Oleg Paliy

Genome-Wide Transcriptional Responses of Escherichia coli K-12 to Continuous Osmotic and Heat Stresses

Osmotic stress is known to increase the thermotolerance and oxidative-stress resistance of bacteria by a mechanism that is not adequately understood. We probed the cross-regulation of continuous osmotic and heat stress responses by characterizing the effects of external osmolarity (0.3 M versus 0.0 M NaCl) and temperature (43 degrees C versus 30 degrees C) on the transcriptome of Escherichia coli K-12. Our most important discovery was that a number of genes in the SoxRS and OxyR oxidative-stress regulons were up-regulated by high osmolarity, high temperature, or a combination of both stresses. This result can explain the previously noted cross-protection of osmotic stress against oxidative and heat stresses. Most of the genes shown in previous studies to be induced during the early phase of adaptation to hyperosmotic shock were found to be also overexpressed under continuous osmotic stress. However, there was a poorer overlap between the heat shock genes that are induced transiently after high temperature shifts and the genes that we found to be chronically up-regulated at 43 degrees C. Supplementation of the high-osmolarity medium with the osmoprotectant glycine betaine, which reduces the cytoplasmic K(+) pool, did not lead to a universal reduction in the expression of osmotically induced genes. This finding does not support the hypothesis that K(+) is the central osmoregulatory signal in Enterobacteriaceae.

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.

High-Throughput Quantitative Analysis of the Human Intestinal Microbiota with a Phylogenetic Microarray

ABSTRACT Gut microbiota carry out key functions in health and participate in the pathogenesis of a growing number of diseases. The aim of this study was to develop a custom microarray that is able to identify hundreds of intestinal bacterial species. We used the Entrez nucleotide database to compile a data set of bacterial 16S rRNA gene sequences isolated from human intestinal and fecal samples. Identified sequences were clustered into separate phylospecies groups. Representative sequences from each phylospecies were used to develop a microbiota microarray based on the Affymetrix GeneChip platform. The designed microbiota array contains probes to 775 different bacterial phylospecies. In our validation experiments, the array correctly identified genomic DNA from all 15 bacterial species used. Microbiota array has a detection sensitivity of at least 1 pg of genomic DNA and can detect bacteria present at a 0.00025% level of overall sample. Using the developed microarray, fecal samples from two healthy children and two healthy adults were analyzed for bacterial presence. Between 227 and 232 species were detected in fecal samples from children, whereas 191 to 208 species were found in adult stools. The majority of identified phylospecies belonged to the classes Clostridia and Bacteroidetes. The microarray revealed putative differences between the gut microbiota of healthy children and adults: fecal samples from adults had more Clostridia and less Bacteroidetes and Proteobacteria than those from children. A number of other putative differences were found at the genus level.

Sulfur and Nitrogen Limitation in Escherichia coli K-12: Specific Homeostatic Responses

We determined global transcriptional responses of Escherichia coli K-12 to sulfur (S)- or nitrogen (N)-limited growth in adapted batch cultures and cultures subjected to nutrient shifts. Using two limitations helped to distinguish between nutrient-specific changes in mRNA levels and common changes related to the growth rate. Both homeostatic and slow growth responses were amplified upon shifts. This made detection of these responses more reliable and increased the number of genes that were differentially expressed. We analyzed microarray data in several ways: by determining expression changes after use of a statistical normalization algorithm, by hierarchical and k-means clustering, and by visual inspection of aligned genome images. Using these tools, we confirmed known homeostatic responses to global S limitation, which are controlled by the activators CysB and Cbl, and found that S limitation propagated into methionine metabolism, synthesis of FeS clusters, and oxidative stress. In addition, we identified several open reading frames likely to respond specifically to S availability. As predicted from the fact that the ddp operon is activated by NtrC, synthesis of cross-links between diaminopimelate residues in the murein layer was increased under N-limiting conditions, as was the proportion of tripeptides. Both of these effects may allow increased scavenging of N from the dipeptide D-alanine-D-alanine, the substrate of the Ddp system.

Altered gut microbial energy and metabolism in children with non-alcoholic fatty liver disease.

