Bioinformatic Analysis of Staphylococcus Phages: A Key Step for Safe Cocktail Development.

An in-depth analysis of phage genomic sequences is essential for the proposal of a cocktail for therapeutic uses. With the burst of publications on phage isolation and genetic studies during the last decade, several different bioinformatics programs have been used. Here we describe our studies on the genetic organization of phages infecting Staphylococcus aureus, a pathogen of human importance, by using an assembly of tools for gene annotation, identification of expression components, and phylogeny analysis.

Phage Transduction of Staphylococcus aureus.

Bacteriophage transduction is the major mechanism of horizontal gene transfer (HGT) among many bacteria. In Staphylococcus aureus, the phage-mediated acquisition of mobile genetic elements (MGEs) that encode virulence and antibiotic resistance genes largely contribute to its evolutionary adaptation and genetic plasticity. In molecular biology, generalized transduction is routinely used as a technique to manipulate and construct bacterial strains. Here, we describe optimized protocols for generalized transduction, applicable for the transfer of plasmid or chromosomal deoxyribonucleic acid (DNA) from donor to recipient S. aureus strains.

Bacterial cGAS senses a viral RNA to initiate immunity.

Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.

Phage activity against Staphylococcus aureus is impaired in plasma and synovial fluid.

S. aureus is a pathogen that frequently causes severe morbidity and phage therapy is being discussed as an alternative to antibiotics for the treatment of S. aureus infections. In this in vitro and animal study, we demonstrated that the activity of anti-staphylococcal phages is severely impaired in 0.5% plasma or synovial fluid. Despite phage replication in these matrices, lysis of the bacteria was slower than phage propagation, and no reduction of the bacterial population was observed. The inhibition of the phages associated with a reduction in phage adsorption, quantified to 99% at 10% plasma. S. aureus is known to bind multiple coagulation factors, resulting in the formation of aggregates and blood clots that might protect the bacterium from the phages. Here, we show that purified fibrinogen at a sub-physiological concentration of 0.4 mg/ml is sufficient to impair phage activity. In contrast, dissolution of the clots by tissue plasminogen activator (tPA) partially restored phage activity. Consistent with these in vitro findings, phage treatment did not reduce bacterial burdens in a neutropenic mouse S. aureus thigh infection model. In summary, phage treatment of S. aureus infections inside the body may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.

Genomic and proteomic characterization of vB_SauM-UFV_DC4, a novel Staphylococcus jumbo phage.

Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: • vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. • vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. • vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.

Dual pathogenicity island transfer by piggybacking lateral transduction.

Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry.

Structural and biochemical insights into the bacteriophage PlyGRCS endolysin targeting methicillin-resistant Staphylococcus aureus (MRSA) and serendipitous discovery of its interaction with a cold shock protein C (CspC).

Methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening human infections. Bacteriophage-encoded endolysins degrade the cell walls of Gram-positive bacteria by selectively hydrolyzing the peptidoglycan layer and thus are promising candidates to combat bacterial infections. PlyGRCS, the S. aureus-specific bacteriophage endolysin, contains a catalytic CHAP domain and a cell-wall binding SH3_5 domain connected by a linker. Here, we show the crystal structure of full-length PlyGRCS refined to 2.1 Å resolution. In addition, a serendipitous finding revealed that PlyGRCS binds to cold-shock protein C (CspC) by interacting with its CHAP and SH3_5 domains. CspC is an RNA chaperone that plays regulatory roles by conferring bacterial adaptability to various stress conditions. PlyGRCS has substantial lytic activity against S. aureus and showed only minimal change in its lytic activity in the presence of CspC. Whereas the PlyGRCS-CspC complex greatly reduced CspC-nucleic acid binding, the aforesaid complex may downregulate the CspC function during bacterial infection. Overall, the crystal structure and biochemical results of PlyGRCS provide a molecular basis for the bacteriolytic activity of PlyGRCS against S. aureus.

Transcriptional Landscapes of Herelleviridae Bacteriophages and Staphylococcus aureus during Phage Infection: An Overview.

