Background: 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.
Results: From previously surveyed human milk and infant stool samples, nine Gramnegative isolates (Limnobacter spp., L. thiooxidans, Burkholderia glathei) were obtained
and identified using 16S rRNA maximum-likelihood phylogeny. All isolates were Gramnegative, 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.
Conclusions: The findings support a model in which LAB outcompete Limnobacter and
Burkholderia through QS antagonism, reduced activity of QS-regulated effectors, andalterations 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.