Therapeutic Phage Research Articles

Lessons Learned From the First 10 Consecutive Cases of Intravenous Bacteriophage Therapy to Treat Multidrug-Resistant Bacterial Infections at a Single Center in the United States.

BackgroundDue to increasing multidrug-resistant (MDR) infections, there is an interest in assessing the use of bacteriophage therapy (BT) as an antibiotic alternative. After the first successful case of intravenous BT to treat a systemic MDR infection at our institution in 2017, the Center for Innovative Phage Applications and Therapeutics (IPATH) was created at the University of California, San Diego, in June 2018.MethodsWe reviewed IPATH consult requests from June 1, 2018, to April 30, 2020, and reviewed the regulatory process of initiating BT on a compassionate basis in the United States. We also reviewed outcomes of the first 10 cases at our center treated with intravenous BT (from April 1, 2017, onwards).ResultsAmong 785 BT requests to IPATH, BT was administered to 17 of 119 patients in whom it was recommended. One-third of requests were for Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium abscessus. Intravenous BT was safe with a successful outcome in 7/10 antibiotic-recalcitrant infections at our center (6 were before IPATH). BT may be safely self-administered by outpatients, used for infection suppression/prophylaxis, and combined successfully with antibiotics despite antibiotic resistance, and phage resistance may be overcome with new phage(s). Failure occurred in 2 cases despite in vitro phage susceptibility.ConclusionsWe demonstrate the safety and feasibility of intravenous BT for a variety of infections and discuss practical considerations that will be critical for informing future clinical trials.

A Deep Learning-Based Method for Identification of Bacteriophage-Host Interaction.

Multi-drug resistance (MDR) has become one of the greatest threats to human health worldwide, and novel treatment methods of infections caused by MDR bacteria are urgently needed. Phage therapy is a promising alternative to solve this problem, to which the key is correctly matching target pathogenic bacteria with the corresponding therapeutic phage. Deep learning is powerful for mining complex patterns to generate accurate predictions. In this study, we develop PredPHI (Predicting Phage-Host Interactions), a deep learning-based tool capable of predicting the host of phages from sequence data. We collect >3000 phage-host pairs along with their protein sequences from PhagesDB and GenBank databases and extract a set of features. Then we select high-quality negative samples based on the K-Means clustering method and construct a balanced training set. Finally, we employ a deep convolutional neural network to build the predictive model. The results indicate that PredPHI can achieve a predictive performance of 81 percent in terms of the area under the receiver operating characteristic curve on the test set, and the clustering-based method is significantly more robust than that based on randomly selecting negative samples. These results highlight that PredPHI is a useful and accurate tool for identifying phage-host interactions from sequence data.

Isolation, Characterization, and Comparison of Efficiencies of Bacteriophages to Reduce Planktonic and Biofilm-Associated Staphylococcus aureus

Abstract Introduction In the present era, wherein occurrence of antimicrobial resistance compounded with biofilms in disease conditions has rendered present antibiotic therapy ineffective, the need for alternative strategies to treat bacterial infections has brought bacteriophages to the forefront. The antimicrobial activity of phages is often determined by a viable cell reduction assay which focuses only on planktonic forms. The physiology of an organism in biofilm differs from those that are planktonic; hence, there is a need to evaluate the activity of phages both on planktonic forms, as well as on biofilms, to select candidate therapeutic phages. Materials and Methods Bacteriophages for Staphylococcus aureus were isolated from environmental samples and characterized based on growth kinetics and DNA fingerprint patterns. Activity of isolated phages on planktonic forms was determined by viable count reduction assay. Phage ability to prevent biofilm formation and ability to disperse formed biofilms were performed in 96-well microtiter plates and biofilm estimated by crystal violet assay. Results Four bacteriophages designated, that is, P3, PD1, PE1, and PE2, were isolated and characterized. Planktonic cells of S. aureus were found to be sensitive to phages PD1, PE1, and PE2. Phages PD1 and PE2 were efficient in preventing biofilm formation and phages PD1, PE1, and P3 were efficient in dispersing formed biofilms. Conclusion The ability of some phages to disperse biofilms effectively, while unable to show the same efficiency on planktonic cells, indicates that viable count reduction assay alone may not be a sufficient tool to imply bactericidal activity of bacteriophages, especially while trying to eradicate biofilms.

