Whole-Genome Sequence Analysis and Probiotic Characteristics of a Lactococcus lactis subsp. lactis strain Lac3 Isolated from Traditional Fermented Buffalo Milk (Dadih)

Abstract

The intention to conduct the current study was associated with the insufficiency of data availability on the whole-genome sequencing and in vitro characterization of probiotic potential of L. lactis subsp. lactis strain Lac3 in order to select the L. lactis subsp. lactis Lac3 as the future probiotic candidate. Most L. lactis has been included in the GRAS (Generally Recognized As Safe) status by (FDA) (the United States Food and Drug Administration). L. lactis has been considered as a promising probiotic candidate that has the versatile applications in animal, animal feeds, pharmaceuticals, clinical and food industries. L. lactis has been used as the starter cultures in the fermentation of dairy products, like cheese, yoghurt, sauerkraut, as well as in clinical to treat diseases by preventing pathogenic infections from the host. Moreover, L. lactis proved to enrich the foods into vitamins and amino acids and prevent allergy, etc. However, the selection of suitable probiotic candidate should require the assessment of its ability of tolerance against stressful conditions, such as acid, salts, bile salt tolerance and adhesion properties either in the gastrointestinal transit or survival in the niche environments, as well as the safety analysis like antibiotic resistance, pathogenicity, virulency, hemolytic activity and biogenic amines production. Besides, probiotic candidate should be evaluated for its ability to produce antimicrobial compounds. By this reason, we aimed at carrying whole-genome sequence analysis and in vitro characterization of probiotic potential of a L. lactis subsp. lactis Lac3. The results of this study are indispensable to provide information on genome diversity, stability, molecular evolution, safety, and probiotic potential properties which are suitable for the selection of L. lactis subsp. lactis Lac3 as the future probiotic for industrial food exploitation and health. Illumina MiSeq Next-generation sequencer was used to produce the genome reads with the size of ~3.8Mb, each sequence had the length of 150bp, and with an estimated genome size approximately ~7.6Mb. Sequencing was processed by (Novogene Co., Ltd). The de novo assembly was conducted using Unicyler.v assembler algorithm integrated into the Pathosystems Resource Integration Center (PATRIC) platform, and produced 55 contigs. Genome annotation was added by prokaryotic genome annotation pipeline (PGAP) available in NCBI and Rapid Annotations under Subsystem Technology (RAST) online webserver. Specific genome features were annotated within the genome, including antibiotic resistance phenotypes (resistomes), mobilomes (prophages, insertion sequences (ISs), plasmid), virulence factors and CRISPR/Cas system. The specific annotation was done by using various online bioinformatic tools, such as Comprehensive Antibiotic resistance Database (CARD), Resistance Gene identifier (RGI), ResFinder 4.1, PlasmidFinder, PHASTER, VirulenceFinder 2.0 and Mobile genetic element (MGEsFinder). Furthermore, identification was done based on 16S rRNA by performing nucleotide BLAST analysis and scanning electron microscopy. Phylogenetic tree was generated by using MEGA-X, and sequences were aligned by using MUSCLE. The bootstrap was inferred from 1000 replications, and taxonomic connection was inferred by using Neighbor-joining method. Besides, in vitro characterization of L. lactis subsp. lactis Lac3 was conducted to assess the probiotic characteristics of L. lactis subsp. lactis Lac3, including tolerance to acid, bile salts, NaCl, auto-aggregation (adhesion properties) and antibiotic resistance. The genome annotation predicted L. lactis subsp. lactis Lac3 to have the genome size of 2411808bp ~ 2.44Mb, with an average %GC content of 34.85%, 2324 CDSs, 56 RNA genes, (5S (2), 16S, 23S) rRNAs, 48 tRNAs, 4 ncRNAs, and 61 pseudogenes. Specific annotation consisted of 1plasmid, 2prophage regions encompassing 6 most hits phages, 3 CRISPR arrays and 3 most hits insertions sequences (IS 3, IS150, IS6). Genomic analysis revealed the ability of L. lactis subsp. lactis Lac3 to harbor several functional genes conferring its ability to survive in stressful conditions, like L-lactate dehydrogenase (EC 1.1.1.27) and D-lactate dehydrogenase (EC 1.1.1.28); several genes and associated-proteins conferring the adhesion mechanisms, such as Sortase (the surface protein anchoring transpeptidase), the LPXTG motif, and pleiotropic regulator of exopolysaccharide (EPS) (Ftr), etc.; numerous proteins involved in carbohydrates and glycerol metabolisms, and functional genes for vitamins and amino acids biosynthesis. We predicted also probiotic potential genes, such as colicin V as a class II bacteriocin, nisin-resistance protein and heavy metal toxic resistance. Moreover, genomic analysis revealed that L. lactis subsp. lactis Lac3 was not a human pathogen with a probability of being a human pathogen scored 0.21 and matching pathogenic families scored zero. Furthermore, L. lactis subsp. lactis Lac3 showed neither to harbor antibiotic resistance genes nor virulence factors. This confirms that it is safe to be selected as a probiotic candidate for the future application. The in vitro study showed the ability of L. lactis subsp. lactis Lac3 to resist in NaCl concentrations ranged between (1, 2, 4, 5%), acid (pH 2.5) and (pH7.0) and adhere to the epithelial cells of the gastrointestinal tract (GIT) with the auto-aggregation capacity that increased from (6.0±0.76%) to (13.1±3.46%). L. lactis subsp. lactis Lac3 showed tolerance against the bile salt at concentrations of (0.3, 0.5, 1%). However, L. lactis subsp. lactis Lac3 was unable to resist against all tested antibiotics. The inability of the strain Lac3 to grow in the presence of all tested antibiotics explained its safety from not carrying antibiotic resistance genes (resistomes). Dislike L. lactis subsp. lactis Lac3 was able to resist in the presence of nisin at 10ng/mL. Overall, this study highlighted the possibility to select L. lactis subsp. lactis Lac3 as the future probiotic candidate in the food industries and health. There is still a need in vitro investigation and carrying comparative genome analysis to maximize the probiotic properties of L. lactis subsp. lactis Lac3.