Development Of Phage Therapy Research Articles

Low-volume enrichment method supports high throughput bacteriophage screening and isolation from wastewater.

Bacteriophage therapy is a rapidly growing field of study. Narrow host ranges, bacterial resistance, and limited antibiotic availability make lytic phages a feasible therapeutic potential. Phage discovery, a critical step in developing phage therapy, is a pathway to accessible treatment. This has always been a laborious, time-consuming and resource-intensive process. In this paper, we describe a 96-well plate low-volume bacteriophage enrichment method with concentrated environmental sources to rapidly discover and isolate phages targeting multiple organisms simultaneously. Samples from natural water sources, wastewater influent, and activated sludge were tested in large volume enrichment cultures and low-volume 96-well plate format. Each plate has the capacity to run as many as 48 different combinations with multiple bacterial hosts. The time to identify the presence of phage in a sample was 5 to 10 hours in the low-volume format versus a minimum of 2 days in the traditional enrichment method. The labor and expense involved also favor the 96-well plate format. There was some loss of discovered phages using this technique, primarily targeting bacterial species less prevalent in the environment. This is an easily modifiable method that is amenable to automation and a variety of potential phage sources.

Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Actual aspects of treatment and prevention of infectious bovine keratoconjunctivitis

Infectious bovine keratoconjunctivitis is a bacterial eye disease. The disease is characterized by inflammation of the conjunctiva, corneal ulceration and excessive lacrimation. Although this disease does not lead to the death of animals, its economic importance is enormous, since it leads to a decrease in livestock weight gain and a reduction in dairy productivity. The main etiological role in IBK belongs to Moraxella spp. (including in associations with Staphylococcus spp., Streptococcus spp., Pseudomonas aeruginosa, E. coli, Proteus). The use of traditional treatment methods, such as the use of antibacterial chemicals, for Moraxella infection may be ineffective due to the factors of resistance and immunity of these bacteria. The development of a vaccine can be expensive and time-consuming, due to the external localization of the infection; it will be difficult to achieve a protective effect. New strategies for the treatment and prevention of bacterial infection open up prospects for the use of bacteriophages. Due to the growing antibiotic resistance of circulating bacterial pathogens and to increase the effectiveness of therapeutic and preventive measures in IBK, the development of phage therapy is relevant. Fundamental studies of biological properties and exploratory studies of virulent active bacteriophages to zoopathogenic Moraxelles are an urgent problem.

Developing Phage Therapy That Overcomes the Evolution of Bacterial Resistance.

The global rise of antibiotic resistance in bacterial pathogens and the waning efficacy of antibiotics urge consideration of alternative antimicrobial strategies. Phage therapy is a classic approach where bacteriophages (bacteria-specific viruses) are used against bacterial infections, with many recent successes in personalized medicine treatment of intractable infections. However, a perpetual challenge for developing generalized phage therapy is the expectation that viruses will exert selection for target bacteria to deploy defenses against virus attack, causing evolution of phage resistance during patient treatment. Here we review the two main complementary strategies for mitigating bacterial resistance in phage therapy: minimizing the ability for bacterial populations to evolve phage resistance and driving (steering) evolution of phage-resistant bacteria toward clinically favorable outcomes. We discuss future research directions that might further address the phage-resistance problem, to foster widespread development and deployment of therapeutic phage strategies that outsmart evolved bacterial resistance in clinical settings.

Antibacterial effects of single phage and phage cocktail against multidrug-resistant Klebsiella pneumoniae isolated from diabetic foot ulcer.