The issue of antibiotic resistance in healthcare worldwide has led to a pressing need to explore and develop alternative approaches to combat infectious diseases. Among these methods, phage therapy has emerged as a potential solution to tackle this growing challenge. Virulent phages of the Herelleviridae family, known for their ability to cause lysis of Staphylococcus aureus, a clinically significant pathogen frequently associated with multidrug resistance, have proven to be one of the most effective viruses utilized in phage therapy. In order to utilize phages for therapeutic purposes effectively, a thorough investigation into their physiology and mechanisms of action on infected cells is essential. The use of omics technologies, particularly total RNA sequencing, is a promising approach for analyzing the interaction between phages and their hosts, allowing for the assessment of both the behavior of the phage during infection and the cell's response. This review aims to provide a comprehensive overview of the physiology of the Herelleviridae family, utilizing existing analyses of their total phage transcriptomes. Additionally, it sheds light on the changes that occur in the metabolism of S. aureus when infected with virulent bacteriophages, contributing to a deeper understanding of the phage-host interaction.

Isolation and in vitro characterization of novel S. epidermidis phages for therapeutic applications.

S. epidermidis is an important opportunistic pathogen causing chronic prosthetic joint infections associated with biofilm growth. Increased tolerance to antibiotic therapy often requires prolonged treatment or revision surgery. Phage therapy is currently used as compassionate use therapy and continues to be evaluated for its viability as adjunctive therapy to antibiotic treatment or as an alternative treatment for infections caused by S. epidermidis to prevent relapses. In the present study, we report the isolation and in vitro characterization of three novel lytic S. epidermidis phages. Their genome content analysis indicated the absence of antibiotic resistance genes and virulence factors. Detailed investigation of the phage preparation indicated the absence of any prophage-related contamination and demonstrated the importance of selecting appropriate hosts for phage development from the outset. The isolated phages infect a high proportion of clinically relevant S. epidermidis strains and several other coagulase-negative species growing both in planktonic culture and as a biofilm. Clinical strains differing in their biofilm phenotype and antibiotic resistance profile were selected to further identify possible mechanisms behind increased tolerance to isolated phages.

Impact of the calcium concentration on the efficacy of phage phiIPLA-RODI, LysRODIΔAmi and nisin for the elimination of Staphylococcus aureus during lab-scale cheese production.

Staphylococcus aureus is a Gram-positive human opportunistic pathogen that may also cause food poisoning because of the ability of some strains to produce heat stable enterotoxins that can persist in food even after the pathogen is successfully eliminated. In this context, biopreservation may be a forward-looking strategy to help eliminate staphylococcal contamination in dairy products by using natural compounds. However, these antimicrobials exhibit individual limitations that may be overcome by combining them. This work investigates the combination of a virulent bacteriophage, phiIPLA-RODI, a phage-derived engineered lytic protein, LysRODIΔAmi, and the bacteriocin nisin for the elimination of S. aureus during lab-scale cheese production at two CaCl2 concentrations (0.2 % and 0.02 %), and subsequent storage at two different temperatures (4 °C and 12 °C). In most of the assayed conditions, our results demonstrate that the combined action of the antimicrobials led to a greater reduction of the pathogen population than the compounds individually, albeit this effect was additive and not synergistic. However, our results did show synergy between the three antimicrobials for reducing the bacterial load after 14 days of storage at 12 °C, temperature at which there is growth of the S. aureus population. Additionally, we tested the impact of the calcium concentration on the activity of the combination treatment and observed that higher CaCl2 levels led to a notable increase in endolysin activity that allowed the utilization of approximately 10-times less protein to attain the same efficacy. Overall, our data show that the combination of LysRODIΔAmi with nisin and/or phage phiIPLA-RODI, and an increase in the calcium concentration are successful strategies to decrease the amount of protein required for the control of S. aureus contamination in the dairy sector with a low potential for resistance selection, thereby reducing costs.

The Influence of Bacteriophages on the Metabolic Condition of Human Fibroblasts in Light of the Safety of Phage Therapy in Staphylococcal Skin Infections.

