Characterization of a potential probiotic bacterium Lactococcus raffinolactis WiKim0068 isolated from fermented vegetable using genomic and in vitro analyses


 Background: Lactococcus members belonging to lactic acid bacteria are widely used as starter bacteria in the production of fermented dairy products. From kimchi, a Korean food made of fermented vegetables, Lactococcus raffinolactis WiKim0068 was isolated and its genome was analyzed. Results: The complete genome of the strain WiKim0068 consists of one chromosome and two plasmids that comprises 2,292,235 bp, with a G+C content of 39.7 mol%. Analysis of orthoANI values among Lactococcus genome sequences showed that the strain WiKim0068 has > 67% sequence similarity to other species and subspecies. In addition, it displayed no antibiotic resistance and can metabolize nicotinate and nicotinamide (vitamin B3). Conclusion: These results augments our understanding of the genus Lactococcus and suggest that this new strain has potential industrial applications.

derived foods and induce to flatulence and diarrhea. Therefore, fermentation feature of these sugars is a significant advantage for use as a starter in dairy products. In this study, we report the isolation, identification, and characterization of the L. raffinolactis WiKim0068 isolated from fermented cabbage (kimchi). We also evaluated the possibility of using the strain WiKim0068 in dairy products, and the safety of the strain. Further, we analyzed its proteolytic enzymes through complete genome sequence analysis. In vitro assays and predictive gene analysis for antibiotic resistance and adhesion were also performed.

Phylogenetic and phenotypic features of the isolated LAB strain
The bacterial strain, designated WiKim0068, was isolated from a Korean fermented food, kimchi. In order to identify the phylogenetic similarity of the strain, 16S rRNA gene based phylogenetic analysis of strain WiKim0068 was performed and the closely related strains were found to be L. raffinolactis NBRC 100932 T with a similarity of 99.9% (Fig. 1). This result indicated that strain WiKim0068 belongs to L. raffinolactis species. Sugar assimilation/acid formation test conducted using API 50CH revealed positive results for galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, esculin, ferric citrate, salicin, cellobiose, maltose, melibiose, saccharose, trehalose, raffinose, and turanose, whereas H 2 S production and urease were negative. Enzyme detection performed with an API ZYM kit indicated esterase, leucine arylamidase, and naphthol-AS-BI-phosphohydrolase activities.

Phage and pathogenesis-related genes
PHAST analysis was performed to identify prophage contamination in the genome of WiKim0068. The chromosome contained two intact, one incomplete, and one questionable prophage. The first plasmid (pWi-Kim0068-1) contained only one incomplete prophage, while the second plasmid (pWiKim0068-2) contained none ( Supplementary Fig. S2). Intact prophage regions were located between positions 57,319-90,123 and 1, 524,268-1,563,900 bp of the chromosome.

Metabolic pathway of carbon and amino acid
Predicted metabolic pathways in the strain WiKim0068 were associated with diverse phosphotransferase (PTS) systems or permeases that transport various carbohydrates, including D-glucose, D-galactose, D-mannose, trehalose, sucrose, cellobiose, N-acetyl-glucosamine, fructose, maltose, mannitol, galactitol, and lactose. The presence of these transport genes suggested that the strain WiKim0068 uses various carbohydrates for fermentation (Fig. 4). Based on the metabolic pathways, it was confirmed that the strain WiKim0068 had heterofermentative pathways. The amino acid metabolism-related genes of strain WiKim0068 were annotated using the KEGG database. Among 163 genes involved in amino acid metabolism, strain WiKim0068 harbors the most genes involved in the amino acid metabolism of cysteine, methionine, alanine, aspartate, and glutamate ( Fig. 5), suggesting that the strain biosynthesize and utilize various amino acids.

Metabolism of nicotinate and nicotinamide and antibiotics susceptibility
In silico analysis of WiKim0068 genome predicted an almost complete complement metabolic pathway from the genes involved in the metabolism of nicotinate and nicotinamide (Fig. 6 Supplementary Fig. S3). These results indicated that nicotinate and nicotinamide metabolism occurs in strain WiKim0068. For comparison, studied 15 LAB isolated from kimchi; Leuconostoc spp. produced 0.837-1.05 mg L − 1 vitamin B3, and Lactobacillus species, L. sakei, and L. curvatus produced 0.05-0.1 mg L − 1 . The strain WiKim0068 showed susceptibility to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, penicillin, rifampin, tetracycline, and vancomycin (Table 2).

