Small intestinal organoids as a model to study interactions of C. suis with its porcine host

Chlamydia suis is an obligate intracellular bacterium endemic in pig populations and is detected in the gastrointestinal tract, suggesting that the intestine may be an important site of chlamydial colonization. Despite this, intestinal chlamydial infections remain poorly understood, largely due to the lack of models that accurately mimic the interaction of C. suis with the gut epithelium. The aim of this study was to evaluate whether porcine jejunum-derived enteroids constitute a suitable in vitro model to investigate intestinal infection by C. suis and to compare infection dynamics with the closely related human pathogen Chlamydia trachomatis . Porcine enteroid monolayers were exposed to C. suis and C. trachomatis , and bacterial uptake, inclusion formation, and replication were assessed using microscopy-based and molecular approaches. Both C. suis and C. trachomatis efficiently attached to primary intestinal epithelial cells and formed intracellular inclusions, indicating successful bacterial uptake and early intracellular survival. Infection levels increased in a dose-dependent manner, confirming that enteroid-derived monolayers are suitable for studying early host–pathogen interactions. Furthermore, treatment with tetracycline reduced C. suis inclusion formation and extracellular bacterial release, thereby functionally confirming that an active infection had been established and that the model responded as expected to antibiotic treatment. However, unlike the McCoy cell model which supports productive bacterial replication, enteroid cultures did not exhibit a significant increase in intracellular bacterial load over time. Despite this limited replication in enteroids, chlamydial DNA accumulated in the culture supernatants, suggesting extracellular release of bacterial material. In conclusion, porcine intestinal epithelial cells permit chlamydial attachment, internalization, and early inclusion formation. However, under the tested conditions, efficient completion of the chlamydial developmental cycle was not observed. Nevertheless, porcine enteroid-derived cultures represent a physiologically relevant in vitro platform to investigate the early stages of intestinal chlamydial infection and provide a valuable system for studying host–pathogen interactions at the intestinal interface.