Abstract
Probiotics are defined as live organisms in the host that contribute to health benefits. Lactobacillus gasseri LM1065, isolated from human breast milk, was investigated for its probiotic properties based on its genome. Draft genome map and de novo assembly were performed using the PacBio RS II system and hierarchical genome assembly process (HGAP). Probiotic properties were determined by the resistance to gastric conditions, adherence ability, enzyme production, safety assessment and mobile genetic elements. The fungistatic effect and inhibition of hyphae transition were studied using the cell-free supernatant (CFS). L. gasseri LM1065 showed high gastric pepsin tolerance and mild tolerance to bile salts. Auto-aggregation and hydrophobicity were measured to be 61.21% and 61.55%, respectively. The adherence to the human intestinal epithelial cells was measured to be 2.02%. Antibiotic-resistance genes and putative virulence genes were not predicted in the genomic analysis, and antibiotic susceptibility was satisfied by the criteria of the European Food Safety Authority. CFS showed a fungistatic effect and suppressed the tricarboxylic acid cycle in Candida albicans (29.02%). CFS also inhibited the transition to true hyphae and damaged the blastoconidia. This study demonstrates the essential properties of this novel probiotic, L. gasseri LM1065, and potential to inhibit vaginal C. albicans infection.
Similar content being viewed by others
Introduction
Lactobacillus gasseri, previously classified as the Lactobacillus acidophilus complex1,2, is present in the gastrointestinal tract (GIT), oral cavity, vaginal tract2,3, and human milk1,4. L. gasseri is an obligate homofermentative and thermophilic lactic acid bacteria (LAB) strain2, with a reported genomic size of approximately 1.89 Mb. L. gasseri was initially referred to as L. acidophilus and was reclassified as a separate species along with Lactobacillus johnsonii3. Many researchers have investigated L. gasseri as a probiotic, such as a yogurt starter4, for the treatment of vaginal dysbiosis5, inflammation6, oral diseases7, and type 2 diabetes8. In clinical trials, L. gasseri CP2305 has been applied as a heat-inactivated probiotic to study premenstrual symptoms in young women9 and to relieve fatigue- and stress-related symptoms in male runners10.
Vulvovaginal candidiasis (VVC) is a type of genital infection in women caused by excessive Candida genus abundance and imbalance of vaginal microbiome in the reproductive phase11,12,13,14. VVC leads to vulval discomfort and pain accompanied by pruritus, vaginal soreness, and abnormal vaginal discharge12. According to a previous study, VVC can frequently occur in adult women in their lifetime, and 80–90% of VVC is caused by Candida albicans11,14. Despite the high frequency of VVC pathogenesis and recurrent vulvovaginal candidiasis (VVCR), Candida infections in the vaginal tract have not been clearly elucidated. Candida species, including C. albicans, is considered to migrate (like vaginal Lactobacillus species) from the lower GIT to the vaginal tract13. Amphotericin B and nystatin family of polyene antifungal agents are commonly used to treat VVC11,14. However, these antifungal agents affect the microbial environment in the vaginal flora12 and may also lead to the development of VVCR in VVC patients11.
In this study, we aimed to isolate a novel probiotic strain and investigate its probiotic properties based on genomic information. Many probiotic strains have been investigated for their probiotic properties, such as resistance to gastric and intestinal conditions, enzyme production, adhesion to intestinal cells, and safety. Nevertheless, these properties are needed at the genomic level for probiotics. In this study, L. gasseri LM1065 isolated from human breast milk was investigated for its essential probiotic properties and antifungal abilities against C. albicans, along with functional gene annotation.
Results
General genomic feature of Lactobacillus gasseri LM1065
As shown in Fig. 1A, the genomic analysis of L. gasseri LM1065 comprises two contigs (circular form and linear form, respectively) and a circular plasmid. The size of the entire genome sequence of L. gasseri LM1065 was 2,251,884 bp. The plasmid of L. gasseri LM1065 was similar to plasmid of L. gsseri HL70. The whole genome of L. gasseri LM1065 showed a 35.02% GC content. The GC contents of each contig were 34.98% (circular contig), 35.09% (linear contig), and 35.99% (plasmid). In total, 2300 protein-coding sequences (CDS) were identified in L. gasseri LM1065. Contig 1 (circular contig) was identified in 1920 CDS, and contig 2 (linear contig) and plasmid contained 322 and 58 coding sequences, respectively. The L. gasseri LM1065 contained 15 rRNA and 79 tRNA genes. Most rRNA and tRNA genes were identified in contig 1, and one tRNA was found in contig 2. This Whole Genome project has been deposited at DDBJ/ENA/GenBank under the accession JAQOUF000000000.
Circular gene map, phylogenetic relationship, and Gene Ontology of Lactobacillus gasseri LM1065. (A) Circular gene map of three contigs. Each circle from outside to inside indicates protein-coding sequences (CDS) on forward strand, CDS on reverse strand, tRNA, rRNA, GC content, and GC skew. (B) Phylogenetic tree. (C) Average nucleotide identity (ANI) value. (D) Gene Ontology. (E) Bacteriocin gene cluster.
