Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1.

BACKGROUND: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. RESULTS: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. CONCLUSIONS: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

A role for the endometrial microbiome in dysfunctional menstrual bleeding.

This study aimed to characterise the microbial community within the endometrial cavity and endocervix in women with menorrhagia or dysmenorrhea. Paired endocervical and endometrial biopsy samples were collected from women undergoing operative hysteroscopy and/or laparoscopy. Samples were cohorted based on pathology, indications for surgery, and histological dating of the endometrium. Samples were interrogated for the presence of microbial DNA using a two-step next generation sequencing technology approach to exploit the V5-V8 regions of the 16S rRNA gene. Pyrosequencing revealed that the endocervix and endometrium share a minor microbial community, but that each site harbours a separate and distinct microbial population (p = 0.024). This was also the case for women with menorrhagia and dysmenorrhea (p = 0.017). Lactobacillus spp. were the most abundant microbial taxa present in 50% of the cohorts, and across all endocervical groups. Members of the genera Prevotella, Fusobacterium and Jonquetella were the most abundant taxa identified in samples collected from nulliparous women. It can be concluded that the female upper genital tract is not sterile. Microbial community profiling revealed differences in the endometrial microbial community profiles for: (1) the endocervix compared to the endometrium, and (2), women with menorrhagia versus dysmenorrhea. The distinct microbial community profiles in these women may offer insight into the pathology and clinical management of dysfunctional menstrual bleeding.

Genetic diagnostic profiling in axial spondyloarthritis: a real world study.

OBJECTIVES: Spondyloarthritis (SpA) is often diagnosed late in the course of the disease and improved methods for early diagnosis are required. We have tested the ability of genetic profiling to diagnose axial SpA (axSpA) as a whole group, or ankylosing spondylitis (AS) alone, in a cohort of chronic back pain patients. METHODS: 282 patients were recruited from centres in the United Kingdom, Germany, Taiwan, Canada, Columbia and Turkey as part of the ASAS classification criteria for axSpA study (ASAS cohort). Subjects were classified according to the ASAS axSpA criteria, and the modified New York Criteria for AS. Patients were genotyped for ~200,000 immune-mediated disease SNPs using the Illumina Immunochip. RESULTS: We first established the predictive accuracy of genetic data comparing 9,638 healthy controls and 4,428 AS cases from the homogenous International Genetics of AS (IGAS) Consortium Immunochip study which showed excellent predictive power (AUC=0.91). Genetic risk scores had lower predictive power (AUC=0.83) comparing ASAS cohort axSpA cases meeting the ASAS imaging criteria with IGAS controls. Comparing genetic risk scores showed moderate discriminatory capacity between IGAS AS and ASAS imaging positive cases (AUC 0.67±0.05), indicating that significant differences in genetic makeup exist between the cohorts. CONCLUSIONS: In a clinical setting of referred back pain patients suspected to have axial SpA we were unable to use genetic data to construct a predictive model better than that based on existing clinical data. Potential confounding factors include significant heterogeneity in the ASAS cohort, possibly reflecting the disease heterogeneity of axSpA, or differences between centres in ascertainment or classification performance.

Airway dysbiosis: Haemophilus influenzae and Tropheryma in poorly controlled asthma.

Asthma is a chronic inflammatory disorder of the airways where bacteria may act as protagonists of chronic inflammation. Little is known about the relation of airway inflammation to the presence of specific bacterial taxa. We sought to describe the sputum microbiome in adults with poorly controlled asthma.DNA was extracted from induced sputum and microbial communities were profiled using 16S rRNA pyrosequencing. Bacterial species were characterised, and the relationship between microbial populations, asthma inflammatory subtypes and other covariates was explored. Real-time PCR was used to identify Tropheryma whipplei and Haemophilus influenzae in sputum.Adults with neutrophilic asthma had reduced bacterial diversity and species richness. Tropheryma was identified and confirmed with real-time PCR in 12 (40%) participants. Haemophilus occurred most often in a group of younger atopic males with an increased proportion of neutrophils. PCR confirmed the presence of H. influenzae in 35 (76%) participants with poorly controlled asthma.There are phenotype-specific alterations to the airway microbiome in asthma. Reduced bacterial diversity combined with a high prevalence of H. influenzae was observed in neutrophilic asthma, whereas eosinophilic asthma had abundant T. whipplei.

Microbiota at Multiple Body Sites during Pregnancy in a Rural Tanzanian Population and Effects of Moringa-Supplemented Probiotic Yogurt.

