Phylogenetic and Comparative Genomic Analysis of Lactobacillus fermentum Strains and the Key Genes Related to Their Intestinal Anti-inflammatory Effects

Yan Zhao , Chengcheng Zhang , Leilei Yu , Fengwei Tian , Jianxin Zhao , Hao Zhang , Wei Chen , Qixiao Zhai

Engineering ›› 2022, Vol. 17 ›› Issue (10) : 170 -182.

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Engineering ›› 2022, Vol. 17 ›› Issue (10) :170 -182. DOI: 10.1016/j.eng.2020.09.016
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Phylogenetic and Comparative Genomic Analysis of Lactobacillus fermentum Strains and the Key Genes Related to Their Intestinal Anti-inflammatory Effects
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Abstract

Highlights

•Pan-genomic and phylogenetic characterizations of 109 L. fermentum strains were performed.

•No co-evolutionary relationship exists between L. fermentum strains and the geographical origins of their host Immunomodulatory properties of L. fermentum were strain-dependent.

•Some specific genes may account for the anti-inflammatory and immunoregulation of L. fermentum strains.

Emerging evidence shows that some Lactobacillus fermentum strains can contribute to the prevention and treatment of ulcerative colitis (UC). In this study, 105 isolates of L. fermentum strains were separated from fecal samples of populations in different regions in China and their draft genomes were sequenced. Pan-genomic and phylogenetic characterizations of these strains and four model strains (L. fermentum 3872, CECT5716, IFO3956, and VRI003) were performed. Phylogenetic analysis indicated that there was no significant adaptive evolution between the genomes of L. fermentum strains and the geographical location, sex, ethnicity, and age of the hosts. Three L. fermentum strains (FWXBH115, FGDLZR121, and FXJCJ61) from different branches of the phylogenetic tree and strain type L. fermentum CECT5716 were selected and their anti-inflammatory and immune modulatory activities in a dextran sulphate sodium (DSS)-induced colitis mouse model were further investigated. Both L. fermentum FXJCJ61 and CECT5716 significantly alleviated UC by reducing all colitis-associated histological indices, maintaining mucosal integrity, and stimulating replenishment of short-chain fatty acids (SCFAs), while the other two strains failed to offer similar protection. The anti-inflammatory mechanisms of L. fermentum FXJCJ61 and CECT5716 were related to the inhibition of nuclear factor kappa-B (NF-κB) signaling pathway activation and enhancement of interleukin 10 (IL-10) production. Comparative genomic analysis of these strains identified candidate genes that may contribute to the anti-inflammatory effects of specific L. fermentum strains.

Keywords

Lactobacillus fermentum / Ulcerative colitis / Anti-inflammation / Phylogenetic analysis / Comparative genomic analysis

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Yan Zhao, Chengcheng Zhang, Leilei Yu, Fengwei Tian, Jianxin Zhao, Hao Zhang, Wei Chen, Qixiao Zhai. Phylogenetic and Comparative Genomic Analysis of Lactobacillus fermentum Strains and the Key Genes Related to Their Intestinal Anti-inflammatory Effects. Engineering, 2022, 17(10): 170-182 DOI:10.1016/j.eng.2020.09.016

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1. Introduction

Lactobacillus fermentum (L. fermentum) is a small, slender, nonmotile, Gram-positive bacterium in the genus Lactobacillus. It is pervasive in environments such as fermented vegetable feed and food, milk, cheese, artisanal starter cultures, human saliva, the human vagina and the intestinal tract of humans and animals[1–2]. The genome of L. fermentum (IFO3956) was first sequenced in 2008 and genomes of 65 L. fermentum strains can now be obtained from the National Coalition Building Institute (NCBI) microbial genome database↑ [3]. The genome size and G + C content of L. fermentum were (2.1 ± 0.1) Mbp and 51% ± 1%, respectively. With the advent of genome-wide sequencing, a series of genes related to the production of functional molecules (bile salt [4], exopolysaccharides [5], short-chain fatty acid (SCFAs) [6] and so on) in the genome of L. fermentum strains had been identified.

The number of genome-sequenced L. fermentum strains is relatively small when compared with some well-documented Lactobacillus strains such as L. plantarum (467), L. rhamnosus (178) and L. paracasei (178). Phylogenetic and comparative genomic analyses indicate that extensive gene loss and lateral gene transfer has occurred during the co-evolution of Lactobacillus spp. with their habitats [7]. Certain genetic variations in Lactobacillus were predicted to provide the ability to adapt to diverse niches, and efforts were made to explore the links between genome evolution and ecological versatility [8]. Genes involved in sugar metabolism, the proteolytic system and bile-salt hydrolysis were shown to be responsible for the specific habitats to which Lactobacillus strains adapt, such as to the human gut, or dairy products [9].

Until now, studies have been predominantly focused on L. plantarum [10], L. reuteri[11], L. rhamnosus [12] and L. casei[13], and current evidence suggests that these species have co-evolved with their habitats, such as host species [14]. To our knowledge, however, studies focused on the evolutionary characteristics of L. fermentum strains are very limited. A multilocus sequence typing analysis of 203 isolates of L. fermentum, based on the sequences of 11 house-keeping gene fragments, indicated a similar evolutionary tendency in L. fermentum isolates from food sources from the same locations[15]. Typically, L. fermentum is found in fermentation products, but it is also a general symbiont in the human gut [16]. The effects of the geographic location, sex, ethnicity and age of the hosts on the evolution of L. fermentum strains need further study.

An increasing number of studies have indicated that L. fermentum can have a positive effect on the health of its host[17,18]. It has been shown to antagonize pathogenic bacteria [19] and alleviate alcoholic liver disease [20], cardiovascular disease [21] and colitis [22]. L. fermentum was listed as a ‘‘generally recognized as safe” (GRAS) organism by the US Food and Drug Administration (FDA) in 2013. Experimental evidence from animal models has demonstrated L. fermentum plays a crucial role in the amelioration of ulcerative colitis (UC) by affecting the production of proinflammatory cytokines and inhibiting activation of nuclear factor kappa-B (NF-κB) [23]. L. fermentum CECT5716 was shown to trigger recovery of normal concentrations of SCFAs in the intestinal contents and reverse microbiota dysbiosis in mice with dextran sulphate sodium (DSS)-induced colitis [24]. In contrast, another study reported that pathogenic L. fermentum was isolated from the chole-cystostomy aspirate and anaerobic blood culture of an 81-year-old male patient with cholecystitis [25]. Anderson et al. [26] also indicated that human oral isolate L. fermentum AGR1487 can cause a pro-inflammatory response in germ-free rats by increasing inflammatory cells (macrophages, lymphocytes and neutrophils), inducing colonic myeloperoxidase and plasma serum amyloid A and activating Toll-like receptor signaling. These studies indicated that the anti-inflammatory effects of L. fermentum were strain-specific. A Bacteroides thetaiotaomicron mutant lacking a choloylglycine hydrolase gene (BT2086) responsible for bile salt hydrolase (BSH) activity could lower liver and plasma lipid levels in mice [27] and a Clostridium sporogenes mutant lacking genes for the synthesis of branched SCFAs could up-regulate IgA-related immune cells in vivo [28]. Other research has suggested that the bsh gene (coding for bile salt hydrolase) is responsible for the cholesterol-lowering activities of L. fermentum [29]. The pdu–cbi–cob (pdu, propanediol dehydratase genes; cbi, cobinamide biosynthetic genes; cob, cobalamin biosynthetic genes) gene cluster was found to encode for reuterin, and cobalamin was shown to contribute to the antibacterial properties of L. reuteri JCM 1112 [3]. As these reports confirmed the tight association between the function of Lactobacillus spp. and some of its genes, the genes crucial to the activity of L. fermentum strains against inflammatory disease need to be further studied.       