Broadscale phage therapy is unlikely to select for widespread evolution of bacterial resistance to virus infection.

Multi-drug resistant bacterial pathogens are alarmingly on the rise, signaling that the golden age of antibiotics may be over. Phage therapy is a classic approach that often employs strictly lytic bacteriophages (bacteria-specific viruses that kill cells) to combat infections. Recent success in using phages in patient treatment stimulates greater interest in phage therapy among Western physicians. But there is concern that widespread use of phage therapy would eventually lead to global spread of phage-resistant bacteria and widespread failure of the approach. Here, we argue that various mechanisms of horizontal genetic transfer (HGT) have largely contributed to broad acquisition of antibiotic resistance in bacterial populations and species, whereas similar evolution of broad resistance to therapeutic phages is unlikely. The tendency for phages to infect only particular bacterial genotypes limits their broad use in therapy, in turn reducing the likelihood that bacteria could acquire beneficial resistance genes from distant relatives via HGT. We additionally consider whether HGT of clustered regularly interspaced short palindromic repeats (CRISPR) immunity would thwart generalized use of phages in therapy, and argue that phage-specific CRISPR spacer regions from one taxon are unlikely to provide adaptive value if horizontally-transferred to other taxa. For these reasons, we conclude that broadscale phage therapy efforts are unlikely to produce widespread selection for evolution of bacterial resistance.

Fine capsule variation affects bacteriophage susceptibility in Klebsiella pneumoniae ST258.

Multidrug resistant (MDR) carbapenemase-producing (CP) Klebsiella pneumoniae, belonging to clonal group CG258, is capable of causing severe disease in humans and is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as therapeutic alternatives to antibiotics. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the genomes of 18Kumoniae target strains and tested them against novel lytic bacteriophages (n=65). The genomes of K pneumoniae carrying blaNDM and blaKPC were sequenced and CG258 isolates selected for bacteriophage susceptibility testing. The local K pneumoniae CG258 population was dominated by sequence type ST258 clade 1 (86%) with variations in capsular locus (cps) and prophage content. CG258-specific bacteriophages primarily targeted the capsule, but successful infection is also likely blocked in some by immunity conferred by existing prophages. Five tailed bacteriophages against K pneumoniae ST258 clade 1 were selected for further characterization. Our findings show that effective control of K pneumoniae CG258 with bacteriophage will require mixes of diverse lytic viruses targeting relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic bacteriophage candidates against this serious pathogen.

Secondary Bacterial Infections During Pulmonary Viral Disease: Phage Therapeutics as Alternatives to Antibiotics?

Secondary bacterial infections manifest during or after a viral infection(s) and can lead to negative outcomes and sometimes fatal clinical complications. Research and development of clinical interventions is largely focused on the primary pathogen, with research on any secondary infection(s) being neglected. Here we highlight the impact of secondary bacterial infections and in particular those caused by antibiotic-resistant strains, on disease outcomes. We describe possible non-antibiotic treatment options, when small molecule drugs have no effect on the bacterial pathogen and explore the potential of phage therapy and phage-derived therapeutic proteins and strategies in treating secondary bacterial infections, including their application in combination with chemical antibiotics.

Optimizing the Timing and Composition of Therapeutic Phage Cocktails: A Control-Theoretic Approach.

