C-section and the Neonatal Gut Microbiome Acquisition: Consequences for Future Health.

BACKGROUND: The human gut microbiota is assembled during infancy with an increase in diversity and stability. The correct colonization and the establishment of this microbiome are linked to the early and future health status of the individual. It is known that caesarean delivery alters this optimal microbial foundation. C-section (CS) is a common obstetrician surgery; however, it is not without risk for the mother/infant dyad. The World Health Organization recommends not exceeding 10-15% of the total deliveries; nevertheless, this rate has been increasing rapidly worldwide in the last decades. SUMMARY: This review discloses the clinical parameters for correct CS recommendation. Moreover, the major microbial changes in the infant gut microbiome acquisition as a consequence of delivery mode and medical practices surrounding it, as well as, the early and long-lasting effects for both mother and babies are discussed. In addition, some strategies for the gut microbiota restoration are analysed. The aim of this review is to show the need for the development of strategies for minimizing or limiting the impact of caesarean on the microbiome development, favouring future health.

Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG.

AIMS: The purpose of this study was to investigate the heat-shock response at molecular level in Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12 and their heat-tolerant derivatives and to characterize the changes that make the derivatives more robust in terms of heat stress. METHODS AND RESULTS: The study strains were exposed for 2 h to a heat-shock treatment, Bif. animalis subsp. lactis BB-12 and its derivative at 50°C and the Lact. rhamnosus GG and its derivative at 60°C. Protein synthesis before and after heat shock was examined using proteomics and RT-qPCR. The analysis revealed that the regulation of seven proteins in both strain pairs was modified as a response to heat or between the original and the derivative strain. The comparison of wild-type strains and the heat-tolerant derivatives suggests that the acquisition of heat tolerance in the Bif. animalis subsp. lactis BB-12 derivative is due to a slightly increased constitutive level of chaperones, while in Lact. rhamnosus GG derivative, the main reason seems to be a higher ability to induce the production of chaperones. CONCLUSIONS: This study revealed possible markers of heat tolerance in B. lactis and Lact. rhamnosus strains. SIGNIFICANCE AND IMPACT OF STUDY: This study increases our knowledge on how Lactobacillus and Bifidobacterium strains may acquire heat tolerance. These findings may be useful for improving the heat tolerance of existing probiotic strains as well as screening new heat-tolerant strains.

Effect of inulin-type fructans and galactooligosaccharides on cultures of Lactobacillus strains isolated in Algeria from camel's milk and human colostrum.

Bacteria from the genus Lactobacillus are responsible for spontaneous food fermentations. Some species, such as Lactobacillus casei and Lactobacillus brevis, have the "Qualified Presumption of Safety" status recognized by the European Food Safety Authority. Several of their strains are used as probiotics in foods and sometimes are included in synbiotic combinations together with prebiotics. New microbial strains isolated from different sources represent an opportunity to use them for the production of traditional food products. The capacity of three selected strains (one isolated from Camel's milk and identified by partial 16 S rRNA gene sequencing as L. brevis, and two isolated from human colostrum and identified as L. paracasei/L. casei and L. brevis, respectively) was assessed in vitro for the ability to survive in gastrointestinal conditions (low pH and high bile salts concentrations). We also tested the capacity of growth and the production of organic acids and volatile compounds by high-performance liquid chromatography and gas chromatography, respectively, when these bacteria were incubated anaerobically in the presence of inulin, fructooligosaccharides, or galactooligosaccharides as the main carbon sources. The strains were able to survive in simulated gastrointestinal conditions and to grow in inulin, fructooligosaccharides, and galactooligosaccharides. However, they displayed different profiles of organic acids and volatile compounds, mainly depending on the microbial species and the prebiotic used. The influence that the combined use of strains and different prebiotics could exert on the organic acids and volatiles formed in food and in the gut should be assessed for each synbiotic combination and food product.

Establishment and development of lactic acid bacteria and bifidobacteria microbiota in breast-milk and the infant gut.

