Associations Between the Gut Microbiota and Internalizing Behaviors in Preschool Children.

OBJECTIVE: Emerging evidence points toward a connection between mental health and the gut microbiota and its metabolites (e.g., short-chain fatty acids). It is unknown whether the gut microbiota is associated with the development of mental health problems (e.g., internalizing or externalizing behaviors) in preschool children. The objective of this study was to evaluate associations between the gut microbiota and internalizing and externalizing behaviors in preschool-aged children. METHODS: A community sample of 248 typically developing children (3-5 years of age) provided a stool sample for gut microbiota and SCFA analysis. Parents reported child internalizing and externalizing behaviors using the Child Behavior Checklist. Associations between child behaviors and gut microbiota measures were analyzed using Spearman correlations followed by an adjustment for multiple testing, with subanalysis conducted in children clinically "at risk" for behavioral problems compared with those who were not. RESULTS: There was a correlation between Shannon alpha diversity with internalizing behaviors (rs = -0.134, p = .035) and its subscale somatic complaints (rs = -0.144, p = .023). In addition, children clinically "at risk" for internalizing problems had decreased alpha diversity (U = 551, p = .017). Internalizing behaviors correlated with valerate and isobutyrate (rs = -0.147, p = .021; rs = -0.140, p = .028, respectively). Furthermore the somatic complaints subscale additionally correlated with acetate and butyrate (rs = -0.219, p = .001; rs = -0.241, p < .001, respectively). These findings were also present in children "at risk" for internalizing problems (U = 569, p = .026; U = 571, p = .028) and somatic complaints (U = 164, p = .004; U = 145, p = .001). CONCLUSIONS: These analyses reveal novel associations between internalizing behaviors and the gut microbiota in preschool children. Furthermore, a relationship between somatic complaints and acetate and butyrate was identified, indicating that interventions that increase SCFA production warrant future investigation.

foxc1 is required for embryonic head vascular smooth muscle differentiation in zebrafish.

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrß positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.

Host genetics and diet composition interact to modulate gut microbiota and predisposition to metabolic syndrome in spontaneously hypertensive stroke-prone rats.

Metabolic syndrome encompasses obesity, glucose intolerance, hypertension, and dyslipidemia; however, the interactions between diet and host physiology that predispose to metabolic syndrome are incompletely understood. Here, we explored the effects of a high-fat diet (HFD) on energy balance, gut microbiota, and risk factors of metabolic syndrome in spontaneously hypertensive stroke-prone (SHRSP) and Wistar-Kyoto (WKY) rats. We found that the SHRSP rats were hypertensive, hyperphagic, less sensitive to hypophagic effects of exendin-4, and expended more energy with diminished sensitivity to sympathetic blockade compared to WKY rats. Notably, key thermogenic markers in brown and retroperitoneal adipose tissues and skeletal muscle were up-regulated in SHRSP than WKY rats. Although HFD promoted weight gain, adiposity, glucose intolerance, hypertriglyceridemia, hepatic lipidosis, and hyperleptinemia in both SHRSP and WKY rats, the SHRSP rats weighed less but had comparable percent adiposity to WKY rats, which supports the use of HFD-fed SHRSP rats as a unique model for studying the metabolically obese normal weight (MONW) phenotype in humans. Despite distinct strain differences in gut microbiota composition, diet had a preponderant impact on gut flora with some of the taxa being strongly associated with key metabolic parameters. Together, we provide evidence that interactions between host genetics and diet modulate gut microbiota and predispose SHRSP rats to develop metabolic syndrome.-Singh, A., Zapata, R. C., Pezeshki, A., Workentine, M. L., Chelikani, P. K. Host genetics and diet composition interact to modulate gut microbiota and predisposition to metabolic syndrome in spontaneously hypertensive stroke-prone rats.

Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Macrophages play central roles in immunity as early effectors and modulating adaptive immune reponses; we implicated macrophages in the anticolitic effect of infection with the tapeworm Hymenolepis diminuta. Here, gene arrays revealed that H. diminuta antigen (HdAg) evoked a program in murine macrophages distinct from that elicited by IL-4. Further, HdAg suppressed LPS-evoked release of TNF-α and IL-1ß from macrophages via autocrine IL-10 signaling. In assessing the ability of macrophages treated in vitro with an extract of H. diminuta [M(HdAg)] to affect disease, intravenous, but not peritoneal, injection of M(HdAg) protected wild-type but not RAG1-/- mice from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Administration of splenic CD4+ T cells from in vitro cocultures with M(HdAg), but not those cocultured with M(IL-4) cells, inhibited DNBS-induced colitis; fractionation of the T-cell population indicated that the CD4+CD25+ T cells from cocultures with M(HdAg) drove the suppression of DNBS-induced colitis. Use of IL-4-/- or IL-10-/- CD4+ T cells revealed that neither cytokine alone from the donor cells was essential for the anticolitic effect. These data illustrate that HdAg evokes a unique regulatory program in macrophages, identifies HdAg-evoked IL-10 suppression of macrophage activation, and reveals the ability of HdAg-treated macrophages to educate ( i.e., condition) and mobilize CD4+CD25+ T cells, which could be deployed to treat colonic inflammation.-Reyes, J. L., Lopes, F., Leung, G., Jayme, T. S., Matisz, C. E., Shute, A., Burkhard, R., Carneiro, M., Workentine, M. L., Wang, A., Petri, B., Beck, P. L., Geuking, M. B., McKay, D. M., Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

A database for ITS2 sequences from nematodes.