Diabetic foot ulcer (DFU) is associated with long-term hospitalization and amputation. Antibiotic resistance has made the infection eradication more difficult. Hence, seeking alternative therapies such as phage therapy seems necessary. Bacteriophages are viruses targeting specific bacterial species. Klebsiella pneumoniae (K. pneumoniae) is among causative agents of the DFU. In this study, the therapeutic effects of single phage and phage cocktail were investigated against multidrug-resistant (MDR) K. pneumonia isolated from DFU. Bacteriophages were isolated from animal feces and sewage samples, and were enriched and propagated using K. pneumoniae as the host. Thirty K. pneumoniae clinical isolates were collected from hospitalized patients with DFU. The antibiotic susceptibility pattern was determined using agar disk diffusion test. The phages' morphological traits were determined using transmission electron microscopy (TEM). The killing effect of isolated phages was assessed using plaque assay. Four phage types were isolated and recognized including KP1, KP2, KP3, and KP4. The bacterial rapid regrowth was observed following each single phage-host interaction, but not phage cocktail due to the evolution of mutant strains. Phage cocktail demonstrated significantly higher antibacterial activity than each single phage (p < 0.05) without any bacterial regrowth. The employment of phage cocktail was promising for the eradication of MDR-K. pneumoniae isolates. The development of phage therapy in particular, phage cocktail is promising as an efficient approach to eradicate MDR-K. pneumoniae isolated from DFU. The application of a specific phage cocktail can be investigated to try and achieve the eradication of various infections.

Unique relationships between phages and endospore-forming hosts.

As part of their survival strategy under harsh environmental conditions, endospore-forming bacteria can trigger a sporulation developmental program. Although the regulatory cascades that precisely control the transformation of vegetative bacteria into mother cells and resilient spores have been described in detail, less is known about how bacteriophages that prey on endospore-formers exploit sporulation. Herein, we argue that phages infecting these bacteria have evolved several specific molecular mechanisms, not yet known in other bacteria, that manifest from the phage-driven alliance to negative effects on the host. We anticipate that the relationships between phages and endospore-formers outlined here will inspire studies on phage ecology and evolution, and could facilitate important advances in the development of phage therapies against pathogenic spore-formers.

A Transducing Bacteriophage Infecting Staphylococcus epidermidis Contributes to the Expansion of a Novel Siphovirus Genus and Implies the Genus Is Inappropriate for Phage Therapy.

The effort to discover novel phages infecting Staphylococcus epidermidis contributes to both the development of phage therapy and the expansion of genome-based phage phylogeny. Here, we report the genome of an S. epidermidis-infecting phage, Lacachita, and compare its genome with those of five other phages with high sequence identity. These phages represent a novel siphovirus genus, which was recently reported in the literature. The published member of this group was favorably evaluated as a phage therapeutic agent, but Lacachita is capable of transducing antibiotic resistance and conferring phage resistance to transduced cells. Members of this genus may be maintained within their host as extrachromosomal plasmid prophages, through stable lysogeny or pseudolysogeny. Therefore, we conclude that Lacachita may be temperate and members of this novel genus are not suitable for phage therapy. IMPORTANCE This project describes the discovery of a culturable bacteriophage infecting Staphylococcus epidermidis that is a member of a rapidly growing novel siphovirus genus. A member of this genus was recently characterized and proposed for phage therapy, as there are few phages currently available to treat S. epidermidis infections. Our data contradict this, as we show Lacachita is capable of moving DNA from one bacterium to another, and it may be capable of maintaining itself in a plasmid-like state in infected cells. These phages' putative plasmid-like extrachromosomal state appears to be due to a simplified maintenance mechanism found in true plasmids of Staphylococcus and related hosts. We suggest Lacachita and other identified members of this novel genus are not suitable for phage therapy.

Phages for treatment of Escherichia coli infections.

Diseases due to infections by pathogenic Escherichia coli strains are on the rise and with the growing antimicrobial resistance among bacterial pathogens, including this group. Thus, alternative therapeutic options are actively investigated. Among these alternatives is phage therapy. In the case of E. coli, the combination of the well understood biology of this species and its bacteriophages represents a good guiding example for the establishment of phage therapy principles against this and other pathogenic bacteria. In this chapter, the procedures toward the development of phage therapy against pathogenic E. coli with the use of T-even group of phages are discussed. These steps involve the isolation, purification, characterisation and large-scale production of these phages, with formulation of phage cocktails for in vitro and in vivo studies. The main emphasis is made on phage therapy of enteropathogenic E. coli O157:H, which is one of the prominent human pathogens but persists as a commensal bacterium in many food animals. The implementation of phage therapy against E. coli O157:H within the One Health framework in carrier animals and for treatment of meat, vegetables, fruits and other agricultural produce thus would allow controlling and interrupting the transmission routes of this pathogen to the human food chain and preventing human disease. Examples of successful control and elimination of E. coli O157:H are given, while the problems encountered in phage treatment of this pathogen are also discussed.

