Optimizing the analysis of human intestinal microbiota with phylogenetic microarray

Phylogenetic microarrays present an attractive strategy to high-throughput interrogation of complex microbial communities. In this work, we present several approaches to optimize the analysis of intestinal microbiota with the recently developed Microbiota Array. First, we determined how 16S rDNA-specific PCR amplification influenced bacterial detection and the consistency of measured abundance values. Bacterial detection improved with an increase in the number of PCR amplification cycles, but 25 cycles were sufficient to achieve the maximum possible detection. A PCR-caused deviation in the measured abundance values was also observed. We also developed two mathematical algorithms that aimed to account for a predicted cross-hybridization of 16S rDNA fragments among different species, and to adjust the measured hybridization signal based on the number of 16S rRNA gene copies per species genome. The 16S rRNA gene copy adjustment indicated that the presence of members of the class Clostridia might be overestimated in some 16S rDNA-based studies. Finally, we show that the examination of total community RNA with phylogenetic microarray can provide estimates of the relative metabolic activity of individual community members. Complementary profiling of genomic DNA and total RNA isolated from the same sample presents an opportunity to assess population structure and activity in the same microbial community.     

Intestinal microbiota in healthy adults: temporal analysis reveals individual and common core and relation to intestinal symptoms

Background

While our knowledge of the intestinal microbiota during disease is accumulating, basic information of the microbiota in healthy subjects is still scarce. The aim of this study was to characterize the intestinal microbiota of healthy adults and specifically address its temporal stability, core microbiota and relation with intestinal symptoms. We carried out a longitudinal study by following a set of 15 healthy Finnish subjects for seven weeks and regularly assessed their intestinal bacteria and archaea with the Human Intestinal Tract (HIT)Chip, a phylogenetic microarray, in conjunction with qPCR analyses. The health perception and occurrence of intestinal symptoms was recorded by questionnaire at each sampling point.

Principal Findings

A high overall temporal stability of the microbiota was observed. Five subjects showed transient microbiota destabilization, which correlated not only with the intake of antibiotics but also with overseas travelling and temporary illness, expanding the hitherto known factors affecting the intestinal microbiota. We identified significant correlations between the microbiota and common intestinal symptoms, including abdominal pain and bloating. The most striking finding was the inverse correlation between Bifidobacteria and abdominal pain: subjects who experienced pain had over five-fold less Bifidobacteria compared to those without pain. Finally, a novel computational approach was used to define the common core microbiota, highlighting the role of the analysis depth in finding the phylogenetic core and estimating its size. The in-depth analysis suggested that we share a substantial number of our intestinal phylotypes but as they represent highly variable proportions of the total community, many of them often remain undetected.

Conclusions/Significance

A global and high-resolution microbiota analysis was carried out to determine the temporal stability, the associations with intestinal symptoms, and the individual and common core microbiota in healthy adults. The findings provide new approaches to define intestinal health and to further characterize the microbial communities inhabiting the human gut.     

Next-generation sequencing in the analysis of human microbiota: essential considerations for clinical application

The development of next-generation sequencing (NGS) presents an unprecedented opportunity to investigate the complex microbial communities that are associated with the human body. It offers for the first time a basis for detailed temporal and spatial analysis, with the potential to revolutionize our understanding of many clinically important systems. However, while advances continue to be made in areas such as PCR amplification for NGS, sequencing protocols, and data analysis, in many cases the quality of the data generated is undermined by a failure to address fundamental aspects of experimental design. While little is added in terms of time or cost by the analysis of repeat samples, the exclusion of DNA from dead bacterial cells and the extracellular matrix, the use of efficient nucleic acid extraction methodologies, and the implementation of safeguards to minimize the introduction of contaminating nucleic acids, such considerations are essential in achieving an accurate representation of the system being studied. In this review, the chronic bacterial infections that characterize lower respiratory tract infections in cystic fibrosis patients are used as an example system to examine the implications of a failure to address these issues when designing NGS-based analysis of human-associated microbiota. Further, ways in which the impact of these factors can be minimized are discussed.     

Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the endoplasmic reticulum in eukarya or to the inner membrane in prokarya. The crystal structure of the universally conserved RNA-protein core of the Escherichia coli SRP, refined here to 1.5 A resolution, revealed minor groove recognition of the 4.5 S RNA component by the M domain of the Ffh protein. Within the RNA, nucleotides comprising two phylogenetically conserved internal loops create a unique surface for protein recognition. To determine the energetic importance of conserved nucleotides for SRP assembly, we measured the affinity of the M domain for a series of RNA mutants. This analysis reveals how conserved nucleotides within the two internal loop motifs establish the architecture of the macromolecular interface and position essential functional groups for direct recognition by the protein.     