The initial establishment of lactic acid bacteria (LAB) and bifidobacteria in the newborn and the role of breast-milk as a source of these microorganisms are not yet well understood. The establishment of these microorganisms during the first 3 months of life in 20 vaginally delivered breast-fed full-term infants, and the presence of viable Bifidobacterium in the corresponding breast-milk samples was evaluated. In 1 day-old newborns Enterococcus and Streptococcus were the microorganisms most frequently isolated, from 10 days of age until 3 months bifidobacteria become the predominant group. In breast-milk, Streptococcus was the genus most frequently isolated and Lactobacillus and Bifidobacterium were also obtained. Breast-milk contains viable lactobacilli and bifidobacteria that might contribute to the initial establishment of the microbiota in the newborn.

Fermented Dairy Foods: Impact on Intestinal Microbiota and Health-Linked Biomarkers.

The intake of fermented foods is gaining increasing interest due to their health-promoting benefits. Among them, fermented dairy foods have been associated with obesity prevention, and reduction of the risk of metabolic disorders and immune-related pathologies. Fermented foods could lead to these health benefits by providing the consumer with both easily metabolizable nutrients and beneficial microorganisms. Our aim was to evaluate the relationship between the consumption of fermented dairy products and the intestinal microbiota, serum lipid profile, and the pro-oxidant/inflammatory status. 130 healthy adults were evaluated. Dietary fermented food intake was assessed by an annual food frequency questionnaire (FFQ), including 26 fermented dairy products. Levels of the major phylogenetic types of the intestinal microbiota were determined by qPCR, and concentration of fecal short chain fatty acids were assessed by gas chromatography. Serum glucose and lipid profile, as well as serum malondialdehyde (MDA), C-reactive protein (CRP), and leptin levels were determined by standardized protocols. Among fermented dairy foods, natural yogurt, sweetened yogurt and matured/semi-matured cheese were the most consumed. While natural yogurt consumers showed increased fecal levels of Akkermansia with respect to non-consumers, sweetened yogurt intake was associated to lower levels of Bacteroides. Serum levels of CRP were also significantly reduced in yogurt consumers. Our results underline the interest in exploring the potential effects of the different yogurt types and the role the microbiota may play in such effects.

Impact of probiotics on development and behaviour in Drosophila melanogaster - a potential in vivo model to assess probiotics.

In vitro models are frequently used in probiotic research. However, such models often fail to predict in vivo functionality and efficacy. This fact complicates the screening process for selecting the most suitable strains, prior to accomplish expensive animal studies and clinical intervention trials. Therefore, additional sensitive, discriminating and cost-effective models are needed to conduct preliminary assays before undertaking human intervention studies definitely proving efficacy. With this purpose in mind, we explored the potential of axenic Drosophila melanogaster populations as well as of these axenic flies treated with probiotic microbial strains as a model to test the effects of probiotics on a subset of developmental and behavioural traits. An axenic D. melanogaster progeny from the wild-type Canton S strain was obtained and its eggs were further developed until pupae eclosion occurred in growth medium containing either of two probiotic strains: Bifidobacterium animalis subsp. lactis Bb12 or Lactobacillus rhamnosus GG. Whereas B. animalis Bb12 colonised the flies, the capacity of L. rhamnosus LGG to colonise was considerably lower in our experimental conditions. Regarding the influence of microbial load on the flies' development, the axenic condition caused a decrease in egg survival, and lowered adults' average weight with respect to wild-type flies. Both probiotics were able to counteract these effects. An earlier emergence of adults was observed from eggs treated with L. rhamnosus GG in comparison to the other fly populations. The axenic condition did not influence negative geotaxis behaviour in Drosophila; however, flies mono-associated with B. animalis Bb12 moved faster than wild-type. Our results suggest that the use of axenic/probiotic-treated D. melanogaster populations may be an affordable model for preliminary testing of the effects of probiotics on developmental or behavioural aspects.

Degradation of 16S rRNA and attributes of viability of viable but nonculturable probiotic bacteria.

