Utilizing an In Vitro Fermentation Model to Assess Probiotics on Eimeria-Disturbed Cecal Microbiome and Metabolome

Rectifying the microbiome perturbed by <i>Eimeria</i> invasion might alleviate the adverse effects of coccidia on broiler growth. This study employed an in vitro fermentation model to investigate the direct, host-independent effects of two probiotics-<i>Lactobacillus rhamnosus</i> (LR) and <i>Bacillus subtilis</i> (BS)-on the cecal microbiome and metabolome perturbed by <i>Eimeria tenella</i>. Four in vitro fermentation treatments consisted of a healthy control (cecal slurry samples from health broilers), an <i>Eimeria</i>-disturbed control (slurry samples from infected broilers), an LR treatment (<i>Eimeria</i>-infected slurry + 3 × 10⁵ of LR cfu/mL), and a BS treatment (<i>Eimeria</i>-disturbed group + 3 × 10⁵ of BS cfu/mL). 16S rRNA sequencing and metabolomic analysis revealed that <i>Eimeria</i> infection resulted in an increase in microbial alpha diversity, promoted opportunistic pathogens, including <i>Helicobacter</i> and <i>Bacteroides</i>, and suppressed commensals like <i>Lactobacillus</i>, concurrently altering 530 intracellular metabolites. Probiotic supplementation partially restored microbial composition. Notably, LR inoculation rectified 107 metabolites across pathways including galactose metabolism and phosphotransferase systems, primarily affecting membrane phospholipid balance. In contrast, BS addition restored only 64 metabolites, largely related to secondary metabolism. The current in vitro study indicates that LR can directly modulate key metabolic disturbances in a dysbiotic microbiota, while the BS may be more dependent on host-mediated interactions.