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.     

Inhibitory Impact of Bifidobacteria on the Transfer of β-Lactam Resistance among Enterobacteriaceae in the Gnotobiotic Mouse Digestive Tract

While looking for new means to limit the dissemination of antibiotic resistance, we evaluated the role of potentially probiotic bifidobacteria on the transfer of resistance genes between enterobacteria. Transfers of bla genes encoding extended-spectrum β-lactamases (SHV-5 and CTX-M-15) were studied in the absence or presence of bifidobacteria. In vitro, transfer frequencies of these bla genes decreased significantly in the presence of three of five tested strains, i.e., Bifidobacterium longum CUETM-89-215, Bifidobacterium bifidum CIP-56.7T, and Bifidobacterium pseudocatenulatum CIP-104168T. Four transfer experiments were conducted in the digestive tract of gnotobiotic mice, the first three observing the effect of B. longum CUETM-89-215, B. bifidum CIP-56.7T, and B. pseudocatenulatum CIP-104168T on bla_SHV-5 transfer and the fourth experiment studying the effect of B. bifidum CIP-56.7T on bla_CTX-M-15 transfer. These experiments revealed significant decreases in the transconjugant levels (up to 3 logs) in mice having received B. bifidum CIP-56.7T or B. pseudocatenulatum CIP-104168T compared to control mice. Bifidobacteria appear to have an inhibitory impact on the transfer of antibiotic resistance genes. The inhibitory effect is associated to specific bifidobacterial strains and may be related to the production of thermostable metabolites by these strains.     

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Background

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and Principal Findings

Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions

We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.     

Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

Background

We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains.

Objective

The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells.

Results

Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells.

Conclusions

We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.     

Promotion of intestinal peristalsis by Bifidobacterium spp. capable of hydrolysing sennosides in mice

Background

While there are a variety of identifiable causes of constipation, even idiopathic constipation has different possible mechanisms. Sennosides, the main laxative constituents of Daio, an ancient Kampo medicine, are prodrugs that are converted to an active principle, rheinanthrone, by intestinal microbiota. In this study, we aimed to determine the sennoside hydrolysis ability of lactic acid bacterial strains and bifidobacteria in the intestine and to investigate their effect on intestinal peristalsis in mice.

Methodology/Principal Findings

A total of 88 lactic acid bacterial strains and 47 bifidobacterial strains were evaluated for their ability to hydrolyze sennosides. Our results revealed that 4 strains, all belonging to the genus Bifidobacterium, had strong sennoside hydrolysis ability, exhibiting a decrease of >70% of sennoside content. By thin-layer chromatography analysis, rheinanthrone was detected in the medium cultured with B. pseudocatenulatum LKM10070 and B. animalis subsp. lactis LKM512. The fecal sennoside contents significantly (P<0.001) decreased upon oral administration of these strains as compared with the control. Intestinal peristalsis activity was measured by the moved distance of the charcoal powder administered orally. The distance travelled by the charcoal powder in LKM512-treated mice was significantly longer than that of control (P<0.05). Intestinal microbiota were analysed by real-time PCR and terminal-restriction fragment length polymorphism. The diversity of the intestinal microbiota was reduced by kanamycin treatment and the diversity was not recovered by LKM512 treatment.

Conclusion/Significance

We demonstrated that intestinal peristalsis was promoted by rheinanthrone produced by hydrolysis of sennoside by strain LKM512 and LKM10070.     

Role of intestinal bacteria in gliadin-induced changes in intestinal mucosa: study in germ-free rats

Background and Aims

Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that is induced by dietary wheat gluten proteins (gliadins) in genetically predisposed individuals. The overgrowth of potentially pathogenic bacteria and infections has been suggested to contribute to CD pathogenesis. We aimed to study the effects of gliadin and various intestinal bacterial strains on mucosal barrier integrity, gliadin translocation, and cytokine production.

