Campylobacter selective agar

Fecal specimens were inoculated onto MacConkey agar (MC agar; Oxoid) and xylose-lysine-deoxycholate agar (Oxoid) and into selenite broth (Oxoid) and incubated at 37°C for 18–24 hours. Salmonella and Shigella were detected based on their characteristic appearance on xylose-lysine-deoxycholate and MC agar and confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker) and API20E (bioMerieux), following the manufacturer’s guidelines. Campylobacter was identified using Campylobacter selective agar (Oxoid) under microaerophilic conditions, followed by Gram staining and microscopy.
Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Oxoid) for Salmonella and Shigella, and on blood agar containing 5% sheep blood for Campylobacter and interpreted using Clinical and Laboratory Standards Institute guidelines [14 ] (Supplementary Table S1). Multidrug resistance (MDR) was defined as nonsusceptibility to ≥1 agent in ≥3 antimicrobial categories listed in Supplementary Table S1. Microbiology results were reported to the collaborating hospitals within 3 days of sampling.

The fecal samples from humans and chicken manure were grown directly on Campylobacter Selective Agar (Oxoid, UK) for Campylobacter selection and Xylose-Lysin-Desoxycholat Agar (Oxoid, UK), MacConkey (Oxoid, UK), and Sheep Blood Agar Base (Oxoid, UK) for Salmonella selection. Agar plates were incubated at 37°C and checked for the presence of colonies after 24–48 hours. The chicken meat samples were pre-processed according to the ISO 10272-1:2017 guidelines for Campylobacter and ISO 6579-1:2017 guidelines for Salmonella. From all samples, suspected colonies for each of the species were selected for microbiological confirmation by the Vitek 2 NH ID card for Campylobacter or Vitek 2 GN ID card for Salmonella using the Vitek 2 compact automated system (BioMerieux, France). All isolates which were confirmed as Salmonella or Campylobacter by the Vitek 2 microbial testing system were subjected to further confirmation by PCR. Molecular species identification for Campylobacter spp. was performed using the PCR protocol published by Nayak et al. (29 (link)), while for Salmonella spp., the procedure published by Paião et al. was used (30 (link)).

The three isolates of C. jejuni used in the study were 48 (Penner serotype O:19), 75 (serotype O:3), and 111 (serotype O: in 1,44) which originated from the stools of diarrheic patients as stated above. They were found to colonize mouse intestine in previous studies [11 (link), 22 (link)]. Stock cultures were maintained in Brucella broth (Becton & Dickinson, Sparks, MD) with 15% (vol/vol) glycerol at -70°C. Depending upon the purpose, three types of media were used for cultivation. These were: blood agar base (Oxoid, Basingstoke, Hampshire, England) with 5% defibrinated sheep blood; Campylobacter-selective agar with laked horse blood, growth supplement and selective supplement (Oxoid); and a biphasic medium with brain heart infusion agar and broth supplemented with 1% yeast extract [23 (link)]. Cultures were incubated at 42°C for 48 h in a microaerobic atmosphere generated by Campigen (Oxoid). The identity of the bacteria was confirmed by cultural characteristics and molecular methods. The three serotypes generated unique fingerprints by flaA restriction fragment length polymorphism (RFLP) analysis [24 (link)].