Campygen sachet

In a 12-month period from November 2015–2016, C. jejuni isolates (n = 60; S5 Table online) from anonymised clinical samples from Northampton General Hospital, UK were collected by swabbing cultured Charcoal-Cefoperazone-Deoxycholate Agar (CCDA) plates. Amines and charcoal swabs (Thermo Fisher Scientific) were used for collection and transportation of Campylobacter isolates. Bacterial isolates were cultured again within 24 hours of collection and grown on MHA plates at 37°C for 24–48 hours under a microaerobic atmosphere of 5% O₂, 10% CO₂ and 85% N₂. The microaerobic environment was provided by either using CampyGen sachets (Oxoid Limited) in 2.5 L air-tight jars or BOC gas mixture (2% H₂, 5% O₂, 10% CO₂ and 83% N₂) in a Whitley G2 workstation (Don Whitley Scientific).

The meat was sliced with a sterile scalpel in a petri dish and incubated for 18-24 h at 30°C in 10 mL Arcobacter enrichment broth (Oxoid, Basingstoke, England) under microaerophilic conditions (CampyGen sachets, Oxoid). Bacterial cells from the Arcobacter enrichment broth were subsequently cultured on Mueller Hinton agar (Oxoid) with 5% (vol/vol) sheep blood using a filter technique as described by Atabay et al. (Atabay et al., 2003 (link)). The plates were incubated at 35–37°C for 18–24 h under microaerophilic conditions. Plates showing no sign of bacterial growth were incubated for four additional days. The identity of oxidase-positive isolates was assessed by Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics GmbH & Co. KG, Bremen, Germany).

The wild-type C. jejuni strain used in this study is the commonly used 81–176 lab strain and all mutant and complemented strains were constructed on this background. The bacteria were routinely grown on Mueller-Hinton agar plates or broth, supplemented with the selective antibiotics Chloramphenicol and/or Kanamycin as required. Additionally, during mutant and complement construction, plates and broth were routinely supplemented with vancomycin (10 µg/mL) and trimethoprim (5 µg/mL) to prevent contamination. Cultures were routinely grown under microaerophilic conditions using anaerojars and CampyGen sachets (Oxoid) at 42°C.

Isolation of Campylobacter spp. was achieved according to the International Standards Organization (ISO) guidelines [40 ]. Briefly, the enrichment broth containing samples was incubated for 48 h at 42 °C in darkness under microaerophilic conditions (5% O₂, 10% CO₂ and 85% N₂) using CampyGen sachets (Oxoid, Cheshire, UK) and anaerobic jar (Oxoid, Cheshire, UK). After that, 10 µL of the enrichment broth was streaked onto the surface of the selective modified charcoal cefoperazone deoxycholate agar (mCCDA) plates with CCDA selective supplement (Oxoid, Cheshire, UK), and the plates were incubated for 48 h at 42 °C in darkness under microaerophilic conditions. For more purification, suspicious colonies were cultivated onto blood agar (Oxoid, Cheshire, UK) supplemented with 5% sterile defibrinated sheep blood, and the plates were incubated for 48 h under microaerophilic conditions. The Campylobacter isolates were presumptively confirmed via cultural characteristics on mCCDA, Gram’s staining, motility, some biochemical tests such as catalase, oxidase and indoxyl acetate and sodium hippurate hydrolysis tests and finally susceptibility to nalidixic and cephalothin.

For Campylobacter spp. isolation, the obtained specimen in Bolton broth was incubated at 42 °C/24–48 h in darkness in a microaerophilic atmosphere (85% N₂, 10% CO₂, and 5% O₂) utilizing an anaerobic jar (Sigma-Aldrich, St. Louis, MO, USA) and CampyGen sachets (Oxoid, Cambridge, UK). Subsequently, 0.1 mL of the inoculated Bolton broth was inoculated onto the surface of supplemented modified charcoal cefoperazone deoxycholate agar (mCCDA) plates (Oxoid, Cambridge, UK), then the inoculated plates were incubated at 42 °C/48 h in a microaerophilic atmosphere. For further purification, the suspected campylobacter colonies were cultivated on 5% sheep blood agar plates (Oxoid, Cambridge, UK), then incubated at 42 °C/48 h in a microaerophilic atmosphere. Next, the suspected colonies were presumably identified through their culture characters on mCCDA and blood agar, Gram’s staining, motility test, susceptibility to nalidixic acid and cephalothin, and biochemical identification procedures, including oxidase, catalase, rapid sodium hippurate, and indoxyl acetate hydrolysis [23 (link)].

For Campylobacter spp. isolation, the collected samples in Campy-Thio broth were incubated for 24–48 h at 42 °C with less than 1 cm of headspace left in the culture vessels, which were kept with tightly-capped lids in darkness under microaerophilic conditions (85% N₂, 10% CO₂ and 5% O₂) using CampyGen sachets (Oxoid, Cambridge, UK) and the anaerobic jar (Sigma-Aldrich, St. Louis, MI, USA). Following the enrichment step, 0.1 mL of the broth was inoculated onto the surface of modified charcoal cefoperazone deoxycholate agar (mCCDA) with CCDA selective supplement (Oxoid, Cambridge, UK) and the plates were incubated under microaerophilic conditions at 42 °C in darkness for 48 h. Additionally, three to four presumptive campylobacter colonies that had similar colonial morphology were further inoculated onto 5% sheep blood agar plates for 24–48 h at 42 °C under microaerophilic conditions in darkness. After incubation, suspected colonies were identified via Gram’s staining, motility test and biochemical identification using catalase, oxidase and rapid hippurate hydrolysis tests [25 (link),26 ].

To isolate Salmonella and Shigella, a loopful of each stool sample was cultured in Selenite F Broth at 37°C for 24h followed by subculturing onto Salmonella-Shigella agar (Condalab, Spain) at 37°C for 24h. On Salmonella-Shigella agar, Salmonella spp. colonies appeared colorless with a black center, while Shigella spp. colonies were colorless. Antisera from the Salmonella seroquick ID kit (SSI Diagnostica, Denmark) were used for further serotyping of Salmonella typhimurium and enteritidis. Campylobacter spp. were isolated for 48h at 42°C in microaerophilic conditions generated by Campygen sachets (Oxoid, UK) on Karmali agar medium enriched with selective Karmali supplement (Condalab, Spain). Catalase, hippurate hydrolysis, the H₂S test, and gram stain were used to identify and differentiate C. jejuni and C. coli colonies grown on Karmali agar.