Phylogenetic relationship of Lactobacillus gasseri LM1065
The ortholog phylogenetic relationships of L. gasseri LM1065 among the Lactobacillus and Lactiplantibacillus are shown in Fig. 1B and C. According to the phylogenetic network based on ortholog (Fig. 1B), L. gasseri LM1065 was grouped into other L. gasseri and further associated with L. acidophilus complex. In the average nucleotide identity (ANI) level comparison, L. gasseri LM1065 was relatively close to other L. gasseri (93.51–99.93). The L. gasseri strains including L. gasseri LM1065 showed approximately 85% of ANI distance with L. johnsonii ATCC33200 and other L. acidophilus complex strains showed approximately 73% of distance. Lactiplantibacillus, which are different species, showed approximately 65% of ANI distance (Fig. 1C).
Genomic insight into Lactobacillus gasseri LM1065.
Figure 1D shows the predicted gene cluster into different functional categories (biological processes, cellular components, and molecular functions) depending on the Gene Ontology (GO) database. Total 6624 of transcripts were annotated and divided into 18 biological processes, 15 cellular components, and 11 molecular function categories. In total, 2300 CDS in all contigs, adhesion ability, bacteriocin production, enzyme production, and stress response functional genes are shown in Table 1.
Prediction of gassericin gene cluster
Figure 1E shows bacteriocin production gene clusters of L. gasseri LM1065. Total three bacteriocin gene clusters were predicted in contig 1. Gassericin T (start at 595,336 and end at 605,613), gassericin A (start at 1,778,187 and end at 1,787,064) and gassericin S (start at 1,851,350 and end at 1,861,589) were predicted in L. gasseri LM1065.
Comparative genomic analysis
Figure 2 shows multiple alignment of the L. gasseri strains in relation to strains closet in homology using MAUVE. L. gasseri LM1065 was most similar to L. gasseri HL70 comparing to Locally Collinear Blocks (LCBs). A total of 27 LCBs were arranged in L. gasseri LM1065. Especially, LCB 4 showed difference in each L. gasseri such as length and alignment.
Multiple genome alignment and genomic comparison of Lactobacillus gasseri.
Fatty acid composition of cell wall
Table S1 shows the fatty acid composition of the bacterial cell wall of L. gasseri LM1065. Thirteen fatty acids were investigated in L. gasseri LM1065. Oleic acid and cis-10-nonadecenoic acid were measured 68.95 and 16.31%, respectively, in L. gasseri LM1065. Most of the cellular fatty acids in L. gasseri LM1065 were unsaturated fatty acids (USFA, 87.96%), whereas saturated fatty acids (SFA) accounted for 12.04%. The USFA/SFA ratio was 7.30.
Resistance to pepsin and bile salt
The viable cell numbers and survival rates of L. gasseri LM1065 under artificial gastric conditions are shown in Table 2. L. gasseri LM1065 showed an 88.97% survival rate in artificial pepsin for 2 h. The survival rate of L. gasseri LM1065 was not significantly different from that of L. rhamnosus ATCC 53103 (92.46%). Despite its high acid tolerance, L. gasseri LM1065 showed mild resistance in 0.05 and 0.1% of bile salt conditions. The changes of viable cells were showed 7.95 to 6.96 Log CFU/mL and 7.96 to 7.93 Log CFU/mL in 0.1% and 0.05% of bile salt, respectively.
Auto-aggregation, hydrophobicity, and adherence to HT-29 cells
Table 2 shows the auto-aggregation, hydrophobicity, and adherence to human epithelial cells of L. gasseri LM1065. L. gasseri LM1065 showed higher auto-aggregation ability (61.21%) than L. rhamnosus ATCC 53103 (48.46%) (P < 0.01). The hydrophobicity to hexadecane and the ability to adhere to HT-29 were measured 61.55% and 2.02%, respectively. These properties are not significantly different from those of L. rhamnosus ATCC 53103.
Enzyme activity
API ZYM shows the intrinsic enzymatic properties of microorganisms using 19 different substrates. L. gasseri LM1065 showed 11 enzyme activities, including alkaline phosphatase, esterase, leucine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase (Table S2).
Safety evaluation
L. gasseri LM1065 showed antibiotic susceptibility according to ESFA guidelines (ampicillin, 0.5 μg/mL; chloramphenicol, 4 μg/mL; clindamycin, 0.5 μg/mL; erythromycin, 0.25 μg/mL; gentamicin, 4 μg/mL; kanamycin, 64 μg/mL; streptomycin, 16 μg/mL; tetracycline, 2 μg/mL; vancomycin, 0.5 μg/mL). The L. gasseri LM1065 was not detected antibiotics resistance and potential antibiotics resistance genes (ARGs). Putative virulence factors were not predicted by genome sequencing. Hemolysis was not detected on the blood agar (γ-hemolysis) (Table 3).
Mobile genetic elements and genomic island
Mobile genetic elements (MGEs) in L. gasseri LM1065 were shown in Table S3. Total 7 of prophages, 2 of integrative and conjugative elements (ICEs), 57 of transposon insertion-sequence (IS) or IS cluster were detected in L. gasseri LM1065. In genomic island (GI) analysis, pathogenicity island and antibiotics resistance island were not found in L. gasseri LM1065. Plasmid sequences were not detected in L. gasseri LM1065. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) which protect against MGEs were detected in L. gasseri LM1065.
