Karmali agar plates

Prior to the infection C. jejuni strain 81-176 was derived from frozen stocks, inoculated on karmali agar plates (Oxoid, Wesel, Germany) and grown at 37°C under microaerophilic conditions (CampyGen gas packs, Oxoid, Wesel, Germany). After 2 days of incubation one fluently grown karmali plate was chosen for harvest and incorporated in 5 mL sterile phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA). This resulted in an optical density of 0.6 at 600 nm wavelength with a yield of 10⁹ viable bacterial cells. On days 0 and 1, a volume of 0.3 mL of the C. jejuni suspension was used to gavage each SAB IL-10^–/– mouse. The yield of 10⁹ colony-forming units (CFU) was respectively confirmed by serial dilutions and culture of the bacterial suspensions on solid culture media as described recently (Du et al., 2022 (link)).

The virulent C. jejuni strain 81–176 (NC_008787), whose whole genome is available in Genoscope Platform (MicroScope Vallenet et al., 2017 (link)), was selected for the experiments. A loopful of frozen 81–176 cells culture, conserved at −80°C in Brain-Heart Infusion (BHI) broth (Biokar, Beauvais, France) containing 20% sterile glycerol, was cultured on fresh Karmali agar plates (Oxoid, Dardilly, France) (Air Liquid, Paris, France) at 42°C for 48 h in microaerobic conditions (MAC) generated using gas replacement jars operated by MACSmics gassing system (BioMérieux, France) with a gas blend composed of 5% O₂, 10% CO₂, and 85% N₂ (Air Liquid, Paris, France) and 4 filled/flushed cycles at −50 kPa as described in Mace et al. (2015) (link). Cultures were obtained by inoculating 500 mL of BHI broth in a 1-L flask and incubating them for 16 h under MAC at 42°C in a rotary shaker.

Experiments were performed with 12 C. jejuni water isolates, one isolate from raw chicken meat and one human clinical isolate (Table 1). All isolates have different genotype. The cultures were stored at −80°C in Brain-Heart Infusion medium (BHI; Oxoid, United Kingdom) containing 20% glycerol. At the beginning of each experiment, the isolates were resuscitated on Karmali agar plates (Oxoid, United Kingdom) at 42°C for 48 h under a microaerobic atmosphere (MAC; 5% O₂, 10% CO₂, 85% N₂) in an MCO-18M multi-gas incubator (Sanyo, Japan).

In this work, C. jejuni strain 81-176 isolated from a raw milk outbreak (Korlath et al., 1985 (link)) was used. Cells were resuscitated from a stock on Karmali agar plates (Oxoid) at 42°C for 48 hours (h) in stainless steel jars filled with gas mixture containing 5% O₂, 10% CO₂, and 85% N₂ (microaerobic atmosphere). Grown cells were subcultured microaerobically in BHI (Merck) for 48 h at 42°C and 110 rpm, and then used for preparation of final suspension. The final suspension was cultivated at 42°C, with shaking at 110 rpm in microaerobic atmosphere for 7 h to harvest proteins in exponential phase and for 18 h for stationary phase. All experiments were performed in 3 biological replicates.

Stains used in this study are presented Table 1. C. jejuni strains were stored at -80°C in Brain Heart Infusion broth (BHI) containing 20% (vol/vol) glycerol. Prior to each experiment frozen cells were streaked on Karmali agar plates (Oxoid Limited, UK), incubated at 42°C for 24 h under microaerobic conditions in CampyGen sachet (Oxoid Limited, UK): 5% oxygen, 10% carbon dioxide, and 85% nitrogen.
As described previously by Rodrigues et al. (2015) (link), C. jejuni Bf cells can be acclimated to aerobic conditions (namely AAC cells for aerobically acclimated cells). This was performed by sub-culturing three times (once for 48 h and then twice 24 h) on Karmali agar plates under aerobiosis (air; Rodrigues et al., 2015 (link)). In order to maintain the same conditions for all samples, cultures under microaerobiosis were identically performed three times under microaerobiosis (MAC cells for microaerobic conditions).

All 668 C. jejuni isolates used in this study were derived from farm 1, during the third phage application field trial described by Kittler et al. [14] (link). The field trial was carried out in 2012, testing the effect of a phage cocktail to reduce Campylobacter in commercial broiler chickens. Briefly, in each of two broiler chicken flocks 9 individual samples of faeces or caecal content were taken for Campylobacter spp. and phage enumeration. The flocks were naturally colonised by Campylobacter. Samples were taken when birds were 31, 32, 35 and 38 days old.
Depending on the availability of single colonies, up to 100 isolates from each flock and sampling time were collected. If the samples of a sampling time contained less than 100 colonies, all available single colonies were isolated. This altogether resulted in 668 collected isolates from a total of 55 Campylobacter positive samples. Only 15 isolates from flock 1 at day 31 were isolated and >65 isolates for all other samplings. The isolates were stored in skimmed milk as described below. For further examination, isolates were grown in Preston selective broth (Oxoid, Germany) for 48 h under microaerobic conditions and subsequently cultivated on Karmali agar plates (Oxoid, Germany).

Live C. jejuni strain 81–176 bacteria were obtained from thawed frozen stocks and cultivated on solid media (karmali agar; Oxoid, Wesel, Germany). Age and sex matched SAB IL-10^−/− mice (3-month-old littermates) were infected with 10⁹ colony forming units (CFU) of the pathogen on days 0 and 1 by oral gavage. The numbers of viable C. jejuni bacteria were determined in fecal samples every day post-infection (p.i.), and upon necropsy in intraluminal gastrointestinal samples that had been homogenized in sterile PBS (Thermo Fisher Scientific, Waltham, MA, United States). Serial dilutions of fecal and gastrointestinal samples were plated onto karmali agar plates (Oxoid, Wesel, Germany) and incubated for at least 48 h at 37°C under microaerophilic conditions as described previously (Bereswill et al., 2011 (link)). The detection limit of the enteropathogens was 100 CFU per gram sample.