AIMS: To assess the stability of 16S rRNA of viable but nonculturable (VBNC) probiotics during storage when compared with different attributes of viability. METHODS AND RESULTS: Levels of RNA of the probiotic strains Bifidobacterium longum 46, B. longum 2C and B. animalis subsp. lactis Bb-12 were monitored during storage in fermented and nonfermented foods. Cells which gradually lost their culturability in fermented products retained high level of rRNA, whereas rRNA of acid-killed control cells decreased at faster rate. Furthermore, the viability of B. longum 2C was monitored during storage by measuring changes in reductase activity, cytoplasmic membrane integrity and esterase activity using a flow cytometer. All of the culture-independent viability assays suggested that the cells remained viable during storage. In nonfermented media, the observed losses in culturability were smaller, and the changes in cell counts were comparable with the changes in rRNA levels. CONCLUSIONS: Viable but nonculturable probiotics maintain high levels of rRNA and retain properties of viable bacteria including reductase activity. Quantification of 16S rRNA complements culture-independent viability assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Culture-independent viability assays allow the detection of VBNC probiotics, and can be used parallel to conventional culture-dependent methods to obtain accurate information on probiotic viability.

Real-time monitoring of HT29 epithelial cells as an in vitro model for assessing functional differences among intestinal microbiotas from different human population groups.

Several in vitro screening tests have been used for selecting probiotic strains; however they often show low predictive value and only a limited number of strains have demonstrated functionality in vivo. The most used in vitro tests represent a very simplified version of the gut environment, especially since they do not consider the accompanying microbiota. Therefore, there is a need to develop sensitive and discriminating in vitro models including the microbiota. Here we developed an in vitro model to discriminate among microbiotas/fecal waters from different population groups. To this end samples were obtained from seven healthy adults, five IBD-patients, ten full-term and ten preterm newborns. Fecal microbiotas were purified and their impact, as well as that of the fecal waters, on HT29 cells was continuously monitored for 22 h using a real-time cell analyzer (RTCA). The composition of the purified microbiotas was assessed by 16S rRNA gene profiling and qPCR and the levels of short chain fatty acids (SCFA) determined by gas chromatography. The microbiota fractions and SCFA concentrations obtained from IBD-patients, full-term and preterm babies, showed clear differences with regard to those of the control group (healthy adults). Moreover, the purified intestinal microbiotas and fecal waters also differed from the control group in the response induced on the HT29 cells assay developed. In short, we have developed a real-time, impedance-based in vitro model for assessing the functional response induced by purified microbiotas and fecal waters upon intestinal epithelial cells. The capability of the assay for discriminating the functional responses induced, by microbiotas or fecal waters from different human groups, promises to be of help on the search for compounds/strains to restore the functionality of the microbiota-host's interaction.

Maternal breast-milk and intestinal bifidobacteria guide the compositional development of the Bifidobacterium microbiota in infants at risk of allergic disease.

BACKGROUND: The sources and the impact of maternal bacteria on the initial inoculum of the intestinal microflora of newborn infants remain elusive. OBJECTIVE: To assess the association between maternal breast-milk and fecal bifidobacteria and infants' fecal bifidobacteria. METHODS: Sixty-one mother-infant pairs were included, special emphasis being placed on the maternal allergic status. Bifidobacteria were analysed by a direct PCR method in fecal samples from mothers at 30-35 weeks of gestation and from infants at 1 month of age and from breast-milk samples 1 month post-partum. RESULTS: Fecal Bifidobacterium adolescentis and Bifidobacterium bifidum colonization frequencies and counts among mother-infant pairs correlated significantly (P=0.005 and 0.02 for frequencies, respectively, and P=0.002 and 0.01 for counts, respectively). Only infants of allergic, atopic mothers were colonized with B. adolescentis. Each of the breast-milk samples contained bifidobacteria [median 1.4 x 10(3) bacterial cells/mL; interquartile range (IQR) 48.7-3.8 x 10(3)]. Bifidobacterium longum was the most frequently detected species in breast-milk. Allergic mothers had significantly lower amounts of bifidobacteria in breast-milk compared with non-allergic mothers [median 1.3 x 10(3) bacterial cells/mL (IQR 22.4-3.0 x 10(3)) vs. 5.6 x 10(3) bacterial cells/mL (1.8 x 10(3)-1.8 x 10(4)), respectively, (P=0.004)], and their infants had concurrently lower counts of bifidobacteria in feces [3.9 x 10(8) bacterial cells/g (IQR 6.5 x 10(6)-1.5 x 10(9)) in infants of allergic mothers, vs. 2.5 x 10(9) bacterial cells/g (6.5 x 10(8)-3.2 x 10(10)) in infants of non-allergic mothers, P=0.013]. CONCLUSIONS: Breast-milk contains significant numbers of bifidobacteria and the maternal allergic status further deranges the counts of bifidobacteria in breast-milk. Maternal fecal and breast-milk bifidobacterial counts impacted on the infants' fecal Bifidobacterium levels. Breast-milk bacteria should thus be considered an important source of bacteria in the establishment of infantile intestinal microbiota.

