Screening of exopolysaccharide-producing Lactobacillus and Bifidobacterium strains isolated from the human intestinal microbiota

Using phenotypic approaches, we have detected that 17% of human intestinal Lactobacillus and Bifidobacterium strains could be exopolysaccharide (EPS) producers. However, PCR techniques showed that only 7% harbored genes related to the synthesis of heteropolysaccharides. This is the first work to screen the human intestinal ecosystem for the detection of EPS-producing strains.     

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.     

双歧杆菌与乳杆菌原生质体的融合及筛选

目的：通过双歧杆菌与乳杆菌的原生质体融合构建耐氧双岐杆菌,以解决双岐杆菌制剂开发中始终存在的“活菌数低”和存活时间短等问题。方法：采用不同浓度的Mutanolysin分别制备长岐杆菌和保加利亚乳杆菌的原生质体,在电场作用下诱导长双岐杆菌原生质体和经56℃热灭活30min的保加利亚乳杆菌原生质体融合,并在双层再生培养基上筛选融合菌株。结果：成功地构建出几种较为稳定的兼具长双岐杆菌和保加利亚乳杆菌生物学特性的融合菌株;其中融合株F2具有良好的耐氧性;且能在有氧、CO2条件下良好生长。结论：通过原生质体融合技术能改良双歧杆菌的严格厌氧特性,有利于对双岐杆菌的开发和应用。     

Bifidobacterium and Lactobacillus DNA in the human placenta

Aims:  Bifidobacteria and lactobacilli are part of the human normal intestinal microbiota and may possibly be transferred to the placenta. It was hypothesized that intestinal bacteria or their components are present in the placenta and that the foetus may be exposed to them. We investigated the presence of bifidobacteria and lactobacilli and their DNA in the human placenta.
Methods and Results:  We studied 34 human placentae (25 vaginal and nine caesarean deliveries) for the presence Bifidobacterium spp. and Lactobacillus rhamnosus. Cultivation was used for the detection of viable cells and genus and species-specific PCR for the detection of DNA. No bifidobacteria or lactobacilli were found by cultivation. Bifidobacterial DNA was detected in 33 and L. rhamnosus DNA in 31 placenta samples.
Conclusions:  DNA from intestinal bacteria was found in most placenta samples. The results suggest that horizontal transfer of bacterial DNA from mother to foetus may occur via placenta.
Significance and Impact of the Study:  Bacterial DNA contains unmethylated CpG oligodeoxynucleotide motifs which induce immune effects. Specific CpG motifs activate Toll-like receptor 9 and subsequently trigger Th-1-type immune responses. Although the newborn infant is considered immunologically immature, exposure by bacterial DNA may programme the infant's immune development during foetal life earlier than previously considered.     

Bifidobacterium microbiota and parameters of immune function in elderly subjects

Faecal and serum samples were collected over a period of 6 months from 55 institutionalized elderly subjects, who were enrolled in a double-blind placebo-controlled study. Participants were randomized in one of the three treatment groups: intervention (two probiotic Bifidobacterium longum strains: 2C and 46), placebo and commercial control (Bifidobacterium lactis Bb-12). The faecal Bifidobacterium microbiota was characterized by genus and species-specific PCR. Serum levels of the cytokines IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 were determined by enzyme-linked immunosorbent assay. Each participant harboured on average approximately three different bifidobacterial species. The most frequently detected species were B. longum, Bifidobacterium adolescentis and Bifidobacterium bifidum. Depending on the treatment, the intervention resulted in specific changes in the levels of certain Bifidobacterium species, and positive correlations were found between the different species. Negative correlations were observed between the levels of Bifidobacterium species and the pro-inflammatory cytokine TNF-alpha and the regulatory cytokine IL-10. The presence of faecal B. longum and Bifidobacterium animalis correlated with reduced serum IL-10. The anti-inflammatory TGF-beta1 levels were increased over time in all three groups, and the presence of Bifidobacterium breve correlated with higher serum TGF-beta1 levels. This indicates that modulation of the faecal Bifidobacterium microbiota may provide a means of influencing inflammatory responses.     

Experience in early infancy is indispensable for color perception

Early visual experience is indispensable to shape the maturation of cortical circuits during development. Monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception. It was tested whether or not early experience is also necessary for color perception. Infant monkeys were reared for nearly a year in a separate room where the illumination came from only monochromatic lights. After extensive training, they were able to perform color matching. But, their judgment of color similarity was quite different from that of normal animals. Furthermore, they had severe deficits in color constancy; their color vision was very much wavelength dominated, so they could not compensate for the changes in wavelength composition. These results indicate that early visual experience is also indispensable for normal color perception.     