Viruses for health: Bacteriophages

Bacteriophages are naturally occurring viruses that specifically target bacteria. They are widely distributed in the environment. The concept of phage therapy is to isolate phages, characterize them, cultivate them and then purify them to treat bacterial infections. There is currently a revival of phage therapy, and its implementation presupposes the availability of active phages of pharmaceutical quality. From a regulatory point of view, the status of phages is not yet clearly defined by the authorities. The availability of phages produced by the pharmaceutical industry and through academic development programs such as the PHAGEinLYON program represents a breakthrough in the development of phage therapy. Prosthetic joint infections and digestive diseases seem to be relevant indications, but preclinical studies and randomized clinical trials are now needed to be done.

Phascinating Phages.

Treatment of infections caused by bacteria has become more complex due to the increasing number of bacterial strains that are resistant to conventional antimicrobial therapy. A highly promising alternative appears to be bacteriophage (phage) therapy, in which natural predators of bacteria, bacteriophages, play a role. Although these viruses were first discovered in 1917, the development of phage therapy was impacted by the discovery of antibiotics, which spread more quickly and effectively in medical practice. Despite this, phage therapy has a long history in Eastern Europe; however, Western countries are currently striving to reintroduce phage therapy as a tool in the fight against diseases caused by drug-resistant bacteria. This review describes phage biology, bacterial and phage competition mechanisms, and the benefits and drawbacks of phage therapy. The results of various laboratory experiments, and clinical cases where phage therapy was administered, are described.

Temporal Transcriptional Responses of a Vibrio alginolyticus Strain to Podoviridae Phage HH109 Revealed by RNA-Seq.

ABSTRACTPhage are thought to exhibit control over host genes during infection. As a preliminary investigation of the kinetics and magnitude of co-expression between phage and bacteria, we compared the global transcriptional profiles for Vibrio alginolyticus strain E110 and its lytic phage HH109 by using RNA sequencing. In total, 24.7% (1,143/4,620) of the host protein-coding genes were differentially expressed genes during infection (DEGs). Functional analysis of the host DEGs suggests that phage HH109 induced rapid and distinctive changes when compared with 60- and 120-min postinfection (mpi). Based on gene co-expression network analysis, an uncharacterized late gene gp27 encoded by the phage HH109 was predicted to modulate the host’s membrane transport and/or transcriptional regulation. Furthermore, expression of several bacterial virulence genes was downregulated while drug resistance genes were upregulated. This work contributes to an in-depth understanding of the reciprocal interactions of lytic phage HH109 and its pathogenic Vibrio host E110, and can provide new insights into the research and development of phage therapy against pathogenic Vibrio infections in the economically significant aquatic animals.IMPORTANCE Vibrio alginolyticus is a common opportunistic pathogen that causes mass mortality in cultured marine animals. Phage HH109 lyses pathogenic V. alginolyticus strain E110 with high efficiency and thus serves as a useful model to understand the dynamic interplay of a phage and its host. Global transcriptomic responses of strain E110 post-HH109 infection were characterized by using RNA sequencing, elucidating step-by-step control by HH109, an antiphage-like responses, and the elevated expression of drug resistance. This study provides a detailed molecular description phage and V. alginolyticus, providing insight into better prevention and control of vibriosis in aquatic animals.

Enhanced antibacterial effect of a novel Friunavirus phage vWU2001 in combination with colistin against carbapenem-resistant Acinetobacter baumannii

The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) has been increasingly reported, leading to greater challenges in treating infections. With the development of phage therapy and phage-antibiotic combinations, it is promising to improve the treatment of bacterial infections. In the present study, a novel vB_AbaP_WU2001 (vWU2001) phage-specific CRAB with a genome of 40,792 bp was isolated. Genomic analysis disclosed that it belongs to the Autographiviridae family of the order Caudovirales. Phage vWU2001 had a broad host range with a high adsorption rate, short latent period, large burst size and good stability. The phage could reduce preformed biofilms and inhibit biofilm formation. The combination of phage vWU2001 and colistin had significantly higher bacterial growth inhibition activity than that of phage, or colistin alone. The efficacy of the combined treatment was also evaluated in Galleria mellonella. Evaluation of its therapeutic potential showed that the combination of phage and colistin resulted in a significantly greater increase in G. mellonella survival and in bacterial clearance, as compared with that of phage or colistin alone, indicating that the combination was synergistic against CRAB. The results demonstrated that phage vWU2001 has the potential to be developed as an antibacterial agent.