Short communication: effect of exopolysaccharide isolated from "viili" on the adhesion of probiotics and pathogens to intestinal mucus.

The strong ropy character of the Scandinavian fermented milk viili is conferred by the exopolysaccharides (EPS) produced by lactococcal strains. These biopolymers can be responsible for some health benefits. We have assessed the influence of the EPS fraction isolated from commercial viili on the adhesion of some probiotics and pathogens to human intestinal mucus. Concentrations of viili EPS greater than 0.1 mg/mL promoted a decrease in adherence of Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus GG and this effect was dose-dependent. However, no modifications were detected on the adhesion levels of the pathogenic strains tested at a concentration of 1 mg/mL of EPS. Results obtained in the present work should be considered in the design of new probiotic products.

Glucolytic fingerprinting reveals metabolic groups within the genus Bifidobacterium: an exploratory study.

Microorganisms of the genus Bifidobacterium are inhabitants of diverse niches including the digestive tract of humans and animals. The species Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum have qualified presumption of safety status granted by EFSA and several strains are considered probiotic, and are being included in functional dairy fermented products. In the present work we carried out a preliminary exploration of general metabolic characteristics and organic acid production profiles of a reduced number of strains selected from these and other species of the genus Bifidobacterium. The use of resting cells allowed obtaining metabolic fingerprints without interference of metabolites accumulated during growth in culture media. Acetic acid was the most abundant organic acid formed per mol of glucose consumed (from 1.07 ± 0.03 to 1.71 ± 0.22 mol) followed by lactic acid (from 0.34 ± 0.06 to 0.90 ± 0.12 mol), with moderate differences in production among strains; pyruvic, succinic and formic acids were also produced at considerably lower proportions, with variability among strains. The acetic to lactic acid ratio showed lower values in stationary phase as regard to the exponential phase for most, but not all, the microorganisms; this was due to a decrease in acetic acid molar proportions together with increases of lactic acid proportions in stationary phase. A linear discriminant analysis allowed to cluster strains into species with 51-100% probability, evidencing different metabolic profiles, according to the relative production of organic acids from glucose by resting cells, of microorganisms collected at the exponential phase of growth. Looking for a single metabolic marker that could adequately discriminate metabolic groups, we found that groups established by the acetic to lactic acid ratio fit well with differences previously evidenced by the discriminant analysis. The proper establishment of metabolic groups within the genus Bifidobacterium could help to select the best suited probiotic strains for specific applications.

The relationship between phenolic compounds from diet and microbiota: impact on human health.