The nutritional status of pregnant women is vital for healthy outcomes and is a concern for a large proportion of the world's population. The role of the microbiota in pregnancy and nutrition is a promising new area of study with potential health ramifications. In many African countries, maternal and infant death and morbidity are associated with malnutrition. Here, we assess the influence of probiotic yogurt containing Lactobacillus rhamnosus GR-1, supplemented with Moringa plant as a source of micronutrients, on the health and oral, gut, vaginal, and milk microbiotas of 56 pregnant women in Tanzania. In an open-label study design, 26 subjects received yogurt daily, and 30 were untreated during the last two trimesters and for 1 month after birth. Samples were analyzed using 16S rRNA gene sequencing, and dietary recalls were recorded. Women initially categorized as nourished or undernourished consumed similar calories and macronutrients, which may explain why there was no difference in the microbiota at any body site. Consumption of yogurt increased the relative abundance of Bifidobacterium and decreased Enterobacteriaceae in the newborn feces but had no effect on the mother's microbiota at any body site. The microbiota of the oral cavity and GI tract remained stable over pregnancy, but the vaginal microbiota showed a significant increase in diversity leading up to and after birth. In summary, daily micronutrient-supplemented probiotic yogurt provides a safe, affordable food for pregnant women in rural Tanzania, and the resultant improvement in the gut microbial profile of infants is worthy of further study.

From deep sequencing to viral tagging: recent advances in viral metagenomics.

Culture-independent high-throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth's most ubiquitous and diverse inhabitants - the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non-viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently labeled viruses to their host cells via high-throughput flow sorting. Sequencing of such isolated virus-host pairs facilitates cultivation-independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts.

Reestablishment of recipient-associated microbiota in the lung allograft is linked to reduced risk of bronchiolitis obliterans syndrome.

RATIONALE: Bronchiolitis obliterans syndrome (BOS) is the primary limiting factor for long-term survival after lung transplantation, and has previously been associated with microbial infections. OBJECTIVES: To cross-sectionally and longitudinally characterize microbial communities in allografts from transplant recipients with and without BOS using a culture-independent method based on high-throughput sequencing. METHODS: Allografts were sampled by bronchoalveolar lavage, and microbial communities were profiled using 16S rRNA gene amplicon pyrosequencing. Community profiles were compared using the weighted Unifrac metric and the relationship between microbial populations, BOS, and other covariates was explored using PERMANOVA and logistic regression. MEASUREMENTS AND MAIN RESULTS: Microbial communities in transplant patients fell into two main groups: those dominated by Pseudomonas or those dominated by Streptococcus and Veillonella, which seem to be mutually exclusive lung microbiomes. Aspergillus culture was also negatively correlated with the Pseudomonas-dominated group. The reestablishment of dominant populations present in patients pretransplant, notably Pseudomonas in individuals with cystic fibrosis, was negatively correlated with BOS. CONCLUSIONS: Recolonization of the allograft by Pseudomonas in individuals with cystic fibrosis is not associated with BOS. In general, reestablishment of pretransplant lung populations in the allograft seems to have a protective effect against BOS, whereas de novo acquisition of microbial populations often belonging to the same genera may increase the risk of BOS.

Grinder: a versatile amplicon and shotgun sequence simulator.

We introduce Grinder (http://sourceforge.net/projects/biogrinder/), an open-source bioinformatic tool to simulate amplicon and shotgun (genomic, metagenomic, transcriptomic and metatranscriptomic) datasets from reference sequences. This is the first tool to simulate amplicon datasets (e.g. 16S rRNA) widely used by microbial ecologists. Grinder can create sequence libraries with a specific community structure, α and ß diversities and experimental biases (e.g. chimeras, gene copy number variation) for commonly used sequencing platforms. This versatility allows the creation of simple to complex read datasets necessary for hypothesis testing when developing bioinformatic software, benchmarking existing tools or designing sequence-based experiments. Grinder is particularly useful for simulating clinical or environmental microbial communities and complements the use of in vitro mock communities.

Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity.