Inhibition of Candida albicans
Table 4 shows MIC and fungistatic effect of L. gasseri CFS. The MIC of CFS was evaluated 50%. To investigate fungistatic effect of CFS, C. albicans was incubated with different concentrations of CFS. Non-treated C. albicans grew up to 7.20 Log CFU/mL while CFS treated C. albicans was inhibited growth to 3.89 Log CFU/mL for 48 h. The decrease of growth rate was also detected by TCA cycle inhibition. CFS inhibited TCA cycle of C. albicans to 10.45, 23.46 and 29.02% in 0.5, 1.0 and 1.5 × MIC, respectively (P < 0.01) (Fig. 3). Moreover, CFS inhibited yeast to hyphae transition and damaged to blastoconidia in C. albicans (Fig. 4).
Tricarboxylic acid cycle activity in Candida albicans ATCC 11006 treated by cell free supernatant. Significant differences within test groups were analyzed Tukey’s range tests (P < 0.001).
Microscopic features of Candida albicans ATCC 11006 treated by cell free supernatant (observed at the magnification of 400 ×).
Discussion
Probiotics are live microorganisms that contribute to host health when adequate amounts are consumed2,15,16,17. Probiotics are required to survive through the GIT and colonize the small intestine and colon for a long period6 with a safety guarantee18,19. L. gasseri LM1065 exhibited survival under gastric conditions, enzyme production, colonization and adhesion ability without antibiotic resistance, hemolysis, and putative virulence factors compared to functional gene annotation.
Probiotics are exposed to various types of stress during freeze-drying20, containing a food matrix and passing through the GIT16. These external stresses can damage cell membranes, induce the release of internal beneficial enzymes, and inhibit the colonization of the intestine6,20. In the present study, stress-responsive functional genes, including acid shock proteins, were investigated in L. gasseri LM1065 (Table 1). L. gasseri LM1065 showed high acid and mild bile salt tolerance (Table 2). Tang et al.21 reported that the bile salt concentrations vary from 0.03 to 0.3% in the small intestine during food digestion. L. gasseri LM1065 can survive in low concentrations of bile salts within the small intestine. Li et al.17 suggested that mild bile salt tolerance results from the microenvironment of the microbial niche. Lactobacillus taiwanensis, isolated from Peyer’s patches, also showed mild tolerance to bile salt because Peyer’s patches show mild bile salt conditions compared to other small intestinal sites. These properties were also observed in Limosilactobacillus fermentum 4LB16 and 10LB1 isolated from the human vaginal tract22.
In general, bacterial adhesion to the human intestinal tract is mediated by cell surface components. In Lactobacillus, surface-layer proteins, cell wall-anchored mucus-binding proteins, cell-surface collagen-binding proteins, and mannose-specific adhesins have been reported to adhere to host molecules and mechanisms23. The cell wall-anchored protein (CWAP) contains five amino acid motifs, LPXTG, which is constructed from Leu-Pro-any amino acid-Thr-Gly24. Gram-positive bacteria utilize sortase to cleave surface proteins, particularly the Thr-Gly residue of LPXTG. The cleaved threonine residue mediates cell wall attachment25. Zhang et al.23 studied the transformation of LPXTG into Lactococcus lactis, which confers adhesion to human epithelial cells. L. gasseri LM1065 showed higher auto-aggregation and adherence than L. rhamnosus ATCC 53103 (Table 2). LPXTG and sortase were predicted in L. gasseri LM1065 (Table 1), and these genetic properties contribute to its essential probiotic properties.
Gut-microbiomes produce enzymes that behave in complementary ways in human metabolism26. In the gut microbiome niche, LAB play critical roles in digestion, nutrient absorption, and improvement of nutritional value using numerous enzymes27. Most represent enzymes in Lactobacillus are lactase, β-galactosidase, glycosidase, protease, lipase, esterase and phytase26,27. Otherwise β-glucuronidase, which is released by Escherichia, Clostridium, and Staphylococcus26, is associated with potential carcinogenic metabolite conversion19,26. L. gasseri LM1065 released 11 enzymes, including β-galactosidase whereas β-glucuronidase activity was not found (Table S2). β-Galactosidase is an important biotechnological source in the food industry. β-Galactosidase is applied to ice cream to prevent undesirable crystallization and improve its creaminess. In addition, β-galactosidase is used in bakeries to improve sweetness27. Lactose intolerance occurred in a person lacking β-galactosidase in the intestine and caused a lack of β-galactosidase accumulation of lactose. Excessive lactose affects osmotic pressure, resulting in digestive disorders26. Thus, β-galactosidase produced by gut-microbes attenuate digestive disorders in lactose intolerance consumers26,27. Interestingly, L. gasseri LM1065 was predicted to contain β-galactosidase and glycoside hydrolase family 1 involved in lactose hydrolysis (Table 1).