Discussion
The phylogenetic and genomic analysis of strain WiKim0068 confirmed that strain WiKim0068 is closely related to L. raffinolactis NBRC 100932 T ( Fig. 1 and Fig. 2). The G + C content of strain WiKim0068 was 39.7 mol%, which is within the range of 35.5-46.4% reported for Lactococcus species [13], and similar to the 39.25 mol% observed in two L. raffinolactis strains, 4877 (CALL00000000) and NBRC 100932 T (BCVN00000000). In addition, the orthoANI analysis showed that strain WiKim0068 is mostly similar to L. raffinolactis NBRC 100932 T (98.73%). In the RAST analysis, various genes were identified in the genome of strain WiKim0068. Biotin, riboflavin, and folate are related to human health and digestion and cause various symptoms when deficient [14]. Bacteriocins are antimicrobial peptides produced by bacteria [15] and an alternative to treat antibiotic resistant bacteria. Significantly, bacteriocins production have been regarded as an important feature in the selection of probiotic strains. These were associated with the presence of useful probiotic characteristics, which play important roles in the food and pharmaceutical industries [16][17][18].
Hexoses (glucose, fructose, and mannose) are converted to lactate, ethanol, and carbon dioxide. Additionally, Dand L-lactate are produced from the reduction of pyruvate by D-lactate dehydrogenase (D-LDH) (EC 1.1.1.28) and Llactate dehydrogenase (L-LDH) (EC 1.1.1.27), respectively. However, strain WiKim0068 harbors only L-LDH (locus tag: CMV25_RS07125). Notably, as shown in a previous   report, L-LDH was identified in Lactococcus lactis, which belongs to the same genus as the strain WiKim0068 [19]. Since D-lactate produced by LAB may induce D-lactate acidosis in some individuals [20], it is important to develop LAB for the production of dairy products that produce only L-lactate. Therefore, the lack of D-LDH is an advantage that makes the strain WiKim0068 suitable for potential applications in the dairy industry.
Vitamin B3 production of strain WiKim0068 was identified through in in silico and in vitro analysis. Vitamin B3, one of the 8 B-vitamins, is also known as nicotinate or niacin. This endogenous metabolite is an effective antioxidant that prevents oxidative damage [21]. In general, nicotinamide and nicotinate metabolites are frequently reported in Lactobacillus strains [22][23][24], while Lactococcus members were not known to produce these metabolites until now. On the other hand, the capacity to adhere to mucosal surfaces is a useful assay to determine whether probiotic strains have beneficial health effects [25]. Strain WiKim0068 was bound to Caco-2 cell cultures, and its adhesion did not significantly differ from that of L. rhamnosus GG (Welch's t-test, P > 0.05) (data not shown). The extracellular proteins of lactobacilli play important roles mediating interactions with the host or the environment [26]. Cell surface proteins of strain WiKim0068 include glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, trehalose and maltose hydrolases (possible phosphorylases), betagalactosidase, lipoprotein signal peptidase, and sortase (surface protein transpeptidase), which have been implicated in adhesion or binding to other cells [27].
Recently, interest in foods as mediators of antibiotics resistance has been increasing. LAB, which are widely used in probiotics and as starter cultures, have the potential to serve as hosts for antibiotic resistance genes, and present the risk of transferring genes from various LAB and bacterial pathogens [28]. Although the strain WiKim0068 was predicted to have vancomycin resistant gene in the genome, antibiotics test confirmed that it was sensitive to vancomycin. This result based on the antibiotic resistance gene prediction can be obtained from a cluster of vancomycin resistant genes. Strain WiKim0068 had only vanW gene among the vancomycin resistance gene cluster, and the function of this gene is still unknown. The safety of L. lactis strains has not yet been assured through the comparison of antibiotic susceptibility profiles and the presence of the genes putatively encoding antibiotic resistance-related proteins [29,30]. The analysis of L. raffinolactis WiKim0068 based on ResFinder 3.0 did not detect antimicrobial resistance genes against aminoglycoside, beta-lactam, colistin, fluoroquinolone, fosfomycin, fusidic acid, glycopeptide, macrolide-lincosamidestreptogramin B, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, or trimethoprim. The safety against antibiotic resistance of L. raffinolactis WiKim0068 could be confirmed by the antibiotic susceptibility test and antibiotic resistance gene prediction.

Conclusions
The complete genome of L. raffinolactis WiKim0068 revealed its general genomic features, carbon metabolic pathway, and its ability to produce and utilize nicotinate and nicotinamide. In addition, in vitro analysis indicated that the strain possesses beneficial health effects such as vitamin B3 production. These results suggest that L. raffinolactis WiKim0068 could be utilized in comparative genome analysis with other Lactococcus strains.

Isolation and characterization of the bacterial strain
The strain WiKim0068 was isolated from kimchi, a Korean fermented food, in Gwangju, Korea using the dilution plating method, and incubated on De Man, Rogosa and Sharpe (MRS) agar (MB cell, LA, USA) at 30°C for 48 h under anaerobic conditions (BD GasPak™ EZ Anaerobe Container Systems, New Jersey, USA). Physiological characteristics (acid production, carbonsource utilization, enzyme activity, and biochemical feature) were determined using the API 50CH, API ZYM, and API 20E galleries (bioMérieux, France), according to the manufacturer's instructions [31,32], while the bacteria were incubated at 30°C for 48 h under anaerobic conditions. Anaerobic conditions were maintained using mineral oil.