Isolation and Characterization of Novel Phages Targeting Pathogenic Klebsiella pneumoniae.

Klebsiella pneumoniae is a dominant cause of community-acquired and nosocomial infections, specifically among immunocompromised individuals. The increasing occurrence of multidrug-resistant (MDR) isolates has significantly impacted the effectiveness of antimicrobial agents. As antibiotic resistance is becoming increasingly prevalent worldwide, the use of bacteriophages to treat pathogenic bacterial infections has recently gained attention. Elucidating the details of phage-bacteria interactions will provide insights into phage biology and the better development of phage therapy. In this study, a total of 22 K. pneumoniae isolates were assessed for their genetic and phenotypic relatedness by multi-locus sequence typing (MLST), endonuclease S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), and in vitro antibiotic susceptibility testing. In addition, the beta-lactamase gene (bla KPC) was characterized to determine the spread and outbreak of K. pneumoniae carbapenemase (KPC)-producing enterobacterial pathogens. Using these ST11 carbapenem-resistant K. pneumoniae isolates, three phages (NL_ZS_1, NL_ZS_2, and NL_ZS_3) from the family of Podoviridae were isolated and characterized to evaluate the application of lytic phages against the MDR K. pneumoniae isolates. In vitro inhibition assays with three phages and K. pneumoniae strain ZS15 demonstrated the strong lytic potential of the phages, however, followed by the rapid growth of phage-resistant and phage-sensitive mutants, suggesting several anti-phage mechanisms had developed in the host populations. Together, this data adds more comprehensive knowledge to known phage biology and further emphasizes their complexity and future challenges to overcome prior to using phages for controlling this important MDR bacterium.

Past and Future of Phage Therapy and Phage-Derived Proteins in Patients with Bone and Joint Infection.

Phage-derived therapies comprise phage therapy and the use of phage-derived proteins as anti-bacterial therapy. Bacteriophages are natural viruses that target specific bacteria. They were proposed to be used to treat bacterial infections in the 1920s, before the discovery and widespread over-commercialized use of antibiotics. Phage therapy was totally abandoned in Western countries, whereas it is still used in Poland, Georgia and Russia. We review here the history of phage therapy by focusing on bone and joint infection, and on the development of phage therapy in France in this indication. We discuss the rationale of its use in bacterial infection and show the feasibility of phage therapy in the 2020s, based on several patients with complex bone and joint infection who recently received phages as compassionate therapy. Although the status of phage therapy remains to be clarified by health care authorities, obtaining pharmaceutical-grade therapeutic phages (i.e., following good manufacturing practice guidelines or being “GMP-like”) targeting bacterial species of concern is essential. Moreover, multidisciplinary clinical expertise has to determine what could be the relevant indications to perform clinical trials. Finally “phage therapy 2.0” has to integrate the following steps: (i) follow the status of phage therapy, that is not settled and defined; (ii) develop in each country a close relationship with the national health care authority; (iii) develop industrial–academic partnerships; (iv) create academic reference centers; (v) identify relevant clinical indications; (vi) use GMP/GMP-like phages with guaranteed quality bioproduction; (vii) start as salvage therapy; (vii) combine with antibiotics and adequate surgery; and (viii) perform clinical trials, to finally (ix) demonstrate in which clinical settings phage therapy provides benefit. Phage-derived proteins such as peptidoglycan hydrolases, polysaccharide depolymerases or lysins are enzymes that also have anti-biofilm activity. In contrast to phages, their development has to follow the classical process of medicinal products. Phage therapy and phage-derived products also have a huge potential to treat biofilm-associated bacterial diseases, and this is of crucial importance in the worldwide spread of antimicrobial resistance.

Bacteriophages as Potential Tools for Use in Antimicrobial Therapy and Vaccine Development.

