Morphological, biochemical, and molecular characterization of the selected isolate L. salivarius F14
Twenty-five bacterial isolates were selected after random sampling of human stool samples. Based on the results of initial morphological, biochemical, and 16S rRNA sequencing, four isolates were shortlisted for further characterization. The results of 16S rRNA sequencing revealed one of the isolate (colony number F14) to be closely related to Lactobacillus salivarius (Fig. S1A). In this study, we have characterized this gut-derived isolate for its complete probiotics and molecular features. The morphological features of this bacteria indicated that it is round in shape with irregular margins and off-white in color (Fig. S1B). Preliminary Gram staining revealed it as a rod-shaped gram-positive bacteria (Fig. S1C). The result of the catalase test showed that it was catalase-negative similar to other lactic acid bacteria. The sugar fermentation pattern was obtained for the isolate following the instructions on the Himedia KB020 kit for identification and differentiation of genus Lactobacillus and the results are shown in Table S1. The pattern of carbohydrate utilization is in agreement with those of other Lactobacillus and could not be compared to that of the salivarius species due to unavailability of species level interpretation for this species in the instruction manual. This isolate of Lactobacillus salivarius has been termed F14 henceforth in this article.
Characterization Of The Probiotic Attributes As Per The Icmr-dbt Guidelines
The F14 isolate survived at pH 3, and the growth at this pH was as good as at the physiological pH of 7.4, as there was no significant difference in growth at the two pH values (Fig. 1A). However, the number of viable cells decreased significantly with time in acidic pH (between first hour and second hour, *p < 0.05; between first hour and third hour, ***p < 0.001). Overall, the isolate grew well in the acidic environment till 3 h of incubation as there was significant change between pH 3 and pH 7.4 after that time point (*p < 0.05). Similarly, the isolate grew in culture medium containing 0.3% bile, showing tolerance to bile salt (Fig. 1B). The presence of bile salt hydrolase (BSH) was qualitatively determined from the appearance of zone of inhibition on the MRS agar plates supplemented with sodium taurodeoxycholic acid (Fig. S2). The isolate was susceptible to different minimum dosages of most of the antibiotics used against enteric infections (Table S2). It showed resistance against metronidazole and vancomycin (Fig. 1C). Antimicrobial properties against a broad range of pathogens are desirable characteristics for probiotics. This isolate showed antimicrobial activity against P. aeruginosa, S. aureus, Salmonella typhimurium ST-Xen 33, and K. pneumoniae, as was evident from the zone of inhibition observed in the agar well diffusion assay (Fig. 1D).
Antimicrobial Activity Using Cell-free Supernatant (Cfs) And Co-culture With Pathogen Bioluminescent St-xen 33
The antimicrobial activity against ST-Xen 33 was assessed using the CFS in a well diffusion and co-culture method. F14, when co-cultured with pathogen ST-Xen 33, efficiently impeded the growth of the pathogen in a time-dependent manner and completely abolished its growth in the culture at 24 h (Fig. 1E). To characterize the nature of the bactericidal activity of the CFS, the acidity was neutralized and the CFS was heated to 95°C for 15 min. The heat-denatured CFS was able to retain its antimicrobial effects, whereas the effect was abolished in the pH-neutralized CFS, suggesting that the acidic nature of the soup contributes considerably towards the antibacterial effects (Fig. 1F). Using different dilutions of the CFS, we observed that 10% of the soup was the minimum inhibitory percentage (MIP) required to suppress the growth in liquid culture medium (Fig. 1G). To further confirm the bactericidal effect, we performed the agar spot assay using the different wells containing the pathogen; the results showed that the MIP of 10% CFS had only bacteriostatic effect, whereas 20% CFS showed bactericidal activity on ST-Xen 33 (Fig. 1H).
Evaluation Of The Safety Of The Isolate
For evaluating the in vitro safety of the isolate, the hemolytic and DNase activities were checked, which confirmed its non-pathogenic nature. Absence of any clear zone on the sheep blood agar plate confirmed that the F14 isolate did not possess any hemolytic activity (Fig. S3). Similarly, absence of any pinkish zone around colonies confirmed the absence of DNase activity (Fig. S4).
Attachment Efficiency And Pathogen Exclusion On Caco2 Cell Surface
One of the mechanisms via which the probiotics confer antimicrobial activity in the gut is by excluding the attachment of pathogenic bacteria to the intestinal epithelial surface (van Zyl, Deane et al. 2020). In this study, the co-culture of the F14 isolate with Caco2 cells for 2 h resulted in the attachment of the isolate to the Caco2 cells. The representative figure shows the SEM image of Caco2 cells only and isolate F14 attached to the Caco2 cell surface (Fig. 2A). The percentage of the bacterial isolate attached per well-differentiated Caco2 cells was 32–40 (Fig. 2B). The number of pathogen attachment reduced significantly when the pathogen and F14 were co-cultured with the Caco2 cells, (***p < 0.001). However, when the Caco2 cells were pre-treated with isolate F14 1 h prior to the pathogen treatment, the attachment efficiency of the pathogen to the Caco2 cell surface further decreased (7-fold decrease vs. 26-fold decrease) (Fig. 2C).
Effect of F14 cell lysate on NF-κB activity in murine macrophage cell line (RAW 264.7) Lactobacillus species are known to exert immunomodulatory effects (Abedin-Do, Taherian-Esfahani et al. 2015). Hence, we adapted a cell line-based assay system to evaluate the effect of the F14 cell lysate on NF-κB activity in RAW 264.7-NF-κB reporter cells (Mahapatra, Jain et al. 2018, Jain, Dash et al. 2019). The LPS-treated RAW 264.7-NF-κB cells were treated with different concentrations of the bacterial cell lysate; we observed that the NF-κB activity was significantly reduced in the presence of the cell lysate, proving its anti-inflammatory potential (Fig. 2D). In addition, the MTT assay indicated the absence of cytotoxic effect on the cells after treatment with the lysate (data not shown).
Molecular identification and whole genome sequencing (WGS) features of the L. salivarius F14
The whole genome of F14 was sequenced using IlluminaNovaSeq and annotated using the NCBI Prokaryotic Genome Annotation Pipeline. The complete genome sequence contains a single circular chromosome of 1,887,930 bp, with an average GC content of 32.94% (Fig. 3 and Table 1). Analysis of the WGS data using Plasmidfinder confirmed the absence of plasmids in the sequenced data (Carattoli and Hasman 2020). The general features are shown in Table 1. Out of the 1,813 coding DNA sequences (CDSs), 1,761 were identified as protein CDSs. The chromosome contained 3 rRNAs and 39 t-RNA encoding sequences. From the results of preliminary 16S rRNA sequencing, the isolate F14 was identified as Lactobacillus salivarius. It was closely related to Lactobacillus salivarius str Ren chromosome, with 99% similarity as inferred from the phylogenetic relationship using neighbor joining method (Fig. S1A). The sequence of isolate F14 was compared with 15 other genomes of Ligilactobacillus type species (https://lpsn.dsmz.de/genus/ligilactobacillus) publicly available in the NCBI database (Table S3). A non-recombinant maximum parsimony tree of core genes was constructed, which showed that the F14 strain was closely related to Ligilactobacillus salivarius CGMCC 3606 (Fig. 4). From the ANI analysis, values more than 95% were considered to belong to the same species. The analysis also supports the relatedness between Ligilactobacillus salivarius F14 and Ligilactobacillus salivarius CGMCC 3606 suggesting the fact that they share a single origin (Fig. S5). BLAST search across the complete genomes of all species of genus Ligilactobacillus was performed to identify the species closest to the F14 isolate. The genome of L. salivarius strain Ren. has been found to be most similar to that of the F14 strain, with BLAST percentage identity ranging from 70−10 % (Fig. 3).