The human oropharynx is a reservoir for many potential pathogens, including streptococcal species that cause endocarditis. Although oropharyngeal microbes have been well described, viral communities are essentially uncharacterized. We conducted a metagenomic study to determine the composition of oropharyngeal DNA viral communities (both phage and eukaryotic viruses) in healthy individuals and to evaluate oropharyngeal swabs as a rapid method for viral detection. Viral DNA was extracted from 19 pooled oropharyngeal swabs and sequenced. Viral communities consisted almost exclusively of phage, and complete genomes of several phage were recovered, including Escherichia coli phage T3, Propionibacterium acnes phage PA6, and Streptococcus mitis phage SM1. Phage relative abundances changed dramatically depending on whether samples were chloroform treated or filtered to remove microbial contamination. pblA and pblB genes of phage SM1 were detected in the metagenomes. pblA and pblB mediate the attachment of S. mitis to platelets and play a significant role in S. mitis virulence in the endocardium, but have never previously been detected in the oral cavity. These genes were also identified in salivary metagenomes from three individuals at three time points and in individual saliva samples by PCR. Additionally, we demonstrate that phage SM1 can be induced by commonly ingested substances. Our results indicate that the oral cavity is a reservoir for pblA and pblB genes and for phage SM1 itself. Further studies will determine the association between pblA and pblB genes in the oral cavity and the risk of endocarditis.

Case studies of the spatial heterogeneity of DNA viruses in the cystic fibrosis lung.

Microbial communities in the lungs of patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) have been shown to be spatially heterogeneous. Viral communities may also vary spatially, leading to localized viral populations and infections. Here, we characterized viral communities from multiple areas of the lungs of two patients with late-stage CF using metagenomics, that is, the explanted lungs from a transplant patient and lungs acquired postmortem. All regions harbored eukaryotic viruses that may infect the human host, notably herpesviruses, anelloviruses, and papillomaviruses. In the highly diseased apical lobes of explant lungs, viral diversity was extremely low, and only eukaryotic viruses were present. The absence of phage suggests that CF-associated microbial biofilms may escape top-down controls by phage predation. The phages present in other lobes of explant lungs and in all lobes of postmortem lungs comprised distinct communities, and encoded genes for clinically important microbial phenotypes, including small colony variants and antibiotic resistance. Based on the these observations, we postulate that viral communities in CF lungs are spatially distinct and contribute to CF pathology by augmenting the metabolic potential of resident microbes, as well as by directly damaging lung tissue via carcinomas and herpesviral outbreaks.

Oxygen minimum zones harbour novel viral communities with low diversity.

Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral-to-microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non-redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non-redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.

Limitations of 16S rRNA Gene Sequencing to Characterize Lactobacillus Species in the Upper Genital Tract.

The endometrial cavity is an upper genital tract site previously thought as sterile, however, advances in culture-independent, next-generation sequencing technology have revealed that this low-biomass site harbors a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most abundant non-pathogenic genital tract commensals. Next-generation sequencing of the female lower genital tract has revealed significant variation amongst microbial community composition with respect to Lactobacillus sp. in samples collected from healthy women and women with urogenital conditions. The aim of this study was to evaluate our ability to characterize members of the genital tract microbial community to species-level taxonomy using variable regions of the 16S rRNA gene. Samples were interrogated for the presence of microbial DNA using next-generation sequencing technology that targets the V5-V8 regions of the 16S rRNA gene and compared to speciation using qPCR. We also performed re-analysis of published data using alternate variable regions of the 16S rRNA gene. In this analysis, we explore next-generation sequencing of clinical genital tract isolates as a method for high throughput identification to species-level of key Lactobacillus sp. Data revealed that characterization of genital tract taxa is hindered by a lack of a consensus protocol and 16S rRNA gene region target allowing comparison between studies.

An antimicrobial peptide that targets DNA repair intermediates in vitro inhibits Salmonella growth within murine macrophages.

The hexapeptide WRWYCR was previously identified on the basis of its ability to inhibit bacteriophage lambda integrase-mediated recombination by trapping and preventing resolution of the Holliday junction intermediate. This peptide inhibits several unrelated DNA repair enzymes that bind to and process Holliday junctions and branched DNA substrates. WRWYCR and its d stereoisomer, wrwycr, are bactericidal against both Gram-positive and Gram-negative bacteria, causing the accumulation of DNA breaks, chromosome segregation defects, and the filamentation of cells. DNA repair is a novel target of antibiotics. In the present study, we examined the ability of the peptides to inhibit the growth of Salmonella in mammalian cells. J774A.1 macrophage-like cells and murine peritoneal macrophages were infected with Salmonella enterica serovar Typhimurium and grown in the presence or absence of peptide. We found that peptide wrwycr reduced the number of Salmonella cells recovered after 24 h growth in J774A.1 cells by 100 to 1,000 times, depending on the multiplicity of infection. The peptide also inhibited Salmonella growth in peritoneal macrophages, and although higher doses were required, these were not toxic to the host cells. The apparent lower level of potency of the peptide paralleled the lower level of replication of Salmonella and the lower level of permeation of the peptide in the peritoneal macrophages than in the J774.1 cells. Treatment with peptide wrwycr elicited the SOS response in a significant fraction of the intracellular bacteria, as would be expected if the mechanism of bacterial killing was the same in pure culture and in host cells. These results represent a proof of principle of the antimicrobial activities of compounds that target DNA repair.