Clostridium innocuum: a glucoseureide-splitting inhabitant of the human intestinal tract

Glycosylureides were recently described as non-invasive markers of intestinal transit time. The underlying principle is an enzymatic splitting of (13)C-labelled ureides by intestinal bacteria. The (13)CO(2) released from the urea moiety of the glycosylureides can be measured in breath samples when the ingested tracer substrate reaches the caecum that is colonised with microbes. To date, the microbes that degrade glycosylureides are unknown. In order to identify the glucoseureide (GU)-splitting bacteria, 174 different strains of intestinal microbes obtained from five healthy adults were checked for their ability to degrade GU. The results of the microbial cultures and thin layer chromatography revealed that GU was exclusively degraded by Clostridium innocuum, belonging to the normal human intestinal microflora. C. innocuum probably synthesises a yet unknown enzyme that splits the glucose-urea bond. We suggest that the term glucoseureidehydrolase is the appropriate designation for this enzyme.     

ArrayFusion: a web application for multi-dimensional analysis of CGH, SNP and microarray data

ArrayFusion annotates conventional CGH results and various types of microarray data from a range of platforms (cDNA, expression, exon, SNP, array-CGH and ChIP-on-chip) and converts them into standard formats which can be visualized in genome browsers (Affymetrix Integrated Genome Browser and GBrowse in the HapMap Project). Converted files can then be imported simultaneously into a single genome browser to benefit a collective interpretation between different array results. ArrayFusion therefore provides a new type of tool facilitating the integration of CGH and array results to provide new experimental directions. AVAILABILITY: http://microarray.ym.edu.tw/tools/arrayfusion     

Mutational analysis of Saccharomyces cerevisiae nuclear RNase P: randomization of universally conserved positions in the RNA subunit.

Three regions in the Saccharomyces cerevisiae RNase P RNA have been identified, at positions Sce 87-94, Sce 309-316, and Sce 339-349, that contain nucleotides that are invariant in identity and position among all the known RNase P RNAs. To study the importance of these conserved RPR1 RNA regions in enzyme function, three independent mutational libraries were created in which the positions of invariant nucleotides were randomized simultaneously. Screening in vivo was used to identify viable RPR1 variants when reconstituted into holoenzyme in cells. Despite the universal evolutionary conservation, most of these positions tolerate certain sequence changes without severely affecting function. Most changes, however, produced subtle defects in cell growth and RNase P function, supporting the importance of these conserved regions. Isolation of conditional growth mutants allowed the characterization of the effects of mutations on cell growth, RPR1 RNA maturation, and activity of the holoenzyme in vitro. Kinetic analysis showed that viable variants were usually more defective in catalytic rate (Kcat) than in substrate recognition (Km).     

老年与中青年患者下呼吸道感染病原菌的对比分析

目的对比分析老年与中青年患者下呼吸道感染病原菌的分布规律及其耐药性特点,以指导临床合理用药。方法将研究对象分为老年(≥60岁)和中青年(20～59岁)2组;采用API系统进行菌种鉴定;采用K-B法进行药敏试验;采用纸片扩散表型确证法进行超广谱β-内酰胺酶(ESBLs)测定;采用SPSS 13.0进行χ2检验。结果老年组的真菌分离率显著高于中青年组,以白色假丝酵母菌最多;中青年组的G-杆菌分离率显著高于老年组,以铜绿假单胞菌最多;老年组主要致病菌对多数药物的耐药率比中青年组有增高趋势,但差异多无统计学意义;老年组肺炎克雷伯菌的ESBLs阳性率显著高于中青年组。结论老年与中青年患者下呼吸道感染的病原菌分布及耐药性存在一定差异。     

Resident,transient and uropathogenicE.coli strains in the intestinal tract of healthy and hospitalized adults

Samples of faeces were obtained at weekly intervals from 12 patients in a traumatology ward. Each time 20 colonies ofE.coli were typed. The results were compared with those of similar investigations in 10 healthy adults. No difference was found between the two groups as regards changes in the residentE. coli flora, nor in the frequency of occurrence of strains often associated with urinary tract infection.     

Selection of bacteria originating from a human intestinal microbiota in the gut of previously germ-free rats

Denaturing gradient gel electrophoresis (DGGE) was applied to separate PCR-amplified 16S rRNA genes originating from human microbiota associated (HMA) rat faeces as well as from the human faecal sample used for inoculation of the animals. Subsequently, a total of 15 dominant bands were excised from the DGGE gels, cloned and sequenced. Comparison of the obtained sequences with the Ribosomal Database revealed that species of Bacteroides/Prevotella and Faecalibacterium gave rise to the majority of the dominant bands in the human sample and in the HMA rats. In the HMA rats, two dominant bands, which were not present in the human DGGE profile, originated from species of Ruminococcus. With the exception of the Ruminococcus sequences, sequences originating from both rats and human samples were represented in all major branches of a maximum parsimony tree, indicating that the rat feed and gut environment allows colonization of the dominant taxonomic units from the human microbiota, but additionally selects for Ruminococci. Bands representing Prevotella and Faecalibacterium, which were found in identical positions of the DGGE gels originating from human and HMA rat faecal samples, originated from completely identical sequences, indicating that the same strains of these species were dominating in the human and rat samples.     

