Therapeutic Phage Research Articles

Isolation of Bacteriophages for Fastidious Bacteria.

One of the most important factors for successful bacteriophage therapy is, undoubtedly, the isolation of excellent therapeutic candidate bacteriophages. There are only a few reports about active bacteriophages in the fastidious bacteria Helicobacter pylori. In this chapter, we describe a method for isolating and purifying KHP30-like bacteriophages in H. pylori, which have lytic and pseudolysogenic life cycles.

Advanced therapies push regulatory boundaries: Novel therapeutic approaches require more regulatory flexibility and transparency.

The regulation of new medical applications has always been lagging behind the science that enabled these therapies in the first place, but emerging therapeutic approaches are pushing the boundaries harder than ever. Although fundamental principles around safety, efficacy and quality are cast in stone, new interpretations and greater flexibility are required for the clinical use of advanced medicines and treatments without introducing unacceptable risks. There are also ethical considerations that, although not new, are amplified by the potential of emerging therapies to address previously untreatable conditions. All major regulatory bodies, such as the US Food and Drug Administration (FDA) and the European Medical Agency (EMA), have for some years been aware of the challenges posed in particular by Advanced Therapy Medicinal Products (ATMPs). These roughly include three categories: gene therapy medicinal products (GTMPs), somatic cell therapy medicinal products (SCTMPs) and tissue‐engineered products (TEPs), many of which have emerged from the “omics” revolution and stem cell research. > All major regulatory bodies […] have for some years been aware of the challenges posed in particular by Advanced Therapy Medicinal Products ### Advanced Therapy Medicinal Products Both GTMPs and SCTMPs involve ex vivo manipulation of cells, but with a crucial difference in terms of manufacture and function. GTMPs deliver recombinant DNA to specific target cells in the body, whereas SCTMPs are whole cells or tissues genetically manipulated to change their biological characteristics or functions for preventing, diagnosing or treating disease. These cells can be autologous, coming from the patient, allogeneic from another human being or xenogeneic from animals. One recent example of GMTP is recombinant viral vaccines against cancer engineered from an immunogenic virus particle to express tumour antigens, cytokines or both [1]. SCTMPs were the first AMPs to be tested clinically in 1997 for corneal epithelial stem cell transplantation. Since then, the technique has been …

Guidelines to Compose an Ideal Bacteriophage Cocktail.

Correctly designed bacteriophage therapeutics are the cornerstone for a successful outcome of bacteriophage therapy. Here we overview strategies on how to choose bacteriophages and their bacterial hosts at different steps of a bacteriophage cocktail development in order to comply with all quality and safety requirements based on the already existing essentially empirical experience in bacteriophage therapy and current accomplishments in modern biomedical sciences. A modification of the classic Appelmans' method (1922) to assess stability of bacteriophage activity in liquid media is presented in order to improve the overall performance of therapeutic bacteriophages individually and collectively in the cocktail.

Characterization of vB_SauM-fRuSau02, a Twort-Like Bacteriophage Isolated from a Therapeutic Phage Cocktail.

Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus. The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus.

Stability of bacteriophages in burn wound care products.

Bacteriophages could be used along with burn wound care products to enhance antimicrobial pressure during treatment. However, some of the components of the topical antimicrobials that are traditionally used for the prevention and treatment of burn wound infection might affect the activity of phages. Therefore, it is imperative to determine the counteraction of therapeutic phage preparations by burn wound care products before application in patients. Five phages, representatives of two morphological families (Myoviridae and Podoviridae) and active against 3 common bacterial burn wound pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus) were tested against 13 different products commonly used in the treatment of burn wounds. The inactivation of the phages was quite variable for different phages and different products. Majority of the anti-infective products affected phage activity negatively either immediately or in the course of time, although impact was not always significant. Products with high acidity had the most adverse effect on phages. Our findings demonstrate that during combined treatment the choice of phages and wound care products must be carefully defined in advance.

Adsorption of Staphylococcus viruses S13' and S24-1 on Staphylococcus aureus strains with different glycosidic linkage patterns of wall teichoic acids.

