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Microbiota dysbiosis and impaired barrier function are among the most prominent features of inflammatory bowel disease. In the gastrointestinal tract, hydrogen sulfide (H2S) is an important regulator of mucosal homeostasis. We hypothesized that H2S promotes resolution of colonic inflammation through actions on microbiota biofilm and the mucus barrier.MethodsWe used mice genetically deficient for a key enzyme for H2S production (cystathionine γ-lyase) and pharmacologically inhibited that enzyme during colitis in wild-type mice. We tested the effects of administering an H2S donor (diallyl disulfide) to rodents during hapten-induced colitis. Colonic microbiota biofilm was visualized by fluorescent in situ hybridization, and mucus granules were quantified with periodic acid–alcian blue staining. We exposed human microbiota biofilms and planktonic bacteria to H2S donors ex vivo to determine changes in their growth, viability, and biomass.ResultsIntestinal microbiota formed linear biofilms in the colon of healthy rodents. During colitis, microbiota biofilms were fragmented and mucus granule production decreased. Endogenous production of H2S had beneficial effects on establishment of microbiota biofilms and colonic mucus production. Therapeutic delivery of H2S into the colon reduced inflammation, restored the microbiota biofilm, and increased the production of mucus granules. In ex vivo human microbiota, H2S not only promoted biofilm formation but also reduced growth of planktonic bacteria.ConclusionsOur results suggest that H2S donors could be used therapeutically during colitis, facilitating correction of microbiota biofilm dysbiosis and mucus layer reconstitution.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lower airways of patients with cystic fibrosis. Throughout the course of infection this organism undergoes adaptations that contribute to its long-term persistence in the airways. While P. aeruginosa diversity has been documented, it is less clear to what extent within-patient diversity contributes to the overall population structure as most studies have been limited to the analysis of only a few isolates per patient per time point. To examine P. aeruginosa population structure in more detail we collected multiple isolates from individual sputum samples of a patient chronically colonized with P. aeruginosa. This strain collection, comprised of 169 clonal isolates and representing three pulmonary exacerbations as well as clinically stable periods, was assayed for a wide selection of phenotypes. These phenotypes included colony morphology, motility, quorum sensing, protease activity, auxotrophy, siderophore levels, antibiotic resistance, and growth profiles. Each phenotype displayed significant variation even within isolates of the same colony morphotype from the same sample. Isolates demonstrated a large degree of individuality across phenotypes, despite being part of a single clonal lineage, suggesting that the P. aeruginosa population in the cystic fibrosis airways is being significantly under-sampled.

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.

The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs. First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Microglia play an important role in the pathogenesis of multiple sclerosis and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). To more fully understand the role of microglia in EAE we characterized microglial transcriptomes before the onset of motor symptoms (pre-onset) and during symptomatic EAE. We compared the transcriptome in brain, where behavioral changes are initiated, and spinal cord, where damage is revealed as motor and sensory deficits. We used a RiboTag strategy to characterize ribosome-bound mRNA only in microglia without incurring possible transcriptional changes after cell isolation. Brain and spinal cord samples clustered separately at both stages of EAE, indicating regional heterogeneity. Differences in gene expression were observed in the brain and spinal cord of pre-onset and symptomatic animals with most profound effects in the spinal cord of symptomatic animals. Canonical pathway analysis revealed changes in neuroinflammatory pathways, immune functions and enhanced cell division in both pre-onset and symptomatic brain and spinal cord. We also observed a continuum of many pathways at pre-onset stage that continue into the symptomatic stage of EAE. Our results provide additional evidence of regional and temporal heterogeneity in microglial gene expression patterns that may help in understanding mechanisms underlying various symptomology in MS.

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. BZUULjYHP9-uSx-sQtCLaZ Video Abstract

Background Aztreonam lysine for inhalation (AZLI) is an inhaled antibiotic used to treat chronic Pseudomonas aeruginosa infection in CF. AZLI improves lung function and quality of life, and reduces exacerbations-improvements attributed to its antipseudomonal activity. Given the extremely high aztreonam concentrations achieved in the lower airways by nebulization, we speculate this may extend its spectrum of activity to other organisms. As such, we sought to determine if AZLI affects the CF lung microbiome and whether community constituents can be used to predict treatment responsiveness. Methods Patients were included if they had chronic P. aeruginosa infection and repeated sputum samples collected before and after AZLI. Sputum DNA was extracted, and the V3-hypervariable region of the 16S ribosomal RNA (rRNA) gene amplified and sequenced. Results Twenty-four patients naïve to AZLI contributed 162 samples. The cohort had a median age of 37.1 years, and a  median FEV 1 of 44% predicted. Fourteen patients were a priori defined as responders for achieving ≥3% FEV 1 improvement following initiation. No significant changes in alpha diversity were noted following AZLI. Furthermore, beta diversity demonstrated clustering with respect to patients, but had no association with AZLI use. However, we did observe a decline in the relative abundance of several individual operational taxonomic units (OTUs) following AZLI initiation suggesting that specific sub-populations of organisms may be impacted. Patients with higher abundance of Staphylococcus and anaerobic organisms including Prevotella and Fusobacterium were less likely to respond to therapy. Conclusions Results from our study suggest potential alternate/additional mechanisms by which AZLI functions. Moreover, our study suggests that the CF microbiota may be used as a biomarker to predict patient responsiveness to therapy suggesting the microbiome may be harnessed for the personalization of therapies.

is the archetypal cystic fibrosis (CF) pathogen. However, the clinical course experienced by infected individuals varies markedly. Understanding these differences is imperative if further improvements in outcomes are to be achieved. Multiple studies have found that patients infected with epidemic (ePA) strains may have a worse clinical prognosis than those infected with unique, non-clonal strains. Additionally, the traditionally uncultured CF lung bacterial community (i.e., CF microbiome) may further influence the outcome. We sought to identify if these two important variables, not identified through routine culture, associate and together may contribute to disease pathogenesis. Patients were classified as being infected with Prairie Epidemic ePA (PES) or a non-clonal strain, unique PA strains (uPA), through a retrospective assessment of a comprehensive strain biobank using a combination of PFGE and PES-specific PCR. Patients were matched to age, sex, time-period controls and sputum samples from equivalent time periods were identified from a sputum biobank. Bacterial 16S rRNA gene profiling and qPCR was used to characterize the respiratory microbiome. We identified 31 patients infected with PES and matched them with uPA controls. Patients infected with PES at baseline have lower microbial diversity (P = 0.02) and higher relative abundance ( < 0.005). Microbial community structure was found to cluster by PA strain type, although it was not the main determinant of community structure as additional factors were also found to be drivers of CF community structure. Communities from PES infected individuals were enriched with and OTUs. The disproportionate disease experienced by ePA infected CF patients may be mediated through a combination of pathogen-pathogen factors as opposed to strictly enhanced virulence of infecting strains.

Background Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia . Methods With MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance. Results Our hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia . Conclusions The ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia . Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.

BackgroundComplex 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.MethodsWe 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.ResultsBased 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.ConclusionsWe 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.