In this work, 105 L. fermentum strains were isolated from fecal samples of human subjects in China, and the draft genome sequences of these strains were obtained. The aim of the study was to evaluate whether genomic distinctions occurred in L. fermentum strains in populations from different geographic regions and having different physiological characteristics, to reveal the effects of L. fermentum strains from different branches of the phylogenetic tree on DSS-induced colitis in mice, and to identify the functional genes potentially responsible for the various levels of anti-inflammatory protection offered by these strains. 

2. Materials and methods

2.1. Chemicals and reagents

DSS (36–50 kDa; MP Biomedicals, USA), Fast DNA spin kit for feces (MP Biomedicals, USA), TRIzol reagent (Invitrogen, USA), QIAquick gel extraction kit (QIAGEN, Germany), BCA protein assay kit (Beyotime Biotechnology, China), RIPA lysis buffer (Beyotime Biotechnology, China), ultrapure RNA kit (CWBIO, China), protease inhibitor cocktail and phosphatase inhibitor cocktail I (MedChemExpress, USA), RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific, USA), iTaqTM universal SYBR® green supermix (Bio-Rad, USA), ELISA kits for interleukin (IL)-10, IL-6, IL-4, IL-1β and tumor necrosis factor (TNF)-alpha (R&D Systems, USA), antiNF-κB p65 and anti-NF-κB p65 (Abcam, UK), goat anti-rabbit IgG antibody (GenScript, China), goat anti-mouse IgG antibody (H&L) (GenScript, China) and β-Actin Antibody (GenScript, China), ELISA kit for lactate (MLBio, China).

2.2. L. fermentum isolates and growth conditions

L. fermentum strains were isolated by a modified LAMVAB (Lactobacillus anaerobic Man–Rogosa–Sharpe (MRS) with vancomycin and bromocresol green) medium [30] developed by our laboratory (Table S1 in Appendix A). A total of 105 strains were separated from the feces of populations from 11 provinces (Anhui, Fujian, Hunan, Gansu, Guangdong, Guangxi, Jiangsu, Jiangxi, Qinghai, Shandong, Sichuan), Xinjiang autonomous region, Ningxia autonomous region, Inner Mongolia autonomous region, Shanghai municipality and Chongqing municipality of China (Appendix A Table S2). For in vitro experiments, L. fermentum strains were incubated at 37 °C on MRS agar for 18–24 h. For animal experiments, final concentrations of L. fermentum strains (5 ×109 colony-forming unit per milliliter (CFU·mL–1 )) suspended in saline solution were adjusted and the gavage volume dosed to each mouse was 100 μL. 

2.3. Comparative genome analysis and phylogenetic analysis of L. fermentum

Genomes of 109 L. fermentum strains (four from NCBI and 105 sequenced in this work) were subject to pan-genome analysis using PGAP v1.2.1 and average nucleotide identity (ANI) analysis using Python. Protein coding sequences (CDS) were predicted using Glimmer v3.02. Orthologous genes were generated using OrthoMCL v1.4 and a maximum-likelihood tree was constructed based on core genes of all L. fermentum strains and 11 housekeeping genes [15] using MAFFT v7.313. For the identification of differences in the functional genes of strains, genomes were subject to BLAST against annotated full-length CAZyme proteins in the Carbohydrate-Active Enzyme (CAZy) database and proteins in the Clusters of Orthologous Groups (COG) protein database [31]. 

2.4. Animals and experiment design

All procedures involving animals were approved by the Ethics Committee of Jiangnan University, China (JN. No. 20180615b0950901(164)). BALB/c mice (male, eight weeks old, Shanghai SLAC Laboratory Animal Co., Ltd., China) were used for our in vitro experiment and each group comprised 10 mice. The mice were allowed to acclimate for one week before the experiment. Normal drinking water was given to mice in the normal group and water containing 4% DSS (w/v) was given to mice for 10 consecutive days in the other groups in the experiment. In the normal group and DSS group, each mouse was orally administered 100 μL saline solution per day. In the four DSS + L. fermentum groups, mice received 100 μL L. fermentum suspension (a concentration of 5 ×109 CFU·mL–1 ) via oral gavage per day [32].

During the treatment, body weight and bloody stools were assessed every day. At the end of the experiment, mice were intraperitoneally injected with 1% pentobarbital sodium solution and euthanized [33]. The length of colons was recorded, with part of the colon used for histological observation and the remaining part stored at –80 °C. The disease activity index (DAI) of mice was evaluated according to a previous study [34].

2.5. Histological assessment

The histological assessment of distal sections of the colon was performed based on hematoxylin–eosin (H&E) staining, as previously reported [35]. The histological scores were assessed based on inflammatory infiltrates, loss of goblet cells and mucosal hyperplasia by two researchers who were blinded to the details of each section.

2.6. ELISA and immunoblotting

The concentrations of IL-4, IL-6, IL-1β, TNF-α and IL-10 in the colons of mice were measured using ELISA kits (R&D Systems, USA). The expression of total p65 and phosphorylated p65 (p-p65) in the colon of mice was determined by western blot assay, as previously described [36]. The protein expression was visualized using AlphaView software v3.4.0.0.

2.7. Gene expression of tight junction protein in the colon of mice

Total RNA in the colon of mice was extracted using an Ultrapure RNA Kit (CWBIO, China) and complementary DNA was synthesized using a RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific, USA). Gene expression concentrations of occludin, claudin-1, zonula occludens (ZO)-1 and ZO-2 were evaluated by real-time quantitative polymerase chain reaction (qPCR) [37]. Information for primer sequences is shown in Table 1.

2.8. SCAFs analyses

Concentrations of SCFAs (acetic acid, propionic acid, pentanoic acid, butyric acid and isobutyric acid) in the colonic contents were determined by gas chromatography-coupled mass spectrometry (GC-MS), as previously described [38].

2.9. Gut microbiome of mice

The DNA of bacteria in fecal samples was extracted with a fast DNA spin kit for feces (MP Biomedicals, USA) and amplification of the 16S ribosomal RNA (rRNA) gene sequences (V3–V4 regions) was performed as previously reported [39]. The DNA amplicons were sequenced by Illumina MiSeq platform and clustered into operational taxonomic units (OTUs) with 97% similarity using UCLUST v11.

2.10. Species-specific qPCR

To examine the total amount of L. fermentum strains in fecal samples from mice, a species-specific qPCR method was used [40]. A standard curve was generally used to react the interactions of cycling threshold (Ct) value and cell counts of L. fermentum. A total of 0.04 g fecal sample of each mice was weighted and fecal DNA was extracted as mentioned in Section 2.9 [41].

Table 1 Primers used for qPCR.

