This study investigates the effects of bacteriophage therapy on the gut microbiome of 4-week-old weaned piglets during enterotoxigenic Escherichia coli (ETEC) infection

This study investigates the effects of bacteriophage therapy on the gut microbiome of 4-week-old weaned piglets during enterotoxigenic Escherichia coli (ETEC) infection. Shotgun metagenomic sequencing was performed on piglet fecal samples from a controlled intervention trial with three treatment groups: 1. ETEC infection alone 2. ETEC infection with bacteriophage intervention (ETEC_PHAGES) 3. Bacteriophages alone without ETEC (PHAGES) Fecal samples were collected at three timepoints (Day 2, 6, and 8 post-treatment) to assess temporal dynamics of the gut microbiome response to phage therapy. Methods: Microbial genomic DNA was extracted using DNeasy PowerSoil Pro Kit (Qiagen, cat. no. 47014) from 125 mg of piglet fecal material following the manufacturer's instructions. DNA concentration and purity were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Negative controls were included for both DNA isolation and library preparation to detect potential contamination. Sequencing libraries were prepared from 100 ng of genomic DNA using the Illumina DNA prep protocol. Samples were dual indexed using IDT for Illumina UD Indexes. The PCR step used 7 cycles, and DNA was pooled and purified using Illumina's SPB bead purification. The library pool was sequenced at 9 pM on the AVITI sequencer (Element Biosciences) using the AVITI 2x150 Sequencing kit, generating 150 bp paired-end reads.