The group of phages belonging to the family Podoviridae, genus P68virus, including Staphylococcus viruses S13' and S24-1, are important because of their benefits in phage therapy against Staphylococcus aureus infections. The O-glycosidic linkage patterns of wall teichoic acids (WTAs) in S. aureus cell walls seem to be important for adsorption of this phage group. In this study, the adsorption of Staphylococcus viruses S13' and S24-1 to S. aureus was examined using strains with modified WTA glycosidic linkage patterns. We found that the β-O-N-acetylglucosamine of WTAs was essential for S13' adsorption, while N-acetylglucosamine, regardless of the α- and β-O-glycosidic linkages of the WTAs, was essential for S24-1 adsorption. Next, examining the binding activities of their receptor-binding proteins (RBPs) to cell walls with different WTA glycosidic patterns, the β-O-N-acetylglucosamine of the WTAs was essential for S13' RBP binding, while N-acetylglucosamine, regardless of the α- and β-O-glycosidic linkages of the WTAs, was essential for S24-1 RBP binding. Therefore, the results of the RBP binding assays were consistent with those of the phage adsorption assays. Bioinformatic analysis suggested that the RBPs of Staphylococcus viruses S13' and S24-1 were structurally similar to the RBPs of phage phi11 of thefamily Siphoviridae. Phylogenetic analysis of the RBPs indicated that two phylogenetic subclusters in the family Podoviridae were related to the glycosidic linkage patterns required for phage adsorption, possibly mediated by RBPs. We hope that this study will encourage the future development of therapeutic phages.

Synergy between the Host Immune System and Bacteriophage Is Essential for Successful Phage Therapy against an Acute Respiratory Pathogen

The rise of multi-drug-resistant (MDR) bacteria has spurred renewed interest in the use of bacteriophages in therapy. However, mechanisms contributing to phage-mediated bacterial clearance in an animal host remain unclear. We investigated the effects of host immunity on the efficacy of phage therapy for acute pneumonia caused by MDR Pseudomonas aeruginosa in a mouse model. Comparing efficacies of phage-curative and prophylactic treatments in healthy immunocompetent, MyD88-deficient, lymphocyte-deficient, and neutrophil-depleted murine hosts revealed that neutrophil-phage synergy is essential for the resolution of pneumonia. Population modeling of invivo results further showed thatneutrophils are required to control both phage-sensitive and emergent phage-resistant variants to clear infection. This "immunophage synergy" contrasts with the paradigm that phage therapy success is largely due to bacterial permissiveness to phage killing. Lastly, therapeutic phages were not cleared by pulmonary immune effector cells and were immunologically well tolerated by lung tissues.

Engineered bacteriophage therapeutics against multidrug‐resistant pathogens

Since the introduction of penicillin over 70 years ago, the number of patient deaths caused by infectious diseases has dramatically decreased and antibiotics have become among the most commonly prescribed drugs. However, the selective pressure imposed by antibiotic overuse has led to the emergence of bacterial strains resistant to virtually all antimicrobials and has renewed interest in the development of lytic bacteriophage (or phage) therapeutic and prophylactic agents. The advantages of using phage to combat infectious diseases include minimal disruption of resident flora, lack of cross‐resistance with antibiotics, low toxicity and self‐limiting dosing. Despite these attractive features, translational development of natural phage has been hindered mainly by the difficulty of accessing bacterial hosts within biofilms, the rapid emergence of resistant bacteria to a single phage, and above all, by the narrow host specificity of phage compared to antibiotics. The need for customized and complex combinations of natural phage to achieve adequate host range activity has made their development as licensed therapeutics very difficult. Genetic engineering of phage genomes can overcome these hurdles; however, broadly applicable methods for efficient construction of defined mutations in virulent phage genomes are still in their infancy. Thus, we developed a completely cell‐free phage engineering method that allows rapid and iterative editing of viral genomic DNA. In parallel, we sequenced and annotated the genomes of ~300 MDR P. aeruginosa clinical isolates, and subsequently determined the susceptibility of each isolate to specific phages. Through bioinformatics analysis in combination with our engineering platform, we have successfully collapsed the host range of a family into a representative phage. We further engineered wide host range phage to express secondary payloads, such as biofilm degrading enzymes and antimicrobial moieties, and demonstrated that these phages have improved activity compared to natural phage in P. aeruginosa models of infection.Support or Funding InformationSynthetic Genomics Vaccines, Inc.NIAID R21AI121531

Means to Facilitate the Overcoming of Gastric Juice Barrier by a Therapeutic Staphylococcal Bacteriophage A5/80.

