Research Paper Oral Phage Therapy of Acute Bacterial Diarrhea With Two Coliphage Preparations: A Randomized Trial in Children From Bangladesh Shafiqul Alam Sarker a , Shamima Sultana a , Gloria Reuteler b , Deborah Moine c , Patrick Descombes c , Florence Charton b , Gilles Bourdin b , Shawna McCallin b , Catherine Ngom-Bru b , Tara Neville b , Mahmuda Akter a , Sayeeda Huq a , Firdausi Qadri a , Kaisar Talukdar a , Mohamed Kassam c , Michèle Delley b , Chloe Loiseau b , Ying Deng b , Sahar El Aidy b , Bernard Berger b , Harald Brüssow b, ⁎ a International Centre for Diarrheal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212, Bangladesh, b Nestlé Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland c Nestlé Institute of Health Science, EPFL Innovation Park, CH-1015 Lausanne, Switzerland abstract article info Article history: Received 19 November 2015 Received in revised form 23 December 2015 Accepted 27 December 2015 Available online 5 January 2016 Background: Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative. Method: T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quan- titative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced. Findings: No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes. Interpretation: Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage–bacterium interaction and the role of E. coli in childhood diarrhea for successful PT. Funding: The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Bacteriophages Diarrhea Escherichia coli Streptococcus Bifidobacterium Children Bangladesh 1. Introduction In view of the growing threat of antibiotic resistance, WHO has warned for a return into a pre-antibiotic era (Martinez, 2012). Alterna- tives to antibiotics are thus urgently needed (Piddock, 2012; Stanton, 2013). Phage therapy (PT), the use of bacterial viruses (phages) against pathogens, is a potentially attractive option for the prevention and treat- ment of some bacterial infections (Sulakvelidze et al., 2001; Brüssow, 2005, 2012). Indeed, a large, randomized and placebo-controlled trial conducted in children from Tbilisi/Republic of Georgia during the 1960s EBioMedicine 4 (2016) 124–137 Abbreviations: Cfu, colony forming unit; ETEC, enterotoxigenic E. coli; EPEC, enteropathogenic E. coli; EAEC, enteroaggregative E. coli; M, ColiProteus phage cocktail from Microgen; ORS, oral rehydration solution; P, placebo; pfu, plaque forming unit; PT, phage therapy; qPCR, quantitative polymerase chain reaction; RCT, randomized controlled trial; T, T4 phage cocktail from NRC. ⁎ Corresponding author. E-mail address: harald.bruessow@rdls.nestle.com (H. Brüssow). http://dx.doi.org/10.1016/j.ebiom.2015.12.023 2352-3964/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect EBioMedicine journal homepage: www.ebiomedicine.com