Abstract 1339 Genetic and Phenotypic Equivalence Between Two Strains of L. plantarum Isolated in Separate Parts of the Planet Supports the Nomadism of Some Lactic Acid Bacteria

Lactiplantibacillus plantarum isolates can adapt to a variety of conditions and are distributed in very diverse ecosystems. However, genomic evolution of these strains seems to be unlinked to the specific niche they inhabit. In this study, we performed deep comparative genomic and phenotypic analyses aiming to demonstrate the equivalence between two L. plantarum isolates collected from two different habitats. L. plantarum strain DR7 was isolated from cow's milk in Malaysia (Min 2019) while L. plantarum KABP051 (CECT7481) is of South America human origin. The genomes of the two strains were obtained by PacBio sequencing and subjected to comparative genomic analyses. Average Nucleotide Identity (ANI) of whole genome sequences (WGS) was determined with JSpeciesWS and compared to other L. plantarum strains. Synteny between DR7 and KABP051 genomes was evaluated by visual inspection using MAUVE and putative plasmids were annotated and compared using pLannotate. Macrorestriction profile (PFGE) was obtained using restriction enzymes SfiI and SmaI. Comparative phenotypic analyses were also conducted to support bioequivalence. Resistance of DR7 and KABP051 to simulated gastrointestinal (GI) conditions was tested in artificial gastric (pH3) and bile salt (0.3%) solutions using. L. plantarum WCFS1 as a control. Adhesion to intestinal epithelial Caco-2 cells was determined in vitro and compared to the control strain L. casei ATCC334. Culture supernatants of L. plantarum strains were characterized by LC-MS following untargeted metabolomic approaches. Finally, an absolute quantification of the two beneficial compounds 2-hydroxyisocapric acid (HICA) and 3-phenyllactic acid (PLA) was performed by targeted analyses. PacBio sequencing revealed that DR7 and KABP051 genomes are identical in size and contain the same single plasmid. Pairwise comparison analysis revealed that only DR7-KABP051 pair displayed 100% ANI at both chromosome and plasmid levels, while other pairs ranged 98.5-99.7%. MAUVE alignment revealed a perfect synteny between DR7 and KABP051 genomes, showing no genomic reorganizations. PFGE analyses revealed that both strains show identical macrorestriction pattern after digestion with either of the two enzymes. Strains DR7 and KABP051 exhibited a similar GI survival rate, with higher bile salt tolerance but equivalent acid resistance compared to the control strain WCFS1. L. plantarum DR7 and KABP051 clustered together based on untargeted metabolomics data. Culture supernatants of DR7 and KABP051 contained the same amount of HICA and PLA molecules (92±1 μg/mL) but showed clear differences to other L. plantarum strains which produced lower amounts. Finally, DR7 and KABP051 also showed the same CaCo-2 adhesion capacity (64.1±0.5%). Together, we conclude that isolates DR7 and KABP051, but not other L. plantarum strains, display identical genomic characteristics, behave equally in the intestinal environment and are metabolically the same as confirmed by LC-MS, confirming the bioequivalence of these two isolates from different niches. These results contribute to demonstrating the nomadic behavior of some lactic acid bacteria. This study was funded by AB-BIOTICS SA (Kaneka Corp.)