Phage therapy has been successfully used as an experimental therapy in the treatment of multidrug-resistant strains of Staphylococcus aureus (MDRSA)-caused skin infections and is seen as the most promising alternative to antibiotics. However, in recent years a number of reports indicating that phages can interact with eukaryotic cells emerged. Therefore, there is a need to re-evaluate phage therapy in light of safety. It is important to analyze not only the cytotoxicity of phages alone but also the impact their lytic activity against bacteria may have on human cells. As progeny virions rupture the cell wall, lipoteichoic acids are released in high quantities. It has been shown that they act as inflammatory agents and their presence could lead to the worsening of the patient's condition and influence their recovery. In our work, we have tested if the treatment of normal human fibroblasts with staphylococcal phages will influence the metabolic state of the cell and the integrity of cell membranes. We have also analyzed the effectiveness of bacteriophages in reducing the number of MDRSA attached to human fibroblasts and the influence of the lytic activity of phages on cell viability. We observed that, out of three tested anti-Staphylococcal phages-vB_SauM-A, vB_SauM-C and vB_SauM-D-high concentrations (109 PFU/mL) of two, vB_SauM-A and vB_SauM-D, showed a negative impact on the viability of human fibroblasts. However, a dose of 107 PFU/mL had no effect on the metabolic activity or membrane integrity of the cells. We also observed that the addition of phages alleviated the negative effect of the MDRSA infection on fibroblasts' viability, as phages were able to effectively reduce the number of bacteria in the co-culture. We believe that these results will contribute to a better understanding of the influence of phage therapy on human cells and encourage even more studies on this topic.

WGS analysis of two Staphylococcus aureus bacteriophages from sewage in China provides insights into the genetic feature of highly efficient lytic phages.

The study of bacteriophages is experiencing a resurgence with the increasing development of antimicrobial resistance in Staphylococcus aureus. Nonetheless, the genetic features of highly efficient lytic S. aureus phage remain to be explored. In this study, two lytic S. aureus phages, SapYZU11 and SapYZU15, were isolated from sewage samples from Yangzhou, China. The phage morphology, one-step growth, host spectrum and lytic activity of these phages were examined, and their whole-genome sequences were analysed and compared with 280 published genomes of staphylococcal phages. The structural organisation and genetic contents of SapYZU11 and SapYZU15 were investigated. The Podoviridae phage SapYZU11 and Herelleviridae phage SapYZU15 effectively lysed all of the 53 S. aureus strains isolated from various sources. However, SapYZU15 exhibited a shorter latent period, larger burst size and stronger bactericidal ability with an anti-bacterial rate of approximately 99.9999% for 24 h. Phylogenetic analysis revealed that Herelleviridae phages formed the most ancestral clades and the S. aureus Podoviridae phages were clustered in the staphylococcal Siphoviridae phage clade. Moreover, phages in different morphology families contain distinct types of genes associated with host cell lysis, DNA packaging and lysogeny. Notably, SapYZU15 harboured 13 DNA metabolism-related genes, 5 lysin genes, 1 holin gene and 1 DNA packaging gene. The data suggest that S. aureus Podoviridae and Siphoviridae phages originated from staphylococcal Herelleviridae phages, and the module exchange of S. aureus phages occurred in the same morphology family. Moreover, the extraordinary lytic capacity of SapYZU15 was likely due to the presence of specific genes associated with DNA replication, DNA packaging and the lytic cycle.

Phage and Antibiotic Combinations Reduce Staphylococcus aureus in Static and Dynamic Biofilms Grown on an Implant Material.

