Richard Agans

Distal gut microbiota of adolescent children is different from that of adults

Human intestinal microbiota plays a number of important roles in human health and is also implicated in several gastrointestinal disorders. Although the diversity of human gut microbiota in adults and in young children has been examined, few reports of microbiota composition are available for adolescents. In this work, we used Microbiota Array for high-throughput analysis of distal gut microbiota in adolescent children 11-18 years of age. Samples obtained from healthy adults were used for comparison. Adolescent and adult groups could be separated in the principal components analysis space based on the relative species abundance of their distal gut microbiota. All samples were dominated by class Clostridia. A core microbiome of 46 species that were detected in all examined samples was established; members of genera Ruminococcus, Faecalibacterium, and Roseburia were well represented among core species. Comparison of intestinal microbiota composition between adolescents and adults revealed a statistically significantly higher abundance of genera Bifidobacterium and Clostridium among adolescent samples. The number of detected species was similar between sample groups, indicating that it was the relative abundances of the genera and not the presence or absence of a specific genus that differentiated adolescent and adult samples. In summary, contrary to the current belief, this study suggests that the gut microbiome of adolescent children is different from that of adults.

Quantitative Profiling of Gut Microbiota of Children with Diarrhea-Predominant Irritable Bowel Syndrome

OBJECTIVES:Human intestinal microbiota has a number of important roles in human health and is also implicated in several gastrointestinal disorders. The goal of this study was to determine the gut microbiota in two groups of pre- and adolescent children: healthy volunteers and children diagnosed with diarrhea-predominant irritable bowel syndrome (IBS-D).METHODS:Phylogenetic Microbiota Array was used to obtain quantitative measurements of bacterial presence and abundance in subjects’ fecal samples. We utilized high-throughput DNA sequencing, quantitative PCR, and fluorescent in situ hybridization to confirm microarray findings.RESULTS:Both sample groups were dominated by the phyla Firmicutes, Bacteroidetes, and Actinobacteria, which cumulatively constituted 91% of overall sample composition on average. A core microbiome shared among analyzed samples encompassed 55 bacterial phylotypes dominated by genus Ruminococcus; members of genera Clostridium, Faecalibacterium, Roseburia, Streptococcus, and Bacteroides were also present. Several genera were found to be differentially abundant in the gut of healthy and IBS groups: levels of Veillonella, Prevotella, Lactobacillus, and Parasporobacterium were increased in children diagnosed with IBS, whereas members of Bifidobacterium and Verrucomicrobium were less abundant in those individuals. By calculating a nonparametric correlation matrix among abundances of different genera in all samples, we also examined potential associations among intestinal microbes. Strong positive correlations were found between abundances of Veillonella and both Haemophilus and Streptococcus, between Anaerovorax and Verrucomicrobium, and between Tannerella and Anaerophaga.CONCLUSIONS:Although at the higher taxonomical level gut microbiota was similar between healthy and IBS-D children, specific differences in the abundances of several bacterial genera were revealed. Core microbiome in children was dominated by Clostridia. Putative relationships identified among microbial genera provide testable hypotheses of cross-species associations among members of human gut microbiota.

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.

Application of phylogenetic microarrays to interrogation of human microbiota

Human-associated microbiota is recognized to play vital roles in maintaining host health, and it is implicated in many disease states. While the initial surge in the profiling of these microbial communities was achieved with Sanger and next-generation sequencing, many oligonucleotide microarrays have also been developed recently for this purpose. Containing probes complementary to small ribosomal subunit RNA gene sequences of community members, such phylogenetic arrays provide direct quantitative comparisons of microbiota composition among samples and between sample groups. Some of the developed microarrays including PhyloChip, Microbiota Array, and HITChip can simultaneously measure the presence and abundance of hundreds and thousands of phylotypes in a single sample. This review describes the currently available phylogenetic microarrays that can be used to analyze human microbiota, delineates the approaches for the optimization of microarray use, and provides examples of recent findings based on microarray interrogation of human-associated microbial communities.

