Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132^T and B. longum JCM1217^T) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.     

低出生体重儿肠道微生态构建影响因素的 动态监测

目的动态监测低出生体重儿肠道菌群,分析不同体重、不同喂养方式及疾病状态等因素对患儿肠道微生态的影响,为规范临床低出生体重儿宫外营养支持措施及治疗手段提供依据。方法应用16SrRNA荧光定量PCR技术检测正常新生儿和低出生体重儿生后第1、3、7天粪便中大肠埃希菌、肠球菌、乳杆菌及双歧杆菌的含量。结果 (1)在生后7d内,无论正常新生儿还是低出生体重儿,其粪便中大肠埃希菌、肠球菌、乳杆菌和双歧杆菌的含量均随日龄的增加而增加,且生后7d内正常新生儿的粪便中大肠埃希菌、肠球菌、乳杆菌和双歧杆菌的含量均显著高于低出生体重儿(P0.05),正常新生儿生后7d内粪便中各细菌的增长率均高于低出生体重儿。(2)体重2 000~2 500g的低出生体重儿粪便中大肠埃希菌和肠球菌在各日龄中的含量明显高于体重2 000g的新生儿(P0.05);同时其粪便中双歧杆菌和乳杆菌含量在3日龄和7日龄阶段明显高于体重2 000g的新生儿(P0.05)。(3)3日龄和7日龄母乳喂养组的低出生体重儿粪便中双歧杆菌和乳杆菌含量明显高于乳制品喂养组(P0.05);且母乳喂养组新生儿生后7日内粪便中大肠埃希菌、乳杆菌和双歧杆菌含量的增长率均高于乳制品喂养组,尤其是双歧杆菌的增长率(126.49%vs 54.81%)。(4)合并并发症的3日龄和7日龄的低出生体重儿,粪便中乳杆菌和双歧杆菌含量均明显低于无合并症的低出生体重儿(P0.05);且无并发症组的低出生体重儿其粪便中肠球菌、乳杆菌和双歧杆菌的增长率均高于有并发症组的低出生体重儿,大肠埃希菌增长率则低于有并发症组。结论低出生体重儿肠道菌群的定植时间晚且数量少,体重、喂养方式及有无并发症是影响新生儿肠道菌群丰度的重要因素。母乳喂养可促进低出生体重儿肠道中益生菌的定植。疾病因素会导致肠道菌群丰度的降低,使肠道菌群紊乱,其程度可能与病情的严重程度相关。     

Modelling the effect of birth and feeding modes on the development of human gut microbiota

The human gut microbiota is transmitted from mother to infant through vaginal birth and breastfeeding. Bifidobacterium, a genus that dominates the infants’ gut, is adapted to breast milk in its ability to metabolize human milk oligosaccharides; it is regarded as a mutualist owing to its involvement in the development of the immune system. The composition of microbiota, including the abundance of Bifidobacteria, is highly variable between individuals and some microbial profiles are associated with diseases. However, whether and how birth and feeding practices contribute to such variation remains unclear. To understand how early events affect the establishment of microbiota, we develop a mathematical model of two types of Bifidobacteria and a generic compartment of commensal competitors. We show how early events affect competition between mutualists and commensals and microbe-host-immune interactions to cause long-term alterations in gut microbial profiles. Bifidobacteria associated with breast milk can trigger immune responses with lasting effects on the microbial community structure. Our model shows that, in response to a change in birth environment, competition alone can produce two distinct microbial profiles post-weaning. Adding immune regulation to our competition model allows for variations in microbial profiles in response to different feeding practices. This analysis highlights the importance of microbe–microbe and microbe–host interactions in shaping the gut populations following different birth and feeding modes.     

Probiotic characterization of Lactiplantibacillus plantarum HOM3204 and its restoration effect on antibiotic-induced dysbiosis in mice

The purpose of this study was to evaluate the probiotic characteristics of Lactiplantibacillus plantarum HOM3204 isolated from homemade pickled cabbage and to examine its restoration effect on antibiotic-induced dysbiosis in mice. Lact. plantarum HOM3204 tolerated simulated gastric and intestinal juices with a 99·38% survival rate. It also showed strong adhesion ability (3·45%) to Caco-2 cells and excellent antimicrobial activity against foodborne pathogens in vitro. For safety (antibiotic susceptibility) of this strain, it was susceptible to all the tested seven antibiotics. Lact. plantarum HOM3204 had good stability during storage, especially in cold and frozen conditions. Furthermore, Lact. plantarum HOM3204 significantly restored the gut microbiota composition by increasing the abundance of Lactobacilli and Bifidobacteria and decreasing Enterococci, and improved antioxidative function by raising the concentrations of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in serum of antibiotic-induced dysbiosis in mice. These results suggest that Lact. plantarum HOM3204 could be a potential probiotic as a functional food ingredient.     

