Lessons from the genomes of bifidobacteria

The gut microbiota is a complex ecosystem composed of hundreds of different bacterial species that altogether play an important role in the physiology of their host. In the past few years the complete genome sequence of a number of bacterial strains isolated from the human gastrointestinal tract has been established including that of Bifidobacterium longum NCC2705 isolated from the feces of a healthy infant. Bifidobacteria are among the first species to colonise the human gastrointestinal tract and as such are believed to play an important role in gut homeostasis and normal development. The genome sequence of NCC2705 has revealed a number of features that suggest how this bacterium has adapted to its environment and that could help understanding how it interacts with its host. Here, we review general features of bifidobacteria and illustrate how genome-based approaches can help us better understand the biology of these organisms.     

Lactoferrin and bifidobacteria

We herein summarized the effects of lactoferrin (LF) on bifidobacteria. Many in vitro studies previously reported the growth-promoting (bifidogenic) effects of LF on bifidobacteria. The involvement of bound iron, sugar chains, and LF peptides has been proposed in this bifidogenic mechanism. Peptides in the LF pepsin hydrolysate (LFH) showed stronger bifidogenic activity than natural LF; therefore, we speculated that peptides may be the bifidogenic active principle of LF. LF or its peptides may be recognized by LF-binding proteins on the surface of bifidobacterial cells, and the cationic nature or disulfide bonds of LF or its peptides may play a crucial role in its recognition by these proteins. Of the bifidobacterial species so far identified, human LF and peptides in human LFH were more likely to show bifidogenic activity especially to Bifidobacterium bifidum, and bovine LF (bLF) and peptides in bovine LFH (bLFH) to B. breve and B. infantis. In animal studies, the administration of LF to mice or piglets increased bifidobacteria levels in the intestine. In human trials, the administration of LF-containing formula to infants increased bifidobacteria levels in the feces; however, human milk achieved better results than LF-containing formula. In the case of breast-fed infants, LF may show bifidogenic activity synergistically with other milk components such as human milk oligosaccharides. As bLFH showed stronger bifidogenic activity than natural bLF, especially to B. breve and B. infantis in vitro, and these species are known to be infant-specific species, bLFH may be a beneficial ingredient in formula.     

Antimicrobial susceptibility of bifidobacteria

Eighteen Bifidobacterium strains were tested for their susceptibility to a range of antimicrobial agents. All the strains tested, including the reference culture Lactobacillus acidophilus CH2, were susceptible to several groups of antimicrobial agents, they were cephalosporin (cefamandole, cefazolin, cefaperazone, cefoxitin), polypeptide (bacitracin), macrolide (erythromycin), penicillin (amoxicillin), phenicol (chloramphenicol) and beta-lactam (imipenem). Fourteen strains were resistant to more than 10 antibiotics. The reference culture was resistant to only three antibiotics. The results showed that bifidobacteria are resistant to a wide range of antimicrobial agents.     

Characteristics of the soluble antigens of bifidobacteria

The immunological study of aqueous buffer extracts obtained from 45 strains of bifidobacteria belonging to the species B. bifidum, B. longum, B. adducens, B. breve, B. infantis and B. parvulorum was made. This study revealed 3 levels of the immunological specificity of soluble bifidobacterial proteins: common to the genus Bifidobacterium, common to a limited number of strains belonging to one or several species of bifidobacteria and strain-specific.     

Understanding the acid tolerance response of bifidobacteria

Aims: To investigate the effect of pH on the viability and the acid tolerance response (ATR) of bifidobacteria. Methods and Results: The impact of low pH on the viability of five species of bifidobacteria was examined under conditions of strict anaerobiosis. Although differences in the ability to resist the lethal effects of low pH were apparent among the species, cell viability could be improved by the provision of fermentable substrate during an acidic pH stress or through the use of stationary phase cells. While a stationary phase ATR was found to occur in two species of bifidobacteria, there was no adaptive response in exponential phase cells. Proteomic analysis of exponential phase Bifidobacterium longum subjected to a mild acid pre‐exposure (pH 4·5, 2 h) prior to an acid challenge revealed a substantial loss in the total number of cellular proteins. In contrast, proteomic analysis of stationary phase cells revealed an increased abundance of proteins associated with the general stress response as well as the β‐subunit of the F₀F₁‐ATPase, known to be important in bifidobacteria acid tolerance. Conclusion: Neither Bif. longum or Bifidobacterium breve possesses an inducible exponential phase ATR. Significance and Impact of the Study: These findings provide further insights into the impact of pH on the viability of bifidobacteria and may partially explain the loss in viability associated with their storage in acid foods.     