Fatty acid composition of Bifidobacterium and Lactobacillus strains

Normal C(14), C(16), and C(18) saturated acids and C(16) and C(18) monoenoic acids are the main fatty acids of nine strains of Bifidobacterium. Their lactobacillic acid content was less than 5%. Lactobacillus strains contained the same fatty acids as main compounds except for octadecanoic acid, which was present only in very low amounts. Eight of nine Lactobacillus strains contained in the stationary phase more than 15% lactobacillic acid. No correlation was observed between the fatty acid composition and other physiological characteristics used in the literature for classification of strains of one genus. Aging of the culture, which involved a decrease of the pH, caused a lengthening of the chain length of the fatty acids of B. bifidum var. pennsylvanicus but only a conversion of octadecenoic to lactobacillic acid in the lactobacilli. Lowering of the temperature of cultivation decreased the chain length of the fatty acids of B. bifidum var. pennsylvanicus. L. lactis did not show any influence of the temperature on the chain length of the fatty acids. The percentage of unsaturated acids was temperature independent in both organisms.     

In vitro fermentation of rice bran combined with Lactobacillus acidophilus 14 150B or Bifidobacterium longum 05 by the canine faecal microbiota

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.     

金双歧辅助治疗小儿扁桃体炎的临床研究

目的探讨金双歧(双歧杆菌乳杆菌三联活菌片)辅助治疗小儿扁桃体炎的疗效及安全性。方法纳入2013年1月至2014年7月在崇州市人民医院儿科住院诊断为扁桃体炎患儿337例,用随机数字表随机分为试验组和对照组。试验组在常规给予抗生素和对症治疗的基础上,给予金双歧口服辅助治疗,出院后继续服用4周;对照组常规给予抗生素和对症治疗。观察并比较两组患儿的临床效果,住院期间发生抗生素相关性腹泻的差异。出院后3个月内定期随访,观察两组患儿出院后3个月内发生呼吸道感染和肠道感染的差异。结果试验组治疗后总有效率为98.00%,对照组治疗后总有效率为93.33%。试验组总有效率明显优于对照组,差异有统计学意义(χ2=3.940,P=0.0470.05)。住院期间试验组抗生素相关性腹泻发生率为8.67%,对照组为19.33%,差异有统计学意义(t=7.087,P=0.0080.01)。出院后3个月内呼吸道感染以及肠道感染的发生率试验组低于对照组,差异有统计学意义(1个月肠道感染χ2=4.624,呼吸道感染χ2=8.549;2个月肠道感染χ2=5.684,呼吸道感染χ2=11.712;3个月肠道感染χ2=7.545,呼吸道感染χ2=4.559,P0.05)。试验组服用金双歧期间,未见明显药物相关不良反应。结论金双歧辅助治疗小儿扁桃体炎可提高临床疗效,降低抗生素相关性腹泻的发生率,降低扁桃体炎患儿出院3个月内呼吸道和肠道感染的发生率,且服用安全。     

Effects of Lactobacillus amylovorus and Bifidobacterium breve on cholesterol

To determine the validity of the hypothesis of assimilation and/or precipitation of cholesterol by Lactobacillus and Bifidobacterium species, culture were undertaken in TPY medium containing oxgall or taurocholic acid. In the case of growing cells, both strains were able to remove cholesterol in the presence of bile salts. Nevertheless, the behaviour was different according to the kind of bile salt. In the presence of taurocholic acid, the removal of cholesterol was due to both bacterial uptake and precipitation. In the presence of Oxgall, bacterial uptake and precipitation were observed for Lactobacillus but only precipitation occurred for Bifidobacterium.     

Biphasic assembly of the murine intestinal microbiota during early development

The birth canal provides mammals with a primary maternal inoculum, which develops into distinctive body site-specific microbial communities post-natally. We characterized the distal gut microbiota from birth to weaning in mice. One-day-old mice had colonic microbiota that resembled maternal vaginal communities, but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of Lactobacillus. By weaning (21 days), diverse intestinal bacteria had established, including strict anaerobes. Our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from Streptococcus to Lactobacillus, followed by an increase in diversity of anaerobes, after the introduction of solid food. Mammalian newborns are born highly susceptible to colonization, and lactation may control microbiome assembly during early development.     

