Bioprospecting and quorum-sensing-pathway–focused in silico docking reveal why LAB outcompete Limnobacter/Burkholderia in the neonatal gut

Abstract

Milk-borne lactic acid bacteria (LAB) and Gram-negative co-colonizers, such as Limnobacter and Burkholderia, often coexist in early life. However, the mechanisms underlying the dominance of LAB in these niches remain unclear. This study aimed to elucidate potential factors contributing to LAB predominance by integrating physiological, molecular, and computational analyses of Gram-negative isolates alongside LAB antagonism assays. From previously surveyed human milk and infant stool samples, nine Gram-negative isolates (Limnobacter spp., L. thiooxidans, Burkholderia glathei) were obtained and identified using 16 S rRNA maximum-likelihood phylogeny. All isolates were Gram-negative, motile, catalase-positive, non-sporulating mesophilic rods exhibiting oxidative metabolism utilizing tricarboxylic acid intermediates and glutamate. The isolates survived but did not proliferate at pH 3.0 and exhibited 0.3–0.6 log growth penalties in 0.5% bile, indicating moderate tolerance to gut-relevant stressors. They were broadly susceptible to tested antibiotics. In vitro assays showed consistent inhibition by LAB, particularly Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus, which produced inhibition zones of approximately 10–12 mm. In silico docking analyses suggested that lactic acid can bind within quorum-sensing (QS)–associated and other functional pockets, consistent with interference in coordinated growth mechanisms. Plantaricin GZ1-27 exhibited plausible binding both to a QS-adjacent receptor and to a site near the membrane/peptidoglycan interface, indicating a potential “two-hit” mode of action combining QS disruption and envelope stress. Docking to sphingomyelin–protein complexes (e.g., 1EEI and 1O7V) revealed interactions with sphingomyelin headgroups, suggesting possible modulation of membrane microdomains. The findings support a model in which LAB outcompete Limnobacter and Burkholderia through QS antagonism, reduced activity of QS-regulated effectors, and alterations in envelope or lipid context. These insights provide design principles for developing infant-compatible postbiotics that emphasize QS interference, targeted envelope stress, and consideration of membrane interactions. Combinations of lactate and plantaricin may represent promising postbiotic mixture formulations for modulating early-life microbiota composition at physiologically safe doses.