阿莫西林干预对婴儿优势菌在无菌小鼠体内定植的影响

目的考察阿莫西林干预对婴儿优势菌双歧杆菌及乳杆菌在无菌小鼠体内定植的影响。方法 1日龄Balb/c无菌乳鼠接种婴儿粪便悬液。饲养至7~21日龄灌胃阿莫西林(100mg/kg),对照组在同日龄给予等体积的生理盐水。利用qRT-PCR检测小鼠粪便中双歧杆菌、乳杆菌的含量。结果阿莫西林处理可显著降低乳杆菌(P0.05)、双歧杆菌(P0.05)在无菌小鼠体内定植数量,但停药后饲养至成年(53日龄)二者定植数量与对照组小鼠比较差异无统计学意义(P0.05)。结论哺乳期阿莫西林干预会导致乳杆菌、双歧杆菌在小鼠体内定植量下降,但停药后小鼠饲养至成年二者可达到正常定植量,婴儿菌群定植小鼠模型可以模拟与现有动物模型一致的阿莫西林对双歧杆菌、乳杆菌定植的影响规律。     

Microbiota profile in feces of breast- and formula-fed newborns by using fluorescence in situ hybridization (FISH)

The development of the gut is controlled and modulated by different interacting mechanisms such as, genetic endowment, intrinsic biological regulatory functions, environment influences and last but no least, the diet influence. Considered together with other endogenous and exogenous factors the type of feeding may interfere greatly in the regulation of the intestinal microbiota. During the last years molecular methods offer a complementarity to the classic culture-based knowledge. FISH has been applied for molecular evaluation of the microbiota in newborns delivered by vaginal delivery. Eleven probes/probe combinations for specific groups of faecal bacteria were used to determine the bacterial composition in faecal samples of newborns infants under different types of feeding. Breast-fed infants harbor a fecal microbiota by more than two times increased in numbers of Bifidobacterium cells when compared to formula-fed infants. After formula-feeding, Atopobium was found in significant counts and the numbers of Bifidobacterium dropped followed by increasing numbers in Bacteroides population. Moreover, under formula feeding the infants microbiota was more diverse.     

Proteomic comparison of the cytosolic proteins of three <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> human isolates and <Emphasis Type="Italic">B. longum</Emphasis> NCC2705

Background  

Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are currently used as probiotic components in functional food products. However, the probiotic effects have been reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced model strain B. longum NCC2705 at the chromosome and proteome levels.     

Recent insight into oligosaccharide uptake and metabolism in probiotic bacteria

Abstract

In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota.

In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics, enzymology and protein characterization proved fruitful in elucidating the protein transport and catabolic machinery conferring the utilization of glucosides, galactosides, and xylosides in the two clinically validated probiotic strains Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Importantly, we have been able to identify and in some cases validate the specificity of several transport systems, which are otherwise poorly annotated. Further, we have demonstrated for the first time that non-naturally occurring tri- and tetra-saccharides are internalized and efficiently utilized by probiotic bacteria in some cases better than well-established natural prebiotics.

Selected highlights of these data are presented, emphasising the importance and the diversity of oligosaccharide transport in probiotic bacteria.     

小檗碱与肠道菌群的相互作用研究

目的研究人体肠道菌群对小檗碱的代谢和小檗碱对人体肠道菌群生长的影响。方法采用UPLC-Q-TOF/MS技术分析小檗碱经肠道菌群转化的代谢产物;并应用浊度测定法测定不同浓度小檗碱对4类肠道细菌(乳杆菌、双歧杆菌、肠杆菌和肠球菌)生长的影响。结果小檗碱主要被代谢为去甲氧基和氢化产物,混合菌群具有去甲氧基和还原作用,在所有检测的单株细菌中,Enterococcus sp.45去甲氧基作用较强;此外,小檗碱明显抑制病原菌肠杆菌和肠球菌的生长,促进有益菌乳杆菌和双歧杆菌的生长。结论本实验对于小檗碱的体内代谢过程及药效的深入研究具有良好的指导意义。     

Molecular analysis of autochthonous microbiota along the digestive tract of juvenile grouper Epinephelus coioides following probiotic Bacillus pumilus administration

