Bolton broth selective supplement

The preparation of the samples and isolation of Campylobacter spp. from the examined samples was done according to FDA [14 ]. The pH of the samples was adjusted to 7.5±0.2, and then centrifugation of 50 g portion at 20,000 ×g for 40 min was attained. Supernatant was discarded and pellets were dissolved in 10 ml Bolton broth (supplemented with Bolton broth Selective Supplement and Laked Horse Blood, Oxoid) and then was transmitted to 90 ml enrichment broth and incubated at 42°C for 48 h in an anaerobic jar containing a gas generating Kit (Oxoid). The Campylobacter blood free selective agar (mCCDA-Preston, Oxoid) which was supplemented with CCDA selective supplement (Oxoid), were then streaked with a loopful of each enrichment broth, and subsequently, incubated at 42°C for 48 h under microaerobic condition. From 2 to 3 presumptive Campylobacter colonies were purified on Columbia blood agar (containing 7% defibrinated sheep blood)without supplement. About 100 Campylobacter isolates were submitted to Gram-stain, oxidase, catalase, inability to grow aerobically at 25°C, hippurate hydrolysis and resistance to naladixic acid and cephalothin to exclude Campylobacter spp. except C. jejuni and C. coli.

One gram of each sample was homogenized with 9 mL of Bolton broth containing 5% hemolyzed horse blood and a Bolton broth selective supplement (Oxoid, United Kingdom). The samples were incubated at 42°C for 48 h microaerobically. On the following day, one loop of broth was streaked onto modified charcoal cefoperazone deoxycholate agar (mCCDA) containing CCDA selective supplement (Oxoid), and then the samples were incubated at 42°C for 48 h microaerobically. Next, two to six suspected colonies from the mCCDA plates were picked, subcultured in blood agar, and incubated microaerobically at 42°C for 48 h. To identify C. jejuni isolates, multiplex PCR targeting the 16S rRNA gene and cj0414 and singleplex PCR targeting hipO were performed using the DNA templates, according to methods described previously (Yamazaki-Matsune et al., 2007 (link); Supplementary Table S2).

This study was carried out between March 2017 and September 2018 (18 months) at Zagazig city, Sharkia Governorate, Egypt. A total of 210 samples were collected from chicken (n = 175) and human (n = 35) sources. Ten samples from chickens of 6 weeks of age were collected per month from multiple retail outlets (n = 16, 11 samples from each outlet) including cloacal swabs, breast muscles, liver, chicken franks and chicken luncheon meats (n = 35 each), while human stool samples were obtained as swabs from diseased children with diarrhea from private laboratories at Zagazig city. Cloacal and stool swabs were transferred directly into a sterile tube containing 9 mL of Bolton broth with Bolton broth selective supplement (Oxoid, UK) with 20 mm space lift in the tube to achieve microaerophilic conditions, while other samples were transferred into ice boxes for laboratory isolation and identification of Campylobacter spp.