Comparison of intestinal microflora in healthy infants and infants with allergic colitis

Twenty-eight exclusively breast-fed healthy infants and 16 infants also exclusively breast-fed with allergic colitis (aged 85 +/- 60 and 98 +/- 58 d, respectively) were screened for differences in fecal flora. Bifidobacteria were detected in 23 healthy infants and only in 4 fecal samples of infants with allergic colitis. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. Infants with allergy colitis possessed significantly lower counts of bifidobacteria and total anaerobes and significantly higher counts of clostridia in their feces. In healthy infants, Bifidobacterium longum was the most frequently found species (54.5% of the samples), followed by B. adolescentis (20.0), B. breve (18.2), B. bifidum (16.4), B. dentium (10.9) and B. pseudocatenulatum (1.80). Bifidobacterial isolates from two babies with allergic colitis were identified as B. longum, one child from patients group contained species B. dentium and one baby B. adolescentis. Our results suggest that there are significantly lower counts of bifidobacteria in infants with allergic colitis than in healthy infants.     

Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Adhesion of Bifidobacterium spp. to human intestinal mucus

Twenty-four Bifidobacterium strains were examined for their ability to bind to immobilized human and bovine intestinal mucus glycoproteins. Each of the tested bacteria exhibited its characteristic adhesion to human and bovine fecal mucus. No significant differences were found among the taxonomic species. Among the tested bacteria, B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum and B. pseudocatenulatum adhered to human fecal mucus better than bovine fecal mucus, while the binding of B. animalis and B. lactis was not preferential. These results suggest that the mucosal adhesive properties of bifidobacteria may be a strain dependent feature, and the mucosal binding of the human bifidobacteria may be more host specific.     

Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Bifidobacterium suppresses IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells

Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro . The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6–56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41–55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis ( P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.     

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     

Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

The intestinal microflora of infants: fecal flora of infants with vitamin K deficiency

The fecal flora of 10 infants with vitamin K deficiency (VKD) and 10 healthy infants was examined. All the infants were breast-fed. In the infants with VKD, the total counts (P less than 0.01) and the numbers of bifidobacteria (P less than 0.001) were lower than in the healthy infants, whereas bacteroides, veillonella and enterococci were present in greater numbers in the feces of infants with VKD. The incidence of the Bacteroides fragilis group was higher (P less than 0.05) in the infants with VKD than in the healthy infants. A significant reduction (P less than 0.05) in Bifidobacterium breve was shown in the infants with VKD. The data emphasize the abnormal flora in infants with VKD.     

婴幼儿腹泻患者肠道菌群分布特点研究

目的研究不同年龄段腹泻患儿肠道菌群分布特点,探讨不同年龄腹泻患儿肠道菌群与疾病的关联。方法选取9例符合临床诊断标准的0~1岁婴儿腹泻患者和8例符合临床诊断标准的1~3岁幼儿腹泻患者的粪便样本,同时于健康儿童中随机选取6例粪便样本作为对照,提取各组对象粪便总DNA,采用PCR-DGGE进行菌群多样性与差异性分析。结果 0~1岁腹泻患儿肠道菌群与健康对照组相比,肠道菌群构成差异显著,条件致病菌巴黎链球菌数量显著增加。1~3岁腹泻患儿肠道菌群与健康对照组相比,条件致病菌解没食子酸链球菌、屎肠球菌数量显著增加,长双歧杆菌数量下降。结论婴幼儿腹泻患者肠道菌群的构成与健康对照组相比差异显著,该特点可作为婴幼儿腹泻早期诊断的实验依据。     

Allergic Patients with Long-Term Asthma Display Low Levels of Bifidobacterium adolescentis

Accumulated evidence suggests a relationship between specific allergic processes, such as atopic eczema in children, and an aberrant fecal microbiota. However, little is known about the complete microbiota profile of adult individuals suffering from asthma. We determined the fecal microbiota in 21 adult patients suffering allergic asthma (age 39.43 ± 10.98 years old) and compare it with the fecal microbiota of 22 healthy controls (age 39.29 ± 9.21 years old) using culture independent techniques. An Ion-Torrent 16S rRNA gene-based amplification and sequencing protocol was used to determine the fecal microbiota profile of the individuals. Sequence microbiota analysis showed that the microbial alpha-diversity was not significantly different between healthy and allergic individuals and no clear clustering of the samples was obtained using an unsupervised principal component analysis. However, the analysis of specific bacterial groups allowed us to detect significantly lower levels of bifidobacteria in patients with long-term asthma. Also, in allergic individuals the Bifidobacterium adolescentis species prevailed within the bifidobacterial population. The reduction in the levels on bifidobacteria in patients with long-term asthma suggests a new target in allergy research and opens possibilities for the therapeutic modulation of the gut microbiota in this group of patients.     