Methodology/Principal Findings

Changes in gut mucosa were assessed in the intestinal loops of inbred Wistar-AVN rats that were reared under germ-free conditions in the presence of various intestinal bacteria (enterobacteria and bifidobacteria isolated from CD patients and healthy children, respectively) and CD-triggering agents (gliadin and IFN-γ) by histology, scanning electron microscopy, immunofluorescence, and a rat cytokine antibody array. Adhesion of the bacterial strains to the IEC-6 rat cell line was evaluated in vitro.Gliadin fragments alone or together with the proinflammatory cytokine interferon (IFN)-γ significantly decreased the number of goblet cells in the small intestine; this effect was more pronounced in the presence of Escherichia coli CBL2 and Shigella CBD8. Shigella CBD8 and IFN-γ induced the highest mucin secretion and greatest impairment in tight junctions and, consequently, translocation of gliadin fragments into the lamina propria. Shigella CBD8 and E. coli CBL2 strongly adhered to IEC-6 epithelial cells. The number of goblet cells in small intestine increased by the simultaneous incubation of Bifidobacterium bifidum IATA-ES2 with gliadin, IFN-γ and enterobacteria. B. bifidum IATA-ES2 also enhanced the production of chemotactic factors and inhibitors of metalloproteinases, which can contribute to gut mucosal protection.

Conclusions

Our results suggest that the composition of the intestinal microbiota affects the permeability of the intestinal mucosa and, consequently, could be involved in the early stages of CD pathogenesis.     

Inter-species differences in the growth of bifidobacteria cultured on human milk oligosaccharides

Human milk (HM) contains as the third most abundant component around 200 different structures of human milk oligosaccharides (HMOs). HMOs are the first and irreplaceable prebiotics for infants, supporting bifidobacteria as the most important bacterial group in an infant intestine. The aim of our study was to test the growth of bifidobacteria in HM and on HMOs. Bifidobacteria were isolated from two groups of infants. The first one (eight strains) were isolated from infants who had bifidobacteria in their feces but, after a short period of time (4 to 24 days), bifidobacteria were no longer detected in their feces (disappeared bifidobacteria [DB]). The second group of bifidobacteria (eight strains) originated from infants with continual presence of bifidobacteria in their feces (persistent bifidobacteria [PB]). There were significant differences (p?Bifidobacterium bifidum and B. breve species were able to utilize HMOs, while B. adolescentis and B. longum subsp. longum species did not. The ability to grow in HM and to utilize HMOs seem to be important properties of bifidobacteria which are able to colonize infant intestinal tract.     

Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR

The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut.     

Identification and characterization of the serine/threonine protein kinases in Bifidobacterium

Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.     

Early colonization with a group of Lactobacilli decreases the risk for allergy at five years of age despite allergic heredity

Background

Microbial deprivation early in life can potentially influence immune mediated disease development such as allergy. The aims of this study were to investigate the influence of parental allergy on the infant gut colonization and associations between infant gut microbiota and allergic disease at five years of age.

Methods and Findings

Fecal samples were collected from 58 infants, with allergic or non-allergic parents respectively, at one and two weeks as well as at one, two and twelve months of life. DNA was extracted from the fecal samples and Real time PCR, using species-specific primers, was used for detection of Bifidobacterium (B.) adolescentis, B. breve, B. bifidum, Clostridium (C.) difficile, a group of Lactobacilli (Lactobacillus (L.) casei, L. paracasei and L. rhamnosus) as well as Staphylococcus (S.) aureus. Infants with non-allergic parents were more frequently colonized by Lactobacilli compared to infants with allergic parents (p = 0.014). However, non-allergic five-year olds acquired Lactobacilli more frequently during their first weeks of life, than their allergic counterparts, irrespectively of parental allergy (p = 0.009, p = 0.028). Further the non-allergic children were colonized with Lactobacilli on more occasions during the first two months of life (p = 0.038). Also, significantly more non-allergic children were colonized with B. bifidum at one week of age than the children allergic at five years (p = 0.048).

Conclusion

In this study we show that heredity for allergy has an impact on the gut microbiota in infants but also that early Lactobacilli (L. casei, L. paracasei, L. rhamnosus) colonization seems to decrease the risk for allergy at five years of age despite allergic heredity.     