Top-down control of microbial activity and biomass in an Arctic soil ecosystem.

Globally, soil microbes preside over vast carbon stores, and both microbial biomass and activity are known to be regulated by bottom-up controls, that is, limitation by nutrients and energy. However, there is evidence that grazing by protozoans exerts top-down controls on biomass. Here, we investigate top-down control by phage on soil microbes using an experimental site near Barrow, Alaska (71 degrees N, 157 degrees W) during the 2007 growing season. Soil measurements were taken from sites that covered a range of microtopographical features within a drained and thawed lake basin including high- and low-centred ice-wedge polygons to estimate the availability of carbon and nitrogen for microbes. Using both field and laboratory experiments, we successfully increased both microbial biomass and respiration by decreasing phage populations. The addition of carbon and nutrients to soils had no significant effects on biomass or respiration, indicating a lack of bottom-up controls. Additionally, we present the first use of tea extracts as a potent anti-phage agent in soils. Our results suggest that top-down controls, such as phage predation, are critical to regulation of microbial activities in Arctic soils.

The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

Metagenomic signatures of 86 microbial and viral metagenomes.

Previous studies have shown that dinucleotide abundances capture the majority of variation in genome signatures and are useful for quantifying lateral gene transfer and building molecular phylogenies. Metagenomes contain a mixture of individual genomes, and might be expected to lack compositional signatures. In many metagenomic data sets the majority of sequences have no significant similarities to known sequences and are effectively excluded from subsequent analyses. To circumvent this limitation, di-, tri- and tetranucleotide abundances of 86 microbial and viral metagenomes consisting of short pyrosequencing reads were analysed to provide a method which includes all sequences that can be used in combination with other analysis to increase our knowledge about microbial and viral communities. Both principal component analysis and hierarchical clustering showed definitive groupings of metagenomes drawn from similar environments. Together these analyses showed that dinucleotide composition, as opposed to tri- and tetranucleotides, defines a metagenomic signature which can explain up to 80% of the variance between biomes, which is comparable to that obtained by functional genomics. Metagenomes with anomalous content were also identified using dinucleotide abundances. Subsequent analyses determined that these metagenomes were contaminated with exogenous DNA, suggesting that this approach is a useful metric for quality control. The predictive strength of the dinucleotide composition also opens the possibility of assigning ecological classifications to unknown fragments. Environmental selection may be responsible for this dinucleotide signature through direct selection of specific compositional signals; however, simulations suggest that the environment may select indirectly by promoting the increased abundance of a few dominant taxa.

The fallopian tube microbiome: implications for reproductive health.

OBJECTIVE: There is a paucity of data characterizing the microbiota of the female upper genital tract, which controversially is described as a sterile site. We examine whether the fallopian tube harbours an endogenous microbial community. DESIGN: This prospective study collected from women undergoing total hysterectomy or salpingectomy-oophorectomy. SETTING: Private hospital gynaecology department. PATIENTS: Fallopian tubes were collected from women diagnosed with benign disease or for prophylaxis. INTERVENTIONS: Samples were interrogated for the presence of microbial DNA using a next generation sequencing technology approach to exploit the V5 to V9 regions of the 16S rRNA gene. MAIN OUTCOME MEASURES: The fallopian tube microbiota was characterized using traditional culture techniques and next generation sequencing. RESULTS: Bacteria were isolated from 50% of cultured samples, and 100% of samples returned positive PCR results. Only 68% of the culture isolates could be confidently identified using automated diagnostic equipment in a clinical microbiology laboratory. Monomicrobial communities were identified only for cultured isolates (50%). Pyrosequencing revealed that all communities were polymicrobial. Lactobacillus spp. were not present in all groups, nor were they the most dominant isolates. Distinct differences in the microbial communities were evident for left compared to right fallopian tubes, ampulla versus isthmus, pre- and post- menopausal tissue, and in secretory phase fallopian tubes with and without Mirena intrauterine devices in situ (all p < 0.05). CONCLUSION: The female upper genital tract is not sterile. Distinct microbial community profiles in the fallopian tubes of healthy women suggest that this genital tract site supports an endogenous microbiota.