BACKGROUND: Marker gene surveys have a wide variety of applications in species identification, population genetics, and molecular epidemiology. As these methods expand to new types of organisms and additional markers beyond 16S and 18S rRNA genes, comprehensive databases are a critical requirement for proper analysis of these data. RESULTS: Here we present an ITS2 rDNA database for marker gene surveys of both free-living and parasitic nematode populations and the software used to build the database. This is currently the most complete and up-to-date ITS2 database for nematodes and is able to reproduce previous analysis that used a smaller database. CONCLUSIONS: This database is an important resource for researchers working on nematodes and also provides a tool to create ITS2 databases for any given taxonomy.

Sputum microbiota is predictive of long-term clinical outcomes in young adults with cystic fibrosis.

BACKGROUND: Complex polymicrobial communities infect cystic fibrosis (CF) lower airways. Generally, communities with low diversity, dominated by classical CF pathogens, associate with worsened patient status at sample collection. However, it is not known if the microbiome can predict future outcomes. We sought to determine if the microbiome could be adapted as a biomarker for patient prognostication. METHODS: We retrospectively assessed prospectively collected sputum from a cohort of 104 individuals aged 18-22 to determine factors associated with progression to early end-stage lung disease (eESLD; death/transplantation <25 years) and rapid pulmonary function decline (>-3%/year FEV1 over the ensuing 5 years). Illumina MiSeq paired-end sequencing of the V3-V4 region of the 16S rRNA was used to define the airway microbiome. RESULTS: Based on the primary outcome analysed, 17 individuals (16%) subsequently progressed to eESLD. They were more likely to have sputum with low alpha diversity, dominated by specific pathogens including Pseudomonas. Communities with abundant Streptococcus were observed to be protective. Microbial communities clustered together by baseline lung disease stage and subsequent progression to eESLD. Multivariable analysis identified baseline lung function and alpha diversity as independent predictors of eESLD. For the secondary outcomes, 58 and 47 patients were classified as rapid progressors based on absolute and relative definitions of lung function decline, respectively. Patients with low alpha diversity were similarly more likely to be classified as experiencing rapid lung function decline over the ensuing 5 years when adjusted for baseline lung function. CONCLUSIONS: We observed that the diversity of microbial communities in CF airways is predictive of progression to eESLD and disproportionate lung function decline and may therefore represent a novel biomarker.

Genomic analysis of the multi-host pathogen Erysipelothrix rhusiopathiae reveals extensive recombination as well as the existence of three generalist clades with wide geographic distribution.

BACKGROUND: Knowledge about how bacterial populations are structured is an important prerequisite for studying their ecology and evolutionary history and facilitates inquiry into host specificity, pathogenicity, geographic dispersal and molecular epidemiology. Erysipelothrix rhusiopathiae is an opportunistic pathogen that is currently reemerging in both the swine and poultry industries globally. This bacterium sporadically causes mortalities in captive marine mammals, and has recently been implicated in large-scale wildlife die-offs. However, despite its economic relevance and broad geographic and host distribution, including zoonotic potential, the global diversity, recombination rates, and population structure of this bacterium remain poorly characterized. In this study, we conducted a broad-scale genomic comparison of E. rhusiopathiae based on a diverse collection of isolates in order to address these knowledge gaps. RESULTS: Eighty-three E. rhusiopathiae isolates from a range of host species and geographic origins, isolated between 1958 and 2014, were sequenced and assembled using both reference-based mapping and de novo assembly. We found that a high proportion of the core genome (58 %) had undergone recombination. Therefore, we used three independent methods robust to the presence of recombination to define the population structure of this species: a phylogenetic tree based on a set of conserved protein sequences, in silico chromosome painting, and network analysis. All three methods were broadly concordant and supported the existence of three distinct clades within the species E. rhusiopathiae. Although we found some evidence of host and geographical clustering, each clade included isolates from diverse host species and from multiple continents. CONCLUSIONS: Using whole genome sequence data, we confirm recent suggestions that E. rhusiopathiae is a weakly clonal species that has been shaped extensively by homologous recombination. Despite frequent recombination, we can reliably identify three distinct clades that do not clearly segregate by host species or geographic origin. Our results provide an essential baseline for future molecular epidemiological, ecological and evolutionary studies of E. rhusiopathiae and facilitate comparisons to other recombinogenic, multi-host bacteria.

