherichia coli Staphylococcus phage phiSa2wa_st80 Staphylococcus phage 3MRA Staphylococcus phage phiSa2wa_st5 S16 Toxin Phage protein; Pathogenicity island protein SDREKAGILFEELAHNK Staphylococcus aureus Escherichia coli Staphylococcus argenteus Staphylococcus sp. HMSC58E11 Staphylococcus phage 3MRA Staphylococcus phage phiSa2wa_st5 Staphylococcus phage phiSa2wa_st80 Staphylococcus phage phiJB Staphylococcus phage phi7401PVL S6 PBSX family phage terminase QADNTYVHHSTYLNNP FISKQFIQEAESAKQR Staphylococccus spp. S11 PBSX family phage terminase QGVSHLFKVTKSPM*R Staphylococcus aureus Staphylococcus lentus Staphylococcus sciuri S20 Phage-related cell wall hydrolase; Peptidase C51; CHAP domainEVPNEPDYIVIDVC*EDYSASK Staphylococcus argenteus Staphylococcus sp. HMSC36F05 Staphylococcus virus IPLA88 Staphylococcus virus phiNM2 Staphylococcus phage SAP40 Staphylococcus phage phi 53 Staphylococcus virus phiNM4 Staphylococcus phage SA12 Staphylococcus virus 69 Staphylococcus phage SA97 Staphylococcus phage TEM123 Staphylococcus virus 11 Staphylococcus virus phiMR25 Staphylococcus virus 53 Staphylococcus phage SAP33 S5 Phage antirepressor Ant QDWLAM*EVLPAIR Staphylococcus aureus, Staphylococcus simulans Staphylococcus argenteus Staphylococcus pseudintermedius Staphylococcus phage SA75 Staphylococcus phage SA13 S11 Phage capsid protein M*AEETNSNVTEETEVNE Staphylococcus, aureus Staphylococcus spp. S4 Phage encoded lipoprotein IHDKELDDPSEEESKLTQEEENSI Staphylococcus aureus, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus cohnii, Staphylococcus haemolyticus Staphylococcus phage SPbeta-like S2 Phage head morphogenesis protein KDVQRIVSHVT Staphylococcus aureus Staphylococcus argenteus S9 YhgE/Pip, Phage infection protein LNEYM*PNIEKLLN VASNDIPAQFPK Staphylococcusaureus, Staphylococcus haemolyticus Staphylococcus sp. HMSC34C02 S14 Minor structural protein KTTSEALKEVLSDT Staphylococcus aureus S4 Phage portal protein EPKPVDATGADDPLKPDDRM* ITNFHANLVDQKVSY Staphylococcus aureus Foods 2021,10, 799 9 of 21 Table 1. Cont. Strain Protein Peptide Bacteria with 100% Homology Based on the NCBI Protein Database Phages with 100% Homology Based on the NCBI Protein Database S5 Phage protein VHISEFKYPLYM*DFLGTKGELE Staphylococcusaureus Staphylococcus haemolyticus S15 Phage protein MSHNALTTGIGIGAGAG Staphylococcus aureus S2 Phage protein EITDGEISSVLTM*M* Staphylococcus aureus, Staphylococcus hominis Staphylococcus epidermidis S20 Phage recombination protein Bet KSSTTYEVNGETVK Staphylococcus aureus, Staphylococcus sciuri S2 Phage resistance protein ESVDTGEITANTTRTVK Staphylococcus aureus Staphylococcus fleurettii Staphylococcus pasteuri Staphylococcus epidermidis Staphylococcus warneri Staphylococcus schleiferi Escherichia coli S13 Tail tape measure protein GM*PTGTNVYAVKGGIADK Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus pseudoxylosus Staphylococcus phage phiSa2wa_st5 Staphylococcus phage phi3A Staphylococcus phage SH-St 15,644 Staphylococcus virus 3a S3 Tail tape measure protein VQHPGKLVNKVM* SGLNINFGGGANATAK Staphylococcus aureus S4 Tail tape measure protein QM*MEGLSGVMDLAAVSGEDLG AVSDIVTDGLTAFGLKAKDSG Staphylococcus aureus S2 Tail tape measure protein AEEAGVTVKQL Staphylococcus aureus Staphylococcus cohnii Staphylococcus sp. HMSC061H04 Staphylococcus hominis Staphylococcus capitis Staphylococcus cohnii Staphylococcus sp. HMSC061H04 Staphylococcus sp. HMSC067G10 Staphylococcus Staphylococcus haemolyticus Enterococcus faecium Staphylococcus epidermidis Staphylococcus sp. HMSC067G10 Staphylococcus haemolyticus Enterococcus faecium Staphylococcus epidermidis Staphylococcus phage SPbeta-like S10 Phage repressor, Cro/CI family QKNVLNYANEQLDEQNKV Staphylococcus aureus, Bacilli, Staphylococcus hyicus Staphylococcus epidermidis Staphylococcus virus phiNM2 Staphylococcus virus 53 Staphylococcus virus 80alpha S13 Phage protein KSNVEAFSNAVK Staphylococcus aureus Staphylococcus virus 80alpha Staphylococcus virus phiNM1 Staphylococcus virus phiNM2 S11 Phage protein PYHDLSDERIM*EELKK Staphylococcus aureus Staphylococcus argenteus taphylococcus schweitzeri Staphylococcus virus phiETA2 Staphylococcus phage P630 Staphylococcus virus SAP26 Staphylococcus phage B236 Staphylococcus virus 88 Staphylococcus prophage phiPV83 S4 Minor structural protein LNDNISNINTIV Pseudomonas aeruginosa E. coli Pararheinheimera mesophila Staphylococcus pseudintermedius Staphylococcus epidermidis, Staphylococcus sp. KY49P Staphylococcus argenteus Staphylococcus schleiferi Staphylococcus hyicus Staphylococcus sp. HMSC063H12 Staphylococcus aureus Staphylococcus virus 77 Staphylococcus phage P630 Staphylococcus phage