Cyclic di-GMP Regulation of Antibiotic-Induced Biofilm Formation: A Systematic Review

Background/Objectives: Biofilm formation is a major bacterial survival strategy in response to antibiotic stress. The bacterial second messenger cyclic di-GMP (c-di-GMP) is a central regulator of biofilm development. Sub-minimum inhibitory concentrations (sub-MICs) of antibiotics act as biological active environmental cues that reprogram bacterial physiology and promote biofilm development. This systematic review synthesizes the current literature on sub-MIC antibiotic-induced biofilm formation across different pathogenic bacterial genera and critically evaluates the role of c-di-GMP. Methods: A systematic literature search was conducted in PubMed and Scopus for articles published from inception to 18 December 2025 using terms including "sub-MIC antibiotic" AND ("biofilm" OR "antibiotic-induced biofilm") and a second search using "sub-MIC antibiotic" AND "biofilm" AND "c-di-GMP". Reviews, commentaries, opinions, meta-analyses, systematic reviews, and non-English-language articles were excluded. Results: Collectively, 28 identified studies demonstrate that sub-MIC antibiotics modulate biofilm formation through diverse mechanisms that intersect with c-di-GMP activity, including modulation of diguanylate cyclases and phosphodiesterases and through regulatory networks that intersect with quorum sensing and different two-component systems. These responses influence multiple components of biofilm matrix- and biofilm-associated physiology, including exopolysaccharides, extracellular DNA, cell surface proteins, secretion systems, and stress-adaptive multicellular behavior. Conclusions: By positioning c-di-GMP as an integrative, but context-dependent, signaling hub linking antibiotic-induced stress, metabolic adaptation, persistence, and biofilm development, this review highlights critical mechanistic gaps and identifies potential therapeutic targets for disrupting biofilms and improving antibiotic efficacy.