Twenty-five-year outbreak of Pseudomonas aeruginosa infecting individuals with cystic fibrosis: identification of the prairie epidemic strain.

Transmissible strains of Pseudomonas aeruginosa have been described for cystic fibrosis (CF) and may be associated with a worse prognosis. Using a comprehensive strain biobank spanning 3 decades, we sought to determine the prevalence and stability of chronic P. aeruginosa infection in an adult population. P. aeruginosa isolates from sputum samples collected at initial enrollment in our adult clinic and at the most recent clinic visit were examined by a combination of pulsed-field gel electrophoresis and multilocus sequence typing and compared against a collection of established transmissible and local non-CF bronchiectasis (nCFB) isolates. A total of 372 isolates from 107 patients, spanning 674 patient-years, including 66 patients with matched isolates from initial and final encounters, were screened. A novel clone with increased antibacterial resistance, termed the prairie epidemic strain (PES), was found in 29% (31/107 patients) of chronically infected patients referred from multiple prairie-based CF centers. This isolate was not found in those diagnosed with CF as adults or in a control population with nCFB. While 90% (60/66 patients) of patients had stable infection over a mean of 10.8 years, five patients experienced strain displacement of unique isolates, with PES occurring within 2 years of transitioning to adult care. PES has been present in our cohort since at least 1987, is unique to CF, generally establishes chronic infection during childhood, and has been found in patients at the time of transition of patients from multiple prairie-based CF clinics, suggesting broad endemicity. Studies are under way to evaluate the clinical implications of PES infection.

Cluster-specific associations between the gut microbiota and behavioral outcomes in preschool-aged children.

BACKGROUND: The gut microbiota is recognized as a regulator of brain development and behavioral outcomes during childhood. Nonetheless, associations between the gut microbiota and behavior are often inconsistent among studies in humans, perhaps because many host-microbe relationships vary widely between individuals. This study aims to stratify children based on their gut microbiota composition (i.e., clusters) and to identify novel gut microbiome cluster-specific associations between the stool metabolomic pathways and child behavioral outcomes. METHODS: Stool samples were collected from a community sample of 248 typically developing children (3-5 years). The gut microbiota was analyzed using 16S sequencing while LC-MS/MS was used for untargeted metabolomics. Parent-reported behavioral outcomes (i.e., Adaptive Skills, Internalizing, Externalizing, Behavioral Symptoms, Developmental Social Disorders) were assessed using the Behavior Assessment System for Children (BASC-2). Children were grouped based on their gut microbiota composition using the Dirichlet multinomial method, after which differences in the metabolome and behavioral outcomes were investigated. RESULTS: Four different gut microbiota clusters were identified, where the cluster enriched in both Bacteroides and Bifidobacterium (Ba2) had the most distinct stool metabolome. The cluster characterized by high Bifidobacterium abundance (Bif), as well as cluster Ba2, were associated with lower Adaptive Skill scores and its subcomponent Social Skills. Cluster Ba2 also had significantly lower stool histidine to urocanate turnover, which in turn was associated with lower Social Skill scores in a cluster-dependent manner. Finally, cluster Ba2 had increased levels of compounds involved in Galactose metabolism (i.e., stachyose, raffinose, alpha-D-glucose), where alpha-D-glucose was associated with the Adaptive Skill subcomponent Daily Living scores (i.e., ability to perform basic everyday tasks) in a cluster-dependent manner. CONCLUSIONS: These data show novel associations between the gut microbiota, its metabolites, and behavioral outcomes in typically developing preschool-aged children. Our results support the concept that cluster-based groupings could be used to develop more personalized interventions to support child behavioral outcomes. Video Abstract.

Spatial distributions of Pseudomonas fluorescens colony variants in mixed-culture biofilms.

BACKGROUND: The emergence of colony morphology variants in structured environments is being recognized as important to both niche specialization and stress tolerance. Pseudomonas fluorescens demonstrates diversity in both its natural environment, the rhizosphere, and in laboratory grown biofilms. Sub-populations of these variants within a biofilm have been suggested as important contributors to antimicrobial stress tolerance given their altered susceptibility to various agents. As such it is of interest to determine how these variants might be distributed in the biofilm environment. RESULTS: Here we present an analysis of the spatial distribution of Pseudomonas fluorescens colony morphology variants in mixed-culture biofilms with the wildtype phenotype. These findings reveal that two variant colony morphotypes demonstrate a significant growth advantage over the wildtype morphotype in the biofilm environment. The two variant morphotypes out-grew the wildtype across the entire biofilm and this occurred within 24 h and was maintained through to 96 h. This competitive advantage was not observed in homogeneous broth culture. CONCLUSIONS: The significant advantage that the variants demonstrate in biofilm colonization over the wildtype denotes the importance of this phenotype in structured environments.