Fecal microbiota fermenting simple organic carbon substrates in vitro as microbial factories capable of distinguishing Crohn’s disease from healthy states

Crohn’s disease (CD) is characterized by low microbial richness and diversity of the gut microbiome, shifts in the abundance of specific taxa, reduced presence of C2-C6 organic acid producers, especially butyrate-forming bacteria, and alterations in gut metabolites. This study aimed to demonstrate differences in the dynamics and fermentation activity of the fecal microbiota of CD patients and healthy individuals (HIs) grown in vitro on glucose or a mixture of acetate and lactate (fecal microbiota batch cultures). Glucose was used as a substrate for glycolytic fermentation, whereas a mixture of acetate and lactate supported related pathways leading to the production of C2–C6 organic acids, particularly butyrate via the conversion of lactate and acetate. HI fecal microbiota cultures produced butyrate mainly through lactate and acetate transformation rather than via glucose fermentation. This pathway was impaired in the CD fecal microbiota cultures, which exhibited reduced synthesis of butyrate, valerate, caproate and propionate, and excessive production of ethanol and certain amino acids. These distinct fermentation activities stemmed from differences in the original CD and HI fecal microbiota composition that were further accentuated in batch cultures. The number of beneficial commensal bacteria (e.g., Coprococcus catus , Ruminococcus torques , Gemmiger formicilis , Eubacterium rectale , Fusicatenibacter saccharivoransi , Faecalibacterium prausnitzii ) were significantly lower in the CD fecal microbiota cultures and correlated with reduced butyrate, valerate and caproate levels. Conversely, an overabundance of the recognized CD dysbiosis-associated bacteria, such as Escherichia coli , was reflected in elevated ethanol and amino acid levels in post-fermentation liquids. Metabolic potential analysis further indicated an enrichment of genes encoding enzymes involved in ethanol and amino acid biosynthesis in CD fecal microbiota cultures and highlighted the metabolic versatility of E. coli . Fermentation patterns of fecal microbiotas in batch cultures can distinguish CD-associated dysbiosis from a healthy microbiome, with particular emphasis on lactate and acetate conversion to butyrate as a key pathway of butyrate production. The differences are observed under standardized in vitro conditions without the need to reconstruct the intestinal environment. These findings, pending further validation, may offer novel diagnostic opportunities and have implications for strategies aimed at restoring a healthy gut microbiome.