Staphylococcus aureus causes the majority of implant-related infections. These infections present as biofilms, in which bacteria adhere to the surface of foreign materials and form robust communities that are resilient to the human immune system and antibiotic drugs. The heavy use of broad-spectrum antibiotics against these pathogens disturbs the host's microbiome and contributes to the growing problem of antibiotic-resistant infections. The use of bacteriophages as antibacterial agents is a potential alternative therapy. In this study, bioluminescent strains of S. aureus were grown to form 48-h biofilms on polyether ether ketone (PEEK), a material used to manufacture orthopaedic implants, in either static or dynamic growth conditions. Biofilms were treated with vancomycin, staphylococcal phage, or a combination of the two. We showed that vancomycin and staph phages were able to independently reduce the total bacterial load. Most phage-antibiotic combinations produced greater log reductions in surviving bacteria compared to single-agent treatments, suggesting antimicrobial synergism. In addition to demonstrating the efficacy of combining vancomycin and staph phage, our results demonstrate the importance of growth conditions in phage-antibiotic combination studies. Dynamic biofilms were found to have a substantial impact on apparent treatment efficacy, as they were more resilient to combination treatments than static biofilms.

Characterization of KMSP1, a newly isolated virulent bacteriophage infecting Staphylococcus aureus, and its application to dairy products.

Staphylococcus aureus is one of the major pathogens causing foodborne outbreaks and severe infections worldwide. Generally, various physical and chemical treatments have been applied to control S. aureus in the food industry. However, conventional treatments usually affected food quality and often produced toxic compounds. Therefore, bacteriophage (phage), a natural antimicrobial agent, has been suggested as an alternative strategy to control foodborne pathogens including S. aureus. In this study, KMSP1, a bacteriophage infecting S. aureus was isolated from a raw milk sample and characterized. Transmission electron microscopy (TEM) analysis revealed that phage KMSP1 belongs to the Myoviridae family. Phage KMSP1 efficiently inhibited bacterial growth for >28 h post-infection. In addition, phage KMSP1 could infect a broad spectrum of S. aureus strains, including methicillin-resistant S. aureus (MRSA) strains. Whole-genome sequence analysis showed that KMSP1 is a lytic phage with the absence of genes related to lysogen formation, toxin production, and antibiotics resistance, respectively. In the genome of KMSP1, the presence of putative tail lysin containing a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain could be one of the reasons for the effective antimicrobial activity of KMSP1. Furthermore, high stability of phage KMSP1 at temperature ranging from 4 to 55 °C and pH ranging from 5 to 11, suggested its potential use in various food systems. Receptor analysis revealed that KMSP1 utilized cell wall teichoic acid (WTA), one of the major virulence factors of S. aureus, as a host receptor. Application of phage KMSP1 at an MOI of 104 achieved a significant reduction of log 8.8 CFU/mL of viable cell number in pasteurized milk and log 4.3 CFU/cm2 in sliced cheddar cheese after 24 h. Taken together, the strong antimicrobial activity of phage KMSP1 suggested that it could be developed as a biocontrol agent in dairy products to control S. aureus contamination.

Identification and Molecular Modification of Staphylococcus aureus Bacteriophage Lysin LysDZ25.

With the continuous emergence and spread of drug-resistant and multi-drug-resistant Staphylococcus aureus, traditional antibiotic treatment has gradually lost its effect. There is an urgent need to develop and study new and effective bio-green inhibitors to control S. aureus. In this study, the S. aureus phage DZ25 was isolated from milk and the lysin LysDZ25 with excellent tolerance to serum and NaCl solution was identified. Subsequently, to improve the lytic activity and thermal stability of LysDZ25, RoseTTAFold was used to construct three-dimensional (3D) structures, molecular dynamics (MD) simulation was used for conformational acquisition, and the MDL strategy previously developed in our lab was used to rationally design variants. After two rounds of rational design, the optimal variant with improved thermal stability, S333V/N245R/D299L, was obtained, and its half-life time was 4.0-fold that of wild-type LysDZ25. At 37, 40, 45, and 50 °C, the lytic activity of the optimal triple-point variant S333V/N245R/D299L was increased by 17.3-, 26.7-, 20.2-, and 50.1-fold compared with that of the wild-type LysDZ25, respectively. Finally, cell count was used to evaluate the lytic activity, and the results showed that the optimal variant S333V/N245R/D299L could drop about 3.5 log 10 values compared with the control and about 2.6 log 10 values compared with the wild-type LysDZ25.