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10⁶ to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10⁶ cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Comparison of compositions and metabolic activities of fecal microbiotas in young adults and in antibiotic-treated and non-antibiotic-treated elderly subjects

The colonic microbiota mediates many cellular and molecular events in the host that are important to health. These processes can be affected in the elderly, because in some individuals, the composition and metabolic activities of the microbiota change with age. Detailed characterizations of the major groups of fecal bacteria in healthy young adults, in healthy elderly people, and in hospitalized elderly patients receiving antibiotics were made in this study, together with measurements of their metabolic activities, by analysis of fecal organic acid and ammonia concentrations. The results showed that total anaerobe numbers remained relatively constant in old people; however, individual bacterial genera changed markedly with age. Reductions in numbers of bacteroides and bifidobacteria in both elderly groups were accompanied by reduced species diversity. Bifidobacterial populations in particular showed marked variations in the dominant species, with Bifidobacterium angulatum and Bifidobacterium adolescentis being frequently isolated from the elderly and Bifidobacterium longum, Bifidobacterium catenulatum, Bifidobacterium boum, and Bifidobacterium infantis being detected only from the healthy young volunteers. Reductions in amylolytic activities of bacterial isolates in healthy elderly subjects and reduced short-chain fatty acid concentrations supported these findings, since bifidobacteria and bacteroides are important saccharolytic groups in the colon. Conversely, higher numbers of proteolytic bacteria were observed with feces samples from the antibiotic-treated elderly group, which were also associated with increased proteolytic species diversity (fusobacteria, clostridia, and propionibacteria). Other differences in the intestinal ecosystem in elderly subjects were observed, with alterations in the dominant clostridial species in combination with greater numbers of facultative anaerobes.     

Mother-to-Infant Transmission of Intestinal Bifidobacterial Strains Has an Impact on the Early Development of Vaginally Delivered Infant's Microbiota

Objectives

Bifidobacterium species are one of the major components of the infant''s intestine microbiota. Colonization with bifidobacteria in early infancy is suggested to be important for health in later life. However, information remains limited regarding the source of these microbes. Here, we investigated whether specific strains of bifidobacteria in the maternal intestinal flora are transmitted to their infant''s intestine.

Materials and Methods

Fecal samples were collected from healthy 17 mother and infant pairs (Vaginal delivery: 12; Cesarean section delivery: 5). Mother''s feces were collected twice before delivery. Infant''s feces were collected at 0 (meconium), 3, 7, 30, 90 days after birth. Bifidobacteria isolated from feces were genotyped by multilocus sequencing typing, and the transitions of bifidobacteria counts in infant''s feces were analyzed by quantitative real-time PCR.

Results

Stains belonging to Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacterium longum subsp. longum, and Bifidobacterium pseudocatenulatum, were identified to be monophyletic between mother''s and infant''s intestine. Eleven out of 12 vaginal delivered infants carried at least one monophyletic strain. The bifidobacterial counts of the species to which the monophyletic strains belong, increased predominantly in the infant''s intestine within 3 days after birth. Among infants delivered by C-section, monophyletic strains were not observed. Moreover, the bifidobacterial counts were significantly lower than the vaginal delivered infants until 7 days of age.

Conclusions

Among infants born vaginally, several Bifidobacterium strains transmit from the mother and colonize the infant''s intestine shortly after birth. Our data suggest that the mother''s intestine is an important source for the vaginal delivered infant''s intestinal microbiota.     

Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples.

Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.     

Distribution of bifidobacteria in the gastrointestinal tract of calves

Development of gastrointestinal microflora of calves with special reference to bifidobacteria was investigated; fecal bacteria were enumerated in calves aged 3 days to 7 weeks. Bacteria were detected by using selective media, bifidobacteria using modified TPY agar with an addition of mupirocin and acetic acid and by fluorescence in situ hybridization (FISH). Bifidobacteria were dominant group of fecal flora of calves after 7 d of life, constituting 10 % of total bacterial counts. The highest bacterial concentrations were observed in rumen, cecum, and colon, the lowest in abomasum and duodenum. Bifidobacteria and lactobacilli exhibited the highest survival ability during stomach passage and dominated in all parts of the digestive tract. Bifidobacteria counts determined by FISH were significantly higher than those provided by cultivation. Modified TPY agar was highly selective and suitable for bifidobacteria isolation but FISH was shown to be a more precise method for their enumeration. Our results show that gastrointestinal microflora of calves in the milk-feeding period is similar to breast-fed infants with respect to the occurrence of bifidobacteria as a dominant bacterial group. The use of Bifidobacterium strains offers a promising way for providing beneficial effectors for calves in the milk-feeding period.     