The human intestinal tract is home to a complex microbial community called microbiota. This gut microbiota, whilst playing essential roles in the maintenance of the health of the host, is exposed to the impact of external factors such as the use of medication or dietary patterns. Alterations in the composition and/or function of the microbiota have been described in several disease states, underlining the role of the gut microbiota in keeping the health status. Among the different dietary compounds, polyphenols constitute a very interesting group as some of them have been found to possess important biological activities, including antioxidant, anticarcinogenic or antimicrobial activities. The term polyphenol comprises thousands of molecules presenting a phenol ring and are widely distributed in plant foods. The bioactivity of these compounds is highly dependent on their intestinal absorption and often they are ingested as non-absorbable precursors that are transformed into bioactive forms by specific microorganisms in the intestine. Some of these microorganisms have been identified and the enzymatic steps involved have been elucidated. However, little is known about the impact of these ingested polyphenols upon the human gut microbiota. The heterogeneity of the polyphenol compounds and their food sources, as well as their coexistence with other bioactive compounds within a normal diet, together with the complexity of the human gut microbiota make difficult the understanding of the interactions between dietary polyphenols and gut microbes. This is, however, an important area of research which promises to expand our knowledge on the food functionality area through understanding the microbiota-food component interaction.

Production of immune response mediators by HT-29 intestinal cell-lines in the presence of Bifidobacterium-treated infant microbiota.

The colonisation and establishment of the intestinal microbiota starts immediately at birth and is essential for the development of the intestine and the immune system. This microbial community gradually increases in number and diversity until the age of two or three years when it becomes a stable ecosystem resembling that of adults. This period constitutes a unique window of opportunity to modulate it through probiotic action, with a potential impact in later health. In the present work we have investigated how putative bifidobacterial probiotics modify the metabolic profiles and immune-modulatory properties of faecal microbiotas. An in vitro pH-controlled single-stage continuous-culture system (CCS) inoculated with infant faeces was employed to characterise the effects of two Bifidobacterium species on the intestinal microbiotas in three children, together with the effects of these modified microbiotas on cytokine production by HT-29 cells. Intestinal bacterial communities, production of short-chain fatty acids and lactate were determined by quantitative PCR and gas chromatography, respectively. Cytokines production by HT-29 cells was measured by ELISA. The combination of CCS with infant faeces and human intestinal cells provided a suitable model to evaluate the specific modulation of the intestinal microbiota and immune system by probiotics. In the CCS, infant faecal microbiotas were influenced by the addition of bifidobacteria, resulting in changes in their ability to induce the production of immune mediators by HT-29 cells. The different metabolic and immunological responses induced by the bifidobacterial species tested indicate the need to assess potential probiotics in model systems including complex intestinal microbiotas. Potential probiotic bifidobacteria can modulate the infant microbiota and its ability to induce the production of mediators of the immune response by intestinal cells.

Metagenomics and probiotics.

The development of extensive sequencing methods has allowed metagenomic studies on the human gut microbiome to be carried out. This has tremendously increased our knowledge on gut microbiota composition and activity, allowing microbiota aberrations related to different diseases to be identified. These aberrations constitute targets for the development of probiotics directed to correct them. Probiotics are extensively used to modulate gut microbiota. Nevertheless, metagenomic studies on the effects of probiotics are still very scarce. In the near future, the use of metagenomics promises to expand our understanding of probiotic action.

Quantitative assessment of faecal bifidobacterial populations by real-time PCR using lanthanide probes.

AIM: To develop real-time quantitative PCR methods, based on the use of probes labelled with a stable fluorescent lanthanide chelate, for the quantification of different human faecal bifidobacterial populations. METHODS AND RESULTS: The designed quantitative PCR assays were found to be specific for the corresponding Bifidobacterium species or groups (Bifidobacterium longum group, Bifidobacterium catenulatum group, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium angulatum, Bifidobacterium bifidum and Bifidobacterium dentium). The detection limits of the methodologies used ranged between 2 x 10(5) and 9 x 10(3) cells g(-1) of faeces. The applicability of the developed assays was tested by analysing 20 human faecal samples. Bif. longum group was found to be the qualitatively and quantitatively predominant bifidobacterial group. CONCLUSIONS: The real-time PCR procedures developed here are specific, accurate, rapid and easy methods for the quantification of Bifidobacterium groups or species in human faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed procedures will facilitate rapid and objective counting of large numbers of samples increasing our knowledge on the role of gut bifidobacterial microbiota in health and disease. This will contribute to the efficient use of intestinal bacterial assays in research, food and pharmaceutical development as well as in the assessment of dietary management of diseases.