Genome sequencing and annotation
Genomic DNA extraction was performed using the QIAcube system with a QIAamp DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The genome was sequenced using the PacBio RS II sequencing system (Pacific Biosciences, Menlo Park, CA). The reads were assembled de novo using Hierarchical Genome Assembly Process  3.0., as described by Jang et al. [33]. The complete genome sequence was annotated using the combined results of the automatic National Center for Biotechnology Information (NCBI) Prokaryotic Genomes Annotation Pipeline 4.1 [34] and the RAST server [35]. Phylogenetic tree based on 16S rRNA gene sequences extracted from the genome, were constructed, as described by Ismaeil et al. [36], using the neighbor-joining [37], minimumevolution [38], and maximum likelihood [39] methods, based on 1000 randomly generated trees. Protein functions were grouped according to COG using WebMGA on-line tools (for carbohydrate metabolism, antibiotic resistance-related genes, adhesion, proteolytic enzymes, and amino acid metabolism) [40]. Nicotinate and nicotinamide metabolic pathway was mapped using the Kyoto Encyclopedia of Genes and Genomes (KEGG) [41]. The fermentative metabolic pathways were constructed based on predicted KEGG pathways and BLASTP analysis using reference gene sequences. Antimicrobial resistance genes were identified using ResFinder 3.0, available from the Center for Genomic Epidemiology (http://genomicepidemiology.org/). Prophage identification was performed using the PHAge Search Tool (PHAST) [42]. The complete genome sequences have been deposited to the DNA databank of Japan/the European Molecular Biology Laboratory/Gen-Bank under the accession numbers CP023392-CP023394.

Carbon metabolic pathway
The fermentative metabolic pathways of L. raffinolactis WiKim0068 were constructed based on predicted KEGG pathways and BLASTP analysis. In detail, the genes of L. raffinolactis WiKim0068 were mapped to the five KEGG pathways (pentose phosphate pathway, fructose and mannose metabolism, pathways for pyruvate, galactose, starch, and sucrose metabolism). Then, only mapped genes were used to draw one pathway (Fig. 4), and the functions of the individual genes were reconfirmed using BLASTP.

Comparative genomic analysis
For comparative genomic analysis of strain WiKim0068, the genome sequences of two other Lactococcus raffinolactis strains: L. raffinolactis 4877 (CALL00000000.1) and L. raffinolactis NBRC 100932 T (BCVN00000000.1) were obtained from GenBank and used as references. To determine the similarity between genome sequences, OrthoANI values of L. raffinolactis WiKim0068 and related strains in the genus Lactococcus were calculated using the orthologous average nucleotide identity tool (OAT software, www.ezbiocloud.net/sw/oat; ChunLab) [43]. Circular comparison map of the genomic sequences was created using Blast Ring Image Generator (BRIG) software [44]. Clustered regularly interspaced short palindromic repeats (CRISPRs) were analyzed using CRISPRFinder [45]. When the algorithm was detected exactly three identical (repeated and sequential) repeating regions separated by a variable order, it was considered "confirmed CRISPR".

Adhesion assay
Human colorectal adenocarcinoma cell line Caco-2 (HTB-37) was obtained from the Korea Collection for Type Culture (KCTC). Caco-2 cells were grown in minimum essential medium (MEM) according to KCTC guidelines. Adhesion of bacteria to Caco-2 cells was tested as previously described [47]. Briefly, the strains were added to confluent cell layers (10 6 CFU well − 1 ) in antibiotic-free cell media. After 2 h of incubation, the cell layer was washed to remove non-adherent bacteria and lysed by the addition of 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). The viable adhered bacteria were plated on LAB Petrifilm (3 M Company, St. Paul, MN, USA) and the cell number was counted after incubation at 30°C for 48 h. Adhesion experiments were performed in triplicate and Lactobacillus rhamnosus GG (KCTC 5033) was used as a control. Statistical evaluation was performed using GraphPad Prism 6.0 (Graph-Pad Software Inc., La Jolla, CA, USA). Differences were considered statistically significant when P < 0.05.

Quantitative vitamin B analysis
The strain WiKim0068 was cultured at 30°C for 48 h in MRS broth under anaerobic conditions. The cell-free supernatant was collected using a 0.22 μm syringe filter. Two microliters of the cell-free supernatant was injected into the HPLC system. Vitamin B levels were determined with a NexeraX2 HPLC (Shimadzu, Japan) equipped with an LCMS-2020 LC/MS System (Shimadzu). The compounds were separated on an Aegispak C-8 column (150 mm × 2 mm, 3 μm; Young Jin Biochrom, Korea) at 40°C. Mobile phase A was a 0.1% formic acid in distilled water and mobile phase B was 0.1% formic acid in acetonitrile.
The gradient elution was as follows: from 0 to 1 min isocratic elution with 100% of mobile phase A, then the mobile phase B content was increased linearly to 75% in 20 min. Finally, the isocratic elution (25% A and 75% B) was continued for 7 min. Solvents were delivered at a total flow rate of 0.25 mL min − 1 . The re-equilibration time was 5 min. Optimal operating conditions for LC-MS/MS analysis were applied according to the method reported by Wirkus et al. [48]. Reference vitamin B group standards with 99% purity supplied by the Sigma-Aldrich were used. All experiments were repeated at least three times. Statistical analysis was performed using Tukey's honest significant difference (HSD) test carried out in the "agricolae" package of the R program for group comparisons. Results with p < 0.05 were regarded as statistically significant.