SA780ruMSSAST101 Staphylococcus phage phiSa119 Staphylococcus phage phiN315 Staphylococcus phage SA7 Staphylococcus phage JS01 Staphylococcus phage StauST398-4 Staphylococcus virus 13 Staphylococcus phage 23MRA Staphylococcus virus 108PVL Staphylococcus phage phiBU01 Staphylococcus phage PVL Staphylococcus phage tp310-1 Staphylococcus phage P954 Staphylococcus phage SA345ruMSSAST8 Staphylococcus phage phiNM3 Staphylococcus virus 77 Staphylococcus phage phiSa2wa_st22 Staphylococcus phage SA1014ruMSSAST7 Staphylococcus phage P282 Staphylococcus prophage phiPV83 Staphylococcus phage 3 AJ-2017 Staphylococcus phage SAP090B Staphylococcus phage IME1346_01 Staphylococcus phage phi5967PVL Staphylococcus phage P1105 Staphylococcus phage IME1361_01 Foods 2021,10, 799 16 of 21 Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , brown-filled diamond HAGYVRC*KLF (cluster A-specific), Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , red=outlined diamond AVAELLKEINR (cluster A.2-specific), Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , pink-filled diamond KSNVEAFSNAVK (cluster A.1), Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), Foods 2021, 10, x FOR PEER REVIEW 15 of 23 Figure 2. Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brownfilled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster , dark gray-filled diamond KLYIIEEYVKQGM (cluster A.1-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , green-filled diamond LLHALPTGNDSGGDKLLPK (clusterD-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , purple-filled diamond LYVGVFNPEATK (cluster D-specific, Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , orange-outlined diamond KAMIKASPK (cluster D-specific) and Foods 2021, 10, x FOR PEER REVIEW 16 of 23 A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Table 2. Phage biomarker peptides that belong to bacteriophages and phylogenomic tree clusters. Relationships between specific phage biomarker peptides and phylogenomic tree clusters. Protein Peptide Phages Cluster Located Major capsid protein VSYTLDDDDFITDVETAK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage tp310-2 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus virus 3a Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein LLHALPTGNDSGGDKLLPK Staphylococcus phage phiSa2wa_st72 Staphylococcus phage phiSa2wa_st121mssa Staphylococcus phage vB_SauS_phi2 Staphylococcus phage StauST398-2 Staphylococcus phage LH1 Staphylococcus phage phiSa2wa_st30 Staphylococcus virus phi12 Staphylococcus virus 3ª Staphylococcus virus phiSLT Staphylococcus phage tp310-2 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage R4 Staphylococcus phage vB_SauS_fPfSau02 Staphylococcus phage SA137ruMSSAST121PVL Cluster D Major capsid protein RVSYTLDDDDFITDVETAKELKL Staphylococcus phage LH1 Staphylococcus phage StauST398-2 Staphylococcus phage vB_SauS_phi2 Staphylococcus phage R4 Cluster D Major tail protein LYVGVFNPEATK Staphylococcus phage vB_SauS_ phi2 Staphylococcus virus phi12 Staphylococcus virus phiSLT Staphylococcus phage R4 Staphylococcus phage vB_SauS_JS02 Staphylococcus phage SH-St 15644 Staphylococcus virus 3a Staphylococcus phage P240 Cluster D Phage repressor, Cro/CI family ELAEAIGVSQPTVSNWIQQTK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Phage repressor, Cro/CI family IQQLADYFNVPK Staphylococcus virus IPLA35 Staphylococcus phage SMSAP5 Staphylococcus phage vB_SauS_phi2 Cluster D Major tail protein AYINITGLGFAK Staphylococcus phage phiNM3 Staphylococcus phage StauST398-4 Staphylococcus phage P282 Staphylococcus phage phiN315 Staphylococcus phage phi7247PVL Staphylococcus phage phiSa2wa_st22 Staphylococcus virus 77 Staphylococcus phage P954 Cluster B.1 Major capsid protein IYDRNSDTLDGLPVVNLK Staphylococcus virus 85 Staphylococcus phage SP5 Cluster A.1 , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific). Foods 2021,10, 799 17 of 21 4. Discussion LC-MS/MS-based methods for bacteriophage identification offer several advantages compared with other approaches, since bacteriophages can be directly identified with this method without using genomic tools, which provides a new strategy for drawing the appropriate conclusions. In addition, the method proposed here may be applied for further analyses without the requirement of growing bacteria, since the samples can be collected directly from foodstuffs. The study of noninduced prophages provides a fast analysis and can detect specific temperate phage proteins produced by S. aureus while integrated in the bacterial genome or by phages that are infecting the bacteria. Both cases provide the identification of specific S. aureus species