Analysis of phospholipids in bifidobacteria

Methods of preparative chromatography on silica gel columns were used for obtaining preparations of polar lipids of bifidobacteria. Studies of the preparations by one-dimensional and two-dimensional TLC demonstrated that diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were the predominant phospholipids; minor phospholipids (phosphorus-containing components present in considerably lower amounts) included phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). Parameters of qualitative composition of phospholipids and glycolipids may serve as a set of chemotaxonomic markers in modem procedures for identifying Bifidobacterium species.     

Analysis of phospholipids in bifidobacteria

Methods of preparative chromatography on silica gel columns were used for obtaining preparations of polar lipids of bifidobacteria. Studies of the preparations by one-dimensional and two-dimensional TLC demonstrated that diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were the predominant phospholipids; minor phospholipids (phosphorus-containing components present in considerably lower amounts) included phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). Parameters of qualitative composition of phospholipids and glycolipids may serve as a set of chemotaxonomic markers in modern procedures for identifying Bifidobacterium species.     

Isoleucine production in bifidobacteria

Summary About 300 strains of bifidobacteria were examined for their capacity to release L-isoleucine in the fermentation broth. A strain of the speciesBifidobacterium ruminale was selected as the best producer. After treatment of this strain with NTG a DL--aminobutyric acid-resistant mutant capable of producing about 5 mg/ml of L-isoleucine in presence of 1.5 % DL--aminobutyric acid was obtained. Cultural conditions and acetohydroxyacid synthetase activity were studied.     

Identification of lactobacilli and bifidobacteria

Selective culture media and phenotypic tests enable lactobacilli to be differentiated from morphologically similar bacteria. The accurate identification of Lactobacillus species can be accomplished by reference to 16S rRNA gene sequences. Species-specific, PCR primers that target the 16S-23S rRNA spacer region are available for a limited number of Lactobacillus species. Molecular methods for the comprehensive identification of Bifidobacterium species are not yet available. Only DNA-DNA reassociation provides a reliable means of species identification for this genus at present. Bifidobacteria can be differentiated from morphologically similar bacteria by the use of genus-specific, PCR primers or oligonucleotide probes.     

Susceptibility of bifidobacteria to nisin

A collection of 17 bifidobacteria was tested for sensitivity or resistance to lantibiotic nisin. Minimal inhibitory concentration of the strain tested was highly variable, ranging from 4·88 to 10 000 IU ml⁻¹. In general, strains isolated from faecal samples were more resistant than those purchased from culture collections. These results could be useful in the production of foods containing both bifidobacteria and nisin.     

双歧杆菌体外对Caco-2的黏附及其表面性质分析

【目的】体外测定双歧杆菌的黏附能力并对其表面性质进行分析。【方法】利用Caco-2细胞作为黏附模型体外测定七株菌的黏附能力,同时分析其自动聚集能力和表面疏水性,通过采用不同酶及化学物质处理双歧杆菌菌体细胞表面初步确定双歧杆菌细胞表面黏附相关化合物的类型,并对双歧杆菌表面蛋白进行电泳分析。【结果】自动聚集能力和表面疏水性均高的双歧杆菌菌株,其黏附能力高于自动聚集能力和表面疏水性均低的菌株,表现出明显的正相关。此外,受试菌株的黏附能力对蛋白酶和高碘酸钠敏感,利用LiCl对菌体表面蛋白进行提取后,其黏附能力明显下降,SDS-PAGE结果表明LiCl提取物中含有分子量大小不等的多个蛋白。【结论】双歧杆菌体外对Caco-2细胞的黏附具有菌株特异性,其黏附能力与表面疏水性质和自动聚集能力相关,此外,推测双歧杆菌表面可能含有能调节其黏附的糖蛋白类物质。     

Metabolism of chicory fructooligosaccharides by bifidobacteria

Two types of chicory fructooligosaccharides (Fibruline Instant and Fibrulose F97) were metabolised by Bifidobacterium longum, B. infantis and B. angulatum. Chromatographic analysis of the medium after 120 h revealed a consumption of all the fructose oligomers present in the commercial chicory fructooligosaccharide mixtures for all the strains. Maximum measurable degree of polymerisation of the substrates before fermentation was 41. The higher biomass production was reached with B. infantis (1.4 and 1.7 g dry wt l^–1) for its cultivation on medium complemented, respectively, with Fibruline Instant and Fibrulose F97 as substrate. These results give the opportunity to use chicory fructooligosaccharides as a prebiotic.     

