Toni Ann Mistretta

Gastrointestinal Microbiome Signatures of Pediatric Patients With Irritable Bowel Syndrome

BACKGROUND & AIMS The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS. METHODS We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7-12 years, by 16S ribosomal RNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8741 bacterial taxa (16S rRNA PhyloChip). RESULTS Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class γ-proteobacteria (0.07% vs 0.89% of total bacteria, respectively; P < .05); 1 prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes. CONCLUSIONS Using 16S metagenomics by PhyloChip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.

A Metagenomic Approach to Characterization of the Vaginal Microbiome Signature in Pregnancy

While current major national research efforts (i.e., the NIH Human Microbiome Project) will enable comprehensive metagenomic characterization of the adult human microbiota, how and when these diverse microbial communities take up residence in the host and during reproductive life are unexplored at a population level. Because microbial abundance and diversity might differ in pregnancy, we sought to generate comparative metagenomic signatures across gestational age strata. DNA was isolated from the vagina (introitus, posterior fornix, midvagina) and the V5V3 region of bacterial 16S rRNA genes were sequenced (454FLX Titanium platform). Sixty-eight samples from 24 healthy gravidae (18 to 40 confirmed weeks) were compared with 301 non-pregnant controls (60 subjects). Generated sequence data were quality filtered, taxonomically binned, normalized, and organized by phylogeny and into operational taxonomic units (OTU); principal coordinates analysis (PCoA) of the resultant beta diversity measures were used for visualization and analysis in association with sample clinical metadata. Altogether, 1.4 gigabytes of data containing >2.5 million reads (averaging 6,837 sequences/sample of 493 nt in length) were generated for computational analyses. Although gravidae were not excluded by virtue of a posterior fornix pH >4.5 at the time of screening, unique vaginal microbiome signature encompassing several specific OTUs and higher-level clades was nevertheless observed and confirmed using a combination of phylogenetic, non-phylogenetic, supervised, and unsupervised approaches. Both overall diversity and richness were reduced in pregnancy, with dominance of Lactobacillus species (L. iners crispatus, jensenii and johnsonii, and the orders Lactobacillales (and Lactobacillaceae family), Clostridiales, Bacteroidales, and Actinomycetales. This intergroup comparison using rigorous standardized sampling protocols and analytical methodologies provides robust initial evidence that the vaginal microbial 16S rRNA gene catalogue uniquely differs in pregnancy, with variance of taxa across vaginal subsite and gestational age.

Exploring Metabolic Pathway Reconstruction and Genome-Wide Expression Profiling in Lactobacillus reuteri to Define Functional Probiotic Features

The genomes of four Lactobacillus reuteri strains isolated from human breast milk and the gastrointestinal tract have been recently sequenced as part of the Human Microbiome Project. Preliminary genome comparisons suggested that these strains belong to two different clades, previously shown to differ with respect to antimicrobial production, biofilm formation, and immunomodulation. To explain possible mechanisms of survival in the host and probiosis, we completed a detailed genomic comparison of two breast milk–derived isolates representative of each group: an established probiotic strain (L. reuteri ATCC 55730) and a strain with promising probiotic features (L. reuteri ATCC PTA 6475). Transcriptomes of L. reuteri strains in different growth phases were monitored using strain-specific microarrays, and compared using a pan-metabolic model representing all known metabolic reactions present in these strains. Both strains contained candidate genes involved in the survival and persistence in the gut such as mucus-binding proteins and enzymes scavenging reactive oxygen species. A large operon predicted to encode the synthesis of an exopolysaccharide was identified in strain 55730. Both strains were predicted to produce health-promoting factors, including antimicrobial agents and vitamins (folate, vitamin B12). Additionally, a complete pathway for thiamine biosynthesis was predicted in strain 55730 for the first time in this species. Candidate genes responsible for immunomodulatory properties of each strain were identified by transcriptomic comparisons. The production of bioactive metabolites by human-derived probiotics may be predicted using metabolic modeling and transcriptomics. Such strategies may facilitate selection and optimization of probiotics for health promotion, disease prevention and amelioration.

Host Response to Probiotics Determined by Nutritional Status of Rotavirus-infected Neonatal Mice

