Hippurate hydrolysis test

All strains isolated were unfrozen and revived (Columbia Blood Agar, Oxoid Ltd., England, UK). Plates were incubated at 41.5 °C for 44 ± 4 h in modified atmosphere (5% O₂, 85% N₂, 10% CO₂, CampyGen, Oxoid, England, UK). Finally, the hippurate hydrolysis test (Oxoid, Madrid, Spain) was used to determine the species of the Campylobacter [33 (link),34 (link)].

Enumeration of Campylobacter in samples was performed according to ISO/TS 10272-2:2017. As required by the ISO method, tenfold dilutions of samples on BPW were performed. Then, 0.1 mL from each inoculum was plated onto mCCDA (Modified Charcoal Cefoperazone Deoxycholate Agar, Oxoid, Dardilly, France) and incubated at 41.5 ± 1 °C in a micro-aerobic atmosphere (84% N₂, 10% CO₂, 6% O₂) for 44 h ± 4 h. For further confirmation analysis, five Campylobacter-like colonies were purified on blood agar (AES La-boratories^®, Bruz Cedex, France) at 41.5 ± 1 °C in a microaerobic atmosphere (84% N₂, 10% CO₂, 6% O₂) over 24 h. Then, colony morphology and motility under dark field microscopy were evaluated. Confirmation of presumptive Campylobacter colonies was assessed by oxidase and catalase tests and plating at different temperatures and atmospheres (25 °C under aerobic conditions and 41.5 °C under microaerophilic conditions for 24 h) in Columbia blood agar (AES Laboratories).
For Campylobacter speciation, isolated strains were plated on Columbia blood agar (Oxoid Ltd., Basingstoke, UK) and incubated at 41.5 °C for 44 ± 4 h in a modified atmosphere (5% O₂, 85% N₂, 10% CO₂, CampyGen, Oxoid, Basingstoke, UK). Finally, the hippurate hydrolysis test (Oxoid, Madrid, Spain) was used to determine the species of the Campylobacter [38 (link),42 (link)].