Table 1
Complete genome features of Ligilactobacillus salivarius F14
No. of sequences | 1 chromosome |
Number of reads/contigs | 40 |
Genome size | 464753 bp |
Total assembly length | 1887930 |
(G + C)s | 32.94% |
N50 length | 108273 |
Total reads | 14267822 |
Genes (total) | 1,859 |
CDSs (total) | 1,813 |
Genes (coding) | 1,761 |
CDSs (with protein) | 1,761 |
Genes (RNA) | 46 |
rRNAs (5S, 16S, 23S) | 3 (1, 1, 1) |
tRNAs | 39 |
ncRNAs | 4 |
Pseudo genes | 52 |
CRISPR Arrays | 1 |
Phylogenetic Position And Strain Level Identification Of F14
Phylogenetic relations among the species of genus Ligilactobacillus have shown that the F14 strain clustered in a single clade with L. salivarius str. Ren (Fig. 4), highlighting that the F14 strain was closest to Ligilactobacillus salivarius. This phylogenetic inference is consistent with the results of whole genome average nucleotide identity (ANI) matrix (Fig. S5), where the F14 genome shows > 96% ANI identity with L. salivarius, clearly suggesting that F14 belongs to the salivarius species (Richter and Rossello-Mora 2009). In addition in silico DNA-DNA-hybridization (DDH) using the GGDC 2.0 server (Meier-Kolthoff, Auch et al. 2013) has shown that the F14 strain shares 86.70% similarity with the reference genome of L. salivarius strain Ren. These similarities were also observed where L. salivarius strain Ren. shows high BLAST hits (80–100%) with L. salivarius F14. Thus, based on the similarity of > 70%, we concluded that F14 is a member of the L. salivarius species.
Core and pan genome analysis of the L. salivarius F14 strains
Core and pan genome analysis was performed to evaluate the genomic diversity within the different strains of L. salivarius species. In total, 989 core genes were shared by all the 15 strains considered in this study, and strain-specific unique genes varied from 68 gene clusters in the strain Ligilactobacillus salivarius CECT5713 to 254 gene clusters specific for Ligilactobacillus salivarius ZLS006 (Fig. S6). Ligilactobacillus salivarius F14 contains 137 unique genes, including 25 proteins with known functions and 112 hypothetical proteins of unknown function on the basis of Prokka annotation (Seemann 2014). Furthermore, clusters of orthologous groups (COG) annotation of 137 unique genes have demonstrated the strain-specific abundance of glycosyltransferase and phosphotransferase functions in the F14 genome. Abundance of glycosyltransferases might have conferred the F14 strain a selective advantage for adherence and colonization in the GI tract (Brockhausen 2014). The presence of a phosphotransferase unique to the F14 isolate suggests that this strain might possess additional capabilities for transport and phosphorylation of sugar derivatives, and metabolic regulation of complex carbohydrates (Deutscher, Francke et al. 2006). Other unique genes associated with a broad spectrum of functions have also been identified, which may contribute to the fitness of the F14 strain (Table S4).
Functional Classification Of Cog Proteins
Amongst the 1,959 CDSs, 1,656 CDSs (84.53%) were categorized into COG functions. The annotated genes mainly belonged to the following categories: carbohydrate transport and metabolism (7.8%), amino acid transport and metabolism (8.1%), translation, ribosomal structure, and biogenesis (9.0%), replication, recombination, and repair (7.9%), cell wall/membrane/envelope biogenesis (6.6%), transcription (7.9%), defense mechanisms (2%), intracellular trafficking, secretion, and vesicular transport (1.4%), secondary metabolite biosynthesis, transport, and catabolism (0.9%), cell cycle control, cell division, and chromosome partitioning (1.7%), post-translational modification, protein turnover, chaperones (3.1%), energy production and conversion (4.0%), Nucleotide transport and metabolism (4.3%), coenzyme transport and metabolism (2.6%), lipid transport and metabolism (2.8%), signal transduction mechanisms (3.41%), and unknown function (9.69%) (Fig. S7).
Secreted Metabolites In F14 Cfs
The cell free supernatant (CFS) of the F14 growth media was analyzed using HRMS. The various antibacterial metabolites secreted by the F14 strain were pyroglutamic acid, lactic acid, 2-hydroxyisocaproic acid, glutathione, and homoserine. Probiotics are known to secrete various essential amino acids and vitamins. The F14 strain secreted essential amino acids such as leucine, isoleucine, threonine, tryptophan, valine, phenylalanine, and methionine and vitamins such as D3, dihydrofolic acid, dethiobiotin and retinoic acid. Various SCFAs such as acetic acid, propionic acid, butyric acid, isobutyric acid and lactic acid, which act as immunomodulators, were also found in the CFS of F14 (Table 2).
