MyoviridaePhagePDXKills EnteroaggregativeEscherichia coliwithout Human Microbiome Dysbiosis

Abstract

AbstractPurposeTo identify therapeutic a bacteriophage that kills diarrheagenic enteroaggregativeEscherichia coli(EAEC) while leaving the human microbiome intact.MethodologyPhages 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 EAECin vivo. Anaerobic culture in the presence of human fecal bacteria determined whether the phage could kill EAECin vitro, and to assess whether the microbiome had been altered.ResultsThe isolated phage, termedEscherichia virus PDX, is a member of the strictly lyticMyoviridaefamily of viruses. PhagePDXkilled 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 ofPDX, 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 five-day period. PhagePDXalso killed EAEC EN1E-0227 when cultured anaerobicallyin vitroin the presence of human fecal bacteria. While the addition of EAEC EN1E-0227 reduced the α-diversity of the human microbiota, that of the cultures with either feces alone, feces with EAEC andPDX, or with just thePDXphage were not different statistically, as measured by Chao1 and Shannon diversity indices. Additionally, β-diversity and differential abundance analyses show that conditions withPDXadded were not different from feces alone, but all groups were significantly different from feces + EAEC.ConclusionsThe strictly bacteriolytic,Myoviridae Escherichia virus PDXkilled EAEC isolate EN1E-0227 bacteria bothin vivoandin vitro, while simultaneously not altering the diversity of normal human microbiota in anaerobic culture. Thus, thePDXphage 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,PDXcould also provide therapeutic relief for these individuals, particularly in light of the growing crisis of antibiotic resistances.