Basics for Improved Use of Phages for Therapy

Blood-borne therapeutic phages and phage capsids increasingly reach therapeutic targets as they acquire more persistence, i.e., become more resistant to non-targeted removal from blood. Pathogenic bacteria are targets during classical phage therapy. Metastatic tumors are potential future targets, during use of drug delivery vehicles (DDVs) that are phage derived. Phage therapy has, to date, only sometimes been successful. One cause of failure is low phage persistence. A three-step strategy for increasing persistence is to increase (1) the speed of lytic phage isolation, (2) the diversity of phages isolated, and (3) the effectiveness and speed of screening phages for high persistence. The importance of high persistence-screening is illustrated by our finding here of persistence dramatically higher for coliphage T3 than for its relative, coliphage T7, in murine blood. Coliphage T4 is more persistent, long-term than T3. Pseudomonas chlororaphis phage 201phi2-1 has relatively low persistence. These data are obtained with phages co-inoculated and separately assayed. In addition, highly persistent phage T3 undergoes dispersal to several murine organs and displays tumor tropism in epithelial tissue (xenografted human oral squamous cell carcinoma). Dispersal is an asset for phage therapy, but a liability for phage-based DDVs. We propose increased focus on phage persistence—and dispersal—screening.

Bacteriophages: from isolation to application

: Bacteriophages are considered as a potential alternative to fight pathogenic bacteria during the antibiotic resistance era. With their high specificity, they are being widely used in various applications: medicine, food industry, agriculture, animal farms, biotechnology, diagnosis, etc. Many techniques have been designed by different researchers for phage isolation, purification, and amplification, each of which has strengths and weaknesses. However, all aim at having a reasonably pure phage sample that can be further characterized. Phages can be characterized based on their physiological, morphological or inactivation tests. Microscopy, in particular, has opened a wide gate not only for visualizing phage morphological structure, but also for monitoring biochemistry and behavior. Meanwhile, computational analysis of phage genomes provides more details about phage history, lifestyle, and potential for toxigenic or lysogenic conversion, which translate to safety in biocontrol and phage therapy applications. This review summarizes phage application pipelines at different levels and addresses specific restrictions and knowledge gaps in the field. Recently developed computational approaches, which are used in phage genome analysis, are critically assessed. We hope that this assessment provides researchers with useful insights for selection of suitable approaches for Phage-related research aims and applications.

A Modified High-Throughput Screening Protocol to Isolate Bacteriophages from Environmental Samples

In the post antimicrobial era, increasing attention is paid towards using bacteriophage (phage in short) therapy to control antibiotic-resistant bacteria. The first step in phage therapy applications is isolating highly efficient lytic phages or phage cocktails from various sources. When a double-layer- agar with around 0.7% agar in top agar is employed, it results in a low number of phage isolation with a poor resolution, and in many cases, you miss the phage. To address this problem, a low concentration of agar in top agar is examined for better phage isolation. Here, our results proved the efficiency of isolating phage upon formulating a double-layer agar with 0.3% agar in top agar. A sewage sample was collected then phages were isolated, purified, and spotted on a top layer agar with 0.3% agar. The results showed the possibility of isolating a higher number of phages on 0.3% top agar than 0.7%. The finding advocates using 0.3% top agar for the double-layer agar, as it will provide fast, better, and easy phage screening and isolation.

A Modified High-Throughput Screening Protocol to Isolate Bacteriophages from Environmental Samples

In the post antimicrobial era, increasing attention is paid towards using bacteriophage (phage in short) therapy to control antibiotic-resistant bacteria. The first step in phage therapy applications is isolating highly efficient lytic phages or phage cocktails from various sources. When a double-layer- agar with around 0.7% agar in top agar is employed, it results in a low number of phage isolation with a poor resolution, and in many cases, you miss the phage. To address this problem, a low concentration of agar in top agar is examined for better phage isolation. Here, our results proved the efficiency of isolating phage upon formulating a double-layer agar with 0.3% agar in top agar. A sewage sample was collected then phages were isolated, purified, and spotted on a top layer agar with 0.3% agar. The results showed the possibility of isolating a higher number of phages on 0.3% top agar than 0.7%. The finding advocates using 0.3% top agar for the double-layer agar, as it will provide fast, better, and easy phage screening and isolation.