In the host immune system, pattern recognition receptors (PRRs) play a critical role in recognizing pathogens from the external environment via pathogen-associated molecular patterns (PAMPs)28,29. The cell wall of C. albicans consists of two parts: mannosylated protein (outer layer) and β-1,3-glucan with underlying chitin (inner layer). Dectin-1 is a major PRR that recognizes β-1,3-glucan as a PAMP29. β-1,3-glucan is not only a PAMP but also a biofilm component that resists stress30. Therefore, β-1,3-glucan regulation is considered an effective strategy for the treatment of C. albicans and VVC30,31. L. gasseri LM1065 was predicted to contain the β-glucanase gene (Table 1), which inhibits the growth and hyphae transition of C. albicans ATCC 11006 (Fig. 4). Additionally, L. gasseri LM1065 was predicted to produce gassericin T (Table 1 and Fig. 1E).
Conclusions
L. gasseri LM1065, isolated from human breast milk, has essential probiotic properties, including resistance to gastric conditions and adherence to intestinal cells. L. gasseri LM1065 satisfied safety requirements, including antibiotic resistance and putative virulence factor genes. Additionally, L. gasseri LM1065 was suggested as a probiotic for women, as the bacteria can inhibit candidiasis by suppressing the TCA cycle in C. albicans and blocking its transition to hyphae.
Materials and methods
Subjects and isolation of Lactobacillus gasseri LM1065
The collection of human breast milk were approved by Institutional Review Board of the Lactomason according to Enforcement Decree of Bioethics and Safety Act in Korea. All donors provided written informed consent before enrollment in the study and all methods were carried out in accordance with the Declaration of Helsinki. Three human breast milk samples were donated by three healthy women (25–40 years old) living in Gyeongsangnam-do on May 24, 2017. The participants did not have any underlying conditions and took any antibiotics or probiotics prior to the study for at least three months18. Human breast milk specimens were placed in a sterile container and diluted 10 times with phosphate-buffered saline (PBS, pH 7.2). The diluted specimens were spread on de Man–Rogosa–Sharpe (MRS) agar (for Lactobacillus) (BD Biosciences, Franklin Lakes, NJ, USA), M17 agar (for lactic Streptococcus and Lactococcus) (MBcell, Seoul, South Korea), and Bifidobacterium selective agar (for Bifidobacterium spp.) (MBcell). The spread agar plates were then incubated at 37 °C for 48 h. After 48 h, colonies isolated from MRS agar were spread on bromocresol purple (BCP) agar, and yellow colonies on BCP agar were further purified in newly prepared MRS agar until a single colony was obtained. Single and pure colonies were enriched in MRS broth for Gram staining and catalase reactions. The isolate was identified as a Gram-positive catalase-negative rod-type strain. The isolated strain was named LM1065 based on the institution’s naming system and identified by 16S rRNA sequencing as L. gasseri. L. gasseri LM1065 was stored in MRS containing 20% glycerol at − 80 °C until use32.
Microorganisms and conditions
L. rhamnosus ATCC 53103 and C. albicans ATCC 11006 were purchased from the American Type Culture Collection (ATCC). L. gasseri LM1065 and L. rhamnosus ATCC 53103 were cultured in MRS broth at 37 °C with aerobic condition and sub-cultured three times every 12 h until use. C. albicans ATCC 11006 was cultured in yeast mold (YM) broth at 24 °C for 48 h with aerobic condition until use.
Extraction of genomic DNA and genome analysis
L. gasseri LM1065 was harvested by centrifugation at 12,000 rpm at 4 °C for 10 min. The harvested bacterial cells were washed with PBS and genomic DNA (gDNA) was extracted. gDNA was extracted from L. gasseri LM1065 using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit (Takara Bio, Kusatsu, Japan) according to the manufacturer’s guidelines.
A total of 5 μg of the gDNA sample was used for library preparation. A DNA library was constructed and sequenced using single molecular real-time (SMRT) sequencing technology (Pacific Biosciences, Menlo Park, CA, USA). The SMRTbell library preparation was performed with the SMRTbell Template Prep Kit 1.0, and DNA/Polymerase Biding kit P6. The SMRT library was sequenced using 1 SMRT cell using C4 chemistry (DNA sequencing Reagent 4.0) and 240-min movies were captured for each SMRT cell using the PacBio RS II system by Insilicogen (Yongin, South Korea). The genome coverage (depth of coverage) was 195 × and HiFi long-read sequencing perform for analysis.
De novo assembly was performed in the hierarchical genome assembly process (HGAP) including consensus polishing workflow using Quiver, and 1,929,825 bp of N50 contig and 2,251,884 bp of total contig were obtained. Sequence alignment and contig formation were performed using MUMmer 3.5. The coding sequence was predicted using GLIMMER 3.0, and the GO analysis was performed using Blast2GO33. The plasmid was predicted using nucleotide BLAST and bacteriocin gene clusters were predicted and visualized by antiSMASH bacterial version 7.0.034.
Multiple alignment and genomic comparison were analyzed using MAUVE35.
Phylogenetic analysis based on orthologous gene and comparing of average nucleotide identity
Phylogenetic relationships of L. gasseri LM1065 were constructed based on ortholog gene sequences. The whole genome sequences of Lactobacillus and Lactiplantibacillus were obtained from the National Center for Biotechnology Information (NCBI) database. Ortholog analysis was performed using OrthoFinder v2.5.4 and species tree was inferred using STAG algorithm and rooted using STRIDE algorithm in OrthoFinder36. The species tree was illustrated by Dendroscope 337.