Alterations of the gut microbiota in high-fat diet mice is strongly linked to oxidative stress

Alterations of the gut microbiota induced by diet exert a strong influence on the development of metabolic syndrome. In this study, we prove the hypothesis that the long-term high-fat diet (HFD) may influence gut microbiota directly and/or indirectly by changing the redox state. Lipoic acid (LA), as a universal antioxidant, was used to improve the redox state. Reactive oxygen species (ROS), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were analyzed to profile oxidative stress states. PCR-denaturing gradient gel electrophoresis (DGGE) was used to describe gut flora structures, while plate count was employed for the quantitative analysis of Escherichia coli, lactobacilli, and enterococcus. The influence of redox state on the vitality of gut-derived bacteria was measured in vitro. ROS and MDA, which significantly decreased in LA mice compared with HFD mice, showed a strong positive association with E. coli and enterococcus (P?<?0.05) and a negative association with lactobacilli (P?<?0.05). Increased T-AOC in LA mice showed a high positive association with lactobacilli (P?<?0.05) and a negative correlation with E. coli and enterococcus. These correlations implied that the dietary effects on the gut microbiota were conferred, at least in part, through an effect on oxidative stress. This study provides evidence that modulation of the redox state by an antioxidant has the potential to improve gut microbiota, which has relevance for metabolic health.     

Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.     

Comparison of the Phospholipid Composition of Bifidobacterium and Lactobacillus Strains

Phospholipid composition of 10 Bifidobacterium strains of human intestinal origin and of 9 Lactobacillus strains was determined by quantitative two-dimensional thin-layer chromatography. Phospholipids of three Bifidobacterium strains from honey bees and of two strains from bovine rumen liquor were qualitatively investigated. Diphosphatidylglycerol and phosphatidylglycerol were present in strains of both genera. All Bifidobacterium strains contained as specific phospholipids a new polyglycerolphospholipid, compound 15, and its lyso derivatives, earlier detected in B. bifidum var. pennsylvanicus. Also, lyso compounds of diphosphatidylglycerol and alanyl phosphatidylglycerol were only present in this genus in variable amounts. Lysyl phosphatidylglycerol was the only ninhydrin-positive phospholipid in seven Lactobacillus strains. In L. delbrückii and L. helveticus it was absent and partially replaced by an unidentified ninhydrin-negative phospholipid. The differences in phospholipid composition between bifidobacteria and lactobacilli may be another argument to differentiate these two genera.     

Distribution of salivary Lactobacillus and Bifidobacterium species in periodontal health and disease

We surveyed the distribution of salivary Lactobacillus and Bifidobacterium species in periodontitis patients and healthy subjects. Approximately 700 lactobacilli and 300 bifidobacterial isolates were obtained from 16 young, orally healthy subjects (mean age +/- standard deviation: 21.0+/-2.0 y), 16 periodontitis patients (51.6+/-13.8 y), and 14 well-maintained former periodontitis patients (60.2+/-9.6 y). Among eleven Lactobacillus species detected in saliva, L. salivarius, L. gasseri, and L. fermentum were prevalent, but no species was specifically associated with periodontal health. In contrast, of four Bifidobacterium species, B. adolescentis was specifically (P<0.05) prevalent in young healthy subjects compared with the other two groups. Furthermore, the bifidobacterial count of the well-maintained subjects was the highest (P<0.05) among the groups. These results suggest that bifidobacterial count and species might be associated with periodontal health status and/or age.     

儿童肠道双歧杆菌和乳杆菌种群结构分析

以21例2～5岁中国儿童的肠道菌群为研究对象,利用传统培养计数法和分子生物学技术,对此年龄段健康儿童的肠道菌群分布及其中关键益生菌的种群结构进行了定量研究。实验表明,儿童肠道厌氧菌的数量高达109CFUg(湿重),其肠道菌群的定植抗力(平均BE=2.38)较强;不同的个体之间所能检测到的关键益生菌的种类有所不同,一般能检测到其中的1～4种双歧杆菌和1～5种乳杆菌;长双歧杆菌和假小链双歧杆菌的平均数量多达107CFUg(湿重),检出率分别为90.48%和85.71%,为儿童肠道内双歧杆菌的优势菌种;L.mucosae和发酵乳杆菌的数量较多,平均为3.68log10CFUg(湿重)和3.97log10CFUg(湿重),检出率分别为71.43%和52.38%,为稳定定植于儿童肠道内的优势乳杆菌;不同种类的益生菌在不同样本之间的数量组成均存在有很大差异,双歧杆菌的样本差异为1.86～3.85,乳杆菌的为2.43～4.07。     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.