Sublethal Rifampicin Enhances the Tolerance of Lactobacillus Delbrueckii Subsp. Bulgaricus to Food Processing and Intestinal Retention Through Protein Aggregation

Lactic acid bacteria in fermented foods and the human gut are chronically exposed to environmental stressors that can trigger adaptive responses. Bacterial dormancy allows non-spore-forming bacteria to withstand adverse conditions through a metabolically suppressed, reversible state. This study aimed to investigate whether sublethal antibiotic stress unexpectedly triggers adaptive responses in Lactobacillus delbrueckii subsp. bulgaricus, a widely used probiotic and starter culture, and to elucidate the underlying mechanisms and functional consequences. Sublethal rifampicin-induced a dormant state in Lactobacillus delbrueckii subsp. bulgaricus sp1.1, characterized by reduced ATP levels and impaired cell division, with colony recovery upon stress removal. This state enhanced cross-tolerance to acid, alkali, and heat stress, prolonged intestinal retention in mice, and increased the impact on gut microbial community structure. Disruption of protein aggregates by pH adjustment or 1,6-hexanediol treatment abolished the enhanced tolerance. Rifampicin triggered a metabolic shift toward transcriptional and translational inhibition. Protein aggregates selectively enriched translation, RNA metabolism, and DNA repair proteins. Two disordered proteins, Gene1622 and Gene1909, were upregulated in whole cell and enriched in the aggregates. Single-cell RNA sequencing identified a cluster that sustained alaS and gatA expression under global translational suppression, contrasting with their downregulation in bulk RNA-seq, revealing population heterogeneity under stress. Our results support a model in which protein aggregates function as “molecular safe houses” that sequester and protect translation-and DNA-repair-related proteins, thereby locally enhancing the efficiency of key protein translation and DNA repair while also serving as a protein reservoir for subsequent growth recovery. This reversible dormancy provides a theoretical basis for engineering robust starter cultures, while also raising ecological considerations regarding stress-induced adaptation in the food chain, including potential impacts on gut microbial homeostasis. Schematic diagram of the mechanism by which sublethal rifampicin induces dormancy and enhances bacterial tolerance and intestinal retention capacity.