Evaluation of intestinal microbiota,short-chain fatty acids,and immunoglobulin a in diversion colitis

It is reported that an increase in aerobic bacteria, a lack of short-chain fatty acids (SCFAs), and immune disorders in the diverted colon are major causes of diversion colitis. However, the precise pathogenesis of this condition remains unclear. The aim of the present study was to examine the microbiota, intestinal SCFAs, and immunoglobulin A (IgA) in the diverted colon. Eight patients underwent operative procedures for colostomies. We assessed the diverted colon using endoscopy and obtained intestinal samples from the diverted colon and oral colon in these patients. We analyzed the microbiota and SCFAs of the intestinal samples. The bacterial communities were investigated using a 16S rRNA gene sequencing method. The microbiota demonstrated a change in the proportion of some species, especially Lactobacillus, which significantly decreased in the diverted colon at the genus level. We also showed that intestinal SCFA values were significantly decreased in the diverted colon. Furthermore, intestinal IgA levels were significantly increased in the diverted colon. This study was the first to show that intestinal SCFAs were significantly decreased and intestinal IgA was significantly increased in the diverted colon. Our data suggest that SCFAs affect the microbiota and may play an immunological role in diversion colitis.     

Molecular analysis of the luminal- and mucosal-associated intestinal microbiota in diarrhea-predominant irritable bowel syndrome

Alterations in the intestinal microbiota have been suggested as an etiological factor in the pathogenesis of irritable bowel syndrome (IBS). This study used a molecular fingerprinting technique to compare the composition and biodiversity of the microbiota within fecal and mucosal niches between patients with diarrhea-predominant IBS (D-IBS) and healthy controls. Terminal-restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the bacterial 16S rRNA gene was used to perform microbial community composition analyses on fecal and mucosal samples from patients with D-IBS (n = 16) and healthy controls (n = 21). Molecular fingerprinting of the microbiota from fecal and colonic mucosal samples revealed differences in the contribution of T-RFs to the microbiota between D-IBS patients and healthy controls. Further analysis revealed a significantly lower (1.2-fold) biodiversity of microbes within fecal samples from D-IBS patients than healthy controls (P = 0.008). No difference in biodiversity in mucosal samples was detected between D-IBS patients and healthy controls. Multivariate analysis of T-RFLP profiles demonstrated distinct microbial communities between luminal and mucosal niches in all samples. Our findings of compositional differences in the luminal- and mucosal-associated microbiota between D-IBS patients and healthy controls and diminished microbial biodiversity in D-IBS fecal samples further support the hypothesis that alterations in the intestinal microbiota may have an etiological role in the pathogenesis of D-IBS and suggest that luminal and mucosal niches need to be investigated.     

Barcoded pyrosequencing analysis of the microbial community in a simulator of the human gastrointestinal tract showed a colon region-specific microbiota modulation for two plant-derived polysaccharide blends

The combination of a Simulator of the Human Intestinal Microbial Ecosystem with ad hoc molecular techniques (i.e. pyrosequencing, denaturing gradient gel electrophoresis and quantitative PCR) allowed an evaluation of the extent to which two plant polysaccharide supplements could modify a complex gut microbial community. The presence of Aloe vera gel powder and algae extract in product B as compared to the standard blend (product A) improved its fermentation along the entire simulated colon. The potential extended effect of product B in the simulated distal colon, as compared to product A, was confirmed by: (i) the separate clustering of the samples before and after the treatment in the phylogenetic-based dendrogram and OTU-based PCoA plot only for product B; (ii) a higher richness estimator (+33 vs. ?36 % of product A); and (iii) a higher dynamic parameter (21 vs. 13 %). These data show that the combination of well designed in vitro simulators with barcoded pyrosequencing is a powerful tool for characterizing changes occurring in the gut microbiota following a treatment. However, for the quantification of low-abundance species—of interest because of their relationship to potential positive health effects (i.e. bifidobacteria or lactobacilli)—conventional molecular ecological approaches, such as PCR–DGGE and qPCR, still remain a very useful complementary tool.     

