Bacteriophage Therapy Testing against Shigella flexneri in a Novel Human Intestinal Organoid-Derived Infection Model

Objective: Enteric bacterial pathogens cause diarrheal disease and mortality at significant rates throughout the world, particularly in children under the age of 5 years. Our ability to combat bacterial pathogens has been hindered antibiotic resistance, a lack of effective vaccines, and accurate models of infection. With the renewed interest in bacteriophage therapy, we sought to use a novel human intestinal model to investigate the efficacy of a newly isolated bacteriophage against Shigella flexneri. Methods: A S. flexneri 2457T-specific bacteriophage was isolated and assessed through kill curve experiments and infection assays with colorectal adenocarcinoma HT-29 cells and a novel human intestinal-derived organoid monolayer model. In our treatment protocol, organoids were generated from intestinal crypt stem cells, expanded in culture, and seeded onto transwells to establish two-dimensional monolayers that differentiate into intestinal cells. Results: The isolated bacteriophage efficiently killed S. flexneri 2457T, other S. flexneri strains, and a strain of 2457T harboring an antibiotic resistance cassette. Analyses with laboratory and commensal Escherichia coli strains demonstrated that the bacteriophage was specific to S. flexneri, as observed under co-culture conditions. Importantly, the bacteriophage prevented both S. flexneri 2457T epithelial cell adherence and invasion in both infection models. Conclusions: Bacteriophages offer feasible alternatives to antibiotics for eliminating enteric pathogens, confirmed here by the bacteriophage-targeted killing of S. flexneri. Furthermore, application of the organoid model has provided important insight into Shigella pathogenesis and bacteriophage-dependent intervention strategies. The screening platform described herein provides proof-of-concept analysis for the development of novel bacteriophage therapies to target antibiotic-resistant pathogens. Address correspondence and reprint requests to Christina S. Faherty, PhD, Address: 114 16th Street (114-3503), Charlestown, MA 02129-4404 (E-mail: csfaherty@mgh.harvard.edu). Received 27 June, 2018 Accepted 15 October, 2018 Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal's Web site (www.jpgn.org). Funding Sources: Funding was provided through the Bill and Melinda Gates Foundation grant OPP1139856. Additional financial support was provided by the National Institute of Allergy and Infectious Diseases grants U19-AI082655 (Cooperative Center on Human Immunology to AF) and K22AI104755 (to CSF). The Lu lab acknowledges additional financial support by the National Science Foundation (MCB-1350625, #1521925), the National Institutes of Health (5-P50-GM098792-05 and 4-R33-AI121669-03), the Office of Naval Research (N00014-13-l-0424), the Army Research Office (W911NF-ll-1-0281), the Broad Institute, the Koch Institute, the Defense Threat Reduction Agency (HDTRAl-14-1-0007), the Kenneth Rainin Foundation (2016-3066), the Ellison Foundation (AG-NS-0948-12), the Wertheimer Fund, the Institute for Soldier Nanotechnologies (W9111NF-13-D-0001), J-WAFS, Novartis (14081955), the Desphande Center at MIT, the Singapore-MIT Alliance for Research and Technology, the Center for Microbiome Informatics and Technology (15127713), and Excet, Inc. (17033835). The TEM core at MGH is supported by National Institute of Neurological Disorders and Stroke P30NS045776. Support for the Philly Dake Electron Microscope Facility was provided by the National Institutes of Health grant 1S10RR023594S10 and by funds from the Dake Family Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. The funders had no role in the study decision, data collection and analysis, decision to publish, or preparation of the manuscript. Conflicts of Interest and Source of Funding: Alejandro Llanos-Chea: None Robert J. Citorik: None Kourtney P. Nickerson: None Laura Ingano: None Gloria Serena: None Stefania Senger: None Timothy K. Lu: Stock/equity holder in Ampliphi and Eligo Biosciences Alessio Fasano: None Christina S. Faherty: None Author's Roles: Alejandro Llanos-Chea: Designed and performed experiments, analyzed data, and contributed to writing the manuscript Robert J. Citorik: Designed and performed experiments, analyzed data, and contributed to writing the manuscript; Kourtney P. Nickerson: Performed experiments and analyzed data Laura Ingano: Performed experiments and analyzed data Gloria Serena: Performed experiments and analyzed data Stefania Senger: Performed experiments, analyzed data, and contributed reagents, materials, and analysis tools Timothy K. Lu: Designed experiments, contributed reagents, materials, and analysis tools; contributed to writing the manuscript Alessio Fasano: Contributed reagents, materials, analysis tools; and contributed to writing the manuscript Christina S. Faherty: Designed experiments, analyzed data, contributed reagents, materials, and analysis tools, and contributed to writing the manuscript Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.jpgn.org). © 2018 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,

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