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.

Escherichia virus, microbiome, dysbiosis antibiotic alternatives. AbstractPurpose. To identify therapeutic a bacteriophage that kills diarrheagenic enteroaggregative Escherichia coli (EAEC) while leaving the human microbiome intact.Methodology. Phages from wastewater in Portland, OR, were screened for bacteriolytic activity using an overlay assay, and isolated in a sequential procedure to enrich for the recognition of core bacterial antigens. Electron microscopy and genome sequencing were performed to classify the isolated phage, and the host range was determined by spot tests and plaque assays. One-step growth curves and time-kill assays were conducted to characterize the life cycle of the phage, and to interrogate the multiplicity of infection (MOI) necessary for killing. A mouse model of infection was used to determine whether the phage could be used therapeutically against EAEC in vivo. Anaerobic culture in the presence of human fecal bacteria determined whether the phage could kill EAEC in vitro, and to assess whether the microbiome had been altered.Results. The isolated phage, termed Escherichia virus PDX, is a member of the strictly lytic Myoviridae family of viruses. Phage PDX killed EAEC isolate EN1E-0227, a case-associated isolate from a child in rural Tennessee, in a dose-dependent manner, and also formed plaques on case-associated clinical EAEC isolates from Columbian children suffering from diarrhea. A single dose of PDX, at a MOI of 100, one day post infection, reduced the population of recovered EAEC isolate EN1E-0227 bacteria in fecal pellets in a mouse model of colonization, over a fiveday period. Phage PDX also killed EAEC EN1E-0227 when cultured anaerobically in vitro in the presence of human fecal bacteria. While the addition of EAEC EN1E-0227 reduced the adiversity of the human microbiota, that of the cultures with either feces alone, feces with EAEC and PDX, or with just the PDX phage were not different statistically, as measured by Chao1 and Shannon diversity indices. Additionally, b-diversity and differential abundance analyses show that conditions with PDX added were not different from feces alone, but all groups were significantly different from feces + EAEC.Conclusions. The strictly bacteriolytic, Myoviridae Escherichia virus PDX killed EAEC isolate EN1E-0227 bacteria both in vivo and in vitro, while simultaneously not altering the diversity of normal human microbiota in anaerobic culture. Thus, the PDX phage could be part of an effective therapeutic intervention for children in developing countries who suffer from acute, or persistent EAEC-mediated diarrhea, and to help reduce the serious effects of environmental enteropathy. Because the emerging pathogen EAEC is now the second leading cause of traveler's diarrhea, PDX could also provide therapeutic relief for these individuals, particularly in light of the growing crisis of antibiotic resistances.