Salmonella Bacteriophage Diversity According to Most Prevalent Salmonella Serovars in Layer and Broiler Poultry Farms from Eastern Spain

The exploration of novel nonantibiotic interventions in the field, such as the use of bacteriophages, is necessary to avoid the presence of Salmonella. Bacteriophages are a group of viruses widely distributed in nature, strictly associated with the prokaryotic cell. Researchers have demonstrated the success of phage therapy in reducing Salmonella counts in poultry products. However, the impact that phage concentration in the environment may have against certain Salmonella serovars is not well understood. Therefore, the aim of this study was to assess Salmonella phage prevalence in commercial poultry farms in terms of the production type: layers or broilers. The most prevalent Salmonella serovars isolated in poultry production were used for phage isolation. Salmonella specific phages were isolated from 141 layer and broiler farms located in the Valencia region during 2019. Analysis of the samples revealed that 100% presented Salmonella phages, the most prevalent being against serovar S. Enteritidis (93%), followed by S. Virchow (59%), S. Typhimurium (55%), S. Infantis (52%) and S. Ohio (51%). These results indicate that poultry farms could represent an important source of Salmonella phages. Nevertheless, further studies are needed to assess the epidemiology of phages against other serovars present in other countries and their diversity from the point of view of molecular studies.

The Israeli Phage Bank (IPB)

A key element in phage therapy is the establishment of large phage collections, termed herein “banks”, where many well-characterized phages, ready to be used in the clinic, are stored. These phage banks serve for both research and clinical purposes. Phage banks are also a key element in clinical phage microbiology, the prior treatment matching of phages and antibiotics to specific bacterial targets. A worldwide network of phage banks can promote a phage-based solution for any isolated bacteria. Herein, we describe the Israeli Phage Bank (IPB) established in the Hebrew University, Jerusalem, which currently has over 300 phages matching 16 bacteria, mainly pathogens. The phage bank is constantly isolating new phages and developing methods for phage isolation and characterization. The information on the phages and bacteria stored in the bank is available online.

Application of Campylobacter jejuni Phages: Challenges and Perspectives

Bacteriophages (phages) are the most abundant and diverse biological entities in the biosphere. Due to the rise of multi-drug resistant bacterial strains during the past decade, phages are currently experiencing a renewed interest. Bacteriophages and their derivatives are being actively researched for their potential in the medical and biotechnology fields. Phage applications targeting pathogenic food-borne bacteria are currently being utilized for decontamination and therapy of live farm animals and as a biocontrol measure at the post-harvest level. For this indication, the United States Food and Drug Administration (FDA) has approved several phage products targeting Listeria sp., Salmonella sp. and Escherichia coli. Phage-based applications against Campylobacter jejuni could potentially be used in ways similar to those against Salmonella sp. and Listeria sp.; however, only very few Campylobacter phage products have been approved anywhere to date. The research on Campylobacter phages conducted thus far indicates that highly diverse subpopulations of C. jejuni as well as phage isolation and enrichment procedures influence the specificity and efficacy of Campylobacter phages. This review paper emphasizes conclusions from previous findings instrumental in facilitating isolation of Campylobacter phages and improving specificity and efficacy of the isolates.

The Perfect Bacteriophage for Therapeutic Applications—A Quick Guide

The alarming spread of multiresistant infections has kick-started the quest for alternative antimicrobials. In a way, given the steady increase in untreatable infectious diseases, success in this endeavor has become a matter of life and death. Perhaps we should stop searching for an antibacterial panacea and explore a multifaceted strategy in which a wide range of compounds are available on demand depending on the specific situation. In the context of this novel tailor-made approach to combating bacterial pathogens, the once forgotten phage therapy is undergoing a revival. Indeed, the compassionate use of bacteriophages against seemingly incurable infections has been attracting a lot of media attention lately. However, in order to take full advantage of this strategy, bacteria’s natural predators must be taken from their environment and then carefully selected to suit our needs. In this review, we have explored the vast literature regarding phage isolation and characterization for therapeutic purposes, paying special attention to the most recent studies, in search of findings that hint at the most efficient strategies to identify suitable candidates. From this information, we will list and discuss the traits that, at the moment, are considered particularly valuable in phages destined for antimicrobial therapy applications. Due to the growing importance given to biofilms in the context of bacterial infections, we will dedicate a specific section to those characteristics that indicate the suitability of a bacteriophage as an antibiofilm agent. Overall, the objective is not just to have a large collection of phages, but to have the best possible candidates to guarantee elimination of the target pathogens.