Objectives: Beneficial microbes and probiotics are promising agents for the prevention and treatment of enteric and diarrheal diseases in children; however, little is known about their in vivo mechanisms of action. We used a neonatal mouse model of rotavirus diarrhea to gain insight into how probiotics ameliorate acute gastroenteritis. Methods: Rotavirus-infected mice were treated with 1 of 2 strains of human-derived Lactobacillus reuteri. We assessed intestinal microbiome composition with 16S metagenomic sequencing, enterocyte migration and proliferation with 5-bromo-2′-deoxyuridine, and antibody and cytokine concentrations with multiplex analyses of intestinal explant cultures. Results: Probiotics reduced diarrhea duration, improved intestinal histopathology, and enhanced intestinal microbiome richness and phylogenetic diversity. The magnitude of reduction of diarrhea by probiotics was strain specific and influenced by nutritional status. L reuteri DSM 17938 reduced diarrhea duration by 0, 1, and 2 days in underweight, normal weight, and overweight pups, respectively. The magnitude of reduction of diarrhea duration correlated with increased enterocyte proliferation and migration. Strain ATCC PTA 6475 reduced diarrhea duration by 1 day in all of the mice without increasing enterocyte proliferation. Both probiotic strains decreased concentrations of proinflammatory cytokines, including macrophage inflammatory protein-1&agr; and interleukin-1&bgr;, in all of the animals, and increased rotavirus-specific antibodies in all but the underweight animals. Body weight also influenced the host response to rotavirus, in terms of diarrhea duration, enterocyte turnover, and antibody production. Conclusions: These data suggest that probiotic enhancement of enterocyte proliferation, villus repopulation, and virus-specific antibodies may contribute to diarrhea resolution, and that nutritional status influences the host response to both beneficial microbes and pathogens.

Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans

BackgroundPolymicrobial infections are responsible for significant mortality and morbidity in adults and children. Staphylococcus epidermidis and Candida albicans are the most frequent combination of organisms isolated from polymicrobial infections. Vascular indwelling catheters are sites for mixed species biofilm formation and pose a significant risk for polymicrobial infections. We hypothesized that enhancement of biofilms in a mixed species environment increases patient mortality and morbidity.ResultsMixed species biofilms of S. epidermidis and C. albicans were evaluated in vitro and in a subcutaneous catheter infection model in vivo. Mixed species biofilms were enhanced compared to single species biofilms of either S. epidermidis or C. albicans. A mixed species environment increased catheter infection and increased dissemination of S. epidermidis in mice. Microarrays were used to explore differential gene expression of S. epidermidis in the mixed species biofilms. In mixed species biofilms, compared to single species S. epidermidis biofilms, 2.7% of S. epidermidis genes were upregulated and 6% were down regulated. Staphylococcal autolysis repressors lrgA and lrgB were down regulated 36-fold and 27-fold respectively. The role of biofilm extracellular DNA was investigated by quantitation and by evaluating the effects of DNAse in a concentration and time dependent manner. S. epidermidis specific eDNA was increased in mixed species biofilms and further confirmed by degradation with DNAse.ConclusionsMixed-species biofilms are enhanced and associated with increased S. epidermidis-specific eDNA in vitro and greater systemic dissemination of S. epidermidis in vivo. Down regulation of the lrg operon, a repressor of autolysis, associated with increased eDNA suggests a possible role for bacterial autolysis in mixed species biofilms. Enhancement and systemic dissemination of S. epidermidis may explain adverse outcomes after clinical polymicrobial infections of S. epidermidis and C. albicans.

From Prediction to Function Using Evolutionary Genomics: Human-Specific Ecotypes of Lactobacillus reuteri Have Diverse Probiotic Functions

The vertebrate gut symbiont Lactobacillus reuteri has diversified into separate clades reflecting host origin. Strains show evidence of host adaptation, but how host–microbe coevolution influences microbial-derived effects on hosts is poorly understood. Emphasizing human-derived strains of L. reuteri, we combined comparative genomic analyses with functional assays to examine variations in host interaction among genetically distinct ecotypes. Within clade II or VI, the genomes of human-derived L. reuteri strains are highly conserved in gene content and at the nucleotide level. Nevertheless, they share only 70–90% of total gene content, indicating differences in functional capacity. Human-associated lineages are distinguished by genes related to bacteriophages, vitamin biosynthesis, antimicrobial production, and immunomodulation. Differential production of reuterin, histamine, and folate by 23 clade II and VI strains was demonstrated. These strains also differed with respect to their ability to modulate human cytokine production (tumor necrosis factor, monocyte chemoattractant protein-1, interleukin [IL]-1β, IL-5, IL-7, IL-12, and IL-13) by myeloid cells. Microarray analysis of representative clade II and clade VI strains revealed global regulation of genes within the reuterin, vitamin B12, folate, and arginine catabolism gene clusters by the AraC family transcriptional regulator, PocR. Thus, human-derived L. reuteri clade II and VI strains are genetically distinct and their differences affect their functional repertoires and probiotic features. These findings highlight the biological impact of microbe:host coevolution and illustrate the functional significance of subspecies differences in the human microbiome. Consideration of host origin and functional differences at the subspecies level may have major impacts on probiotic strain selection and considerations of microbial ecology in mammalian species.