2.11. Resistance of L. fermentum strains to gastrointestinal environment

The tolerance of L. fermentum strains to acid and bile salt was studied [42]. Bacterial culture solution was harvested (6000 revolutions per minute (rpm), 2 min) and washed with sterile saline solution. Cell pellets were exposed to simulated gastric juice (3 g·L–1 pepsin in sterile saline, pH 3.0) for 3 h and subsequently to simulated small intestinal juice (trypsin (1 g·L–1 ) and bile salts (3 g·L–1 ) in sterile saline, pH 8.0) for 4 h. The viable bacterial count was determined at 0, 3, and 7 h.

2.12. Lactate production by L. fermentum in vitro

Quantification of lactate production was determined as described [43]. L. fermentum strains were pre-cultivated twice using MRS broth at 37 °C for 48 h. The concentration of lactate in the supernatant was quantitatively determined by the ELISA kit for lactate (MLBio, China).

2.13. Statistics

All data were described as mean ± standard error of mean (SEM). Difference analyses of DAI values and body weight of mice were carried out using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The difference analysis of other biomarkers was analyzed using one-way ANOVA followed by Tukey’s test (P < 0.05, P < 0.01, and P < 0.001). Statistical analyses in this study were performed using the GraphPad Prism v6.0.

3. Results

3.1. Genetic diversity and evolution of L. fermentum strains

The genomic information and detailed sources of 105 L. fermentum strains isolated from human fecal samples of healthy Chinese subjects (41 male, 62 female, 2 unknown; aged 0–100 years) were provided in Fig. 1(a) and Appendix A Table S2. Taking the four additional type strains (3872, CECT5716, IFO3956 and VRI003) into consideration, the genome size of L. fermentum was approximately 2.0 Mbp (G + C content ranged from 50.56% to 52.50%). Pangenome analysis reveals 11 579 gene families corresponding to 109 L. fermentum genomes and the pan-genome curve increased sharply as the number of genomes increases. Conversely, the number of core genes decreased gradually as the number of genomes increased, reaching 1179 for 109 genomes (Fig. 1(b)). The ANI values of whole genomes between homologous regions shared by any two genomes are generally greater than 97% (Appendix A Fig. S1). 

The 109 L. fermentum strains shared 1303 orthologous genes (Fig. 1(c)). Phylogenetic analysis of L. fermentum strains based on 11 housekeeping genes was annotated with different shapes and colors representing several factors, including the geographical location, sex, ethnicity and age. L. fermentum strains with common characteristics did not cluster together and this revealed that no direct relationships existed between the genomes of L. fermentum strains and the geographical location, sex, ethnicity and age of the hosts (Figs. 2(a) and (b)). Phylogenetic analysis based on 1303 core genes of the 109 strains indicated that while all L. fermentum isolates were divided into three distinct branches, no single selective pressure factor significantly affected the evolution of L. fermentum strains (Fig. 2(c)). Three L. fermentum strains (FWXBH115, FGDLZR121, and FXJCJ61, abbreviated as WX115, GD121 and XC61 respectively in Figs. 1 and 2) from different branches and the type strain L. fermentum CECT5716 (marked with black maple leaf in Fig. 2(c)) were selected to study their effects on DSSinduced colitis in BALB/c mice.

Fig. 1. The genomic and genetic characteristics of L. fermentum strains isolated from fecal samples of Chinese populations: (a) number of strains isolated from different provinces or municipalities; (b) pan-genome and core genome (the curve represents the pan-genome and core-genome plotted against the number of genomes of L. fermentum); and (c) Venn diagram of the homologous clusters shared among the core genes (the number in the outer ring means counts of specific genes in each strain).

Fig. 2. Phylogenetic analysis of 109 L. fermentum strains: (a) maximum likelihood tree based on 11 housekeeping genes (each label annotated with various shapes and colors corresponds to certain characteristics; bootstrap confidence values were marked in the branches of the tree; I, II, and III represent three separate phylogenetic clades); (b) information on 11 housekeeping genes; and (c) maximum likelihood phylogeny derived from 1303 core genes across 109 strains.

3.2. Effects of L. fermentum supplementation on body weight, DAI value, and colonic histopathology of DSS-treated mice

Symptoms such as loss of body weight (Fig. 3(a)), apparent diarrhea and rectal bleeding were induced by DSS treatment. DSS treatment also significantly raised the DAI scores (Fig. 3(b)) and shortened the colon (Fig. 3(c)). Oral gavage with L. fermentum FXJCJ61 and the type strain L. fermentum CECT5716 significantly alleviated these symptoms. And compared with L. fermentum CECT5716, L. fermentum FWXBH115 and FGDLZR121 had little effect on DSS-induced symptoms in mice.

A decreased number of crypts and marked infiltration of inflammatory cells were observed in the DSS group (Fig. 3(e)). Treatment with L. fermentum FXJCJ61 and the type train L. fermentum CECT5716 significantly alleviated these pathological damages in DSS-treated mice, while the other two strains failed to offer similar protection (Fig. 3(f)).

Fig. 3. Effects of L. fermentum supplementation on DSS-treated mice (n = 8): (a) weight changes of mice during the experiment; (b) disease activity index (DAI) of mice during the experiment (DAI was evaluated based on body-weight changes, rectal bleeding and diarrhea); (c) colon length of mice; (d) representative images of the colons; (e) pathological images of the distal colon ((i) normal; (ii) DSS-treated; (iii) L. fermentum FWXBH115-treated; (iv) FGDLZR121-treated; (v) FXJCJ61-treated; and (vi) CECT5716- treated (the images are shown at 20 ×magnification and scale bars are 500 μm)); and (f) histological scores of the colon. All data are expressed as the mean ± SEM. * and # indicate significant differences when compared with normal or DSS groups, respectively (*P < 0.05, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001).

3.3. Effects of L. fermentum supplementation on inflammatory cytokines and NF-κB signaling in colon of DSS-treated mice 

Compared with the DSS group, L. fermentum FXJCJ61 and CECT5716 supplementation significantly reduced the colonic concentration of TNF- α but did not affect the concentrations of IL6, IL-4, and IL-1β (Fig. 4). Unlike the type strain L. fermentum CECT5716, L. fermentum FWXBH115 and FGDLZR121 had no regulatory effect on TNF- α and IL-1 β but increased the colonic concentration of IL-6 (Fig. 4). Oral administration of all of the four L. fermentum strains triggered rapid upregulation in the expression of anti-inflammatory cytokines, such as IL-10, compared with the DSS group (Fig. 4(d)).

The expression of p-p65 in colon tissue increased significantly after treatment with DSS when compared with the normal group. Compared with the DSS group, L. fermentum FXJCJ61 and the type strain L. fermentum CECT5716 markedly decreased the level of p-p65 in the colon of DSS-treated mice. However, L. fermentum FWXBH115 and L. fermentum FGDLZR121 did not have an obvious effect on the expression of p-p65 protein in DSS-treated mice (Figs. 4(f) and (h)). 

Fig. 4. Effect of L. fermentum supplementation on cytokines (n = 8) and NF-κB signaling in colon of colitis mice: the concentration of cytokines (a) TNF- α; (b) IL-6; (c) IL-1 β; (d) IL-10; (e) IL-4; (f) detection of p65 and p-p65 by western blot analysis; (g) expression of p65; and (h) expression of p-p65. Densitometric quantification normalized to actin was calculated as change from the normal group; OD: optical density. For box and whiskers plot in (a)–(e), the middle line represents the median; the boxes represent the interquartile range (IQR); the error bars represent min to max values. All data in (g) and (h) are expressed as the mean ± SEM. * and # indicate significant differences when compared with the normal or DSS-treated group, respectively (*P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001).

