First-Pass Meconium Samples from Healthy Term Vaginally-Delivered Neonates: An Analysis of the Microbiota.

BACKGROUND: Considerable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation; however, this view has recently been challenged. We examined first-pass meconium from healthy term infants to confirm or refute sterility. METHODS: Healthy mothers were approached following vaginal delivery. First-pass meconium stools within 24 hours of delivery were obtained from healthy, breastfed infants with tight inclusion/exclusion criteria including rejecting any known antibiotic exposure - mother within 7 days preceding delivery or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with 16S rRNA-targeted probes including Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification of each bacterial group were calculated. Confirmation of bacterial presence by PCR was undertaken on FISH-positive samples. RESULTS: The mothers of 31 newborn infants were recruited, 15 met inclusion/exclusion criteria and provided a sample within 24 hours of birth, processed in the lab within 4 hours. All babies were 37-40 weeks gestation. 8/15 were male, mean birth weight was 3.4 kg and mean maternal age was 32 years. Meconium samples from 10/15 (66%) infants had evidence of bacteria based on FISH analysis. Of these, PCR was positive in only 1. Positive FISH counts ranged from 2.2-41.8 x 10(4) cells/g with a mean of 15.4 x 10(4) cells/g. (The limit of detection for automated counting is 10(6) cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. One baby was dominated by Enterobacteriaceae. The others contained 2-5 genera, with Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella the most prevalent. There was no association between bacterial counts and rupture of membrane duration, time to passage of meconium or time to lab. CONCLUSION: This study provides evidence that low numbers of bacteria are present in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants. Only two-thirds of meconium samples had detectable bacteria, though at levels too low for automated counting or for reliable confirmation by PCR. This study suggests that gut bacterial colonisation is extremely limited at birth and occurs rapidly thereafter.

Substrate-driven gene expression in Roseburia inulinivorans: importance of inducible enzymes in the utilization of inulin and starch.

Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a ß-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the ß-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans ß-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.

Modificación fíiica del almidón de yuca y evalución de la susceptibilidad a laa hidrólisis enzimática por una alfa miilasa / Physial modificaation of cassava starh and evaluation of susceptibillity to enzyme hydrolysis by alpha amylase / Modificção físicca do amido de mandiocay avaliação do susceptibilidadee à hidrólise enzimática com uma alfa mmilase

Almidón de yuca comercial (variedad MTAI8) se sometió a modificación física por sinéresis, extrusión, gelatinización y secado por rodillos. Al almidón nativo y a los almidones modificados se les determinó su morfología, cristalinidad, distribución molecular y susceptibilidad a la hidrólisis enzimática con una alfa amilasa porcina pancreática. En los almidones modificados físicamente se incrementó el grado de hidrólisis, en comparación con el almidón nativo. Sin embargo no se observaron diferencias estadísticamente significativas en el grado de hidrólisis entre los almidones modificados. El patrón de difracción de rayos X tipo A, presentado por el almidón de yuca nativo y su propiedad birrefringente se alteraron por los pretratamientos, presentándose difractogramas de estructuras amorfas y pérdida de la cruz de malta en los almidones modificados. La microscopía electrónica de barrido (MEB) demostró alteración en la apariencia y estructura del gránulo nativo, dando lugar a partículas de formas irregulares con superficies fragmentadas y rugosas como resultado del proceso de modificación. La cromatografía de exclusión sobre sepharosa 6B confirmó la desaparición de la fracción de alto peso molecular presente en el almidón nativo, con el consecuente incremento de las fracciones de bajo peso molecular en los almidones modificados.

Cassava starch (MTAI 8 variety) was subjected to syneresis, gelatinization, extrusion and processing by drum dryers. The morphology, crystallinity, molecular weight distribution and susceptibility to enzyme hydrolysis by porcine pancreatic a-amylase were determined before and after the physical treatments. The physically modified starches increased the extent of a-amylolysis, compared to the native one. However, there were not significant differences in the degree of amylolisis between the treatments during the procedure. Both the X-ray pattern type-A, presentedinthecassavastarch, and its birrefringent property, observed in polarized light, were altered by the treatments causing amorphous structures and the loss of the Maltese cross. When the modified starches were observed n scann ng electron micrographs (SEM), an alteration in the appearance and structure of the native granule was shown, where particles with irregular shape and fragmented and wrinkled surfaces could be seen as a result of the modification process. The profile of the size exclusion chromatography on sepharose 6B showed two characteristic fractions of high and low molecular weight in the native starch, while in modified starches only one peak was obtained showing low molecular weight. The data showed that the treatments modified the physical structure of the starch granule, allowing more accessibility for the enzyme to the amorphous and crystalline regions of starch.