Viruses that infect bacteria, i.e., bacteriophage or 'phage,' are increasingly considered as treatment options for the control and clearance of bacterial infections, particularly as compassionate use therapy for multi-drug-resistant infections. In practice, clinical use of phage often involves the application of multiple therapeutic phage, either together or sequentially. However, the selection and timing of therapeutic phage delivery remains largely ad hoc. In this study, we evaluate principles underlying why careful application of multiple phage (i.e., a 'cocktail') might lead to therapeutic success in contrast to the failure of single-strain phage therapy to control an infection. First, we use a nonlinear dynamics model of within-host interactions to show that a combination of fast intra-host phage decay, evolution of phage resistance amongst bacteria, and/or compromised immune response might limit the effectiveness of single-strain phage therapy. To resolve these problems, we combine dynamical modeling of phage, bacteria, and host immune cell populations with control-theoretic principles (via optimal control theory) to devise evolutionarily robust phage cocktails and delivery schedules to control the bacterial populations. Our numerical results suggest that optimal administration of single-strain phage therapy may be sufficient for curative outcomes in immunocompetent patients, but may fail in immunodeficient hosts due to phage resistance. We show that optimized treatment with a two-phage cocktail that includes a counter-resistant phage can restore therapeutic efficacy in immunodeficient hosts.

Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research: Personalized Infectious Disease Medicine for the Most Vulnerable At-Risk Patients.

Mutation is the most powerful driver of change for life on Earth. Pathogenic bacteria utilize mutation as a means to survive strong live-die selective pressures generated by chemical antibiotics. As such, the traditional drug-making pipeline, characterized by significant financial and time investment, is insufficient to keep pace with the rapid evolution of bacterial resistance to structurally fixed and chemically unmalleable antibacterial compounds. In contrast, the genetic diversity and adaptive mutability of the bacteriophage can be leveraged to not only overcome resistance but also used for the development of enhanced traits that increase lytic potential and therapeutic efficacy in relevant host microenvironments. This is the fundamental premise behind Baylor College of Medicine's Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research (TAILΦR) initiative. In this perspective, we outline the concept, structure, and process behind TAILΦR's attempt to generate a personalized therapeutic phage that addresses the most clinically challenging of bacterial infections.

The Stabilizing Excipients in Dry State Therapeutic Phage Formulations.

Phage therapy has gained prominence due to the increasing pathogenicity of "super bugs" and the rise of their multidrug resistance to conventional antibiotics. Dry state formulation of therapeutic phage is attractive to improve their "druggability" by increasing their shelf life, improving their ease of handling, and ultimately retaining their long-term potency. The use and selection of excipients are critical to stabilize phage in solid formulations and protect their viability from stresses encountered during the solidification process and long-term storage prior to use. Here, this review focuses on the current classes of excipients used to manufacture dry state phage formulations and their ability to stabilize and protect phage throughout the process, as discussed in the literature. We provide perspective of outstanding challenges involved in the formulation of dry state phage. We suggest strategies to improve excipient identification and selection, optimize the potential excipient combinations to improve phage viability during formulation, and evaluate new methodologies that can provide greater insight into phage-excipient interactions to improve design criteria to improve formulation of dry state phage therapeutics. Addressing these challenges opens up new opportunities to re-design and re-imagine phage formulations for improved efficacy as a pharmaceutical product.

Bacteriophage Therapy: Developments and Directions.

In an era of proliferating multidrug resistant bacterial infections that are exhausting the capacity of existing chemical antibiotics and in which the development of new antibiotics is significantly rarer, Western medicine must seek additional therapeutic options that can be employed to treat these infections. Among the potential antibacterial solutions are bacteriophage therapeutics, which possess very different properties from broad spectrum antibiotics that are currently the standard of care, and which can be used in combination with them and often provide synergies. In this review we summarize the state of the development of bacteriophage therapeutics and discuss potential paths to the implementation of phage therapies in contemporary medicine, focused on fixed phage cocktail therapeutics since these are likely to be the first bacteriophage products licensed for broad use in Western countries.

Parallel Genomics Uncover Novel Enterococcal-Bacteriophage Interactions.

Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

Characterization and application of a novel Aeromonas bacteriophage as treatment for pathogenic Aeromonas hydrophila infection in rainbow trout

Aeromonas hydrophila is an opportunistic pathogen responsible for motile Aeromonas septicemia in several fish species, including rainbow trout. With the emergence of antibiotic-resistant strains and safety concerns on fish products, alternative treatment strategies should be developed. A previously isolated virulent A. hydrophila phage, designated as MJG, meets the main prerequisites for a candidate therapeutic phage based on genomic characterization. In this study, the biological characteristics of MJG were further examined. Results showed that MJG had activity at a broad range of temperature (10 °C–60 °C) and pH (2−10), and its latent and rise periods were 30 and 40 min, respectively. However, MJG exhibited a narrow host range and infected only 4 out of 20 A. hydrophila strains. Compared with PBS injection, MJG injection (3.2 × 106 PFU/fish) at 2 h post A. hydrophila infection reduced the bacterial numbers from 5.4 × 106 to 1.4 × 104 CFU/g in liver after 3 days post infection (dpi). The expression levels of pro-inflammatory cytokines (IL-8 and IL-1β) in the spleen of MJG-treated fish were significantly higher than those in PBS-treated fish at 1 or 2 dpi but significantly lower than those in PBS-treated fish at 3 dpi. MJG treatment would restore liver tissue damages and abolish the clinical signs of infection. MJG administered through injection or immersion completely protected the fish from death due to A. hydrophila infection. Meanwhile, oral feeding with MJG (3.2 × 107 PFU/g) could provide remarkable protection. Our work explores the potential use of phage as an alternative therapeutic agent against A. hydrophila infection in fish.

A Call For a Multidisciplinary Future of Phage Therapy to Combat Multi-drug Resistant Bacterial Infections

A Call For a Multidisciplinary Future of Phage Therapy to Combat Multi-drug Resistant Bacterial Infections

Building Better Bacteriophage with Biofoundries to Combat Antibiotic-Resistant Bacteria.

Resistance to antibiotics is an escalating global crisis, presenting a major health, social, and economic burden. An underexplored alternative to antibiotic treatment is phage therapy whereby bacteriophages are used to infect and kill pathogenic multidrug-resistant (MDR) bacteria. A primary challenge is the highly specific infectivity range of phages that can limit their ability to infect across different bacterial strains. Synthetic biology can enable the design, modification, and synthesis of phages with improved antimicrobial performance and efficacy to help realize novel strategies to study and treat bacterial infectious diseases, including those caused by MDR pathogens. In this perspective article, we discuss the potential for an innovative synthetic biology approach to enhance phage therapeutics and the role a biofoundry can play in bringing phage therapy to fruition.

Treat phage like living antibiotics.

Bacteriophage therapeutics has emerged as one of the few potential beacons that represent possible solutions to the growing global crisis of antimicrobial resistance. Bringing science to the bedside (and vice versa) will maximize the potential of this compelling opportunity.

Approaches to optimize therapeutic bacteriophage and bacteriophage-derived products to combat bacterial infections.

The emerging occurrence of antibiotic-resistant bacterial pathogens leads to a recollection of bacteriophage as antimicrobial therapeutics. This article presents a short overview of the clinical phage application including their use in military medicine and discusses the genotypic and phenotypic properties of a potential “ideal” therapeutic phage. We describe current efforts to engineer phage for their improved usability in pathogen treatment. In addition, phage can be applied for pathogen detection, selective drug delivery, vaccine development, or food and surface decontamination. Instead of viable phage, (engineered) phage-derived enzymes, such as polysaccharide depolymerases or peptidoglycan-degrading enzymes, are considered as promising therapeutic candidates. Finally, we briefly summarize the use of phage for the detection and treatment of “Category A priority pathogens”.

Myoviridae phage PDX kills enteroaggregative Escherichia coli without human microbiome dysbiosis.