3.4. Effect of L. fermentum supplementation on the concentrations of intestinal SCFAs in DSS-treated mice

The effect of L. fermentum supplementation on SCFA concentrations in DSS-induced colitis mice was shown in Fig. 5. DSS treatment significantly reduced the production of propionic acid and pentanoic acid compared with the normal group. All groups of L. fermentum strains significantly increased (P < 0.05) the concentrations of acetic acid and propionic acid compared with the DSS group (Figs. 5(a) and (b)). Besides, L. fermentum FXJCJ61, the strain with the most similar regulatory effects to the type strain L. fermentum CECT5716, was the most effective orally administered strain for improving the concentrations of pentanoic acid (Fig. 5(c)) and butyric acid (Fig. 5(d)). 

Fig. 5. Effect of L. fermentum supplementation on the intestinal concentrations of SCFAs (a) acetic acid, (b) propionic acid, (c) pentanoic acid, (d) butyric acid, and (e) isobutyric acid in DSS-treated mice (n = 8). For boxes and whiskers, the middle line represents the median; the boxes represent the interquartile range (IQR); the error bars represent min to max values. * and # indicate significant differences when compared with the normal or DSS-treated group, respectively (*P < 0.05, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001).

3.5. Effect of L. fermentum supplementation on mRNA expression of occludin, claudin-1, ZO-1, and ZO-2 in DSS-treated mice

Compared with the DSS group, L. fermentum FXJCJ61 and the type strain L. fermentum CECT5716 enabled substantial recovery of the expression of occludin, claudin-1 and ZO-1 in DSS-treated mice. In contrast, L. fermentum FWXBH115 and L. fermentum FGDLZR121 had no significant effects on the expression of ZO-1 and claudin-1 in the colon of DSS-treated mice. Oral administration of L. fermentum FXJCJ61 increased the expression of claudin-1 and ZO-1 in the colon of DSS-induced mice more than L. fermentum CECT5716. No L. fermentum strains had significant effects on the expression of ZO-2 (Fig. 6).

Fig. 6. Effect of L. fermentum supplementation on mRNA expression of four tight junction proteins (a) ZO-1, (b) ZO-2, (c) claudin-1, and (d) occludin in the colon of DSStreated mice (n = 8). All data are expressed as the mean ± SEM. * and # indicate significant differences when compared with the normal or DSS-treated group, respectively (*P < 0.05, ***P < 0.001, #P < 0.05, ##P < 0.01 and ###P < 0.001).

3.6. Effects of L. fermentum on fecal microbiota in DSS-treated mice

The Shannon index indicated that alpha diversity for the DSS and L. fermentum groups increased significantly compared with the normal group (Fig. 7(a)). Though there was no obvious separation trend among normal, DSS-treated and L. fermentum supplementation groups in the principal component analysis (PCA) plot, permutational multivariate analysis of variance (PERMANOVA) and pairwise comparison results showed that treatment with L. fermentum FWXBH115, FXJCJ61 and CECT5716 changed the structure of the gut microbiota compared with the DSS group (Fig. 7(b) and Appendix A Table S3). Linear discriminant analysis effect size (LEfSe) analysis and a column diagram show that there are 33 dominant operational taxonomic units (OTUs) from the six groups (Fig. 7(c)). As can be seen, at genus level, L. fermentum FXJCJ61 treatment markedly increased the level of Lactobacillus and Bacteroides compared with DSS groups (Fig. 7(d)).

Fig. 7. Effect of L. fermentum supplementation on the gut microbiota composition of DSS-treated mice (n = 8): (a) Shannon diversity index; (b) PCA for gut microbiota; (c) column diagram of microbial composition (Lefse analysis); and (d) effects of L. fermentum on the structure of fecal microbiota at genus level (Bacteroides, Lactobacillus) in DSS-treated mice. * indicates a significant difference (*P < 0.05, **P < 0.01 and ***P < 0.001); ns: not significant.

3.7. Comparative genomic analysis of the specific genes in the four L. fermentum strains

In Fig. 8(a), the number of genes shared between the four L. fermentum isolates ranges from 1635/3160 (51.74%) to 1747/3160 (55.28%), and 1497 core genes are shared in the genome of these strains. The genomes of the four L. fermentum CECT5716 strains displayed an average of 2 055 461 bp and the G + C content is 51.72% (Fig. 8(b)). The L. fermentum genomes were predicted by comparison with a protein database (COG), and the results showed that 11 COG families were present only in a subset of the examined L. fermentum FXJCJ61 and CECT5716 genomes and 17 additional COGs were unique to a single strain of L. fermentum FXJCJ61 (Fig. 8(c)–(e) and 8 Appendix A Table S4). Except for an average of 182 genes assigned to the COG ‘‘general function prediction only”, the genes were predicted to be mainly associated with amino acid metabolism, translation, replication and repair. Based on carbohydrate active enzymes database, our research showed that relatively large concentrations of glycoside hydrolases (GH2, GH25, GH43, GH78) and glycosyl transferases (GT14, GT83) existed in L. fermentum FXJCJ61 (Appendix A Fig. S2).   

Fig. 8. Comparative genomic analyses of L. fermentum: (a) Venn diagram of core, distributed and unique gene numbers between FWXBH115, FGDLZR121, FXJCJ61, and CECT5716; (b) genomic information of FWXBH115, FGDLZR121, FXJCJ61, and CECT5716 (CDS: coding sequence; rRNA: ribosomal RNA; tRNA: transfer RNA; CRISPR: clustered regularly interspaced short palindromic repeats); (c) functional annotation based on Clusters of Orthologous Groups (COG) database; (d) functional annotation of FWXBH115, FGDLZR121, FXJCJ61, and CECT5716 based on Clusters of Orthologous Groups (COG) database (number of genes associated with COG functional categories for all three sequenced strains and CECT5716 is shown); and (e) COG categories that are contained only in FXJCJ61 and CECT5716.

3.8. Detection of L. fermentum in fecal samples in mice

The detection limit of L. fermentum was considered to be 104 CFU·mL–1 , as estimated from the standard curve (Appendix A Fig. S3). In the DSS, normal and L. fermentum FWXBH115 groups, L. fermentum strains were not detected. L. fermentum strains were detected in the FXJCJ61, FGDLZR121, and CECT5716 groups. And the concentration of L. fermentum strains in feces of mice in the FXJCJ61-treated group was significantly greater compared with all other groups (Table 2).

Table 2 Qualitative detection of L. fermentum in fecal samples in mice (n = 5).

a Values below detection limit are denoted as < 6.3 and other data are described as mean ± SEM.

3.9. Survival of L. fermentum in simulated gastric juices and simulated small intestinal juices

L. fermentum FWXBH115, FGDLZR121, FXJCJ61, and CECT5716 were successively exposed to simulated gastric juices and simulated small intestinal juices. The viable cell count was measured by plate counting and the survival rate of the strains was calculated by comparison of the initial counts with those after exposure to simulated gastric juices and simulated small intestinal juices. The data in Table 3 show that all L. fermentum strains were tolerant to simulated gastric juices (survival rate was greater than 85%) and L. fermentum FXJCJ61 had the highest survival rate of 98.33% ± 3.51%. In the simulated small intestinal juices, L. fermentum FXJCJ61 also showed significantly higher tolerance to 3 g·L–1 bile salts compared with the other strains. The survival rate of FGDLZR121 in simulated small intestinal juices was significantly higher than L. fermentum CECT5716 (Table 3).