The ANI values were estimated using the OrthoANI algorithm in EzBioCloud (https://www.ezbiocloud.net/tools/ani)38. The result of ANI distance was generated using the heatmap plot function of the TBtools39.
Cellular fatty acid analysis of Lactobacillus gasseri LM1065
Cellular fatty acid extraction of L. gasseri LM1065 was performed using the Bligh and Dyer method with modifications20. In brief, 200 μL of chloroform/methanol solution (2:1, v/v) and 300 μL of 0.6 M hydrochloric acid solution (in methanol) were added to 20 mg of lyophilized L. gasseri LM1065. The mixture was shaken vigorously for 2 min and then incubated at 85 °C for 60 min. The reaction mixture was cooled at 25 °C for 20 min, and fatty acid methyl esters (FAME) were extracted using n-hexane for 60–120 min. The FAME extracted layer (n-hexane layer) was transferred into a clear vial and stored at − 20 °C until analysis.
Cellular fatty acid analysis was performed using Gas Chromatography/Mass selective detector (GC/MSD). The GC/MSD system was composed of an Agilent 8890 gas chromatography system coupled with a 5977 B mass selective detector (MSD) and a 7693A automated liquid sampler (Agilent, Santa Clara, CA, USA). An Agilent J&W DB-FastFAME capillary column packed with cyanopropyl (30 m × 0.25 mm, 0.25 μm) was employed. The injection port temperature was 250 °C under constant flow, and 1 μL of the sample was injected using the split mode of 20:1. Ultrapure helium was used as the carrier gas at a flow rate of 1 mL/min. The initial oven temperature was 60 °C for 1 min, raised from 60 to 165 °C at a rate of 60 °C/min, held for 1 min at 165 °C, raised from 165 to 230 °C at a rate of 5 °C/min, and maintained for 3 min. The temperatures of the ion source and transfer line were 230 °C and 250 °C, respectively. Mass spectra were obtained using electron ionization (EI) at 70 eV and recorded m/z 40–550 of mass range. Methyl undecanoate was used as an internal standard40.
Tolerance to pepsin and bile salt
The resistance properties of L. gassseri LM1065 to artificial gastric conditions were investigated in a previous study with modifications2,15,16,17,21,22. L. gasseri LM1065 was inoculated into MRS containing 0.3% pepsin (pH 2.5) or oxgall (0.05, 0.1, 0.2, and 0.3%) and incubated at 37 °C. After 2 and 24 h of incubation, viable cells in pepsin- and oxgall-containing MRS broths were measured by spreading on MRS agar. L. rhamnosus ATCC 53103 was used as the control.
Auto-aggregation and cell surface hydrophobicity
L. gasseri LM 1065 was harvested by centrifugation at 12,000 rpm and 4 °C for 10 min and washed twice with PBS. The washed cells were resuspended in PBS and adjusted to an OD600 of 0.5.
To evaluate the auto-aggregation of L. gasseri LM1065, the adjusted bacterial suspension was allowed to stand and incubated at 37 °C. The upper suspension was collected (4 and 24 h), and the absorbance was measured at 600 nm. Auto-aggregation was calculated using the following equation:
where A0 is the initial absorbance (0.5), and Atime is the absorbance of the supernatant at 4 and 24 h, respectively2,11. L. rhamnosus ATCC 53103 was used as the control.
To evaluate the hydrophobicity of L. gasseri LM1065, 2 mL of the adjusted bacterial suspension was mixed with 1 mL hexadecane. The mixture was then allowed to stand at 25 °C for 30 min. After incubation, the aqueous phase was separated, and its absorbance was measured at 600 nm. The hydrophobicity was calculated using the following equation.
where A0 is the initial absorbance (0.5) and A30 is the absorbance of the aqueous phase at 30 min18,40. L. rhamnosus ATCC 53103 was used as the control.
Adhesion to human intestinal epithelial cell
Adhesion ability of L. gasseri LM1065 was investigated in human intestinal epithelial cell (HT-29)15,18,41. HT-29 human intestinal epithelial cells were purchased from Korean Cell Line Bank (Seoul, South Korea). The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO2. During incubation, the media were changed every 2–3 days, and cells were grown to 80% confluence. When HT-29 cells reached 80% confluence, the adherent cells were trypsinized with trypsin–EDTA solution (0.25%) and harvested by centrifugation. Harvested cells were seeded in 24-well plates (1 × 105 cells/well) and incubated with changing media to form a monolayer. Activated L. gasseri LM1065 was diluted to approximately 8 Log CFU/mL, and the HT-29 monolayer was treated with diluted L. gasseri LM1065 for 2 h without antibiotics. After 2 h, non-adherent bacterial cells were washed using PBS, and adherent bacterial cells were collected using 1% (v/v) Triton-X solution. Adherent bacterial cells were spread on MRS agar and viable cells were estimated. L. rhamnosus ATCC 53103 was used as the control.
Enzymatic profile of Lactobacillus gasseri LM1065
The intrinsic enzyme activities of L. gasseri LM1065 were estimated using API ZYM, according to the manufacturer’s guidelines (bioMérieux, Marcy-l’Étoile, France).