In this article we compare the efficacy of different pharmacological agents (ranitidine, and omeprazole) to support phage transit from stomach to distal portions of the gastrointestinal tract in rats. We show that a temporal modification of environment in the animal stomach may protect Twort-like therapeutic antistaphylococcal phage A5/80 (from bacteriophage collection of the Hirszfeld Institute of Immunology and Experimental Therapy PAS in Wroclaw, Poland) from the inactivation by gastric juice effectively enough to enable a significant fraction of orally administered A5/80 to pass to the intestine. Interestingly, we found that yogurt may be a relatively strong in enhancing phage transit. Given the immunomodulating activities of phages our data may suggest that phages and yogurt can act synergistically in mediating their probiotic activities and enhancing the effectiveness of oral phage therapy. We also demonstrate that orally applied phages of similar size, morphology, and sensitivity to acidic environment may differ in their translocation into the bloodstream. This was evident in mice in which a therapeutic staphylococcal phage A5/80 reached the blood upon oral administration combined with antacid agent whilst T4 phage was not detected even when applied in 103 times higher dose. Our findings also suggest that phage penetration from digestive tract to the blood may be species-specific.

The Lysis of Pathogenic Escherichia coli by Bacteriophages Releases Less Endotoxin Than by β-Lactams.

Other than numerous experimental data assessing phage therapy efficacy, questions regarding safety of this approach are not sufficiently addressed. In particular, as phages can kill bacterial cells within <10 minutes, the associated endotoxin release (ER) in severe infections caused by gram-negative bacteria could be a matter of concern. Two therapeutic virulent phages and 4 reference antibiotics were studied in vitro for their ability to kill 2 pathogenic strains of Escherichia coli and generate an ER. The early interaction (first 3 hours) between these actors was assessed over time by studying the instantaneous cell viability, the colony-forming unit count, the concentration of free endotoxin released, and the cell morphology under light microscope. While β-lactams have a relatively slow effect, both tested phages, as well as amikacin, were able to rapidly abolish the bacterial growth. Even when considering the fastest phage (cell lysis in 9 minutes), the concentrations of phage-induced ER never reached the highest values, which were recorded with antibiotic treatments. Cumulative concentrations of endotoxin over time in phage-treated conditions were lower than those observed with β-lactams and close to those observed with amikacin. Whereas β-lactams were responsible for strong cell morphology changes (spheroplast with imipenem, filamentous cells with cefoxitin and ceftriaxone), amikacin and phages did not modify cell shape but produced intracellular inclusion bodies. This work provides important and comforting data regarding the safety of phage therapy. Therapeutically relevant phages, with their low endotoxin release profile and fast bactericidal effect, are not inferior to β-lactams.

Phage therapy: An alternative to antibiotics in the age of multi-drug resistance.

The practice of phage therapy, which uses bacterial viruses (phages) to treat bacterial infections, has been around for almost a century. The universal decline in the effectiveness of antibiotics has generated renewed interest in revisiting this practice. Conventionally, phage therapy relies on the use of naturally-occurring phages to infect and lyse bacteria at the site of infection. Biotechnological advances have further expanded the repertoire of potential phage therapeutics to include novel strategies using bioengineered phages and purified phage lytic proteins. Current research on the use of phages and their lytic proteins, specifically against multidrug-resistant bacterial infections, suggests phage therapy has the potential to be used as either an alternative or a supplement to antibiotic treatments. Antibacterial therapies, whether phage- or antibiotic-based, each have relative advantages and disadvantages; accordingly, many considerations must be taken into account when designing novel therapeutic approaches for preventing and treating bacterial infections. Although much is still unknown about the interactions between phage, bacteria, and human host, the time to take phage therapy seriously seems to be rapidly approaching.