Table 3 Survival rate of L. fermentum strains under simulated gastric juices and simulated small intestinal juicesa .

a All data are described as mean ± SEM. The superscript letters b, c and d indicate statistically significant differences at P < 0.05 among the different groups (one way ANOVA and Tukey’s test). Duplicate samples were included for each treatment and the experiment was repeated three times.

3.10. Quantification of lactate production by L. fermentum in vitro

The production of lactate by four L. fermentum strains was determined in vitro and L. fermentum FWXBH115 showed the least ability to produce lactate (Fig. 9). Compared with L. fermentum FWXBH115, L. fermentum FGDLZR121 and CECT5716 produced higher concentrations of lactate (> 65 μg·L–1 MRS). The highest concentration of lactate was 75 μg·L–1 MRS, which was fermented by L. fermentum FXJCJ61.

Fig. 9. Quantification of lactate production by L. fermentum in vitro. The superscript letters a, b and c indicate statistically significant differences at P values less than 0.05 among the different groups (one way ANOVA and Tukey’s test). Duplicate samples were included for each treatment and the experiment was repeated three times. All data are described as mean ± SEM.

4. Discussion

In this study, we analyzed the population genetic structure and phylogeny of 109 L. fermentum strains (105 isolated from fecal samples of the Chinese population, and four reference strains downloaded from NCBI), based on both 1303 core genes and 11 housekeeping gene fragments. Phylogenetic analyses showed that the evolution of L. fermentum lineages was not associated with geographical location, sex, age or ethnicity of fecal donors. The unequal sample numbers from different geographical location may not be the cause of the deviation in our results, since the samples from a same region (like Jiangsu, Guangdong or Xinjiang) showed no obvious clustering trend in the phylogenetic tree. A previous population genetic structure and phylogeny analysis of L. reuteri strains by Oh et al. [14] indicated that evolution of L. reuteri lineages was adaptive for the different host species, although the sample numbers from different host were unequal (humans(n = 35), mice (n = 35), rats (n = 26), pigs (n = 41), chickens (n = 26), and turkeys (n = 5)). This was consistent with a previous report indicating that L. fermentum strains isolated from food sources did not exhibit a co-evolutionary trend with geographical location or type of food [15]. Previous studies showed that only a small number of gut microbiomes (such as those of Helicobacter pylori, L. reuteri, L. johnsonii and L. acidophilus) evolved in parallel with their hosts, while others (e.g., L. plantarum, L. casei, and L. rhamnosus) did not[44,45].

Our results showed that all these isolates were clearly categorizable into three sub-types. Phylogenetic and functional analyses previously revealed that Lactobacillus species belonging to separate phylogenetic clades exhibited distinct types of metabolism, ecological niches and lifestyles [46]. Genetical difference has been shown to be associated with certain characteristics of a strain and to result in diverse phenotypes [31]. For example, phylogenetic analysis of 39 Akkermansia muciniphila strains derived from human and other mammalian gut microbiotas showed that the genomes were assignable to three phylogroups, and two of these phylogroups were associated with obesity in their hosts [47]. Ottman et al. [48] elucidated that the protein Amuc_1100 of A. muciniphila annotated as a hypothetical protein was an outer membrane pililike protein, which could regulate the immune and metabolic responses by inducing the production of IL-8, IL-1 β, IL-6, and IL10 through activation of Toll-like receptor (TLR) 2 and TLR4.

In this study, L. fermentum FWXBH115, FGDLZR121, and FXJCJ61 were distributed in different evolutionary branches by phylogenetic analysis derived from 1303 core genes and were randomly selected for further study in a mouse model of DSS-induced colitis. Oral administration of L. fermentum FXJCJ61 and CECT5716 showed a protective effect against DSS-induced intestinal inflammation in mice, in the form of reduced disease activity scores, alleviated histopathology symptoms (Fig. 3), regulated relevant inflammatory factors and inhibition of the expression of NF-κB (Fig. 4). Interestingly, L. fermentum FXJCJ61 and CECT5716 belonging to the same evolutionary branch in the cladogram based on core genes had similar protective effects against DSS-induced colitis, but these two strains did not cluster in the maximum likelihood tree based on 11 housekeeping genes (Fig. 2). This may be due to the more comprehensive and representative phylogenetic analysis based on the whole core genes.

It has been pointed out that the reduction of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-4) in mice with colitis could be a logical target for UC therapy [49]. To this end, L. fermentum FXJCJ61 and CECT5716 greatly decreased the concentrations of proinflammatory cytokines (TNF-α) and increased the concentrations of anti-inflammatory cytokines (IL-10) (Fig. 4). T-cellderived cytokines, such as interferon gamma (IFN-) and TNF-α,  were found to upregulate intestinal epithelial TLR4 expression, mediate epithelial cell death and correlate to immunopathology in inflammatory bowel disease (IBD) colitis [50]. Another report indicated that the downregulation of the translocation and transcriptional activity of the nuclear factor of activated T cells could block the production of TNF-α, which could be a therapeutic approach for treating bowel diseases [51]. In addition, IL-10 produced by T cells, dendritic cells, macrophages and B cells has been shown to suppress the expression of TNF-α in immune regulatory processes [52]. It has also been demonstrated that persistent NF-κB activation in epithelial cells can contribute to the development of inflammation and TNF-α could activated the NF-κB pathway[53]. This may suggest that these strains could also play a potential role against a range of inflammatory diseases. 

In our work, L. fermentum FXJCJ61 and CECT5716-treated groups displayed downregulation of the expression of NF-κB (Fig. 4), which may partly explain the regulatory mechanisms of these strains in relieving DSS-induced colitis. Taking all of the above mentioned in vivo biological indicators into consideration, L. fermentum FXJCJ61 and CECT5716 could more significantly reduce DSS-induced colitis than the other tested strains. These findings were instrumental for the clinical application of these strains in the future. Our findings showed the potential of the clinical translation of L. fermentum FXJCJ61 in the treatment of colitis.

Comparative genomic analysis has become a routine method with which to explore specific functions of genes, such as those coding for transporter proteins and catabolic enzymes of bacteria [54]. A comparative genomic analysis of 43 uropathogenic Escherichia coli strains isolated from a cohort of 14 women with urinary tract infections revealed that strains from distinct clades of the phylogenetic tree possessed different pathogenicities [55].