Aims: To evaluate the diversity of dominant autochthonous microbiota along the digestive tract of juvenile Epinephelus coioides following the dietary administration of probiotic Bacillus pumilus for 60 days. Methods and Results: Polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) with subsequently sequencing analysis was used to assess the gut microbiota. Generally similar DGGE patterns were observed in the foregut, midgut and hindgut of E. coioides, while the similarity dendrogram clearly revealed three different clusters depending on the three compartments of the GI tract. Dietary administration of B. pumilus stimulated its colonization in each compartment of the digestive tract. Samples collected from the probiotic group and the control group showed similar DGGE patterns, and no significant difference in the total number of bands and the Shannon index were detected between the probiotic group and the control group, suggested that B. pumilus exert no significant effect on the gut microbiota. However, various potentially beneficial bacteria, such as uncultured Bacillus sp. clone QJNY94‐like, Nitratireductor sp. YCSC5‐like, Methylobacterium hispanicum‐like and Microbacterium sp. YACS1‐like bacteria were stimulated by probiotic B. pumilus, while the potential harmful Staphylococcus saprophyticus‐like bacterium was depressed. Conclusions: Autochthonous gut microbiota of E. coioides was modulated to some degree, not significant, by probiotic B. pumilus, various potentially beneficial bacteria were selectively stimulated, while one potential harmful species was depressed. Significance and Impact of the Study: This work represents the first report that dietary administration of probiotic B. pumilus modulated the gut microbiota of E. coioides. These findings broaden our understanding of probiotic effects at the gut level, which is helpful in understanding the mechanisms that underpin host benefits.     

In vitro Effects of Prebiotics and Synbiotics on Apis cerana Gut Microbiota

This study aimed to investigate in vitro effects of the selected prebiotics alone, and in combination with two potential probiotic Lactobacillus strains on the microbial composition of Apis cerana gut microbiota and acid production. Four prebiotics, inulin, fructo-oligosaccharides, xylo-oligosaccharides, and isomalto-oligosaccharides were chosen, and glucose served as the carbon source. Supplementation of this four prebiotics increased numbers of Bifidobacterium and lactic acid bacteria while decreasing the pH value of in vitro fermentation broth inoculated with A. cerana gut microbiota compared to glucose. Then, two potential probiotics derived from A. cerana gut at different dosages, Lactobacillus helveticus KM7 and Limosilactobacillus reuteri LP4 were added with isomalto-oligosaccharides in fermentation broth inoculated with A. cerana gut microbiota, respectively. The most pronounced impact was observed with isomalto-oligosaccharides. Compared to isomalto-oligosaccharides alone, the combination of isomalto-oligosaccharides with both lactobacilli strains induced the growth of Bifidobacterium, LAB, and total bacteria and reduced the proliferation of Enterococcus and fungi. Consistent with these results, the altered metabolic activity was observed as lowered pH in in vitro culture of gut microbiota supplemented with isomalto-oligosaccharides and lactobacilli strains. The symbiotic impact varied with the types and concentration of Lactobacillus strains and fermentation time. The more effective ability was observed with IMO combined with L. helveticus KM7. These results suggested that isomalto-oligosaccharides could be a potential prebiotic and symbiotic with certain lactobacilli strains on A. cerana gut microbiota.     

Differential Induction of Antimicrobial REGIII by the Intestinal Microbiota and Bifidobacterium breve NCC2950

The intestinal microbiota is a key determinant of gut homeostasis, which is achieved, in part, through regulation of antimicrobial peptide secretion. The aim of this study was to determine the efficiency by which members of the intestinal microbiota induce the antimicrobial peptide REGIII and to elucidate the underlying pathways. We showed that germfree mice have low levels of REGIII-γ in their ileum and colon compared to mice with different intestinal microbiota backgrounds. Colonization with a microbiota of low diversity (altered Schaedler flora) did not induce the expression of REGIII-γ as effectively as a complex community (specific pathogen free). Monocolonization with the probiotic Bifidobacterium breve, but not with the nonprobiotic commensal Escherichia coli JM83, upregulated REGIII-γ expression. Induction of REGIII-γ by B. breve was abrogated in mice lacking MyD88 and Ticam1 signaling. Both live and heat-inactivated B. breve but not spent culture medium from B. breve induced the expression of REGIII-α, the human ortholog and homolog of REGIII-γ, in human colonic epithelial cells (Caco-2). Taken together, the results suggest that REGIII-γ expression in the intestine correlates with the richness of microbiota composition. Also, specific bacteria such as Bifidobacterium breve NCC2950 effectively induce REGIII production in the intestine via the MyD88-Ticam1 pathway. Treatment with this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation.     

Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30–40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people. Received: 7 February 2001 / Accepted: 3 April 2001     

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

Background  

Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals.     