or strains—in this case, an S. aureus mastitis producer. In the proteomic repository of the 20 different S. aureus strains analyzed, 79 peptides from staphylococcal bacteriophages were identified. Among them, eighteen of these phage peptides were S. aureus-specific. As bacteriophages are host-specific, these putative diagnostic peptides could be good diagnostic biomarkers for the detection and characterization of S. aureus and S. aureus phages. The results show that a given specific peptide is present in closely related phages (Table 2). These bacteriophage peptides can be used as specific markers to establish S. aureus bacteriophage relationships (Figure 2). Additionally, phages that show the same peptides and are specific to Staphylococcus spp. are located close to one another in the phylogenomic tree, suggesting that a link does exist between phage phylogeny and bacteriophages that can infect the same bacterial species. The study shown here exemplifies how phylogenomic trees based on the genome analysis provide useful information, and the study corroborates previous investigations, which suggested that viral genomic or subgenomic region analyses provide the best tool for reconstructing viral evolutionary histories [ 48 ]. Nevertheless, the lack of knowledge of the phage genomic content [ 49 ] makes a phage analysis more difficult. The first priority must be the contribution of new large amounts of data for phages infecting bacteria [12]. In addition, there is an urgent need for novel therapies to treat and prevent mastitis [ 50 ]. Bacteriophage therapy is an alternative to the antibiotic treatment of bovine mastitis [ 51 ], with a high specificity and a low probability for bacterial resistance development [ 52 ]. Many studies have demonstrated the effectiveness of bacteriophages in a variety of animal models to fight several mastitis-causing pathogenic bacteria. Some studies have shown how virulent phages such as SPW and SA phages are active against bovine mastitis-associated S. aureus. Moreover, SAJK-IND and MSP phages have specific lytic activity against several strains of S. aureus isolated from mastitis milk samples [ 53 ]. Indeed, mouse-induced mastitis models decreased their bacterial counts after treatment with a vBSM-A1 and vBSP-A2 phage cocktail [ 54 ]. Finally, several temperate phage mixtures have been shown to be more effective than using a single temperate phage for inhibiting S. aureus. According to the data obtained for the different models of mastitis, phage therapy using bacteriophages in this study can be considered an innovative alternative to antibiotics for the treatment of mastitis caused by S. aureus. Finally, the proteomic analysis by LC-ESI-MS/MS performed in this study provides relevant insights into the search for potential phage origin diagnostic peptide biomarkers for mastitis-causing S. aureus. In addition, this method may be useful for searching peptide biomarkers for the identification and characterization of mastitis-causing species and for finding new S. aureus phages useful as possible therapies for mastitis. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/foods10040799/s1: Figure S1: MS/MS spectrums for S. aureus-specific peptide biomarkers. The corresponding peptides were tested for specificity using the BLASTp algorithm. Excel Dataset Supplemental Data 1: Complete nonredundant peptide dataset. Supplemental Data 2: Table S1: Staphylococcus aureus (SA) strains used in this study. Table S2: Linage, authors and accession number of studied bacteriophages [55–88]. Foods 2021,10, 799 18 of 21 Author Contributions: A.G.A. wrote the manuscript; A.G.A., K.B., T.G.V., P.C.-M., B.C., J.B.-V., J.-L.R.R. and M.C. conceptualized, revised and corrected the paper. P.C.-M. and M.C. co-supervised the work. M.C. and P.C.-M. got the funding. All authors listed have made a substantial, direct and intellectual contribution to the work and approved the work for publication. Funding: This work received financial support from the Xunta de Galicia and the European Union (European Social Fund-ESF), from the Spanish Ministry of Economy and Competitivity Project AGL 2.013-48.244-R and from the European Regional Development Fund (ERDF) (2007–2013). This work was also supported by the GAIN-Xunta de Galicia Project (IN607D 2017/01) and the Spanish AEI/EU-FEDER PID2019-103845RB-C21 project. Mónica Carrera was supported by the Ramón y Cajal contract (Ministry of Science and Innovation of Spain). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All relevant data are included in the article. 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