In our work, we compared the genomes of L. fermentum FXJCJ61 and CECT5716, two strains with significant anti-inflammatory abilities, with those of two inactive strains, L. fermentum FWXBH115 and FGDLZR121. As shown in Fig. 8, there are 11 specific genes existing only in the genomes of L. fermentum FXJCJ61 and CECT5716. These specific genes were characterized and assigned to specific pathways. First, three genes associated with the production of SCFAs and lactate were analyzed. Beta-xylosidase (COG3507) is the main enzyme for the production of monomeric xylose and Yang et al. [56] showed that fecal SCFA production was increased in mice fed with fiber xylooligosaccharide. Alcohol dehydrogenase (COG1454) and aldehyde dehydrogenase (ALDH) are involved in the metabolism of alcohol to acetic acid [57]. Supporting these analyses, our work suggests that L. fermentum FXJCJ61 and CECT5716 can effectively trigger the recovery of normal concentrations of SCFAs in the colon of mice (Fig. 5). Since the SCFAs may also play a pivotal role in regulating host metabolism and reduce the risk of cancer and diabetes[58,59], L. fermentum FXJCJ61 and CECT5716 were expected to exhibit protective effects against these diseases. 

Mechanistically, the SCFAs produced by bacterial fermentation were shown to regulate intestinal inflammation by increasing cluster of differentiation 4 + (CD4+ ) T cell frequency and number and IL10 expression [60]. Butyrate, acetate and propionate have been shown to be able to protect the intestinal epithelium from inflammatory damage by increasing tight junctions and reducing intestinal epithelial permeability[61–63]. In our experiments, both L. fermentum FXJCJ61 and CECT5716 increased the expression of occludin, claudin-1 and ZO-1 mRNA compared with the levels in DSS-induced colitis mice, which might correlate with the ability of these strains to increase SCFAs production (Fig. 6). Whether the L. fermentum strains can also increase the expression of tight junction protein in normal mice was unknown and need to be evaluated in the future study. Tight junction proteins in the gut were also associated with irritable bowel syndrome (IBS) and arthritis and this indicated that these strains may have a potential clinical application prospect in the future[64,65].

Candidate genes of malate/L-lactate dehydrogenases (COG2055) have been shown to play a vital role in lactate synthesis and generating the lactate secreted from lactic-acid-producing bacteria (LAB). Lactate was reported to have a protective effect in response to gut injury [66] and it is also one of the precursors in the production of acetate, propionate or butyrate. Moreover, lactate can regulate the proinflammatory cytokines produced by intestinal epithelial cells through TLR-4 and TLR-5 [67]. Our in vitro experiment confirmed that L. fermentum FXJCJ61 had a greater ability to produce lactate than other strains (Fig. 9).

Specific genes (denoted COG0553, COG1468, COG2189, and COG3587) belonging to L. fermentum FXJCJ61 and L. fermentum CECT5716 have been found to be responsible for defense mechanisms, replication, and recombination and repair of DNA, and were predicted to enhance cell survival [68]. Furthermore, genes denoted isopropyl-malate/homocitrate/citramalate synthase (COG0119) and 5,10-methylenetetrahydrofolate reductase (COG0685) belonging to L. fermentum FXJCJ61 were found to be involved in amino acid transport and metabolism (Table S4). Additionally, COG0685 may enable the strains to produce methionine, and COG0119 was demonstrated to be involved in the biosynthesis of lysine [69]. Research also showed that L. casei can synthesize cysteine from methionine and the related metabolism may be associated with the resistance of bacteria to bile-salt stress [70]. The production of lysine by gut microbes has been reported to be associated with their microbial competitive capacities in the gut [71]. Enhancement of stress tolerance and good gut fitness are the foremost criteria for probiotics to survive in the host intestinal environment and play a beneficial role in health [72]. Thus, these specific genes may guarantee the anti-inflammatory effect of L. fermentum FXJCJ61 and CECT5716.

Supporting these analyses, qualitative detection of L. fermentum in fecal samples in mice indicated that L. fermentum FXJCJ61 had a greater intestinal colonization ability than the type strain L. fermentum CECT5716 (Table 2). This also correlates with the high tolerance of L. fermentum FXJCJ61 to a simulated gastrointestinal environment (Table 3). 

Utilization of oligosaccharides (such as xylan) by bacteria can contribute to anti-oxidant and immune-modulatory activities in their host, and the ability to utilize more carbon sources was found to be involved in the adaptive ability of the microbiota [73]. Several genes involving the synthesis of specific carbohydrate active enzymes were found in the genome of L. fermentum FXJCJ61 (Fig. S2). Moreover, genes belonging to COG5416 and COG5523 were found only in L. fermentum FXJCJ61 (Table S4), these were responsible for producing integral membrane proteins and their functions need to be further studied.

In conclusion, the present study showed the strain-specific properties of L. fermentum strains selected from different clades of the phylogenetic tree against DSS-induced intestinal inflammation. L. fermentum FXJCJ61 and CECT5716 offered a more significant protective effect than the other two control strains. Inhibition of NF-κB signaling pathways may have contributed to the reduction of inflammatory cytokine secretions and alleviation of disease symptoms in a UC mouse model. Our study further suggested that specific genes existing only in the genome of L. fermentum FXJCJ61 and CECT5716 were responsible for their colonic SCFAs production and tolerance to the gastrointestinal environment. This may account for the protective effect of L. fermentum FXJCJ61 and CECT5716 in a DSS-induced colitis mouse model.

Acknowledgments

This work was supported by the National Natural Science Foundation of China Program (31820103010, 31530056, and 31871773); National Key Research and Development Project (2018YFC1604206); Projects of Innovation and Development Pillar Program for Key Industries in Southern Xinjiang of Xinjiang Production and Construction Corps (2018DB002); National FirstClass Discipline Program of Food Science and Technology (JUFSTR20180102); BBSRC Newton Fund Joint Centre Award (BB/ J004529/1); and Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province.

Compliance with ethics guidelines

Yan Zhao, Chengcheng Zhang, Leilei Yu, Fengwei Tian, Jianxin Zhao, Hao Zhang, Wei Chen, and Qixiao Zhai declare that they have no conflict of interest or financial conflicts to disclose.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.eng.2020.09.016.

References

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[1]

[2]

Archer AC, Halami PMJAM. Probiotic attributes of Lactobacillus fermentum isolated from human feces and dairy products. Appl Microbiol Biotechnol 2015;99(19):8113–23.
[3]

Lin WH, Yu B, Jang SH, Tsen HY. Different probiotic properties for Lactobacillus fermentum strains isolated from swine and poultry. Anaerobe 2007;13(3– 4):107–13.
[4]

Morita H, Toh H, Fukuda S, Horikawa H, Oshima K, Suzuki T, et al. Comparative genome analysis of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic island for reuterin and cobalamin production. DNA Res 2008;15 (3):151–61.
[5]

Jayashree S, Pooja S, Pushpanathan M, Rajendhran J, Gunasekaran P. Identification and characterization of bile salt hydrolase genes from the genome of Lactobacillus fermentum MTCC 8711. Appl Biochem Biotechnol 2014;174(2):855–66.
[6]

Dan T, Fukuda K, Sugai-Bannai M, Takakuwa N, Motoshima H, Urashima T. Characterization and expression analysis of the exopolysaccharide gene cluster in Lactobacillus fermentum TDS030603. Biosci Biotechnol Biochem 2009;73 (12):2656–64.
[7]

Oloyede AR, Albert OM, Arowosegbe OA. Detection and molecular characterization of butyrate-producing genes in probiotic lactic acid bacteria for use in livestock. Niger J Biotechnol 2017;33(1):58–65.
[8]

Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, et al. Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci USA 2006;103(42):15611–6.
[9]

Mendes-Soares H, Suzuki H, Hickey RJ, Forney LJ. Comparative functional genomics of Lactobacillus spp. reveals possible mechanisms for specialization of vaginal lactobacilli to their environment. J Bacteriol 2014;196(7):1458–70.
[10]

O’Sullivan O, O’Callaghan J, Sangrador-Vegas A, Mcauliffe O, Slattery L, Kaleta P, et al. Comparative genomics of lactic acid bacteria reveals a niche-specific gene set. BMC Microbiol 2009;9(1):50.
[11]

Siezen RJ, Tzeneva VA, Castioni A, Wels M, Phan HTK, Rademaker JLW, et al. Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches. Environ Microbiol Rep 2010;12 (3):758–73.
[12]

Zheng J, Zhao X, Lin XB, Ganzle M. Comparative genomics Lactobacillus reuteri from sourdough reveals adaptation of an intestinal symbiont to food fermentations. Sci Rep 2015;5(1):18234.
[13]

Kant R, Rintahaka J, Yu X, Sigvart-Mattila P, Paulin L, Mecklin JP, et al. A comparative pan-genome perspective of niche-adaptable cell-surface protein phenotypes in Lactobacillus rhamnosus. PLoS ONE 2014;9(7).
[14]

Cai H, Thompson R, Budinich MF, Broadbent JR, Steele JLJGB. Genome sequence and comparative genome analysis of Lactobacillus casei: insights into their niche-associated evolution. Genome Biol Evol 2009;1:239–57.
[15]

Oh PL, Benson AK, Peterson DA, Patil PB, Moriyama EN, Roos S, et al. Diversification of the gut symbiont Lactobacillus reuteri as a result of hostdriven evolution. ISME J 2010;4(3):377–87.
[16]

Dan T, Liu W, Song Y, Xu H, Menghe B, Zhang H, et al. The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST). BMC Microbiol 2015;15(1):107.
[17]

Silvi S, Verdenelli MC, Orpianesi C, Cresci A. Cresci AJJoFE. EU project Crownalife: functional foods, gut microflora and healthy ageing:isolation and identification of Lactobacillus and Bifidobacterium strains from faecal samples of elderly subjects for a possible probiotic use in functional foods. J Food Eng 2003;56(2-3):195–200.
[18]

Morandi S, Brasca M, Lodi R. Technological, phenotypic and genotypic characterisation of wild lactic acid bacteria involved in the production of Bitto PDO Italian cheese. Dairy Sci Technol 2011;91(3):341–59.
[19]

Blomberg L, Henriksson A, Conway PL. Inhibition of adhesion of Escherichia coli K88 to piglet ileal mucus by Lactobacillus spp.. Appl Environ Microbiol 1993;59(1):34–9.
[20]

Lehri B, Seddon AM, Karlyshev AV. Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections. Virulence 2017;8(8):1753–60.
[21]

Kim BK, Lee IO, Tan PL, Eor JY, Hwang JK, Kim SH. Protective effect of Lactobacillus fermentum LA12 in an alcohol-induced rat model of alcoholic steatohepatitis. Korean J Food Sci Anim Resour 2017;37:931–9.
[22]

Kullisaar T, Zilmer K, Salum T, Rehema A, Zilmer M. The use of probiotic L. fermentum ME-3 containing reg’activ cholesterol supplement for 4 weeks has a positive influence on blood lipoprotein profiles and inflammatory cytokines: an open-label preliminary study. Nutr J 2016;15(1):93.
[23]

Chen X, Zhao X, Wang H, Yang Z, Li J, Suo H. Prevent effects of Lactobacillus fermentum HY01 on dextran sulfate sodium-induced colitis in mice. Nutrients 2017;9(6):545.
[24]

Zhao Y, Hong K, Zhao J, Zhang H, Zhai Q, Chen W. Lactobacillus fermentum and its potential immunomodulatory properties. J Funct Foods 2019;56:21–32.
[25]

Rodríguez-Nogales A, Algieri F, Garrido-Mesa J, Vezza T, Utrilla MP, Chueca N, et al. Differential intestinal anti-inflammatory effects of Lactobacillus fermentum and Lactobacillus salivarius in DSS mouse colitis: impact on microRNAs expression and microbiota composition. Mol Nutr Food Res 2017;61(11):1700144.
[26]

Chery J, Dvoskin D, Morato FP, Fahoum B. Lactobacillus fermentum, a pathogen in documented cholecystitis. Int J Surg Case Rep 2013;4(8):662–4.
[27]

Anderson RC, Ulluwishewa D, Young W, Ryan LJ, Henderson G, Meijerink M, et al. Human oral isolate Lactobacillus fermentum AGR1487 induces a proinflammatory response in germ-free rat colons. Sci Rep 2016;6(1):20318.
[28]

Yao L, Seaton SC, Ndousse-Fetter S, Adhikari AA, DiBenedetto N, Mina AI. A selective gut bacterial bile salt hydrolase alters host metabolism. eLife 2018;7: e37182.
[29]

Guo CJ, Allen BM, Hiam KJ, Dodd D, Van Treuren W, Higginbottom S, et al. Depletion of microbiome-derived molecules in the host using Clostridium genetics. Science 2019;366(6471):eaav1282.
[30]

Kumar R, Rajkumar H, Kumar M, Varikuti SR, Athimamula R, Shujauddin M, et al. Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394. Mol Biol Rep 2013;40(8):5057–66.
[31]

Hartemink R, Domenech VR, Rombouts FM. LAMVAB—a new selective medium for the isolation of lactobacilli from faeces. J Microbiol Methods 1997;29 (2):77–84.
[32]

Zheng J, Ruan L, Sun M, Ganzle M. A genomic view of Lactobacilli and Pediococci demonstrates that phylogeny matches ecology and physiology. Appl Environ Microbiol 2015;81(20):7233–43.
[33]

Verma R, Lee C, Jeun EJ, Yi J, Kim KS, Ghosh A. Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of Foxp3+ regulatory T cells. Sci Immunol 2018;3(28):eaat6975.
[34]

Liu HL, Zhang YL, Yang N, Zhang YX, Liu XQ, Li CG, et al. A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt-TSC2-mTOR signaling. Cell Death Dis 2011;2(5).
[35]

Zhang Z, Wu X, Cao S, Wang L, Wang D, Yang H, et al. Caffeic acid ameliorates colitis in association with increased Akkermansia population in the gut microbiota of mice. Oncotarget 2016;7(22):31790–9.
[36]

Zhai Q, Wang G, Zhao J, Liu X, Tian F, Zhang H, et al. Protective effects of Lactobacillus plantarum CCFM8610 against acute cadmium toxicity in mice. Appl Environ Microbiol 2013;79(5):1508–15.
[37]

Tan H, Zhao J, Zhang H, Zhai Q, Chen WJAM. Novel strains of Bacteroides fragilis and Bacteroides ovatus alleviate the LPS-induced inflammation in mice. Appl Microbiol Biotechnol 2019;103(5):2353–65.
[38]

Xu Q, Li X, Wang E, He Y, Yin B, Fang D, et al. A cellular model for screening of lactobacilli that can enhance tight junctions. RSC Adv 2016;6 (113):111812–21.
[39]

Mao B, Li D, Ai C, Zhao J, Zhang H, Chen W. Lactulose differently modulates the composition of luminal and mucosal microbiota in C57BL/6J mice. J Agric Food Chem 2016;64(31):6240–7.
[40]

Sun Y, Jin C, Zhang X, Jia W, Le J, Ye J. Restoration of GLP-1 secretion by Berberine is associated with protection of colon enterocytes from mitochondrial overheating in diet-induced obese mice. Nutr Diabetes 2018;8 (1):53.
[41]

Song YL, Kato N, Liu CX, Matsumiya Y, Kato H, Watanabe KJFML. Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S–23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Microbiol Lett 2000;187(2):167–73.
[42]

Bahl MI, Bergstrom A, Licht TR. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis. FEMS Microbiol Lett 2012;329(2):193–7.
[43]

Li P, Gu Q, Yang L, Yu Y, Wang Y. Characterization of extracellular vitamin B12 producing Lactobacillus plantarum strains and assessment of the probiotic potentials. Food Chem 2017;234:494–501.
[44]

Sulemankhil I, Parent M, Jones ML, Feng Z, Labbe A, Prakash S. In vitro and in vivo characterization and strain safety of Lactobacillus reuteri NCIMB 30253 for probiotic applications. Can J Microbiol 2012;58(6):776–87.
[45]

Moran NA, Sloan DB. The hologenome concept: helpful or hollow? PLoS Biol 2015;13(12).
[46]

Groussin M, Mazel F, Sanders JG, Smillie CS, Lavergne S, Thuiller W, et al. Unraveling the processes shaping mammalian gut microbiomes over evolutionary time. Nat Commun 2017;8(1):14319.
[47]

Duar RM, Lin XB, Zheng J, Martino ME, Grenier T, Perez-Munoz ME, et al. Lifestyles in transition: evolution and natural history of the genus Lactobacillus. FEMS Microbiol Rev 2017;41(Supp 1):S27–48.
[48]

Guo X, Li S, Zhang J, Wu F, Li X, Wu D, et al. Genome sequencing of 39 Akkermansia muciniphila isolates reveals its population structure, genomic and functional diversity, and global distribution in mammalian gut microbiotas. BMC Genomics 2017;18(1):800.
[49]

Ottman N, Reunanen J, Meijerink M, Pietila TE, Kainulainen V, Klievink J, et al. Pili-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function. PLoS ONE 2017;12(3).
[50]

Pandurangan AK, Ismail S, Saadatdoust Z, Esa NM. Allicin alleviates dextran sodium sulfate- (DSS-) induced ulcerative colitis in BALB/c mice. Oxid Med Cell Longev 2015;2015.
[51]

Cario E. Toll-like receptors in inflammatory bowel diseases: a decade later. Inflamm Bowel Dis 2010;16(9):1583–97.
[52]

Takagawa T, Kitani A, Fuss I, Levine B, Brant SR, Peter I, et al. An increase in LRRK2 suppresses autophagy and enhances Dectin-1-induced immunity in a mouse model of colitis. Sci Transl Med 2018;10(444):eaan8162.
[53]

Mittal SK, Roche PA. Suppression of antigen presentation by IL-10. Curr Opin Immunol 2015;34:22–7.
[54]

Onizawa M, Nagaishi T, Kanai T, Nagano K, Oshima S, Nemoto Y, et al. Signaling pathway via TNF-alpha/NF-kappaB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis. Am J Physiol Gastrointest Liver Physiol 2009;296(4):G850–9.
[55]

Price MN, Wetmore KM, Waters RJ, Callaghan M, Ray J, Liu H, et al. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 2018;557(7706):503–9.
[56]

Spaulding CN, Klein RD, Ruer S, Kau AL, Schreiber HL, Cusumano ZT, et al. Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist. Nature 2017;546(7659):528–32.
[57]

Yang J, Li Q, Henning SM, Zhong J, Hsu M, Lee R, et al. Effects of prebiotic fiber xylooligosaccharide in adenine-induced nephropathy in mice. Mol Nutr Food Res 2018;62(15):1800014.
[58]

Adachi O, Fujii Y, Ano Y, Moonmangmee D, Toyama H, Shinagawa E, et al. Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation. Biosci Biotechnol Biochem 2001;65(1):115–25.
[59]

Wen L, Wong FS. Dietary short-chain fatty acids protect against type 1 diabetes. Nat Immunol 2017;18(5):484–6.
[60]

Zagato E, Pozzi C, Bertocchi A, Schioppa T, Saccheri F, et al. Endogenous murine microbiota member Faecalibaculum rodentium and its human homologue protect from intestinal tumour growth. Nat Microbiol 2020;5(3):511–24.
[61]

Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, et al. the microbial metabolites, short-chain fatty acids, regulate colonic treg cell homeostasis. Science 2013;341(6145):569–73.
[62]

Mishiro T, Kusunoki R, Otani A, Ansary MM, Tongu M, Harashima N, et al. Butyric acid attenuates intestinal inflammation in murine DSS-induced colitis model via milk fat globule-EGF factor 8. Lab Invest 2013;93(7):834–43.
[63]

Laffin M, Fedorak R, Zalasky A, Park H, Gill A, Agrawal A, et al. A high-sugar diet rapidly enhances susceptibility to colitis via depletion of luminal short-chain fatty acids in mice. Sci Rep 2019;9(1):12294.
[64]

Wang M, Wichienchot S, He X, Fu X, Huang Q, Zhang B. In vitro colonic fermentation of dietary fibers: fermentation rate, short-chain fatty acid production and changes in microbiota. Trends Food Sci Technol 2019;88:1–9.
[65]

Tajik N, Frech M, Schulz O, Schälter F, Lucas S, Azizov V, et al. Targeting zonulin and intestinal epithelial barrier function to prevent onset of arthritis. Nat Commun 2020;11(1):1995.
[66]

Yang C, Gao J, Zhang J, Luo AL. Enterochromaffin cells in the gut: a distant regulator of brain function? Gut 2018;67(8):1557–8.
[67]

Lee YS, Kim TY, Kim Y, Lee SH, Kim S, Kang SW, et al. Microbiota-derived lactate accelerates intestinal stem-cell-mediated epithelial development. Cell Host Microbe 2018;24(6):833–46.
[68]

Iraporda C, Errea A, Romanin DE, Cayet D, Pereyra E, Pignataro O, et al. Lactate and short chain fatty acids produced by microbial fermentation downregulate proinflammatory responses in intestinal epithelial cells and myeloid cells. Immunobiology 2015;220(10):1161–9.
[69]

Witkin SS, Linhares IM. Why do lactobacilli dominate the human vaginal microbiota? BJOG 2017;124(4):606–11.
[70]

Wulandari AP, Miyazaki J, Kobashi N, Nishiyama M, Hoshino T, Yamane H. Characterization of bacterial homocitrate synthase involved in lysine biosynthesis. FEBS Lett 2002;522(1–3):35–40.
[71]

Wu R, Sun Z, Wu J, Meng H, Zhang H. Effect of bile salts stress on protein synthesis of Lactobacillus casei Zhang revealed by 2-dimensional gel electrophoresis. J Dairy Sci 2010;93(8):3858–68.
[72]

Blount KF, Wang JX, Lim J, Sudarsan N, Breaker RR. Antibacterial lysine analogs that target lysine riboswitches. Nat Chem Biol 2007;3(1):44–9.
[73]

Goh YJ, Barrangou R. Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli. Curr Opin Biotechnol 2019;56:163–71.

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