Safety assessments
Safety assessments were measured based on antibiotic resistance18,22,42, analysis of ARGs, virulence genes19,42, and hemolysis18,22,41.
Antibiotic resistance of L. gasseri LM1065 was evaluated according to the European Food Safety Authority (EFSA) guidelines. Ampicillin, erythromycin, gentamicin, tetracycline, streptomycin, vancomycin, chloramphenicol, kanamycin, and clindamycin susceptibilities were measured by cut-off values using ETEST® strips (bioMérieux).
ARGs and virulence genes were predicted using a nucleotide database. The draft genome sequence of L. gasseri LM1065 was performed to identify genetic variations. The Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/)7,19 and ResFinder (https://cge.food.dtu.dk/services/ResFinder/)42 were used for genome-based analysis of ARGs. Virulence genes were analyzed by comparison with the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/main.htm)42.
Hemolysis activity was determined using Columbia blood agar (Oxoid, Basingstoke, United Kingdom) containing 5% sheep blood.
Mobile genetic elements and genomic island
The MGEs and genomic island were measured for predicting horizontal gene transfer. CRISPR-Cas were investigated by CRISPRCasFinder (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index)43. Plasmid sequences were detected using PlasmidFinder (https://cge.food.dtu.dk/services/PlasmidFinder/)43. Prophage, IS, and GI were analyzed using VRprofile2 (https://tool2-mml.sjtu.edu.cn/VRprofile/home.php)44.
Minimum inhibitory concentration and fungistatic effect of Lactobacillus gasseri LM1065
The cell-free supernatant (CFS) of L. gasseri LM1065 was prepared using a 0.45 μm cellulose acetate membrane filter. To determine the minimum inhibitory concentration (MIC), the CFS was diluted in a 96-well plate using YM broth (MBcell, Seoul, South Korea). After dilution, approximately 6 Log CFU/mL of C. albicans ATCC 11006 was added to each well and further incubated at 24 °C for 48 h. MIC was determined as the lowest concentration that did not show C. albicans growth visually45. To measure the fungistatic effect of CFS, approximately 7 Log CFU/mL of C. albicans ATCC 11006 was treated with different CFS concentrations (0.5, 1.0, and 1.5 × MIC) and incubated at 24 °C for 48 h. After incubation, C. albicans was spread on YM agar, further incubated at 24 °C, and viable cells were counted.
Tricarboxylic acid cycle inhibition and microscopic observation
Tricarboxylic acid (TCA) cycle activity in CFS-treated C. albicans ATCC 11006 was measured using an iodonitrotetrazolium chloride (INT; Sigma-Aldrich, MO, USA) solution44. Briefly, approximately 7 Log CFU/mL of C. albicans ATCC 11006 was treated with 0.5, 1.0 and 1.5 × MIC of CFS and incubated for 24 h. After incubation, C. albicans was collected by centrifugation at 3,000 rpm at 4 °C for 10 min and washed twice with PBS. The harvested C. albicans was diluted to an OD600 of 0.1, and INT solution (1 mM final concentration) was added. The INT solution-treated C. albicans cells were incubated at 37 °C for 30 min. The TCA cycle was assessed by measuring the absorbance of formazan at 630 nm.
For microscopic observation, the harvested cells were stained with 0.4% trypan blue solution (Gibco). The morphology of the CFS-treated C. albicans was observed using a BX53 biological microscope (Olympus, Tokyo, Japan). All observations were performed at a total magnification of 400 × . Microscopic images were obtained using the eXcope software. Morphological information was obtained from a previous study46.
Statistical analysis
Statistical analyses were performed using the SPSS Statistics version 18 software (IBM, Armonk, NY, USA). Mean values were analyzed using the t-test and one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests and Tukey's range test at P < 0.05.
Ethical consideration
The collection of human breast milk were approved by Institutional Review Board of the Lactomason according to Enforcement Decree of Bioethics and Safety Act in Korea. All donors signed an informed consent form before enrollment in the study and voluntarily provided samples for only research purpose in accordance with the Declaration of Helsinki.
Data availability
The draft genome sequence has been deposited at DDBJ/ENA/GenBank under the accession JAQOUF000000000 (https://www.ncbi.nlm.nih.gov/nuccore/JAQOUF000000000). The authors confirm that the data of this study are available within the article.
References
Garcia-Gutierrez, E. et al. Production of multiple bacteriocins, including the novel bacteriocin gassericin M, by Lactobacillus gasseri LM19, a strain isolated from human milk. Appl. Microbiol. Biotechnol. 104, 3869–3884 (2020).
Gunyakti, A. & Asan-Ozusaglam, M. Lactobacillus gasseri from human milk with probiotic potential and some technological properties. LWT Food Sci. Technol. 109, 261–269 (2019).
Zhou, X. et al. Comparative analysis of Lactobacillus gasseri from Chinese subjects reveals a new species-level taxa. BMC Genomics 21, 119 (2020).
Zhou, Q. Y. et al. Lactobacillus gasseri LGZ 1029 in yogurt: Rheological behaviour and volatile compound composition. Int. J. Food Sci. Technol. 56, 2992–3003 (2021).
