Campygen 2.5 l sachet

Stool specimens were cultured for Campylobacter using modified ISO methods (EN ISO 10272-2019) for detecting and enumerating Campylobacter [54 (link)] by direct plating and by filtration of stool prior to plating. For the direct plating, a 10 μl aliquot of each stool specimen was directly plated onto modified charcoal-cefoperazone deoxycholate agar (mCCDA) with cefoperazone and amphotericin B supplements (Oxoid, Hampshire, United Kingdom). In parallel, a 4 ml aliquot of each stool specimen was emulsified in 4 ml of phosphate-buffered saline (PBS) and filtered through a 0.65 μm syringe filter (Sartorius, Göttingen, Germany), before 10 µl was inoculated onto a mCCDA plate. All plates throughout the protocol were incubated in a microaerophilic atmosphere using anaerobic jars with CampyGen 2.5 L sachet (Oxoid, Hampshire, United Kingdom) at 37 °C for 48 h. C. jejuni strain 81116 was used as a positive control throughout the protocol.
Once incubated, up to 30 suspected Campylobacter colonies per patient specimen were sub-cultured onto mCCDA for purification, and further sub-cultured onto Columbia agar with 5% horse blood (Oxoid, Hampshire, United Kingdom). Colony morphology, microscopy, and oxidase test (Thermo Fisher Scientific, Loughborough, United Kingdom) were utilised to confirm presumptive Campylobacter isolates.

Campylobacter jejuni was inoculated in Mueller Hinton broth (BD 275730) supplemented with Campylobacter selective supplement (Skirrow) (SR0069E) according the manufacturer’s directions. After incubating the inoculated broth at 42^°C for 48 h under microaerophilic conditions (5% O₂, 10% CO₂ and 85% N₂) using CampyGen™ 2.5 L Sachet (CN0025A, Oxoid), Mueller Hinton agar (1.5%) plates (90 mm circular plates) were spread with 100 µl of this culture and were allowed to dry. Then, using a sterile 200 µl pipette tip, wells of approximately 5 mm in diameter were created aseptically in the agar. The LAB culture supernatants (both neat and pH neutralized) were added to each well (50 µl/well) and then the plates were left for approximately 30 min until the supernatants were absorbed into agar (wells were empty). These plates were incubated at 42^°C for 24 h under microaerophilic conditions for 24 h. The inhibition zone around the wells was observed and recorded (in mm). The experiment was performed in triplicate.

Nutrient agar (NA, Oxoid) was used for routine selection and maintenance of Escherichia coli strains. For experiments, E. coli cells were grown using Luria–Bertani (LB) medium. Supplements were added as required: 100 μg/ml ampicillin. H. pylori 26695 strain was purchased from National Collection of Type Cultures of Public Health England and maintained on Columbia Blood Agar (CBA) plates containing 5% defibrinated Sheep Blood (Thermo Fisher Scientific) in microaerofilic chambers (Oxoid) in which the atmosphere was maintained with CampyGen 2.5L Sachet (Oxoid). Supplements were added as required: 5 μg/ml chloramphenicol, 10 μg/ml kanamycin. Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich.