Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice.

Unlike most other indigenous bacteria, segmented filamentous bacteria (SFB) are potent activators of the mucosal immune system. SFB are strongly anchored to the epithelial cells of the small intestine where they have a preference for mucosal lymphoid epithelium. Since SFB are only present in high numbers shortly after weaning, it was investigated whether an SFB-induced immune reaction results in the removal of these bacteria from the small intestine. A correlation was found between age and colonization levels in the small intestines of SFB monoassociated Swiss mice. Five-week-old athymic BALB/c (nu/nu) mice showed lower colonization levels than their heterozygous littermates, but the opposite was found at the age of 12 weeks. However, SFB inoculation of germfree Swiss mice resulted in higher colonization levels in 5-week-old mice when compared with 4-month-old mice. We conclude that SFB colonization levels in the small intestine are likely influenced by the activity of the mucosal immune system. However, an additional age-dependent factor that modulates SFB colonization levels cannot be excluded.     

Mono-association of mice with non-cultivable,intestinal, segmented,filamentous bacteria

A technique is described so that mice mono-associated with non-cultivable, segmented filamentous bacteria (SFB's) can be produced for the first time. As SFB donors, mice were used which had an intestinal microflora consisting of both SFB's and bacteria of the genus Clostridium. Recipients were germ-free mice. It was demonstrated that the intraileal inoculation method was more effective than the orogastric route. Therefore, intestinal homogenates of donor mice were treated with filtered ethanol, diluted and administered intraileally to recipient mice. Evidence is presented that cage mates of the recipient mice were mono-associated with SFB's. The availability of these animals, i.e. in vivo monocultures of SFB's, allows taxonomic and functional characterization of SFB's, which was as yet not possible.Abbreviation SFB Segmented filamentous bacterium     

Comparative analysis of the distribution of segmented filamentous bacteria in humans,mice and chickens

Segmented filamentous bacteria (SFB) are indigenous gut commensal bacteria. They are commonly detected in the gastrointestinal tracts of both vertebrates and invertebrates. Despite the significant role they have in the modulation of the development of host immune systems, little information exists regarding the presence of SFB in humans. The aim of this study was to investigate the distribution and diversity of SFB in humans and to determine their phylogenetic relationships with their hosts. Gut contents from 251 humans, 92 mice and 72 chickens were collected for bacterial genomic DNA extraction and subjected to SFB 16S rRNA-specific PCR detection. The results showed SFB colonization to be age-dependent in humans, with the majority of individuals colonized within the first 2 years of life, but this colonization disappeared by the age of 3 years. Results of 16S rRNA sequencing showed that multiple operational taxonomic units of SFB could exist in the same individuals. Cross-species comparison among human, mouse and chicken samples demonstrated that each host possessed an exclusive predominant SFB sequence. In summary, our results showed that SFB display host specificity, and SFB colonization, which occurs early in human life, declines in an age-dependent manner.     

Different degree of ileal colonization by segmented, filamentous bacteria in two strains of mice.

Segmented, filamentous bacteria (SFBs) are autochthonous, apathogenic inhabitants of the ileum of various animal species. Outbred Swiss (Cpb:SE) mice have significantly higher degrees of SFB colonization than do inbred BALB/c mice. The present studies were carried out to identify determinants of this strain difference. In a cross-fostering experiment it was shown that SFB colonization of the pups is determined by the strain of the pups themselves rather than by the strain of the nursing dam. Thus, maternal effects may not be involved in SFB colonization. In a cross-infecting experiment using germ-free and SFB-positive animals of the two mouse strains, it was found that ileal SFB colonization is determined by host characteristics rather than by origin of the SFBs. Thus, SFBs that are specific for a given mouse strain may not exist in the two strains of mice. It is concluded that the mouse strain difference in SFB colonization is determined by host characteristics, which probably have a genetic basis.     

Segmented Filamentous Bacteria Are Indigenous Intestinal Bacteria That Activate Intraepithelial Lymphocytes and Induce MHC Class II Molecules and Fucosyl Asialo GM1 Glycolipids on the Small Intestinal Epithelial Cells in the Ex-Germ-Free Mouse

In ex-germ-free mice conventionalized by association with fecal microorganisms, the induction of major histocompatibility complex class II molecules and fucosylation of asialo GM1 glycolipid occur in the small intestinal epithelial cells (IEC). The intestinal intraepithelial lymphocytes (IEL), especially αβ T-cell receptor-bearing ones, also remarkably expand and show cytolytic activity. In this study, we investigated the immunological and physiological characteristics of the small intestine induced by a kind of indigenous bacteria of the small intestine, segmented filamentous bacteria (SFB), among chloroform-resistant intestinal bacteria. Monoassociation of SFB with germ-free mice was confirmed by the determination of the base sequences of polymerase chain reaction products of 16S rRNA genes of the fecal bacteria of these mice and in situ hybridization using fluorescein-labeled probes based on them. SFB increased the number of αβTCR-bearing IEL and induced Thy-1 expression and cytolytic activity of IEL. The induction of MHC class II molecules and fucosyl asialo GM1 glycolipids and the increases in the mitotic activity and the ratio of the number of columnar cells to those of goblet cells also occurred in the small intestinal epithelial cells on monoassociation of these bacteria. SFB are important indigenous bacteria for the development of the mucosal architecture and immune system in the small intestine, at least in mice.     

The genome of th17 cell-inducing segmented filamentous bacteria reveals extensive auxotrophy and adaptations to the intestinal environment

Perturbations of the composition of the symbiotic intestinal microbiota can have profound consequences for host metabolism and immunity. In mice, segmented filamentous bacteria (SFB) direct the accumulation of potentially proinflammatory Th17 cells in the intestinal lamina propria. We present the genome sequence of SFB isolated from monocolonized mice, which classifies SFB phylogenetically as a unique member of Clostridiales with a highly reduced genome. Annotation analysis demonstrates that SFB depend on their environment for amino acids and essential nutrients and may utilize host and dietary glycans for carbon, nitrogen, and energy. Comparative analyses reveal that SFB are functionally related to members of the genus Clostridium and several pathogenic or commensal "minimal" genera, including Finegoldia, Mycoplasma, Borrelia, and Phytoplasma. However, SFB are functionally distinct from all 1200 examined genomes, indicating a gene complement representing biology relatively unique to their role as a gut commensal closely tied to host metabolism and immunity.     

Colonization and distribution of segmented filamentous bacteria (SFB) in chicken gastrointestinal tract and their relationship with host immunity

Uncultivable segmented filamentous bacteria (SFB) reside in the gastrointestinal (GI) tract of mammals and can boost the host immunity. Immunoglobulin A (IgA) from mother's milk has been previously shown to be a key factor in regulating SFB colonization. Because neonatal chicken cannot acquire IgA from maternal milk, they are a good model to examine the role of IgA in SFB colonization. Here, we used the fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) to monitor the colonization and distribution of SFB in chickens aged from 2-day-old to 6-week-old. Early SFB colonization, which primarily occurred in the ileal mucosa (< 13 days old), was IgA independent. From the age of 17-42 days, there was an increase in IgA in the gut mucosa, which was correlated with a decrease in SFB. To examine the effect of probiotics and immunosuppression on SFB colonization, we treated the chickens by feeding them Lactobacillus delbrueckii or giving them a subcutaneous injection of cyclophosphamide (CTX). Feeding lactobacilli at birth rendered SFB colonization occurring 4 days earlier, while CTX treatment increases the SFB colonization through reducing the other non-SFB bacteria. Altogether, our data suggest that early colonization of SFB in chicken occurs independently of IgA and the population of SFB in the GI tract of chicken may be manipulated from birth via probiotic or CTX treatment.     

Colonization of segmented filamentous bacteria and its interaction with the luminal IgA level in conventional mice

Segmented filamentous bacteria (SFB) colonize in the ileum. They promote the development of intraepithelial lymphocytes and immunoglobulin A (IgA)-producing cells in the small intestine. In SFB-monoassociated mice, changes in SFB colonization of the small intestine were related to the level of IgA derived from maternal milk during the suckling period and self-produced in the small intestine after weaning. In this study, we investigated whether or not maternal and neonatal IgA influence the colonization of SFB in conventional mice from 18 to 105 days old. The pups were forcedly weaned at 20 days old. SFB could be detected in the distal small intestine after day 22, and their number rapidly reached a maximum on day 28. Thereafter, they gradually declined to one-fourth of the maximum level. The lowest concentrations of IgA in the small intestinal and cecal contents were detected on day 22. Thereafter, they increased as the age of the mice increased. The expression of the polymeric immunoglobulin receptor gene in the distal small intestine increased after weaning. These results suggested that the colonization of SFB in the pre-weaning and post-weaning periods might be prevented with IgA derived from maternal milk and self-produced IgA, respectively.     

Complete genome sequences of rat and mouse segmented filamentous bacteria, a potent inducer of th17 cell differentiation

Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E.?coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.     

Microbiology and immunology: An ideal partnership for a tango at the gut surface—A tribute to Philippe Sansonetti

Over the past 20 years, the highly dynamic interactions that take place between hosts and the gut microbiota have emerged as a major determinant in health and disease. The complexity of the gut microbiota represents, however, a considerable challenge, and reductionist approaches are indispensable to define the contribution of individual bacteria to host responses and to dissect molecular mechanisms. In this tribute to Philippe Sansonetti, I would like to show how rewarding collaborations with microbiologists have guided our team of immunologists in the study of host–microbiota interactions and, thanks to the use of controlled colonisation experiments in gnotobiotic mice, toward the demonstration that segmented filamentous bacteria (SFB) are indispensable to drive the post‐natal maturation of the gut immune barrier in mice. The work led with Philippe Sansonetti to set up in vitro culture conditions has been one important milestone that laid the ground for in‐depth characterization of the molecular attributes of this unusual symbiont. Recent suggestions that SFB may be present in the human microbiota encourage further cross‐fertilising interactions between microbiologists and immunologists to define whether results from mice can be translated to humans and, if so, how SFB may be used to promote human intestinal defences against enteropathogens. Nurturing the competences to pursue this inspiring project is one legacy of Philippe Sansonetti.     

Gut immune maturation depends on colonization with a host-specific microbiota

Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.     

Spatial and temporal colonization dynamics of segmented filamentous bacteria is influenced by gender,age and experimental infection with Helicobacter hepaticus in Swiss Webster mice

In this study, we examined colonization dynamics of segmented filamentous bacteria (SFB) in intestine of Swiss Webster (SW) mice infected with Helicobacter hepaticus (Hh). At 8 weeks post-inoculation with Hh (WPI), cecal and colonic SFB levels in the control males were significantly lower compared to those at 16 WPI. Hh infection in both genders did not alter SFB levels in the jejunum and ileum, but increased SFB levels in the cecum and colon of males compared to the controls (P < 0.05) at 8 WPI. At 16 WPI, the Hh-infected females contained lower levels of SFB in the jejunum, cecum and colon compared to the female controls. Irrespective of gender, aging and Hh infection, the Il-17A mRNA levels decreased from the small intestine to the cecum and then to the colon, whereas the Foxp3 mRNA levels were comparable in these intestinal regions. There were significant differences in Il-17A mRNA levels in the ileum (P < 0.05, R² = 0.31), with females having greater Il-17A mRNA levels than males, and higher SFB colonization levels related to more Il-17A mRNA. These results indicate that aging and gender play an important role in colonization dynamics of intestinal SFB and ileal SFB-associated Th17 response.     

Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.     

微贮用植物乳杆菌固定化菌剂的制备

乳酸菌与纤维素降解菌因其可防止微贮饲料酸败、增加秸秆饲料的营养价值等优点,在秸秆微贮过程中起重要作用。但由于乳酸菌的繁殖会抑制纤维素降解菌的活性,如何实现微贮过程中两种微生物分时发挥功能是解决上述问题的关键。文中利用固定化技术将乳酸菌制备成含有玉米秸秆粉的固定化菌剂以达到缓释的目的。首先制作固定化空白小球得出复合固定化载体成球的最佳浓度,利用玉米芯吸附植物乳杆菌S1得到复合固定化载体,以对S1的包埋率、成球效果等为指标,通过对比两种固定化方法 (包埋法与包埋-交联法),得到固定化植物乳杆菌S1的最佳条件。研究表明,使用6%PVA+0.4%SA+0.3%CMC-Na进行包埋-交联时成球效果最好,使用1.2%SA+0.5%CMC-Na进行直接包埋时成球效果最好。通过对比5种固定化工艺,将1.2%SA+0.5%CMC-Na和吸附玉米粉组成的固定化载体混合物逐滴滴入4%氯化钙中直接包埋24 h得到的固定化小球其机械强度以及包埋率均优于其他工艺。因此,利用玉米芯吸附-海藻酸钠包埋的方法可以有效提高植物乳杆菌包埋效率,为使用固定化技术制备微贮饲料菌剂奠定基础。     

Differential effects of two probiotic strains with different bacteriological properties on intestinal gene expression, with special reference to indigenous bacteria

Probiotics are used for the improvement of gut disorders. To explore the potential of probiotics, a gnotobiotic study using BALB/c mice to analyze epithelial gene expression was performed. Microarray analysis of probiotic strain-monoassociated mice showed that Lactobacillus casei Shirota and Bifidobacterium breve Yakult noticeably affected gene expression in the ileal and colonic epithelial cells, respectively, although to a smaller extent than segmented filamentous bacteria (SFB). Lactobacillus casei Shirota enhanced the gene expression involving defense/immune functions and lipid metabolism more strongly than B. breve Yakult. In the colon, expression of a chloride transporter was slightly enhanced, although downregulation of many genes, such as guanine nucleotide-binding protein, was evident in mice with B. breve Yakult compared with the ones with L. casei Shirota. SFB affected gene expression more strongly than the probiotic strains. In particular, alpha(1-2) fucosyltransferase and pancreatitis-associated protein were significantly enhanced only in SFB-monoassociated mice but not probiotic strain-monoassociated mice. Gene expression of SFB-monoassociated mice was either stimulated or repressed in a manner similar to or opposite that of conventional colonized mice. Taken together, probiotic strains of L. casei Shirota and B. breve Yakult differentially affect epithelial gene expression in the small intestine and colon, respectively.     

接种植物乳杆菌(Lactobacillus plantarum)对小规模饲料稻青贮品质的影响

【目的】研究接种植物乳杆菌对小规模饲料稻品质的影响。【方法】以自然发酵的样品为对照,接种不同来源植物乳酸菌发酵饲料稻,发酵30 d后对饲料稻的感官进行评价;通过选择性平板对饲料稻青贮中的不同微生物进行计数;并采用V-Score评价法对发酵品质进行评定。【结果】相对自然发酵的样品而言,接种植物乳杆菌的青贮样品感官评分等级达到优良;乳酸菌为优势菌株,引起腐败变质的好氧菌、霉菌、大肠杆菌等受到抑制;接种发酵的样品中乳酸含量明显增加,氨态氮的产生量为对照的1/2左右,V-Score评分为满分。【结论】供试的植物乳杆菌,尤其是从青饲料和青贮材料中分离的菌株能有效改善饲料稻青贮的品质,可考虑用作青贮饲料稻发酵剂。     

Antigenotoxicity of probiotics and prebiotics on faecal water-induced DNA damage in human colon adenocarcinoma cells

