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.

ABSTRACT Background The endometrial cavity is an upper genital tract site largely heralded as sterile, however, advances in culture-independent, next generation sequencing technology have revealed that this site harbours a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most common 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 and diseased women. The aim of this study was to evaluate the ability of the 16S rRNA gene to characterize genital tract lactobacilli to species-level taxonomy. Methods Samples were interrogated for the presence of microbial DNA using two-step next generation sequencing technology to exploit the V5–V8 regions of the 16S rRNA gene and compared to standard speciation using qPCR. Results The V5-V8 region of the 16S rRNA gene has sufficient sequence variation within frequently encountered genital tract lactobacilli to allow accurate determination of relative abundance within the community, and speciation for several key community members without completing additional experimentation. Conclusions Next-generation sequencing of clinical genital tract isolates is an effective method for high throughput identification to species-level of key Lactobacillus sp. IMPORTANCE Human microbiome experiments, including the low biomass organs such as the upper genital tract, require the development of consensus protocols to ensure accurate comparison between such studies and our data forms an important foundation for future protocols. This paper provides evidence to support the selection of the V5-V8 regions of the 16S rRNA gene improved Lactobacillus speciation using next generation sequencing technology. The choice of variable region for broad-range amplification in microbiome studies is important due to preferential primer binding associated with some genera based on nucleotide sequence patterns. By utilising the V5-V8 region, multiple species of Lactobacillus can be characterised with relative confidence.

The Deblur sOTU counts table for the fecal samples used in the American Gut Project manuscript. The samples were trimmed to a common read length of 125nt, and processed by Deblur (Amir et al mSystems 2017). Blooms were removed (Amir et al mSystems 2017) and any sample with fewer than 1250 sequences was omitted. This table is not rarefied,.

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.

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.

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.

The full American Gut Project mapping file, includes non-fecal samples.

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.

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.

Although a higher maternal body mass index is associated with preterm birth, it is unclear whether excess gestational weight gain (GWG) or obesity drives increased risk. We and others have shown that the placenta harbors microbiota, which is significantly different among preterm births. Our aim in this study was to investigate whether the preterm placental microbiome varies by virtue of obesity or alternately by excess GWG.Placentas (n = 320) were collected from term and preterm pregnancies. Genomic DNA was extracted and subjected to metagenomic sequencing. Data were analyzed by clinical covariates that included the 2009 Institute of Medicine’s GWG guideline and obesity.Analysis of 16S recombinant RNA–based metagenomics revealed no clustering of the microbiome by virtue of obesity (P = .161). Among women who spontaneously delivered preterm, there was again no clustering by obesity (P = .480), but there was significant clustering by excess GWG (P = .022). Moreover, among preterm births, detailed analysis identified microbial genera (family and genus level) and bacterial metabolic gene pathways that varied among pregnancies with excess GWG. Notably, excess GWG was associated with decreased microbial folate biosynthesis pathways and decreased butanoate metabolism (linear discriminate analysis, >3.0-fold).Although there were no significant alterations in the microbiome by virtue of obesity per se, excess GWG was associated with an altered microbiome and its metabolic profile among those women who experienced a preterm birth.

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.

Termites and their microbial gut symbionts are major recyclers of lignocellulosic biomass. This important symbiosis is obligate but relatively open and more complex in comparison to other well-known insect symbioses such as the strict vertical transmission of Buchnera in aphids. The relative roles of vertical inheritance and environmental factors such as diet in shaping the termite gut microbiome are not well understood.

ABSTRACT 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.

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.

Urinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenic Escherichia coli strains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typed Escherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene, fimH. There were nine highly abundant fimH types, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eight E. coli urine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicated E. coli-mediated UTIs, single cultured isolates are diagnostic of the infection. IMPORTANCE In clinical practice, the diagnosis and treatment of acute uncomplicated urinary tract infection (UTI) are based on analysis of a single bacterial isolate cultured from urine, and it is assumed that this isolate represents the dominant UTI pathogen. However, these methods detect only culturable bacteria, and the existence of multiple pathogens as well as strain diversity within a single infection is not examined. Here, we explored bacteria present in acute uncomplicated UTIs using culture-independent sequence-based methods. Escherichia coli was the most common organism identified, and analysis of E. coli dominant UTI samples and their paired clinical isolates revealed that in the majority of infections the cultured isolate was representative of the dominant taxon at both the genus and the strain level. Our data demonstrate that in most cases single cultured isolates are diagnostic of UTI and are consistent with the notion of bottlenecks that limit strain diversity during UTI pathogenesis.

In the title-page footnotes of the article by Jiang et al in the May 2014 issue of Arthritis & Rheumatology (pages 1121–1132), the institutional affiliations of the authors who were designated with the number 2 in the byline were shown incorrectly. The correct affiliations are as follows: Jian Yang, PhD, Peter M. Visscher, PhD: University of Queensland, University of Queensland Diamantina Institute, Translational Research Institute, and Queensland Brain Institute, Brisbane, Queensland, Australia; Gibran Hemani, PhD: University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia; Patrick Danoy, MD, PhD, Dana Willner, MD, PhD, Catherine Cremin, BSc(Hons), Johanna Hadler, Dipl Ing, Matthew A. Brown, MD, PhD: University of Queensland, University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia. We regret the error.