A comparative study of different strategies for removal of endotoxins from bacteriophage preparations

Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography. We show that CsCl density purification of the P. aeruginosa phages, at an initial concentration of 1012–1013pfu/ml, led to the strongest reduction of endotoxins, with an endotoxin removal efficacy of up to 99%, whereas additional purification methods did not result in a complete removal of endotoxins from the phage preparations and only yielded an additional endotoxin removal efficacy of 23 to 99%, sometimes accompanied with strong losses in phage titer.

Two Novel Myoviruses from the North of Iraq Reveal Insights into Clostridium difficile Phage Diversity and Biology.

Bacteriophages (phages) are increasingly being explored as therapeutic agents to combat bacterial diseases, including Clostridium difficile infections. Therapeutic phages need to be able to efficiently target and kill a wide range of clinically relevant strains. While many phage groups have yet to be investigated in detail, those with new and useful properties can potentially be identified when phages from newly studied geographies are characterised. Here, we report the isolation of C. difficile phages from soil samples from the north of Iraq. Two myoviruses, CDKM15 and CDKM9, were selected for detailed sequence analysis on the basis of their broad and potentially useful host range. CDKM9 infects 25/80 strains from 12/20 C. difficile ribotypes, and CDKM15 infects 20/80 strains from 9/20 ribotypes. Both phages can infect the clinically relevant ribotypes R027 and R001. Phylogenetic analysis based on whole genome sequencing revealed that the phages are genetically distinct from each other but closely related to other long-tailed myoviruses. A comparative genomic analysis revealed key differences in the genes predicted to encode for proteins involved in bacterial infection. Notably, CDKM15 carries a clustered regularly interspaced short palindromic repeat (CRISPR) array with spacers that are homologous to sequences in the CDKM9 genome and of phages from diverse localities. The findings presented suggest a possible shared evolutionary past for these phages and provides evidence of their widespread dispersal.

An assessment of the future impact of alternative technologies on antibiotics markets.

BackgroundThe increasing threat of antimicrobial resistance combined with the paucity of new classes of antibiotics represents a serious public health challenge. New treatment technologies could, in theory, have a significant impact on the future use of traditional antibiotics, be it by facilitating rational and responsible use or by product substitution in the existing antibiotics markets, including by reducing the incidence of bacterial infections through preventative approaches. The aim of this paper is to assess the potential of alternative technologies in reducing clinical use of and demand for antibiotics, and to briefly indicate which segments of the antibiotics market that might be impacted by these technologies.MethodsAn initial mapping exercise to identify the alternative technologies was followed by a review of relevant published and grey literature (n = 52). We also carried out stakeholder engagement activities by a round-table discussion with infectious disease specialists and a multi-criteria decision analysis exercise with pharmaceutical industry experts.ResultsTen alternative technologies were identified and analyzed for their potential impact on the antibiotics market. Of these, rapid point-of-care diagnostics, vaccines, fecal microbiota transplantation, and probiotics were considered to have a “high” or “medium” potential impact over a 10-20 year horizon. Therapeutic antibodies, antibiotic biomaterials, bacteriophages, antimicrobial nanoparticles, antimicrobial peptides, and anti-virulence materials were rated as having “low” potential impact.ConclusionDespite the apparent potential of the most promising alternative technologies to reduce demand, that reduction will likely only happen in limited segments of the antibiotics market or, in the case of preventing community acquired streptococcal infections by vaccination, in a low-price generics market segment. Thus, alternative technologies are not expected to represent any disincentive to antibiotics developers. Finally, it is unlikely that alternative technologies will displace the need for new classes, and sub-classes, of antibiotics in the short and medium terms.