Protective effects of bifidobacteria against enteropathogens

Recent major advances in metagenomics and metabolomics technologies have enabled us to collect more data on the gut microbiome and metabolome to evaluate its influence on host health. In this short opinion article, we have chosen to focus on summarizing the protective mechanisms of bifidobacteria, a highly regarded probiotic, and it's metabolite: acetate; against enteropathogens, specifically in the E. coli O157:H7 mice model. We advocate for using a novel approach metabologenomics, which is an integration of metagenomic and metabolomic information on a systems biology-wide approach to better understand this interplay between gut microbiome and host metabolism.     

Survival of bifidobacteria administered to calves

Twenty-five bifidobacteria were isolated from feces of calves. Isolates were identified, and their functional properties and antimicrobial activity were determined. From 10 strains with suitable properties rifampicin-resistant mutants (RRBs) were prepared and mixture of RRBs was administered to 2-d-old calves. These strains were identified by sequencing as Bifidobacterium animalis ssp. animalis (6 strains), B. thermophilum (2 strains), B. choerinum (1 strain) and B. longum ssp. suis (1 strain). The control group was without probiotic treatment. Survival ability of administered bifidobacteria was monitored in fecal samples by cultivation on modified TPY agar supplemented with mupirocin, acetic acid, and rifampicin. Administered bifidobacteria survived in gastrointestinal tract of calves for at least 60 d. Other bacteria were also determined after cultivation using fluorescence in situ hybridization (FISH). Bifidobacteria and lactobacilli dominated in fecal microflora. Significantly lower amounts of E. coli and higher amounts of bifidobacteria and total anaerobes were found in the treated group relative to the control group.     

Unique sugar metabolic pathways of bifidobacteria

Bifidobacteria have many beneficial effects for human health. The gastrointestinal tract, where natural colonization of bifidobacteria occurs, is an environment poor in nutrition and oxygen. Therefore, bifidobacteria have many unique glycosidases, transporters, and metabolic enzymes for sugar fermentation to utilize diverse carbohydrates that are not absorbed by host humans and animals. They have a unique, effective central fermentative pathway called bifid shunt. Recently, a novel metabolic pathway that utilizes both human milk oligosaccharides and host glycoconjugates was found. The galacto-N-biose/lacto-N-biose I metabolic pathway plays a key role in colonization in the infant gastrointestinal tract. These pathways involve many unique enzymes and proteins. This review focuses on their molecular mechanisms, as revealed by biochemical and crystallographic studies.     

Autoaggregation and surface hydrophobicity of bifidobacteria

Thirteen strains of four different Bifidobacterium spp. were observed for their autoaggregation ability and surface hydrophobicity, and correlation between these two traits was determined. Bifidobacteria were classified into high, medium and low autoaggregation strains according to autoaggregation ratio measured from changes in absorbance of media. High autoaggregation strains showed microscopic clustering of cells, whereas low and medium autoaggregation strains showed no such clustering. Autoaggregation ability decreased in high autoaggregation strains but increased in medium and low autoaggregation strains when the assay was performed at higher temperature (37°C compared with 25 and 10°C). Bacterial strains belonging to the high, medium or low autoaggregation group were correlated in terms of their surface hydrophobicity and evaluated based on changes in absorbance of the bacterial suspension before and after extraction with xylene. These results indicate that autoaggregation ability, together with surface hydrophobicity and microscopic image could be used for evaluating the adhesion ability of potential probiotic bifidobacterial strains. Moreover, a synergistic effect of pH and media may be involved in autoaggregation.     

Mortality of bifidobacteria in boiled yogurt

Bifidobacterial strains showed various mortalities during storage at 30°C in boiled yogurt prepared with Streptococcus sp. and Lactobacillus sp. Bifidobacterium breve 203 was most stable, maintaining its initial cell number for more than 5 days. It was also stable at 4 or 10°C in fresh yogurt. B. longum 401 rapidly lost viability in the boiled yogurt and was unstable in the fresh yogurt at any temperature. Lactobacillus sp. remained fully viable for one week or more at 4–30°C while Streptococcus sp. lost viability at 20°C or above. The difference in mortality between B. breve 203 and B. longum 401 was mainly due to their sensitivity to the acidic environment, with temperature during storage having a secondary effect. Effects of lysozyme, pepsin and bile acids on the two strains were also investigated.