Fecal microflora of Greek healthy neonates

This study aimed to explore, in our geographical region, the development of intestinal microflora and the colonization patterns of lactic acid bacteria and bifidobacteria during the first three months of life and to investigate the effect of the mode of delivery. Fecal specimens from 82 healthy, full-term infants were collected prospectively 4, 30 and 90 days after delivery and subcultured on nonselective and selective media. Identification of isolates was performed by microbiological and molecular methods. For the delivery effect, two groups of vaginally or caesarean-delivered exclusively breast-fed infants were studied. Despite the early high total counts of aerobes and anaerobes, colonization of lactobacilli and bifidobacteria was overall limited until 3 months of age. Furthermore, caesarean-delivered infants were less often colonized with lactobacilli at day 4 (4% vs. 59%, p = 0.000) and with bifidobacteria at day 4 (0% vs. 23%, p = 0.015) and 30 (0% vs. 35%, p = 0.042) compared to vaginally delivered ones. No bacterial populations differences were detected to compare colonized infants. Identification results indicated the predominance of Lactobacillus rhamnosus, Lactobacillus johnsonii and Lactobacillus paracasei species in neonatal gut microflora up to the first month of life and diversity of Lactobacillus species in vaginally delivered, colonized newborns, at fourth day. Furthermore, Bifidobacterium longum and Bifidobacterium breve were the most frequently detected Bifidobacterium species in vaginally delivered, breast-fed infants. In conclusion our study revealed a restricted colonization pattern of lactic acid bacteria in Greek infants and a delay in the development of Lactobacillus and Bifidobacterium spp. microbiota after caesarean section. Further analysis of potential consequences of these findings is required.     

Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups

The number of bifidobacteria in faeces and intestinal contents has been shown to be reduced with increasing age of the subject. The adhesion of four Bifidobacterium strains was tested to mucus isolated from subjects of different age. All strains bound significantly less to mucus isolated from elderly subjects, compared to mucus from the other age groups. Two of the tested strains also showed decreased adhesion to mucus isolated from 6-month-old and adult subjects compared to the adhesion to mucus from 2-month-old subjects. The results suggest that reduced adhesion may be a factor involved in the decreasing colonisation of elderly subjects by bifidobacteria.     

Comparison of Compositions and Metabolic Activities of Fecal Microbiotas in Young Adults and in Antibiotic-Treated and Non-Antibiotic-Treated Elderly Subjects

The colonic microbiota mediates many cellular and molecular events in the host that are important to health. These processes can be affected in the elderly, because in some individuals, the composition and metabolic activities of the microbiota change with age. Detailed characterizations of the major groups of fecal bacteria in healthy young adults, in healthy elderly people, and in hospitalized elderly patients receiving antibiotics were made in this study, together with measurements of their metabolic activities, by analysis of fecal organic acid and ammonia concentrations. The results showed that total anaerobe numbers remained relatively constant in old people; however, individual bacterial genera changed markedly with age. Reductions in numbers of bacteroides and bifidobacteria in both elderly groups were accompanied by reduced species diversity. Bifidobacterial populations in particular showed marked variations in the dominant species, with Bifidobacterium angulatum and Bifidobacterium adolescentis being frequently isolated from the elderly and Bifidobacterium longum, Bifidobacterium catenulatum, Bifidobacterium boum, and Bifidobacterium infantis being detected only from the healthy young volunteers. Reductions in amylolytic activities of bacterial isolates in healthy elderly subjects and reduced short-chain fatty acid concentrations supported these findings, since bifidobacteria and bacteroides are important saccharolytic groups in the colon. Conversely, higher numbers of proteolytic bacteria were observed with feces samples from the antibiotic-treated elderly group, which were also associated with increased proteolytic species diversity (fusobacteria, clostridia, and propionibacteria). Other differences in the intestinal ecosystem in elderly subjects were observed, with alterations in the dominant clostridial species in combination with greater numbers of facultative anaerobes.     

Enumeration of bifidobacteria in gastrointestinal samples from piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.