Genbox generators

Upon arrival at the laboratory, samples were inoculated into Bolton Broth (Oxoid, Basingstoke, UK), containing the Bolton selective supplement for enrichment, and then incubated at 42 °C for 24 h in a microaerobic environment (5% O₂, 10% CO₂, and 85% N₂), with GENbox generators (BioMérieux, Craponne, France). After enrichment, putative Campylobacter-positive samples were streaked on Karmali agar (Oxoid, Basingstoke, UK) and incubated under the same conditions as described above for 48 h [24 (link)]. From each sample, suspected colonies were examined for the typical morphology and motility of Campylobacter, under a light microscope and using the oxidase/catalase tests. Thereafter, presumed Campylobacter colonies were subjected to PCR analysis for genus confirmation and species identification. Confirmed Campylobacter isolates were conserved at −80 °C in Mueller–Hinton broth containing 25% glycerol (v/v).

Enumeration and determination of C. jejuni in 1 ml of initial suspension (skin homogenate or culture) were performed according to ISO 10272:2006 methods (ISO, 2006a ,b ). For enumeration of C. jejuni culture, 1 ml of initial culture suspension was applied onto three mCCDA (modified charcoal cefoperazone desoxycholate agar; Oxoid, UK) plates and 0.1 ml of further decimal dilutions up to 10⁻⁸ on single mCCDA plates. For enumeration of skin-homogenate samples, 1 ml of sample and decimal dilutions to 10⁻³ were spread plated. After 40–48 h incubation at 41.5°C in the microaerophilic atmosphere created by the GENbox generators (BioMerieux, France), plates from two successive dilutions with <150 Campylobacter-suspected colonies per plate were counted to obtain the final colony forming units (CFU) per unit of measure (g or ml). To determine the isolates to the species level, the hippurate and indoxyl acetate hydrolysis, catalase and susceptibility to cephalotine and nalidixic acid tests were performed. Two suspected Campylobacter colonies from each sample were randomly selected for identification.

Overall, 590 cloacal swabs were taken from chickens. Samples were inoculated into Bolton Broth (Oxoid, UK) and incubated, for enrichment, at 42°C for 48 h in a microaerobic environment (5% O₂, 10% CO₂ and 85% N₂), created by GENbox generators (BioMerieux, France). A loopful of each enriched sample was streaked on Karmali plates (SIGMA-ALDRICH) and incubated under the same conditions, as described above. From each sample, a total of ten swarming, opaque, white to grey colonies suspected of being campylobacters were subcultured on Karmali agar. Colonies comprising curved or spiral motile rods, when observed by light microscopy, were examined for oxidase/catalase activities and Gram stained. Thereafter, a maximum of three microscopically confirmed Campylobacter isolates per sample were subjected to PCR analyses for genus confirmation and species identification.
Confirmed Campylobacter isolates were stored in brain heart infusion broth (Bio-Rad) with 25% glycerol at −80°C for further analysis. Only one isolate per positive samples was used for further analyses.