Do gut microbial communities differ in pediatric IBS and health

Human gastrointestinal microbial communities are recognized as important determinants of the host health and disease status. We have recently examined the distal gut microbiota of two groups of children: healthy adolescents and those diagnosed with diarrhea-predominant irritable bowel syndrome (IBS). We have revealed the common core of phylotypes shared among all children, identified genera differentially abundant between two groups and surveyed possible relationships among intestinal microbial genera and phylotypes. In this article we explored the use of supervised and unsupervised ordination and classification methods to separate and classify child fecal samples based on their quantitative microbial profile. We observed sample separation according to the participant health status, and this separation could often be attributed to the abundance levels of several specific microbial genera. We also extended our original correlation network analysis of the relative abundances of bacterial genera across samples and determined possible association networks separately for healthy and IBS groups. Interestingly, the number of significant genus abundance associations was drastically lower among the IBS samples, which can potentially be attributed to the existence of multiple routes to microbiota disbalance in IBS or to the loss of microbial interactions during IBS development.

Transcriptional Responses of Uropathogenic Escherichia coli to Increased Environmental Osmolality Caused by Salt or Urea

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections in humans. The majority of urinary infections develop via ascending route through the urethra, where bacterial cells come in contact with human urine prior to reaching the bladder or kidneys. Since urine contains significant amounts of inorganic ions and urea, it imposes osmotic and denaturing stresses on bacterial cells. In this study, we determined the transcriptional adaptive responses of UPEC strain CFT073 to the presence of 0.3 M NaCl or 0.6 M urea in the growth medium. The cell responses to these two osmolytes were drastically different. Although most of the genes of the osmotically inducible regulon were overexpressed in medium with salt, urea failed to stimulate osmotic stress response. At the same time, UPEC colonization genes encoding type 1 and F1C fimbriae and capsule biosynthesis were transcriptionally induced in the presence of urea but did not respond to increased salt concentration. We speculate that urea can potentially be sensed by uropathogenic bacteria to initiate infection program. In addition, several molecular chaperone genes were overexpressed in the presence of urea, whereas adding NaCl to the medium led to an upregulation of a number of anaerobic metabolism pathways.

Advantages of phylogenetic distance based constrained ordination analyses for the examination of microbial communities

Recently developed high throughput molecular techniques such as massively parallel sequencing and phylogenetic microarrays generate vast datasets providing insights into microbial community structure and function. Because of the high dimensionality of these datasets, multivariate ordination analyses are often employed to examine such data. Here, we show how the use of phylogenetic distance based redundancy analysis provides ecological interpretation of microbial community differences. We also extend the previously developed method of principal response curves to incorporate phylogenetic distance measure, and we demonstrate the improved ability of this approach to provide ecologically relevant insights into temporal alterations of microbial communities.

DIETARY FATTY ACIDS SUSTAIN GROWTH OF HUMAN GUT MICROBIOTA

Increased intake of fats in many developed countries has raised awareness of potentially harmful and beneficial effects of high fat consumption on human health. Some dietary fats escape digestion in the small intestine and reach the colon where they can be metabolized by gut microbiota. We show that human gut microbes are able to maintain a complex community when supplied with dietary fatty acids as the only nutrient and carbon sources. Such fatty acid-based growth leads to lower production of short-chain fatty acids and antioxidants by community members, which potentially have negative health consequences on the host. ABSTRACT While a substantial amount of dietary fats escape absorption in the human small intestine and reach the colon, the ability of resident microbiota to utilize these dietary fats for growth has not been investigated in detail. In this study, we used an in vitro multivessel simulator system of the human colon to reveal that the human gut microbiota is able to utilize typically consumed dietary fatty acids to sustain growth. Gut microbiota adapted quickly to a macronutrient switch from a balanced Western diet-type medium to its variant lacking carbohydrates and proteins. We defined specific genera that increased in their abundances on the fats-only medium, including Alistipes, Bilophila, and several genera of the class Gammaproteobacteria. In contrast, the abundances of well-known glycan and protein degraders, including Bacteroides, Clostridium, and Roseburia spp., were reduced under such conditions. The predicted prevalences of microbial genes coding for fatty acid degradation enzymes and anaerobic respiratory reductases were significantly increased in the fats-only environment, whereas the abundance of glycan degradation genes was diminished. These changes also resulted in lower microbial production of short-chain fatty acids and antioxidants. Our findings provide justification for the previously observed alterations in gut microbiota observed in human and animal studies of high-fat diets. IMPORTANCE Increased intake of fats in many developed countries has raised awareness of potentially harmful and beneficial effects of high fat consumption on human health. Some dietary fats escape digestion in the small intestine and reach the colon where they can be metabolized by gut microbiota. We show that human gut microbes are able to maintain a complex community when supplied with dietary fatty acids as the only nutrient and carbon sources. Such fatty acid-based growth leads to lower production of short-chain fatty acids and antioxidants by community members, which potentially have negative health consequences on the host.