The constantly growing number of people suffering from bacterial, viral, or fungal infections, parasitic diseases, and cancers prompts the search for innovative methods of disease prevention and treatment, especially based on vaccines and targeted therapy. An additional problem is the global threat to humanity resulting from the increasing resistance of bacteria to commonly used antibiotics. Conventional vaccines based on bacteria or viruses are common and are generally effective in preventing and controlling various infectious diseases in humans. However, there are problems with the stability of these vaccines, their transport, targeted delivery, safe use, and side effects. In this context, experimental phage therapy based on viruses replicating in bacterial cells currently offers a chance for a breakthrough in the treatment of bacterial infections. Phages are not infectious and pathogenic to eukaryotic cells and do not cause diseases in human body. Furthermore, bacterial viruses are sufficient immuno-stimulators with potential adjuvant abilities, easy to transport, and store. They can also be produced on a large scale with cost reduction. In recent years, they have also provided an ideal platform for the design and production of phage-based vaccines to induce protective host immune responses. The most promising in this group are phage-displayed vaccines, allowing for the display of immunogenic peptides or proteins on the phage surfaces, or phage DNA vaccines responsible for expression of target genes (encoding protective antigens) incorporated into the phage genome. Phage vaccines inducing the production of specific antibodies may in the future protect us against infectious diseases and constitute an effective immune tool to fight cancer. Moreover, personalized phage therapy can represent the greatest medical achievement that saves lives. This review demonstrates the latest advances and developments in the use of phage vaccines to prevent human infectious diseases; phage-based therapy, including clinical trials; and personalized treatment adapted to the patient’s needs and the type of bacterial infection. It highlights the advantages and disadvantages of experimental phage therapy and, at the same time, indicates its great potential in the treatment of various diseases, especially those resistant to commonly used antibiotics. All the analyses performed look at the rich history and development of phage therapy over the past 100 years.

Distribution of CRISPR Types in Fluoroquinolone-Resistant Campylobacter jejuni Isolates.

To aid development of phage therapy against Campylobacter, we investigated the distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) systems in fluoroquinolone (FQ)-resistant Campylobacter jejuni. A total of 100 FQ-resistant C. jejuni strains from different sources were analyzed by PCR and DNA sequencing to determine resistance-conferring mutation in the gyrA gene and the presence of various CRISPR systems. All but one isolate harbored 1–5 point mutations in gyrA, and the most common mutation was the Thr86Ile change. Ninety-five isolates were positive with the CRISPR PCR, and spacer sequences were found in 86 of them. Among the 292 spacer sequences identified in this study, 204 shared 93–100% nucleotide homology to Campylobacter phage D10, 44 showed 100% homology to Campylobacter phage CP39, and 3 had 100% homology with Campylobacter phage CJIE4-5. The remaining 41 spacer sequences did not match with any phages in the database. Based on the results, it was inferred that the FQ-resistant C. jejuni isolates analyzed in this study were potentially resistant to Campylobacter phages D10, CP39, and CJIE4-5 as well as some unidentified phages. These phages should be excluded from cocktails of phages that may be utilized to treat FQ-resistant Campylobacter.

Advancing phage therapy through the lens of virus host-breadth and emergence potential.

Phages are viruses that specifically infect bacteria, and their biodiversity contributes to historical and current development of phage therapy to treat myriad bacterial infections. Phage therapy holds promise as an alternative to failing chemical antibiotics, but there are benefits and costs of this technology. Here, we review the rich history of phage therapy, highlighting reasons (often political) why it was widely rejected by Western medicine until recently. One longstanding idea involves mixing different phages together in cocktails, to increase the probability of killing target pathogenic bacteria without pre-screening for phage susceptibility. By challenging 30 lytic phages to infect 14 strains of the bacteria Pseudomonas aeruginosa, we showed that some phages were "generalists" with broad host-ranges, emphasizing that extreme host-specificity of phages was not necessarily a liability. Using a "greedy algorithm" analysis, we identified the best cocktail mixture of phages to achieve broad bacteria killing. Additionally, we review how virus host-range can evolve and connect lessons learned from virus emergence-including contributions of elevated virus mutation rates in promoting emergence and virus evolutionary transitions from specialized to generalized host-use-as cautionary tales for avoiding risk of "off-target" phage emergence on commensal bacteria in microbiomes. Throughout, we highlight how fundamental understanding of virus ecology and evolution is vital for developing phage therapy; heeding these principles should help in designing therapeutic strategies that do not recapitulate consequences of virus selection to emerge on novel hosts.