Effect of Exposure to Air on 84 strains of Bifidobacteria

Among 84 strains of bifidobacteria tested, 62%, belonging to animal species, decreased in count by less than one log 10 unit after 4 days of contact with air at a temperature of 20°C. When 10 strains each of B. pseudolongum subsp. pseudolongum and B. thermophilum were tested at 4°C under the same conditions, eight of the former and five of the latter survived without any decline during 4 days.     

Inactivation of Adhesion and Invasion of Food-Borne Listeria monocytogenes by Bacteriocin-Producing Bifidobacterium Strains of Human Origin

Three bacteriocin-producing bifidobacterial isolates from newborns were identified as Bifidobacterium thermacidophilum (two strains) and B. thermophilum (one strain). This study was undertaken to evaluate the ability of these strains to compete with food-borne Listeria monocytogenes for adhesion and invasion sites on Caco-2 and HT-29 cells. The bifidobacteria adhered at levels ranging from 4% to 10% of the CFU added, but none of the bifidobacteria were able to invade cells. The abilities of Listeria to adhere to and to invade cells varied widely depending on the strain tested. Three groups of Listeria were identified based on invasiveness: weakly invasive, moderately invasive, and highly invasive strains. One strain from each group was tested in competition with bifidobacteria. B. thermacidophilum RBL70 was the most effective in blocking invasion of Listeria, and the decreases in invasion ranged from 38% to 90%. For all three bifidobacterial strains, contact between the cell monolayer and the bifidobacteria for 1 h before exposure to Listeria increased the degree of inhibition. Finally, visualization of competition for adhesion sites on cells by fluorescent in situ hybridization suggested that the two bacteria tended to adhere in close proximity.     

Screening for and Identification of Starch-, Amylopectin-, and Pullulan-Degrading Activities in Bifidobacterial Strains

Forty-two bifidobacterial strains were screened for α-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.     

Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

Background

In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.

Results

Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.

Conclusions

The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1476-6) contains supplementary material, which is available to authorized users.     

Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I,a Major Building Block of Human Milk Oligosaccharides,in Bifidobacterial Strains

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.Bifidobacteria are gram-positive anaerobic bacteria that naturally colonize the human intestinal tract and are believed to be beneficial to human health (21, 30). Breastfeeding has been shown to be associated with an infant fecal microbiota dominated by bifidobacteria, whereas the fecal microbiota of infants who are consuming alternative diets has been described as being mixed and adult-like (12, 21). It has been suggested that the selective growth of bifidobacteria observed in breast-fed newborns is related to the oligosaccharides and other factors that are contained in human milk (human milk oligosaccharides [HMOs]) (3, 4, 10, 11, 16, 17, 34). Kitaoka et al. (15) have recently found that bifidobacteria possess a unique metabolic pathway that is specific for lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc). LNB is a building block for the type 1 HMOs [such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and lacto-N-difucohexaose I (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc)], and GNB is a core structure of the mucin sugar that is present in the human intestine and milk (18, 27). The GNB/LNB pathway, as previously illustrated by Wada et al. (33), involves proteins/enzymes that are required for the uptake and degradation of disaccharides such as the GNB/LNB transporter (29, 32), galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP; LnpA) (15, 24) (renamed from lacto-N-biose phosphorylase after the finding of phosphorylases specific to GNB [23] and LNB [22]), N-acetylhexosamine 1-kinase (NahK) (25), UDP-glucose-hexose 1-phosphate uridylyltransferase (GalT), and UDP-galactose epimerase (GalE). Some bifidobacteria have been demonstrated to be enzymatically equipped to release LNB from HMOs that have a type 1 structure (lacto-N biosidase; LnbB) (33) or GNB from the core 1-type O-glycans in mucin glycoproteins (endo-α-N-acetylgalatosaminidase) (6, 13, 14). It has been suggested that the presence of the LnbB and GNB/LNB pathways in some bifidobacterial strains could provide a nutritional advantage for these organisms, thereby increasing their populations within the ecosystem of these breast-fed newborns (33).The species that predominantly colonize the infant intestine are the bifidobacterial species B. breve, B. longum subsp. infantis, B. longum subsp. longum, and B. bifidum (21, 28). On the other hand, strains of B. adolescentis, B. catenulatum, B. pseudocatenulatum, and B. longum subsp. longum are frequently isolated from the adult intestine (19), and strains of B. animalis subsp. animalis, B. animalis subsp. lactis, B. thermophilum and B. pseudolongum have been shown to naturally colonize the guts of animals (1, 2, 7, 8). However, it is unclear whether there is a relationship between the differential colonization of the bifidobacterial species and the presence of the GNB/LNB pathway. In the present study, we investigated the ability of individual bifidobacterial strains in the in vitro fermentation of LNB and in addition, we also tried to determine whether or not the GLNBP gene (lnpA), which is a key enzyme of the GNB/LNB pathway, was present.     

