Karmali agar

On days 0 and 1, the microbiota-depleted 3-month-old mice (total number of n = 36) were subjected to 10⁹ colony forming units (CFU) of C. jejuni strain 81–176 (oral gavage). Therefore, the enteropathogens were derived from frozen stocks and streaked onto karmali agar and on columbia agar (supplemented with 5% sheep blood) plates (both from Oxoid, Wesel, Germany) two days prior respective infections (i.e., day-2 and day-1). The culture media were incubated in a box at 37 °C (microaerophilic conditions, CampyGen gas packs, Oxoid, Wesel, Germany). Two days later, bacteria from one fluently grown karmali agar plate were harvested in 5 mL sterile phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) to an approximate McFarland density of 3 (i.e., OD of 0.6 at 600 nm wavelength) resulting in a yield of 10⁹ víable C. jejuni cells in a volume of 0.3 mL used for subsequent gavage. The yields were reconfirmed by cultural analyses of serial dilutions of the bacterial suspensions on respective solid culture media. The following cohorts of age- and sex-matched microbiota-depleted IL-10^−/− mice (individual numbers in parentheses) were finally included in three independent C. jejuni infection experiments: (i) naive (non-infected, non-treated) mice (6/5/5); (ii) C. jejuni-infected, placebo-treated mice (6/6/6); (iii) C. jejuni-infected, butyrate-treated mice (6/6/6).

For infection experiments, C. jejuni strain 81–176 was thawed from a stock and cultivated on columbia agar (supplemented with 5% sheep blood) and karmali agar plates (both from Oxoid, Wesel, Germany). Microbiota-depleted IL-10^−/− mice were infected orally with 10⁹ CFU in a volume of 0.3 mL phosphate buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) by gavage on days 0 and 1, as stated earlier [33 (link)]. For a kinetic assessment of the intestinal colonization efficacies of the pathogen, C. jejuni loads were enumerated in fecal samples every day post-infection, and in luminal samples obtained from the stomach, duodenum, ileum and colon) upon necropsy (i.e., day 6 p.i.) by culture, as described previously [33 (link)]. In brief, serial dilutions of respective samples were streaked onto columbia agar plates containing 5% sheep blood and karmali agar plates (both from Oxoid, Wesel, Germany), and incubated in a jar under microaerophilic conditions for 48 h at 37 °C.

The pathogenic loads were surveyed in fecal samples at defined time points p.i., and upon necropsy in luminal samples derived from the colon, ileum, duodenum and stomach and additionally, in ex vivo biopsies taken from mesenteric lymph nodes (MLN), spleen, lungs, liver, and kidneys by culture as reported earlier [36 (link),38 (link)]. Briefly, by using sterile pestles respective samples were homogenized in sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA). Serial dilutions were then streaked onto karmali agar and Columbia agar (supplemented with 5% sheep blood; both from Oxoid, Wesel, Germany) and incubated in a jar containing CampyGen gas packs (Oxoid, Wesel, Germany) under microaerophilic conditions at 37 °C for at least 48 h. In order to survey systemic translocation, cardiac blood (0.1 mL) was directly plated onto Columbia agar (supplemented with 5% sheep blood) and incubated likewise. The detection limit of viable pathogens was 100 CFU per g.

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).

After C. jejuni infection, the pathogen loads were determined in fecal samples daily, and upon necropsy in luminal samples from the stomach, duodenum, ileum, and colon by culture as described previously [39 (link),41 (link)]. In brief, intraluminal gastrointestinal samples were homogenized in sterile phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) with a sterile pistil and serial dilutions plated onto Karmali agar (Oxoid, Wesel, Germany) and incubated under microaerophilic conditions for at least 48 h (37 °C). The detection limit of viable pathogens was 100 CFU per g.

For infection, a stock solution of C. jejuni 81–176 strain that had been stored at −80°C was thawed, aliquots streaked onto karmali agar (Oxoid, Wesel, Germany) and incubated in a microaerophilic atmosphere at 37°C for 48 h. Immediately before peroral infection of mice, bacteria were harvested in sterile PBS to a final inoculum of 10⁹ bacterial cells.
Female and male hma mice (4 months of age) were perorally infected by gavage (in a total volume of 0.3 mL PBS) on 2 consecutive days starting on day (d) 0 and d1 (Figure 1). C. jejuni loads were monitored in fecal samples over time post-infection as reported previously (15 (link), 25 (link)). In brief, serial dilutions of fecal samples were dissolved in sterile PBS, streaked onto karmali agar and quantitatively assessed 48 h following incubation in a microaerophilic atmosphere at 37°C. The detection limit of viable pathogens was ≈100 CFU per g.