N-glycan Utilization by Bifidobacterium Gut Symbionts Involves a Specialist β-Mannosidase

Bifidobacteria represent one of the first colonizers of human gut microbiota, providing to this ecosystem better health and nutrition. To maintain a mutualistic relationship, they have enzymes to degrade and use complex carbohydrates non-digestible by their hosts. To succeed in the densely populated gut environment, they evolved molecular strategies that remain poorly understood. Herein, we report a novel mechanism found in probiotic Bifidobacteria for the depolymerization of the ubiquitous 2-acetamido-2-deoxy-4-O-(β-d-mannopyranosyl)-d-glucopyranose (Man-β-1,4-GlcNAc), a disaccharide that composes the universal core of eukaryotic N-glycans. In contrast to Bacteroidetes, these Bifidobacteria have a specialist and strain-specific β-mannosidase that contains three distinctive structural elements conferring high selectivity for Man-β-1,4-GlcNAc: a lid that undergoes conformational changes upon substrate binding, a tryptophan residue swapped between the two dimeric subunits to accommodate the GlcNAc moiety, and a Rossmann fold subdomain strategically located near to the active site pocket. These key structural elements for Man-β-1,4-GlcNAc specificity are highly conserved in Bifidobacterium species adapted to the gut of a wide range of social animals, including bee, pig, rabbit, and human. Together, our findings uncover an unprecedented molecular strategy employed by Bifidobacteria to selectively uptake carbohydrates from N-glycans in social hosts.     

肠黏膜屏障与肠道菌群的相互关系

摘要：人类肠道中微生物群与肠道环境相互作用以维持机体健康。肠黏膜屏障主要由黏液层、肠道菌群、肠道免疫系统和肠上皮细胞本身的完整性等构成。肠道作为直接与大量菌群接触的器官,其屏障功能在肠道健康中的作用尤为显著。肠道菌群与肠道屏障相互作用,保持肠道菌群与肠道屏障相对稳定,肠道菌群参与肠道免疫反应的建立,共同建立机体天然防御系统,在保持肠道免疫的动态平衡中具有重要作用。当两者之间的平衡被打破时,可诱发功能性胃肠病（如肠易激综合征）及免疫相关性疾病（如炎症性肠病）。本文主要阐述肠黏膜屏障与肠道菌群之间的相互关系以及与肠道屏障功能障碍相关的肠道疾病。     

Like Will to Like: Abundances of Closely Related Species Can Predict Susceptibility to Intestinal Colonization by Pathogenic and Commensal Bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM^con21). 16S rRNA sequence analysis comparing LCM, LCM^con21 and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri ^RR strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.     

Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains

Novel oligomers that resist digestion in the upper gut were prepared from oat mixed-linked β-glucan and xylan by enzymatic hydrolysis with lichenase of Bacillus subtilis and xylanase of Trichoderma reesei respectively. The low-molecular-mass hydrolysis products of β-glucan and xylan were compared with fructooligomers and raffinose in their ability to provide growth substrates for probiotic (Lactobacillus and Bifidobacterium) and intestinal (Bacteroides, Clostridium and Escherichia coli) strains in vitro. A degradation profile of each carbohydrate and total sugar consumption were analysed with HPLC, and bacterial growth rate with an automatic turbidometer, the Bioscreen C system. β-Glucooligomers and xylooligomers both enhanced the growth of health-promoting probiotic strains as compared with intestinal bacterial growth, but not to a significant level. Raffinose stimulated the probiotic strains significantly, whereas fructooligomers induced high average growth for intestinal bacteria also. Received: 16 May 1997 / Received revision: 12 September 1997 / Accepted: 19 September 1997     

Intestinal microbiota in exclusively breast-fed infants with blood-streaked stools

Intestinal microbiota in exclusively breast-fed infants with blood-streaked stools and in healthy exclusively breast-fed babies was compared. Total anaerobes, bifidobacteria, lactobacilli, coliform bacteria, enterococci and clostridia were quantified by cultivation methods in feces of 17 full-term exclusively breastfed patients (aged 16.3 ± 7.4 weeks) with blood-streaked stools and in the control group of 22 healthy fullterm exclusively breast-fed infants (13.7 ± 6.4 weeks). Specific fluorescence in situ hybridization kits for Bifidobacterium spp. were used for the quantitative detection of bifidobacteria in samples. Control samples had significantly (p < 0.05) higher counts of total anaerobes. Bifidobacteria were not detected in patients’ samples in 65 % and in controls in 36 % (p < 0.01). Bifidobacteria counts were also significantly higher in the control group (p < 0.01). Furthermore, clostridia strains were detected only in feces from bifidobacteria-negative infants reaching counts >8 log CFU/g. Lactobacilli were not detected in 65 % patients and in 45 % control samples. However, this difference was not significant as well as the difference in lactobacilli counts. Eosinophilia was observed in 35 % of patients, low IgA concentration in 71 % and also low IgG concentration in 71 %. pANCA positivity was found in 53 % of patients. In conclusion a significant low proportion of bifidobacterial microbiota in patients with blood-streaked stools was shown in comparison with controls.