Antimycobacterial Potential of Mycobacteriophage Under Disease-Mimicking Conditions.

Antibiotic resistance continues to be a major global health risk with an increase in multi-drug resistant infections seen across nearly all bacterial diseases. Mycobacterial infections such as Tuberculosis (TB) and Non-Tuberculosis infections have seen a significant increase in the incidence of multi-drug resistant and extensively drug-resistant infections. With this increase in drug-resistant Mycobacteria, mycobacteriophage therapy offers a promising alternative. However, a comprehensive study on the infection dynamics of mycobacteriophage against their host bacteria and the evolution of bacteriophage (phage) resistance in the bacteria remains elusive. We aim to study the infection dynamics of a phage cocktail against Mycobacteria under various pathophysiological conditions such as low pH, low growth rate and hypoxia. We show that mycobacteriophages are effective against M. smegmatis under various conditions and the phage cocktail prevents emergence of resistance for long durations. Although the phages are able to amplify after infection, the initial multiplicity of infection plays an important role in reducing the bacterial growth and prolonging efficacy. Mycobacteriophages are effective against antibiotic-resistant strains of Mycobacterium and show synergy with antibiotics such as rifampicin and isoniazid. Finally, we also show that mycobacteriophages are efficient against M. tuberculosis both under lag and log phase for several weeks. These findings have important implications for developing phage therapy for Mycobacterium.

Isolation and Characterization of Bacteriophages That Infect Citrobacter rodentium, a Model Pathogen for Intestinal Diseases.

Enteropathogenic Escherichia coli (EPEC) is a major pathogen for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse have led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy as an alternative antibacterial therapy. Because EPEC in vivo models have shortcomings, a surrogate is used to study the mouse pathogen Citrobacter rodentium in animal models. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus, and CrRp10 is a new strain within the species Escherichia virus Ime09, in the genus Tequatrovirus. Both phages appear to have independently evolved from E. coli phages, rather than other Citrobacter spp. phages. Neither phage strain carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 is more potent, having a 24-fold faster adsorption rate and shorter lytic cycle when compared to the same properties of CrRp10. However, a lysis curve analysis revealed that CrRp10 prevented growth of C. rodentium for 18 h, whereas resistance developed against CrRp3 within 9 h. We also show that hypoxic (5% oxygen) conditions decreased CrRp3 ability to control bacterial densities in culture. In contrast, low oxygen conditions did not affect CrRp10 ability to replicate on C. rodentium. Together, CrRp10 is likely to be the better candidate for future phage therapy investigations.

The Virulence Index: A Metric for Quantitative Analysis of Phage Virulence.

Background: One of the main challenges in developing phage therapy and manufacturing phage products is the reliable evaluation of their efficacy, performance, and quality. Since phage virulence is intrinsically difficult to fully capture, researchers have turned to rapid but partially inadequate methods for its evaluation. Materials and Methods: This study demonstrates a standardized quantitative method to assess phage virulence based on three parameters: the virulence index (VP )-quantifying the virulence of a phage against a host, the local virulence (vi )-assessing killing potential at given multiplicities of infection (MOIs), and MV50 -the MOI at which the phage achieves 50% of its maximum theoretical virulence. This was shown through comparative analysis of the virulence of phages T4, T5, and T7. Results: Under the conditions tested, phage T7 displayed the highest virulence, followed by phage T4 and, finally, by phage T5. The impact of parameters such as temperature and medium composition on virulence was shown for each phage. The use of the method to evaluate the virulence of combinations of phages-for example, for cocktail formulation-is also shown with phages T5 and T7. Conclusions: The method presented provides a platform for high-throughput quantitative assessment of phage virulence and quality control of phage products. It can also be applied to phage screening, evaluation of phage strains, phage mutants, infection conditions and/or the susceptibility of host strains, and the formulation of phage cocktails.