Atassi, F., Pho Viet Ahn, D. L. & Lievin-Le Moal, V. Diverse expression of antimicrobial activities against bacterial vaginosis and urinary tract infection pathogens by cervicovaginal microbiota strains of Lactobacillus gasseri and Lactobacillus crispatus. Front. Microbiol. 10, 2900 (2019).
Oh, N. S., Joung, J. Y., Lee, J. Y. & Kim, Y. Probiotic and anti-inflammatory potential of Lactobacillus rhamnosus 4B15 and Lactobacillus gasseri 4M13 isolated from infant feces. PLoS ONE 13, e0192021 (2018).
Mann, S. et al. Oral probiotic activities and biosafety of Lactobacillus gasseri HHuMIN D. Microb. Cell Factories 20, 75 (2021).
Niibo, M. et al. Probiotic Lactobacillus gasseri SBT2055 improves insulin secretion in a diabetic rat model. J. Dairy Sci. 102, 997–1006 (2019).
Nishida, K., Sawada, D., Yasui, T., Kuwano, Y. & Rokutan, K. Daily intake of Lactobacillus gasseri CP2305 ameliorates psychological premenstrual symptoms in young women: A randomized, double-blinded, placebo-controlled study. J. Funct. Foods 80, 104426 (2021).
Sawada, D. et al. Daily intake of Lactobacillus gasseri CP2305 relieves fatigue and stress-related symptoms in male university Ekiden runners: A double-blind, randomized, and placebo-controlled clinical trial. J. Funct. Foods 57, 465–476 (2019).
Feng, W. et al. Mrr2 mutations and upregulation are associated with increased fluconazole resistance in Candida albicans isolates from patients with vulvovaginal candidiasis. Lett. Appl. Microbiol. 70, 95–101 (2020).
Li, T., Liu, Z., Zhang, X., Chen, X. & Wang, S. Local probiotic Lactobacillus crispatus and Lactobacillus delbrueckii exhibit strong antifungal effects against vulvovaginal candidiasis in a rat model. Front. Microbiol. 10, 1033 (2019).
Pereira, L. C. et al. Vulvovaginal candidiasis and current perspectives: New risk factors and laboratory diagnosis by using MALDI TOF for identifying species in primary infection and recurrence. Eur. J. Clin. Microbiol. Infect. Dis. 40, 1681–1693 (2021).
Zhang, X., Li, T., Chen, X., Wang, S. & Liu, Z. Nystatin enhances the immune response against Candida albicans and protects the ultrastructure of the vaginal epithelium in a rat model of vulvovaginal candidiasis. BMC Microbiol. 18, 166 (2018).
Cheon, M. J., Lim, S. M., Lee, N. K. & Paik, H. D. Probiotic properties and neuroprotective effects of Lactobacillus buchneri KU200793 isolated from Korean fermented foods. Int. J. Mol. Sci. 21, 1227 (2020).
de Oliveira Vogado, C., dos Santos Leandro, E., de Alencar, E. R., Ginani, V. C. & Zandonadi, R. P. Survival of Lactobacillus paracasei subsp. paracasei LBC 81 in fermented milk enriched with green banana pulp under acid stress and in the presence of bile salts. Probiotics Antimicrob. Proteins 12, 320–324 (2020).
Li, X. Y. et al. Complete genome sequencing of Peyer’s patches-derived Lactobacillus taiwanensis CLG01, a potential probiotic with antibacterial and immunomodulatory activity. BMC Microbiol. 21, 68 (2021).
Kim, T. R., Choi, K. S., Ji, Y., Holzapfel, W. H. & Jeon, M. G. Anti-inflammatory effects of Lactobacillus reuteri LM1071 via MAP kinase pathway in IL-1β-induced HT-29 cells. J. Anim. Sci. Technol. 62, 864–874 (2020).
Kang, M. S., Yeu, J. E. & Hong, S. P. Safety evaluation of oral care probiotics Weissella cibaria CMU and CMS1 by phenotypic and genotypic analysis. Int. J. Mol. Sci. 20, 2693 (2019).
Cheng, Z., Yan, X., Wu, J., Weng, P. & Wu, Z. Effects of freeze drying in complex lyoprotectants on the survival, and membrane fatty acid composition of Lactobacillus plantarum L1 and Lactobacillus fermentum L2. Cryobiology 105, 1–9 (2022).
Tang, W. et al. Probiotic properties and cellular antioxidant activity of Lactobacillus plantarum MA2 isolated from Tibetan kefir grains. Probiotics Antimicrob. Proteins 10, 523–533 (2018).
Ouarabi, L., Chait, Y. A., Seddik, H. A., Drider, D. & Bendali, F. Newly isolated Lactobacilli strains from Algerian human vaginal microbiota: Lactobacillus fermentum strains relevant probiotic’s candidates. Probiotics Antimicrob. Proteins 11, 43–54 (2019).
Zhang, B. et al. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells. Sci. Rep. 5, 14109 (2015).
Yonemoto, K. et al. Redundant and distinct roles of secreted protein Eap and cell wall-anchored protein SasG in biofilm formation and pathogenicity of Staphylococcus aureus. Infect. Immun. 87, e00894-e918 (2019).