Novel methods to design wild bacteriophages into highly lytic and therapeutic bacteriophages to extensively drug-resistant Mycobacterium tuberculosis

Background: The emergence of multiple drug resistant (MDR) Mycobacterium tuberculosis (M.TB) and extensive drug resistant (XDR) M.TB lay huge burden on TB endemic countries such as Iraq.Objectives: Bacteriophage (phage) therapy can be used as alternative approach to tackle this problem. Patients and methods: Forty isolates of M.TB were cultured from TB-positive sputum specimens with three ATCC strains. Phage passaging and biokinetic based techniques were used to optimize wild anti-M.TB phages. Three chemical, non genetic- designing techniques, tween-80, mycobacterial lysis buffer, and xyelen, were used to change the specificity of wild anti-TB phages towards phage-resistant target M.TB.Results: Five wild anti-M.TB phages were isolated and optimized. The optimization techniques were successful in enhancing plaques size, clarity, burst size, and infective ratio. Chemical designing techniques succeeded to reorient specificity of 6 phages to new host bacteria.Conclusions: phage designing opens door wide for endless future applications of phage-based therapy, biocontrol and diagnosis.

A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells.

A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baumannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 × 109 pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required.

Phage Therapy in the Era of Synthetic Biology.

For more than a century, bacteriophage (or phage) research has enabled some of the most important discoveries in biological sciences and has equipped scientists with many of the molecular biology tools that have advanced our understanding of replication, maintenance, and expression of genetic material. Phages have also been recognized and exploited as natural antimicrobial agents and nanovectors for gene therapy, but their potential as therapeutics has not been fully exploited in Western medicine because of challenges such as narrow host range, bacterial resistance, and unique pharmacokinetics. However, increasing concern related to the emergence of bacteria resistant to multiple antibiotics has heightened interest in phage therapy and the development of strategies to overcome hurdles associated with bacteriophage therapeutics. Recent progress in sequencing technologies, DNA manipulation, and synthetic biology allowed scientists to refactor the entire bacterial genome of Mycoplasma mycoides, thereby creating the first synthetic cell. These new strategies for engineering genomes may have the potential to accelerate the construction of designer phage genomes with superior therapeutic potential. Here, we discuss the use of phage as therapeutics, as well as how synthetic biology can create bacteriophage with desirable attributes.

Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

Bacteriophages as Therapeutic and Prophylactic Means: Summary of the Soviet and Post Soviet Experiences.

Bacteriophage (from 'bacteria' and Greek φαγεῖν phagein "to devour" or bacterial eaters) are bacterial viruses that infect and kill bacteria. Bacteriophages (shortly "phages") are among the most common and diverse entities in the biosphere. The estimated number of phages on earth is about 1032. Bacteriophages are often isolated from environmental sources, such as water samples, etc. Felix d'Herelle, one of the discoverers of bacteriophages, was the one who suggested them for therapy of human and animal bacterial infections. This idea was very popular in the world until the advent of antibiotics commercial after which production of therapeutic phages ceased in most of the Western countries, but not in the former Soviet Union. The application of antibiotics in the clinical practice, besides the well-known side effects, entails, in addition, the appearance of the forms of bacteria, resistant to newly synthesized preparations. It was concluded that a European and global strategy to address this gap is urgently needed. Now, faced with the alarming growth of a variety of antibiotic resistant bacterial infections, Western researchers and governments are giving phages a serious look. The phages nowadays are seen as a possible therapy against multi-drug-resistant strains of many bacteria. The therapeutic action of bacteriophages significantly differs from antibiotics, which makes them still active against multi-drug-resistant bacteria. Bacteriophages have a number of other advantages in comparison with antibiotics. First of all, they are efficient against multi-drug-resistant bacteria. The aim of this review was to provide an overview of the past and current experiences in the field of phage therapy in the countries where it has been traditionally applied in the clinical practice. Although the style and quality of old Soviet scientific publications dedicated to phage therapy are not challenging the international standards, there is still valuable information which may not be neglected by modern researchers. This information is especially important as it remained unavailable for the Western scientists before because of the language barriers until now. The goal of this article is to encourage further research on this topic, and facilitate rapid decisions on the development of appropriate regulations, which would ultimately permit the use of phages as therapeutic or preventative medicines in daily clinical practice in the Western countries where multiplying drug-resistant bacteria gradually becomes the greatest life threatening problem.

Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus).

Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.