Fucose permease gene (fucP) and γ-glutamyl transpeptidase gene (ggt) screening and l-fucose/d-glucose utilization experiments with C. jejuni and C. coli strains.

To identify the potential fucose utilization ability of Campylobacter strains isolated in GEMS, we screened for the fucP gene in all GEMS-isolated Campylobacter strains (C. jejuni and C. coli) by colony or genomic DNA PCR using primers designed to amplify the fucP gene of C. jejuni NCTC 11168 (fucP-F/R [CAT GAA AGT GGC TTT TTA CAG/ATT TTT TTC ATC ACC AAG CTT TG]) in a multiplex format with 16S rRNA control primers (16S-F/R [AAT GGC TTA ACC ATT AAA CTG C/AAC TAA ATA CGT GGG TTG CG]), which was confirmed to also work for C. coli strains (data not shown). To verify that a fucP PCR product corresponded to a strain’s ability to use fucose, we performed growth experiments in a subset of the strains lacking fucP and all fucP⁺ strains as described previously (16). Briefly, Campylobacter strains were adjusted to an optical density at 600 nm (OD₆₀₀) of 0.05 and inoculated into 5 ml MEMα (minimum essential medium α; Thermo Fisher Scientific) (with 20 μM iron added) with or without 25 mM l-fucose. The inoculated culture was incubated microaerobically at 37°C for 18 h, and the OD₆₀₀ was measured using the NanoDrop One^C system (Thermo Scientific). Screening for the ggt gene was carried out as previously described (29). To test for d-glucose utilization, we performed growth experiments as described above, using a random subset of 33 strains in DMEM (Dulbecco’s modified Eagle’s medium) without glucose (Thermo Fisher Scientific), and the strains were incubated microaerobically for 48 h. Campylobacter cuniculorum LMG 24588 was used as the positive control.