The Genboree Microbiome Toolset and the analysis of 16S rRNA microbial sequences

BackgroundMicrobial metagenomic analyses rely on an increasing number of publicly available tools. Installation, integration, and maintenance of the tools poses significant burden on many researchers and creates a barrier to adoption of microbiome analysis, particularly in translational settings.MethodsTo address this need we have integrated a rich collection of microbiome analysis tools into the Genboree Microbiome Toolset and exposed them to the scientific community using the Software-as-a-Service model via the Genboree Workbench. The Genboree Microbiome Toolset provides an interactive environment for users at all bioinformatic experience levels in which to conduct microbiome analysis. The Toolset drives hypothesis generation by providing a wide range of analyses including alpha diversity and beta diversity, phylogenetic profiling, supervised machine learning, and feature selection.ResultsWe validate the Toolset in two studies of the gut microbiota, one involving obese and lean twins, and the other involving children suffering from the irritable bowel syndrome.ConclusionsBy lowering the barrier to performing a comprehensive set of microbiome analyses, the Toolset empowers investigators to translate high-volume sequencing data into valuable biomedical discoveries.

Genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups

Despite being the most common liver cancer in children, hepatoblastoma (HB) is a rare neoplasm. Consequently, few pretreatment tumors have been molecularly profiled, and there are no validated prognostic or therapeutic biomarkers for HB patients. We report on the first large‐scale effort to profile pretreatment HBs at diagnosis. Our analysis of 88 clinically annotated HBs revealed three risk‐stratifying molecular subtypes that are characterized by differential activation of hepatic progenitor cell markers and metabolic pathways: high‐risk tumors were characterized by up‐regulated nuclear factor, erythroid 2–like 2 activity; high lin‐28 homolog B, high mobility group AT‐hook 2, spalt‐like transcription factor 4, and alpha‐fetoprotein expression; and high coordinated expression of oncofetal proteins and stem‐cell markers, while low‐risk tumors had low lin‐28 homolog B and lethal‐7 expression and high hepatic nuclear factor 1 alpha activity. Conclusion: Analysis of immunohistochemical assays using antibodies targeting these genes in a prospective study of 35 HBs suggested that these candidate biomarkers have the potential to improve risk stratification and guide treatment decisions for HB patients at diagnosis; our results pave the way for clinical collaborative studies to validate candidate biomarkers and test their potential to improve outcome for HB patients. (Hepatology 2017;65:104‐121).

Gene expression in Barrett's esophagus: Laser capture versus whole tissue

Objective. Diagnosis of Barretts esophagus (BE) is typically done through morphologic analysis of esophageal tissue biopsy. Such samples contain several cell types. Laser capture microdissection (LCM) allows the isolation of specific cells from heterogeneous cell populations. The purpose of this study was to determine the degree of overlap of the two sample types and to define a set of genes that might serve as biochemical markers for BE. Material and methods. Biopsies were obtained from regions of the glandular tissue of BE and normal esophagus from 9 subjects with BE. Samples from 5 subjects were examined as whole tissue (BE [whole]; E [whole]), and in 4 subjects the glandular epithelium of BE was isolated using LCM (BE [LCM]) and compared with the averaged values (E [LCM]) for both basal cell (B [LCM]) and squamous cell (S [LCM]) epithelium. Results. Gene expression revealed 1797 probe sets between BE [whole] and E [whole] (fold change > 2.0; p<0.001). Most of these genes (74%) were also differentially expressed between BE [LCM] and E [LCM], showing that there was high concordance between the two sampling methods. LCM provided a great deal of additional information (2113 genes) about the alterations in gene expression that may represent the BE phenotype. Conclusions. There are differences in gene expression profiles depending on whether specimens are whole tissue biopsies or LCM dissected. Whole tissue biopsies should prove satisfactory for diagnostic purposes. Because the data from LCM samples delineated many more Barretts-specific genes, this procedure might provide more information regarding pathogenesis than would whole tissue material.

Context-specific role of SOX9 in NF-Y mediated gene regulation in colorectal cancer cells

Roles for SOX9 have been extensively studied in development and particular emphasis has been placed on SOX9 roles in cell lineage determination in a number of discrete tissues. Aberrant expression of SOX9 in many cancers, including colorectal cancer, suggests roles in these diseases as well and recent studies have suggested tissue- and context-specific roles of SOX9. Our genome wide approach by chromatin immunoprecipitation sequencing (ChIP-seq) in human colorectal cancer cells identified a number of physiological targets of SOX9, including ubiquitously expressed cell cycle regulatory genes, such as CCNB1 and CCNB2, CDK1, and TOP2A. These novel high affinity-SOX9 binding peaks precisely overlapped with binding sites for histone-fold NF-Y transcription factor. Furthermore, our data showed that SOX9 is recruited by NF-Y to these promoters of cell cycle regulatory genes and that SOX9 is critical for the full function of NF-Y in activation of the cell cycle genes. Mutagenesis analysis and in vitro binding assays provided additional evidence to show that SOX9 affinity is through NF-Y and that SOX9 DNA binding domain is not necessary for SOX9 affinity to those target genes. Collectively, our results reveal possibly a context-dependent, non-classical regulatory role for SOX9.