Al Mamun, A. A. M. et al. A cell wall-anchored glycoprotein confers resistance to cation stress in Actinomyces oris biofilms. Mol. Oral Microbiol. 37, 206–217 (2022).
Maske, B. L. et al. A review on enzyme-producing lactobacilli associated with the human digestive process: From metabolism to application. Enzyme Microb. Technol. 149, 109836 (2021).
García-Cano, I., Rocha-Mendoza, D., Kosmerl, E., Zhang, L. & Jiménez-Flores, R. Technically relevant enzymes and proteins produced by LAB suitable for industrial and biological activity. Appl. Microbiol. Biotechnol. 104, 1401–1422 (2020).
Lee, S., Channappanavar, R. & Kanneganti, T. D. Coronaviruses: Innate immunity, inflammasome activation, inflammatory cell death, and cytokines. Trends Immunol. 41, 1083–1099 (2020).
Yang, M. et al. Control of β-glucan exposure by the endo-1,3-glucanase Eng1 in Candida albicans modulates virulence. PLOS Pathog. 18, e1010192 (2022).
Tan, Y., Ma, S., Leonhard, M., Moser, D. & Schneider-Stickler, B. β-1,3-glucanase disrupts biofilm formation and increases antifungal susceptibility of Candida albicans DAY185. Int. J. Biol. Macromol. 108, 942–946 (2018).
Taher, J. M. & Raheem, N. N. Role of purified beta-glucanase from Lactobacillus acidophilus in disruption of biofilm formation by Candida spp causing of denture stomatitis. Mater. Today Proc. 60, 1507–1512 (2022).
Rajoka, M. S. R. et al. Anticancer potential against cervix cancer (HeLa) cell line of probiotic Lactobacillus casei and Lactobacillus paracasei strains isolated from human breast milk. Food Funct. 9, 2705–2715 (2018).
Choi, Y. et al. Alleviation of periodontal disease using Lactobacillus curvatus SMFM2016-NK. J. Funct. Foods 83, 104531 (2021).
Blin, K. et al. antiSMASH 7.0: New and improved predictions for detection, regulation, chemical structures and visualisation. Nucleic. Acids Res. 51, W46–W50 (2023).
Tenea, G. N. Decoding the gene variants of two native probiotic Lactiplantibacillus plantarum strains through whole-genome resequencing: Insights into bacterial adaptability to stressors and antimicrobial strength. Genes 13, 443 (2022).
Carpi, W. et al. Comprehensive pan-genome analysis of Lactiplantibacillus plantarum complete genomes. J. Appl. Microbiol. 132, 592–604 (2022).
Baek, M. G., Kim, K. W. & Yi, H. Subspecies-level genome comparison of Lactobacillus delbrueckii. Sci. Rep. 13, 3171 (2023).
Lee, C., Kim, Y. O., Park, S. C. & Chun, J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 66, 1100–1103 (2016).
Chen, C. et al. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. plant 13, 1194–1202 (2020).
Liu, W. et al. Effect of Lactobacillus plantarum on functional characteristics and flavor profile of fermented walnut milk. LWT Food Sci. Technol. 160, 113254 (2022).
Sornsenee, P., Singkhamanan, K., Sangkhathat, S., Saengsuwan, P. & Romyasamit, C. Probiotic properties of Lactobacillus species isolated from fermented palm sap in Thailand. Probiotics Antimicrob. Proteins 13, 957–969 (2021).
Chokesajjawatee, N. et al. Safety assessment of a Nham starter culture Lactobacillus plantarum BCC9546 via whole-genome analysis. Sci. Rep. 10, 10241 (2020).
Kandasamy, S. et al. Probiogenomic in-silico analysis and safety assessment of Lactiplantibacillus plantarum DJF10 strain isolated from Korean raw milk. Int. J. Mol. Sci. 23, 14494 (2022).
Bertelli, C., Tilley, K. E. & Brinkman, F. S. L. Microbial genomic island discovery, visualization and analysis. Brief. Biofinrom. 20, 1685–1698 (2019).
Bae, W. Y. et al. Antimicrobial effects of three herbs (Brassica juncea, Forsythia suspensa, and Inula britannica) on membrane permeability and apoptosis in Salmonella. J. Appl. Microbiol. 130, 394–404 (2021).
Corrêa, J. L. et al. Propolis extract has bioactivity on the wall and cell membrane of Candida albicans. J. Ethnopharmacol. 256, 112791 (2020).
Funding
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through (High Value-added Food Technology Development Program), funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant Number 121026-3).
Author information
Authors and Affiliations
Contributions
W.Y.B., S.L.S., and T.R.K. contributed conceptualization of investigation. W.Y.B., Y.L., and W.H.J. analyzed and perfomed experiments. S.L.S., M.S., and T.R.K. contributed data validation and curation. T.R.K. and M.S. supervised the works. W.Y.B. wrote original draft and all authors reviewed and revised manuscript before submission.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Bae, WY., Lee, Y.J., Jung, WH. et al. Draft genome sequence and probiotic functional property analysis of Lactobacillus gasseri LM1065 for food industry applications. Sci Rep 13, 12212 (2023). https://doi.org/10.1038/s41598-023-39454-2
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-023-39454-2
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