Presence of a single abundant storage hexamerin in both larvae and adults of the grasshopper, Schistocerca americana

We identified a single hexameric storage protein in the grasshopper, Schistocerca americana, and monitored its abundance through the last larval instar and up until reproductive competence in adults of both sexes. This storage hexamerin, termed Schistocerca americana Persistent Storage Protein (saPSP) was the most abundant soluble protein in both larvae and adults. In both sexes, saPSP abundance started out low at the onset of the last larval instar and accumulated during feeding, peaking just prior to molting. Adults of both sexes contained significant amounts of saPSP after eclosion. In adult males, saPSP content dropped continuously after eclosion and was lowest once individuals reached reproductive maturity. In contrast, adult females depleted saPSP reserves during the first days of adulthood, but subsequently accumulated significant saPSP stores. In adult females, saPSP stores peaked just prior to the completion of egg provisioning. Given the overall patterns of abundance, saPSP has functions in both larvae and adults. In addition, the observed pattern of storage hexamerin accumulation differs from patterns of accumulation in the other known grasshoppers, Locusta migratoria and Romalea microptera, suggesting that significant functional diversity has evolved in storage hexamerins among the grasshoppers.     

Comparative sequence analysis of a single-gene conserved segment in mouse and human

Comparative mapping and sequencing of the mouse and human genomes have defined large, conserved chromosomal segments in which gene content and order are highly conserved. These regions span megabase-sized intervals and together comprise the vast majority of both genomes. However, the evolutionary relationships among the small remaining portions of these genomes are not as well characterized. Here we describe the sequencing and annotation of a 341-kb region of mouse Chr 2 containing nine genes, including biliverdin reductase A (Blvra), and its comparison with the orthologous regions of the human and rat genomes. These analyses reveal that the known conserved synteny between mouse Chromosome (Chr) 2 and human Chr 7 reflects an interval containing one gene (Blvra/BLVRA) that is, at most, just 34 kb in the mouse genome. In the mouse, this segment is flanked proximally by genes orthologous to human chromosome 15q21 and distally by genes orthologous to human Chr 2q11. The observed differences between the human and mouse genomes likely resulted from one or more rearrangements in the rodent lineage. In addition to the resulting changes in gene order and location, these rearrangements also appear to have included genomic deletions that led to the loss of at least one gene in the rodent lineage. Finally, we also have identified a recent mouse-specific segmental duplication. These finding illustrate that small genomic regions outside the large mouse–human conserved segments can contain a single gene as well as sequences that are apparently unique to one genome. The nucleotide sequence data reported in this paper have been submitted to GenBank and assigned the accession numbers AC074224 and AC074041.     

Clostridium ramosum, an IgA protease-producing species and its ecology in the human intestinal tract

A bacterial strain isolated from feces of a patient with ulcerative colitis, which had been shown to produce a novel immunoglobulin A (IgA) protease (cleaving both the human IgA1 subclass and IgA2 subclass of A2m(1) allotype) extracellularly, was identified as Clostridium ramosum. By using a selective medium (propionate-rifampicin-gentamicin-colimycin-polymyxin medium) devised for C. ramosum, analysis of the population level of this organism was performed to determine its ecology in the human intestinal tract. C. ramosum was isolated in 20 of 25 fecal samples (80%) from patients with inflammatory bowel disease (I.B.D.) and in 112 of 135 samples (83%) from patients without I.B.D. (control group). C. ramosum was also isolated from 6 of 11 biopsy samples (55%) of the inflamed rectal mucosa from patients with ulcerative colitis and from five of 15 samples (33%) from the intact mucosa of the control group. The population levels of C. ramosum in most of the biopsy samples ranged from 2.3 to 5.0 log10 per gram. The IgA protease-positive C. ramosum was found in only four of 135 fecal samples (3%) and one of 15 biopsy samples (6.7%) from the control group. These results indicate that IgA protease-positive C. ramosum is not likely to play a role in the induction of I.B.D., unless the organism is first isolated from the patient with I.B.D.     

Evidence of regio-specific glycosylation in human intestinal mucins: presence of an acidic gradient along the intestinal tract

Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, transverse and sigmoid colon, and rectum) of two individuals with ALeb blood group. A systematic study of the monosaccharides and oligosaccharide alditols released by reductive beta-elimination from mucins was performed using gas chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy techniques. Important variations were observed in the mucin-associated oligosaccharide content with an increasing gradient of sialic acid from the ileum to the colon associated with a reverse gradient of fucose. Moreover, a comparative study of the Sda/Cad and ABH blood group determinants along the gastrointestinal tract showed the same reverse distribution in the two kinds of antigens. In addition, besides their heterogeneity, sialic acids presented considerable variations in the degree of O-acetylation in relation to glycan sialylation level. These data are discussed in view of recent concepts suggesting that the oligosaccharide composition of the gut constitutes a varied ecosystem for microorganisms that are susceptible to adapt there and possess the specific adhesion system and specific enzymes able to provide a carbohydrate nutrient.