Sample transportation, storage, and preparation

All aseptically collected stool samples were put in separate stomacher bags and kept in a cold box containing ice packs, and transported to the laboratory on the day of sample collection. On arrival at the laboratory, the samples were processed using a Standard Operating Procedure (SOP) created for the FOCAL project upon the existing protocol^37,38 and analyzed at the microbiology laboratory of Haramaya University, College of Health, and Medical Sciences (HU_CHMS), Harar, Ethiopia. The samples were gently mixed using vortex and sterile plastic loop; and plated on different agar Medias aerobically (for Salmonella, Shigella spp., and E. coli) and micro-aerobically (for Campylobacter spp.). The remaining stool samples were preserved at −80°C for further meta-genomic analysis (the MG result is not included in this particular manuscript).

Isolation and Identification: for isolation and identification of Salmonella, Shigella spp. and E. coli, samples were plated in triplicate on selective media: Xylose Lysine Deoxycholate (GranuCult™ XLD) and Hektoen Enteric Agar (HE) (Oxoid CM0419B, UK) and MacConkey agar (MAC) and aerobically incubated at 37°C. After overnight incubation the 3 plates were examined for growth and morphological features of each pathogen, characteristic colonies of each pathogen were picked and subjected to gram staining. However, suspected mixed colonies of Salmonella and Shigella spp., and E. coli were sub-cultured on XLD and HE or Brilliant Green agar (BGA) (Oxoid CM0263B, UK), and MAC, respectively before gram staining. From Gram negative rod, E. coli suspected colonies on MAC, a single colony was then transferred to Levine’s eosin-methylene blue (EMB) and colonies showing green metallic sheen morphology on EMB were taken as suspected E. coli isolates.

From the suspected respective pure colonies of Salmonella, Shigella spp., and E. coli, a single colony was then transferred on to Tryptone soya agar (TSA) (Merck, Germany), and followed by biochemical confirmation tests after overnight incubation. Specific biochemical reaction of presumptive colonies for each bacterial pathogen was examined on; Triple sugar iron (TSI) and lysine iron agar (LIA), urease and IMViC (Indole (I), Methyl red (M), Voges proskauer (Vi), and Citrate (C) midia (Merck, Germany), and interpreted according to Andrews et al. ³⁹ Besides, biochemically confirmed E. coli isolates were plated on Sorbitol MacConkey (SMAC) agar (Oxoid CM0813,UK; Lot: 3413831), and only biochemically confirmed, non Sorbitol fermenter (NSF) E. coli isolates were considered as pathogenic E. coli or DEC.

For Campylobacter spp., stool samples were plated on blood free Campylobacter selective (mCCD (Modified charcoal cefoperazone deoxycholate) agar (base) (Merck, Germany; Cat number: 1000700500) containing selective supplement (antibiotics) comprising of amphotericin B and Cefoperazone (Merck, Germany; Cat number: 1000710010) and incubated for 48 hours at 42°C in anaerobic jar 2.5 L (AG0025A, Oxiod; Cat number: 10219202) under a microaerophilic condition provided by Gas generating sachets containing 5% O₂, 10% CO₂, and 85% N₂ (Campy-Gen 2.5 L; SSI Diagnostica, Japan; Cat number: 19438). After the incubation period, suspected colonies were subjected to gram staining and characteristic were taken on to Brain heart infusion (BHI) agar and incubated microaerophilically. ⁴⁰ Finally, presumptive colonies found positive for both catalase and oxidase (Sigma, India; Cat number: 70439) tests were preserved as Campylobacter isolates.