Table 2
List of metabolites produced by Ligilactobacillus salivarius F14
Name | Input Mass | Matched Mass | Delta | Significance |
GABA | 104.071 | 104.0706 | 0.0004 | Neurotransmitter |
Threonine | 120.081 | 120.0655 | 0.0155 | Protein synthesis |
Tryptophan | 205.0972 | 205.0971 | 0.0001 |
Asparagine | 131.0705 | 131.0462 | 0.0243 |
Isoleucine | 132.1019 | 132.1019 | 0 |
Proline | 116.0709 | 116.0706 | 0.0003 |
Valine | 118.0865 | 118.0862 | 0.0003 |
Leucine | 132.1019 | 132.1019 | 0 |
Methionine | 150.0584 | 150.0583 | 0.0001 |
Tyrosine | 182.0813 | 182.0812 | 0.0001 |
Phenylalanine | 166.0863 | 166.0862 | 0.0001 |
Pyroglutamic acid | 130.0864 | 130.0499 | 0.0365 | Antimicrobial, anti-tumoral , preserving the quality and nutritional value of foods |
Uracil | 111.0078 | 111.02 | 0.0122 | Nitrogenous base |
Pyruvic acid | 87.0077 | 87.0088 | 0.0011 | Potential antioxidant and anti-inflammatory agent |
Propionic acid | 73.0284 | 73.0295 | 0.0011 | Inhibits growth of mold and various bacteria |
Isobutyric acid | 87.0077 | 87.0452 | 0.0375 | Antimutagenic activity and energy source for colonocytes |
Butyric acid | 87.0077 | 87.0452 | 0.0375 |
Myristic acid | 227.1037 | 227.2017 | 0.098 | Cardiovascular health improvement |
Lactic acid | 89.0234 | 89.0244 | 0.001 | Antimicrobial, antiviral and immunomodulatory properties |
Succinic acid | 117.0184 | 117.0193 | 0.0009 | Cellular metabolic intermediate |
Oxaloacetic acid | 131.0705 | 130.9986 | 0.0719 | Cellular metabolite |
Acetic acid | 59.0127 | 59.0139 | 0.0012 | Fermentation and acidification activities, increasing cholesterol synthesis |
Citric Acid | 191.0193 | 191.0197 | 0.0004 | Enhances mineral absorption ,rich source of flavonoids, antioxidant and vitamin C |
2-hydroxyisocaproic acid | 131.0705 | 131.0714 | 0.0009 | Antifungal and antibacterial properties |
Vitamin D3 | 385.2444 | 385.3465 | 0.1021 | Maintenance of normal levels of serum calcium and phosphorus in the bloodstream |
Dihydrofolic acid (vitamin B9) | 444.2453 | 444.1626 | 0.0827 | Nucleotide biosynthesis |
Dethiobiotin (vitamin B7) | 215.1391 | 215.139 | 0.0001 | Metabolize fats, carbohydrates, and protein |
Retinoic acid | 301.2485 | 301.2162 | 0.0323 | A nutrient important to vision, growth, cell division, reproduction and immunity |
Fructose | 179.0556 | 179.0561 | 0.0005 | Cellular metabolite |
Glucose | 179.0556 | 179.0561 | 0.0005 |
Deoxyribose | 133.0498 | 133.0506 | 0.0008 |
2,3-Butanediol | 89.0234 | 89.0608 | 0.0374 | Has great potential for diverse industries, including chemical, cosmetics, agriculture, and pharmaceutical areas |
Arabinonic acid | 165.0551 | 165.0405 | 0.0146 | Involved in an organism's growth, development or reproduction. |
Homoserine | 120.081 | 120.0655 | 0.0155 | Antibacteial and anticancer |
Glutathione | 308.2179 | 308.0911 | 0.1268 | Antibacterial and antioxidant |
Betaine | 118.0865 | 118.0862 | 0.0003 | Can significantly prevent/attenuate progressive liver injury by preserving gut integrity and adipose function |
Dihydroxyacetone | 89.0234 | 89.0244 | 0.001 | Cosmetic industry as an artificial suntan, intermediate in lipid biosynthesis and in glycolysis. Dihydroxyacetone phosphate has been investigated for the treatment of Lymphoma |
Acetoin | 87.0077 | 87.0452 | 0.0375 | In food, cosmetics, synthesis of optically active pharmaceuticals |