Root iron plaque formation and characteristics under N2 flushing and its effects on translocation of Zn and Cd in paddy rice seedlings (Oryza sativa)

Background and Aims

Anoxic conditions are seldom considered in root iron plaque induction of wetland plants in hydroponic experiments, but such conditions are essential for root iron plaque formation in the field. Although ferrous ion availability and root radial oxygen loss capacity are generally taken into account, neglect of anoxic conditions in root iron plaque formation might lead to an under- or over-estimate of their functional effects, such as blocking toxic metal uptake. This study hypothesized that anoxic conditions would influence root iron plaque formation characteristics and translocation of Zn and Cd by rice seedlings.

Methods

A hydroponic culture was used to grow rice seedlings and a non-disruptive approach for blocking air exchange between the atmosphere and the induction solution matrix was applied for root iron plaque formation, namely flushing the headspace of the induction solution with N₂ during root iron plaque induction. Zn and Cd were spiked into the solution after root iron plaque formation, and translocation of both metals was determined.

Key Results

Blocking air exchange between the atmosphere and the nutrient solution by N₂ flushing increased root plaque Fe content by between 11 and 77 % (average 31 %). The N₂ flushing treatment generated root iron plaques with a smoother surface than the non-N₂ flushing treatment, as observed by scanning electron microscopy, but Fe oxyhydroxides coating the rice seedling roots were amorphous. The root iron plaques sequestrated Zn and Cd and the N₂ flushing enhanced this effect by approx. 17 % for Zn and 71 % for Cd, calculated by both single and combined additions of Zn and Cd.

Conclusions

Blocking of oxygen intrusion into the nutrient solution via N₂ flushing enhanced root iron plaque formation and increased Cd and Zn sequestration in the iron plaques of rice seedlings. This study suggests that hydroponic studies that do not consider redox potential in the induction matrices might lead to an under-estimate of metal sequestration by root iron plaques of wetland plants.     

Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria

Thirty-four strains of bifidobacteria belonging to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium pseu-docatenulatum were assayed in vitro for the ability to assimilate cholesterol and for bile salt hydrolase (BSH) against glycocholic and taurodeoxycholic acids (GCA and TDCA). Cholesterol assimilation was peculiar characteristic of two strains belonging to the species B. bifidum (B. bifidum MB 107 and B. bifidum MB 109), which removed 81 and 50 mg of cholesterol per gram of biomass, being the median of specific cholesterol absorption by bifidobacteria 19 mg/g. Significant differences in BSH activities were not established among bifidobacterial species. However, the screening resulted in the selection of promising strains able to efficiently deconjugate GCA and TDCA. No relationship was recognized between BSH phenotype and the extent of cholesterol assimilation. On the basis of cholesterol assimilation or BSH_GCA and BSH_TDCA activities, B. bifidum MB 109 (DSMZ 23731), B. breve MB 113 (DSMZ 23732), and B. animalis subsp. lactis MB 2409 (DSMZ 23733) were combined in a probiotic mixture to be fed to hypercholesterolemic rats. The administration of this probiotic formulation resulted in a significant reduction of total cholesterol and low-density cholesterol (LDL-C), whereas it did not affect high-density cholesterol (HDL-C) and HDL-C/LDL-C ratio.