Obesity is becoming the new pediatric epidemic. Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity and has become the most common cause of pediatric liver disease. The gut microbiome is the major metabolic organ and determines how calories are processed, serving as a caloric gate and contributing towards the pathogenesis of NAFLD. The goal of this study is to examine gut microbial profiles in children with NAFLD using phylogenetic, metabolomic, metagenomic and proteomic approaches. Fecal samples were obtained from obese children with or without NAFLD and healthy lean children. Stool specimens were subjected to 16S rRNA gene microarray, shotgun sequencing, mass spectroscopy for proteomics and NMR spectroscopy for metabolite analysis. Children with NAFLD had more abundant Gammaproteobacteria and Prevotella and significantly higher levels of ethanol, with differential effects on short chain fatty acids. This group also had increased genomic and protein abundance for energy production with a reduction in carbohydrate and amino acid metabolism and urea cycle and urea transport systems. The metaproteome and metagenome showed similar findings. The gut microbiome in pediatric NAFLD is distinct from lean healthy children with more alcohol production and pathways allocated to energy metabolism over carbohydrate and amino acid metabolism, which would contribute to development of disease.

Species and genus level resolution analysis of gut microbiota in Clostridium difficile patients following fecal microbiota transplantation

BackgroundClostridium difficile is an opportunistic human intestinal pathogen, and C. difficile infection (CDI) is one of the main causes of antibiotic-induced diarrhea and colitis. One successful approach to combat CDI, particularly recurrent form of CDI, is through transplantation of fecal microbiota from a healthy donor to the infected patient. In this study we investigated the distal gut microbial communities of three CDI patients before and after fecal microbiota transplantation, and we compared these communities to the composition of the donor’s fecal microbiota. We utilized phylogenetic Microbiota Array, high-throughput Illumina sequencing, and fluorescent in situ hybridization to profile microbiota composition down to the genus and species level resolution.ResultsThe original patients’ microbiota had low diversity, was dominated by members of Gammaproteobacteria and Bacilli, and had low numbers of Clostridia and Bacteroidia. At the genus level, fecal samples of CDI patients were rich in members of the Lactobacillus, Streptococcus, and Enterobacter genera. In comparison, the donor community was dominated by Clostridia and had significantly higher diversity and evenness. The patients’ distal gut communities were completely transformed within 3 days following fecal transplantation, and these communities remained stable in each patient for at least 4 months. Despite compositional differences among recipients’ pre-treatment gut microbiota, the transplanted gut communities were highly similar among recipients post-transplantation, were indistinguishable from that of the donor, and were rich in members of Blautia, Coprococcus, and Faecalibacterium. In each case, the gut microbiota restoration led to a complete patient recovery and symptom alleviation.ConclusionWe conclude that C. difficile infection can be successfully treated by fecal microbiota transplantation and that this leads to stable transformation of the distal gut microbial community from the one abundant in aerotolerant species to that dominated by members of the Clostridia.

Application of multivariate statistical techniques in microbial ecology

Recent advances in high‐throughput methods of molecular analyses have led to an explosion of studies generating large‐scale ecological data sets. In particular, noticeable effect has been attained in the field of microbial ecology, where new experimental approaches provided in‐depth assessments of the composition, functions and dynamic changes of complex microbial communities. Because even a single high‐throughput experiment produces large amount of data, powerful statistical techniques of multivariate analysis are well suited to analyse and interpret these data sets. Many different multivariate techniques are available, and often it is not clear which method should be applied to a particular data set. In this review, we describe and compare the most widely used multivariate statistical techniques including exploratory, interpretive and discriminatory procedures. We consider several important limitations and assumptions of these methods, and we present examples of how these approaches have been utilized in recent studies to provide insight into the ecology of the microbial world. Finally, we offer suggestions for the selection of appropriate methods based on the research question and data set structure.

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.

The networks of human gut microbe–metabolite associations are different between health and irritable bowel syndrome

The goal of this study was to determine if fecal metabolite and microbiota profiles can serve as biomarkers of human intestinal diseases, and to uncover possible gut microbe–metabolite associations. We employed proton nuclear magnetic resonance to measure fecal metabolites of healthy children and those diagnosed with diarrhea-predominant irritable bowel syndrome (IBS-D). Metabolite levels were associated with fecal microbial abundances. Using several ordination techniques, healthy and irritable bowel syndrome (IBS) samples could be distinguished based on the metabolite profiles of fecal samples, and such partitioning was congruent with the microbiota-based sample separation. Measurements of individual metabolites indicated that the intestinal environment in IBS-D was characterized by increased proteolysis, incomplete anaerobic fermentation and possible change in methane production. By correlating metabolite levels with abundances of microbial genera, a number of statistically significant metabolite–genus associations were detected in stools of healthy children. No such associations were evident for IBS children. This finding complemented the previously observed reduction in the number of microbe–microbe associations in the distal gut of the same cohort of IBS-D children.