Fallopian tube microbiota: evidence beyond DNA.

AIM: To determine whether cultivation-dependent and -independent analyses identifying fallopian tube bacteria were associated with visually observable microbial cells in situ using scanning electron microscopy. PATIENTS: Fallopian tubes were collected from pre- and postmenopausal women undergoing salpingectomies for benign disease or as prophylaxis. MATERIALS & METHODS: Fresh fallopian tube samples were processed for scanning electron microscopy to characterize fallopian tube ultrastructure. Histopathology was used to exclude fallopian tube abnormalities and for menstrual cycle staging of the endometrium. RESULTS: Scanning electron microscopy revealed observable microbial cells in fallopian tube samples. CONCLUSION: In the absence of inflammatory pathology, the fallopian tube harbors a visually observable microbial population, which correlates with cultivation-dependent and -independent data, further refuting the sterility of this anatomical niche.

Brief Report: Intestinal Dysbiosis in Ankylosing Spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a common, highly heritable immune-mediated arthropathy that occurs in genetically susceptible individuals exposed to an unknown but likely ubiquitous environmental trigger. There is a close relationship between the gut and spondyloarthritis, as exemplified in patients with reactive arthritis, in whom a typically self-limiting arthropathy follows either a gastrointestinal or urogenital infection. Microbial involvement in AS has been suggested; however, no definitive link has been established. The aim of this study was to determine whether the gut in patients with AS carries a distinct microbial signature compared with that in the gut of healthy control subjects. METHODS: Microbial profiles for terminal ileum biopsy specimens obtained from patients with recent-onset tumor necrosis factor antagonist-naive AS and from healthy control subjects were generated using culture-independent 16S ribosomal RNA gene sequencing and analysis techniques. RESULTS: Our results showed that the terminal ileum microbial communities in patients with AS differ significantly (P < 0.001) from those in healthy control subjects, driven by a higher abundance of 5 families of bacteria (Lachnospiraceae [P = 0.001], Ruminococcaceae [P = 0.012], Rikenellaceae [P = 0.004], Porphyromonadaceae [P = 0.001], and Bacteroidaceae [P = 0.001]) and a decrease in the abundance of 2 families of bacteria (Veillonellaceae [P = 0.01] and Prevotellaceae [P = 0.004]). CONCLUSION: We show evidence for a discrete microbial signature in the terminal ileum of patients with AS compared with healthy control subjects. The microbial composition was demonstrated to correlate with disease status, and greater differences were observed between disease groups than within disease groups. These results are consistent with the hypothesis that genes associated with AS act, at least in part, through effects on the gut microbiome.

Do you kiss your mother with that mouth? An authentic large-scale undergraduate research experience in mapping the human oral microbiome.

Clinical microbiology testing is crucial for the diagnosis and treatment of community and hospital-acquired infections. Laboratory scientists need to utilize technical and problem-solving skills to select from a wide array of microbial identification techniques. The inquiry-driven laboratory training required to prepare microbiology graduates for this professional environment can be difficult to replicate within undergraduate curricula, especially in courses that accommodate large student cohorts. We aimed to improve undergraduate scientific training by engaging hundreds of introductory microbiology students in an Authentic Large-Scale Undergraduate Research Experience (ALURE). The ALURE aimed to characterize the microorganisms that reside in the healthy human oral cavity-the oral microbiome-by analyzing hundreds of samples obtained from student volunteers within the course. Students were able to choose from selective and differential culture media, Gram-staining, microscopy, as well as polymerase chain reaction (PCR) and 16S rRNA gene sequencing techniques, in order to collect, analyze, and interpret novel data to determine the collective oral microbiome of the student cohort. Pre- and postsurvey analysis of student learning gains across two iterations of the course (2012-2013) revealed significantly higher student confidence in laboratory skills following the completion of the ALURE (p < 0.05 using the Mann-Whitney U-test). Learning objectives on effective scientific communication were also met through effective student performance in laboratory reports describing the research outcomes of the project. The integration of undergraduate research in clinical microbiology has the capacity to deliver authentic research experiences and improve scientific training for large cohorts of undergraduate students.