Staphylococcus aureus Prophage-Encoded Protein Causes Abortive Infection and Provides Population Immunity against Kayviruses.

Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

A novel Staphylococcus phage, vB_Sau-RP15, and its application in contaminated milk.

The vB_Sau-RP15 phage, selected for its potential use as a phage treatment in milk, was isolated from raw milk using Staphylococcusaureus NP01 as the host. The host range test revealed that the phage was able to lyse 12 strains of Staph. aureus from raw milk. This phage was stable at 4-37°C and pH 6-9 for at least 1 h. The adsorption rate was ~78% within the first 3 min. A low frequency of phage-insensitive mutant induction (4.6 × 10-6) was observed. Genomic analyses revealed that the vB_Sau-RP15 represented a novel species in the genus Silviavirus. Even though no virulence or antibiotic resistance genes were detected, the phage genome carried lysogenic-associated genes. Phage treatments (108 PFU per ml) in pasteurized milk contaminated with low (104 CFU per ml) and high (107 CFU per ml) concentrations of Staph. aureus confirmed the proficiency of the phage in the diminishing of the number of bacterial cells at 4°C and ambient temperature. A Staphylococcus phage, vB_Sau-RP15, could be a promising agent for controlling Staph. aureus contamination in milk.

Characterization of the novel temperate Staphylococcus haemolyticus phage IME1365_01.

The presence of a novel functional prophage, IME1365_01, was predicted from bacterial high-throughput sequencing data and then successfully induced from Staphylococcus haemolyticus by mitomycin C treatment. Transmission electron microscopy showed that phage IME1365_01 has an icosahedral head (43 nm in diameter) and a long tail (172 nm long). This phage possesses a double-stranded DNA genome of 44,875 bp with a G+C content of 35.35%. A total of 63 putative open reading frames (ORFs) were identified in its genome. BLASTn analysis revealed that IME1365_01 is similar to Staphylococcus phage vB_SepS_E72, but with a genome homology coverage of only 26%. The phage genome does not have fixed termini. In ORF24 of phage IME1365_01, a conserved Toll-interleukin-1 receptor domain of the TIR_2 superfamily (accession no. c123749) is located at its N-terminus, and this might serve as a component of an anti-bacterial system. In conclusion, we developed a platform to obtain active temperate phage from prediction, identification, and induction from its bacterial host. After mass screening using this platform, numerous temperate phages and their innate anti-bacterial elements can provide extensive opportunities for therapy against bacterial (especially drug-resistant bacterial) infections.

Phage transcription activator RinA regulates Staphylococcus aureus virulence by governing sarA expression.

BACKGROUND: Staphylococcus aureus is a major human pathogen, that can lead to various community- and hospital-acquired infections. RinA is a transcription activator of S. aureus phage φ 11 involved in phage packaging and virulence gene transfer. However, little is known about the molecular mechanism of RinA in the regulation of virulence. OBJECTIVE: We aimed to explore a novel contribution of RinA in the regulation of virulence and provide a new drug target in the treatment of S. aureus infections. METHODS: The specific functions of RinA in S. aureus were analyzed by the methods of growth curve, real-time quantitative PCR (RT-qPCR), subcellular localization, electrophoretic mobility shift assay (EMSA), infection model of Galleria mellonella larvae and the mouse subcutaneous abscess model. RESULTS: In this study, we demonstrated that RinA is a protein evenly distributed in the cytoplasm of S. aureus, and its deletion could cause the growth defects. RT-qPCR and EMSA determined that rinA could negatively regulate the expression of sarA by directly binding to its promoter, and vice versa. The Galleria mellonella larvae infection and mouse subcutaneous abscess models revealed that the rinA mutant strain exhibited obvious virulence defects. When sarA is knocked out, the virulence of S.aureus had no significantly changes whether rinA is knocked out or not. CONCLUSION: Our fndings demonstrated that phage transcription activator RinA regulates S. aureus virulence by governing sarA expression.

Biological properties of Staphylococcus virus ΦSA012 for phage therapy.

Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48 h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.