Six strains of lactic acid producing bacteria (LAB) were incubated (1 x 10(8)cfu/ml) with genotoxic faecal water from a human subject. HT29 human adenocarcinoma cells were then challenged with the resultant samples and DNA damage measured using the single cell gel electrophoresis (comet) assay. The LAB strains investigated were Bifidobacterium sp. 420, Bifidobacterium Bb12, Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus bulgaricus and Enterococcus faecium. DNA damage was significantly decreased by all bacteria used with the exception of Strep. thermophilus. Bif. Bb12 and Lact. plantarum showed the greatest protective effect against DNA damage. Incubation of faecal water with different concentrations of Bif. Bb12 and Lact. plantarum revealed that the decrease in genotoxicity was related to cell density. Non-viable (heat treated) probiotic cells had no effect on faecal water genotoxicity. In a second study, HT29 cells were cultured in the presence of supernatants of incubations of probiotics with various carbohydrates including known prebiotics; the HT29 cells were then exposed to faecal water. Overall, incubations involving Lact. plantarum with the fructooligosaccharide (FOS)-based prebiotics Inulin, Raftiline, Raftilose and Actilight were the most effective in increasing the cellular resistance to faecal water genotoxicity, whereas fermentations with Elixor (a galactooligosaccharide) and Fibersol (a maltodextrin) were less effective. Substantial reductions in faecal water-induced DNA damage were also seen with supernatants from incubation of prebiotics with Bif. Bb12. The supernatant of fermentations involving Ent. faecium and Bif. sp. 420 generally had less potent effects on genotoxicity although some reductions with Raftiline and Elixor fermentations were apparent.     

'Candidatus Arthromitus' revised: segmented filamentous bacteria in arthropod guts are members of Lachnospiraceae

The name Arthromitus has been applied collectively to conspicuous filamentous bacteria found in the hindguts of termites and other arthropods. First observed by Joseph Leidy in 1849, the identity of these filaments has remained contentious. While Margulis and colleagues declared them to be a life stage of Bacillus cereus, others have assumed them to belong to the same lineage as the segmented filamentous bacteria (SFB) from vertebrate guts, a group that has garnered much attention due to their unique ability to specifically modulate their host's immune response. Both SFB and Arthromitus filaments from arthropod guts were grouped under provisional name 'Candidatus Arthromitus' by Snel and colleagues as they share a striking similarity in terms of their morphology and close contact to the host gut wall. While SFB form a distinct lineage within the family Clostridiaceae, the identity of the filaments from arthropod guts remains elusive. Using whole-genome amplification of single filaments capillary picked from termite guts and fluorescence in situ hybridization of 16S rRNA with group-specific oligonucleotide probes, we show that they represent a monophyletic lineage within the family Lachnospiraceae distinct from that of SFB. Therefore, 'Candidatus Arthromitus' can no longer be used for both groups. Given the historic precedence, we propose to reserve this name for the filaments that were originally described by Leidy. For the SFB from vertebrate guts, we propose the provisional name 'Candidatus Savagella' in honour of the American gut microbiologist Dwayne C. Savage, who was the first to describe that important bacterial group.     

Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated from a Green Olive Fermentation

Twenty-six strains of Lactobacillus plantarum isolated from green olive fermentations were tested for cross-antagonistic activities in an agar drop diffusion test. Cell-free supernatants from four of these strains were shown to inhibit the growth of at least one of the L. plantarum indicator strains. L. plantarum LPCO10 provided the broadest spectrum of activity and was selected for further studies. The inhibitory compound from this strain was active against some gram-positive bacteria, including clostridia and propionibacteria as well as natural competitors of L. plantarum in olive fermentation brines. In contrast, no activity against gram-negative bacteria was detected. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. Since the inhibitory activity of the active supernatant was lost after treatment with various proteolytic enzymes, this substance could be classified as a bacteriocin, designated plantaricin S. Plantaricin S was also sensitive to glycolytic and lipolytic enzymes, suggesting that it was a glycolipoprotein. It exhibited a bactericidal and nonbacteriolytic mode of action against indicator cells. This bacteriocin was heat stable (60 min at 100 degrees C), active in a pH range of 3.0 to 7.0, and also stable in crude culture supernatants during storage. Ultrafiltration studies indicated that plantaricin S occurred as multimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum production of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated plantaricin T) was produced in late-stationary-phase cultures of L. plantarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plantaricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, plantaricin T appeared different from plantaricin S. Curing experiments with L. plantarum LPCO10 resulted in the appearance of variants that no longer produced either of the two bacteriocins but that were still immune to both of them.