Characterizing Phage Genomes for Therapeutic Applications

Philipson, Casandra; Voegtly, Logan J.; Lueder, Matthew R.; Long, Kyle A.; Rice, Gregory K.; Frey, K. G.; Biswas, Biswajit; Cer, Regina Z.; Hamilton, Theron; Bishop‐Lilly, Kimberly A.

doi:10.3390/v10040188

Cited by 133 publications

(107 citation statements)

References 60 publications

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“…These phages were characterized by electron microscopy, one-step growth experiments, and genome sequencing. Sequence analysis confirmed that P1 and P2 were related to strictly lytic phages and that neither phage harbored an identifiable integrase, toxin, or bacterial virulence factor, satisfying several recommended criteria for therapeutic phage selection (14).…”

Section: Resultsmentioning

confidence: 80%

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

Hesse

Rajaure

Wall

et al. 2020

mBio

105

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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.

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Section: Resultsmentioning

confidence: 80%

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

Hesse

Rajaure

Wall

et al. 2020

mBio

105

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“…Therefore, guidelines are needed to analyze and differentiate phages designated for therapy in humans or animals by their lifecycle. Such guidelines do not yet exist [85]. The US FDA, for example, relies on emergency protocols proposed by medical doctors for the case-by-case approval of phage cocktails for therapeutic purposes in special "compassionate" use of Investigational New Drugs (IDEs).…”

Section: Determining Safety Of Phages In the Era Of "Omics"mentioning

confidence: 99%

Application of Campylobacter jejuni Phages: Challenges and Perspectives

Ushanov

Lasareishvili

Janashia

et al. 2020

Animals

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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.

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“…After identifying the endolysin gene E-LM12 (gp159) from the S. aureus vB_SauM-LM12 phage genome 46 , a BLAST analysis revealed that E-LM12 shows high similarity with endolysins from several staphylococcal phages, including Staphylococcus phage MCE-2014 (100% coverage, 95% identity) 47 , Staphylococcus phage phiIPLA-RODI (100% coverage, 94% identity) 48 and Staphylococcus phage vB_SauM_0414_108 (99% coverage, 95% identity) 49 . Also, no similarity was found with endolysins from phages infecting other bacterial genera.…”

Section: Bioinformatics Analysis Of E-lm12mentioning

confidence: 99%

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

Costa

Dias

Melo

et al. 2020

Sci Rep

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Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1-5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs. Bloodstream infections (BSIs) are severe diseases caused by the presence of microorganisms, mainly bacteria, in blood and are characterized by high morbidity and mortality 1,2. The BSIs and associated organ dysfunctions (sepsis or septic shock) remain a life-threating disease due, partially, to the inability to rapidly detect and identify the responsible pathogens 3,4. Staphylococcus spp. are Gram-positive facultative anaerobic bacteria that frequently colonize the human body 5,6. These pathogens are becoming increasingly resistant to antibiotics and are well-established in both community and healthcare environments, being commonly isolated in intensive care units (ICU) 6,7. Staphylococcus aureus is a common cause of a variety of infections, from superficial skin infections to life-threatening diseases, including necrotizing pneumonia 8 , infective endocarditis 9 and BSIs 10. Coagulase-negative staphylococci (CoNS) have also been described as harmful to humans, causing several infections, particularly in patients with implanted medical devices 6. The empirical antibiotic therapy remains the standard of BSIs treatments 11 and its correct use within the first hour after the recognition of the BSI is recommended by the Surviving Sepsis Campaign Guidelines 11 and was reported as having a great impact on the patient survival rate 12. Nevertheless, the extensive use of broad-spectrum antibiotics and the large number of patients having negative blood culture samples and thus receiving unnecessary antibiotic treatment, are important contributors to the increase of antimicrobial resistance 13-15. Thus, sensitive, rapid, cost-efficient and specific ...

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Characterizing Phage Genomes for Therapeutic Applications

Cited by 133 publications

References 60 publications

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

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

Application of Campylobacter jejuni Phages: Challenges and Perspectives

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

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