Multi-omics joint analysis of biofilm formation in kefir-derived mixed strains

The mechanism underlying extracellular matrix formation in kefir grains remains poorly understood. Since biofilms constitute the structural foundation of this extracellular matrix, the present study explored the molecular mechanisms governing the formation of kefir-derived mixed-species biofilms. A co-culture system was established using one strain each of lactic acid bacteria, yeast, and acetic acid bacteria. These microbes were previously isolated from kefir and selected for their strong biofilm-forming capabilities. Samples were collected at different time points during cultivation for metabolomic and metatranscriptomic analysis. The results showed that at 4 h, during the initial stage of biofilm formation, microbial metabolic activity was at its peak, with most metabolic energy directed toward cell proliferation and signal transduction. At 8 h, the biofilm had reached a state of relative maturity, and metabolic energy was primarily allocated to biofilm formation and matrix construction. This phase was characterized by the downregulation of key metabolic pathways, such as the tricarboxylic acid cycle, and a synchronous downregulation of over 90% of genes associated with metabolism. At 12 h, the biofilm transitioned into a steady-state maintenance and disassembly initiation phase. During this period, the expression levels of most genes stabilized, and fluctuations in certain metabolite levels were associated with a decline in biofilm biomass. In the mixed-strain system, when the concentration of autoinducer-2 reached threshold levels, this molecule was detected by microbial cells, triggering the activation or repression of various genes. The enzymes encoded by these genes modulated intracellular metabolic pathways, leading to changes in downstream metabolite concentrations and ultimately affecting biofilm formation. • Metabolomics and metatranscriptomics revealed significant differences in mixed-strain cultures at different time points. • Core energy metabolism pathways were downregulated following 8 h of mixed-strain culture. • Autoinducer-2 attenuation emerged as a key trigger for the maturation of mixed-strain biofilms. • In mixed-strain cultures, signaling molecules altered gene expression, affecting metabolite levels and thereby influencing biofilm formation. • The molecular mechanisms regulating the formation of kefir-derived multispecies biofilms were preliminarily elucidated.