Amido de mandioca comercial (variedade MTAI 8) foi submetido a modificação física por sinérese, extrusão, gelatinização e secado por tambor. Foram determinados para o amido nativo e os amidos modificados a sua morfologia, cristalinidade, distribuição molecular y susceptibilidade à hidrólise enzimática com uma alfa amilase porcina pancreática. Nos amidos modificados fisicamente foi verificado um aumento do nível de hidrólise, em comparação com o amido nativo. Porém, não se observaram diferenças estatisticamente significativas no nível de hidrólise entre os almidões modificados. O padrão de difracção de raios-X tipo A apresentado pelo amido de mandioca nativo e a sua propriedade birrefringente, foram alterados pelos prétratamentos, apresentando-se difractogramas de estruturas amorfas y perda da cruz de malta nos almidões modificados. A microscopia electrónica de barrido (MEB) demonstrou alteração na aparência e estrutura do granulo nativo, dando lugar a partículas de formas irregulares com superfícies fragmentadas e rugosas como resultado do processo de modificação. A cromatografia de exclusão sobre sepharosa 6B corroborou a desaparição da fracção de alto peso molecular presente no amido nativo, com o consequente aumento das fracções de baixo peso molecular nos almidões modificados.

Transfer of conjugative elements from rumen and human Firmicutes bacteria to Roseburia inulinivorans.

Studies on Firmicutes bacteria from the gut are hampered by a lack of gene transfer systems. Here the human colonic anaerobe Roseburia inulinivorans A2-194 was shown to be a transfer recipient for two conjugative transposons, Tn1545 from Eubacterium cellulosolvens and TnK10 from Clostridium saccharolyticum K10.

Cell-associated alpha-amylases of butyrate-producing Firmicute bacteria from the human colon.

Selected butyrate-producing bacteria from the human colon that are related to Roseburia spp. and Butyrivibrio fibrisolvens showed a good ability to utilize a variety of starches for growth when compared with the Gram-negative amylolytic anaerobe Bacteroides thetaiotaomicron. A major cell-associated amylase of high molecular mass (140-210 kDa) was detected in each strain by SDS-PAGE zymogram analysis, and genes corresponding to these enzymes were analysed for two representative strains. Amy13B from But. fibrisolvens 16/4 is a multi-domain enzyme of 144.6 kDa that includes a family 13 glycoside hydrolase domain, and duplicated family 26 carbohydrate-binding modules. Amy13A (182.4 kDa), from Roseburia inulinivorans A2-194, also includes a family 13 domain, which is preceded by two repeat units of approximately 116 aa rich in aromatic residues, an isoamylase N-terminal domain, a pullulanase-associated domain, and an additional unidentified domain. Both Amy13A and Amy13B have N-terminal signal peptides and C-terminal cell-wall sorting signals, including a modified LPXTG motif similar to that involved in interactions with the cell surface in other Gram-positive bacteria, a hydrophobic transmembrane segment, and a basic C terminus. The overexpressed family 13 domains showed an absolute requirement for Mg2+ or Ca2+ for activity, and functioned as 1,4-alpha-glucanohydrolases (alpha-amylases; EC 3.2.1.1). These major starch-degrading enzymes thus appear to be anchored to the cell wall in this important group of human gut bacteria.

Unravelling the genetic diversity of ruminal bacteria belonging to the CFB phylum.

Molecular biology approaches were employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle. By this means we were able to identify cultured strains that represent some of the larger CFB clusters previously identified only by PCR amplification and sequencing. Complete 16S rDNA sequences were obtained for 16 previously isolated rumen strains, including the type strains of Prevotella ruminicola, P. bryantii, P. brevis and P. albensis to represent a wide range of diversity. Phylogenetic analysis of cultured strains revealed the existence of three clusters of ruminal CFB: (i) a cluster of Prevotella strains, which have been found only in the rumen, including the two type strains, P. brevis GA33(T) and P. ruminicola 23(T); (ii) Prevotella spp. that cluster with prevotellas from other ecological niches such as the oral cavity and which include the type strains, P. bryantii B(1)4(T) and P. albensis M384(T); (iii) two Bacteroides spp. strains clustering with B. forsythus of oral origin. In order to establish whether the cultivated isolates cover the whole range of ruminal CFB genetic diversity, 16S rRNA gene sequences were amplified and cloned from DNA extracted from the same rumen samples (one cow in Slovenia, one in Scotland and three in Japan). Sequencing and phylogenetic analysis of 16S rRNA genes confirmed the existence of two superclusters of ruminal Prevotella, one exclusively ruminal and the other including non-ruminal species. In the case of ruminal Bacteroides spp., however, phylogenetic analysis revealed the existence of three new superclusters, one of which has as yet no cultivable counterpart. Interestingly, these Bacteroides clusters were represented almost exclusively by clone libraries from the Japanese cattle and only three sequences were from the European cattle. This study agrees with previous analyses in showing that rumen Prevotella/Bacteroides strains exhibit a remarkable degree of genetic diversity and suggests that different strain groupings may differ greatly in their recovery by cultural methods. The most important conclusion, however, is that cultured strains can be identified that represent some of the larger clusters previously identified only by PCR amplification and sequencing.