Introduction Bacteriophage therapy can be developed to target emerging diarrhoeal pathogens, but doing so in the absence of microbiome disruption, which occurs with antibiotic treatment, has not been established.Aim Identify a therapeutic bacteriophage that kills diarrhoeagenic enteroaggregative Escherichia coli (EAEC) while leaving the human microbiome intact.Methodology Phages from wastewater in Portland, OR, USA were screened for bacteriolytic activity by overlay assay. One isolated phage, PDX, was classified by electron microscopy and genome sequencing. A mouse model of infection determined whether the phage was therapeutic against EAEC. 16S metagenomic analysis of anaerobic cultures determined whether a normal human microbiome was altered by treatment.Results Escherichia virus PDX, a member of the strictly lytic family Myoviridae, killed a case-associated EAEC isolate from a child in rural Tennessee in a dose-dependent manner, and killed EAEC isolates from Columbian children. A single dose of PDX (multiplicity of infection: 100) 1 day post-infection reduced EAEC recovered from mouse faeces. PDX also killed EAEC when cultured anaerobically in the presence of human faecal bacteria. While the addition of EAEC reduced the β-diversity of the human microbiota, that of the cultures with either faeces alone, faeces with EAEC and PDX, or with just PDX phage was not different statistically.Conclusion PDX killed EAEC isolate EN1E-0007 in vivo and in vitro, while not altering the diversity of normal human microbiota in anaerobic culture, and thus could be part of an effective therapy for children in developing countries and those suffering from EAEC-mediated traveller’s diarrhoea without causing dysbiosis.

Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption.

The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design.IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

Bacteriophage Therapeutics: A Primer for Clinicians on Phage-Antibiotic Combinations.

Multidrug-resistant organisms have caused a marked depletion of effective antimicrobials, and the narrow pipeline of antibiotics has demanded the need to find novel therapeutic alternatives including nonantibiotic agents. Bacteriophages (phages) are viruses that use the bacterial machinery to infect, replicate, and kill bacterial cells. Although a marked decline in their use was driven by the discovery of antibiotics, the era of antibiotic resistance has led to a resurgence of phage therapy into clinical practice. The term phage-antibiotic synergy (PAS) was coined just over a decade ago and described that sublethal concentrations of antibiotics could stimulate phage production by bacterial cells. Recent literature has described PAS and other encouraging interactions with various phage and antibiotic combinations against a variety of bacterial strains. The primary objective of this review is to discuss the positive interactions between phage and antibiotic combinations, with an emphasis on PAS, reductions in bacterial growth or minimum inhibitory concentrations, enhanced biofilm eradication, and alterations in the emergence of bacterial resistance. A peer-reviewed literature search was conducted (1890-2019) using the PubMed, Medline, and Google Scholar databases. Although more investigation is certainly needed, the combination of bacteriophages with antibiotics is a promising strategy to target organisms with limited or no therapeutic options. This approach may also foster the ability to lower the antibiotic dose and may reduce the potential for antibiotic resistance emergence during therapy.

Potential of using bacteriophages and nanomaterials for eradicating bacterial diseases in animals

The appearance of new and the recurrence of old bacterial infections in animals, coupled with a simultaneous epidemic increase in the number of multidrug-resistant strains and accompanying diagnostic problems, causes a growing interest in alternative strategies for prevention and treatment of resultant diseases. Technologies based on therapeutic bacteriophages or various kinds of nanomaterials are very promising and increasingly applied in eradicating harmful enzootic and zoonotic pathogens. A new development in these endeavours may be the combined use of phages (particularly M13, MS2, λ and T-even phages) and nanoparticles for a synergistic effect. Mutual interactions between the two factors depend not only on the type and characteristics of a given nanomaterial and on the morphological type of the phage, but also on their quantity, ambient temperature, and time of exposure. Interactions between nanoparticles and bacteriophages are due to electrostatic effects used in creating hybrid phage-nanomaterial constructs that find application in eradicating bacterial pathogens, including drug-resistant and biofilming ones, as well as in directed drug delivery. One of the methods for creating useful phage-nanomaterial complexes is immobilisation by encapsulation. The entrapment of phages in a liposome structure promotes their replication and activity thanks to the small size and positive electric charge. Liposomal encapsulation protected them from the strongly acidic environment in the stomach, significantly prolonged the possibility of their storage, as well as increased their stability and durability in drinking water and feed without changing the sensory properties of water and feed. Phage-nanomaterial complexes can also be a very precise diagnostic tool for detecting bacterial pathogens in the environment, considerably increasing the sensitivity, specificity and rapidity of detection tests.