Early-life nasal microbiota dynamics relate to longitudinal respiratory phenotypes in urban children.

BACKGROUND: Five distinct respiratory phenotypes based on latent classes of longitudinal patterns of wheezing, allergic sensitization. and pulmonary function measured in urban children from ages from 0 to 7 years have previously been described. OBJECTIVE: Our aim was to determine whether distinct respiratory phenotypes are associated with early-life upper respiratory microbiota development and environmental microbial exposures. METHODS: Microbiota profiling was performed using 16S ribosomal RNA-based sequencing of nasal samples collected at age 12 months (n = 120) or age 36 months (n = 142) and paired house dust samples collected at 3 months (12-month, n = 73; 36-month, n = 90) from all 4 centers in the Urban Environment and Childhood Asthma (URECA) cohort. RESULTS: In these high-risk urban children, nasal microbiota increased in diversity between ages 12 and 36 months (ß = 2.04; P = .006). Age-related changes in microbiota evenness differed significantly by respiratory phenotypes (interaction P = .0007), increasing most in the transient wheeze group. At age 12 months, respiratory illness (R2 = 0.055; P = .0001) and dominant bacterial genus (R2 = 0.59; P = .0001) explained variance in nasal microbiota composition, and enrichment of Moraxella and Haemophilus members was associated with both transient and high-wheeze respiratory phenotypes. By age 36 months, nasal microbiota was significantly associated with respiratory phenotypes (R2 = 0.019; P = .0376), and Moraxella-dominated microbiota was associated specifically with atopy-associated phenotypes. Analysis of paired house dust and nasal samples indicated that 12 month olds with low wheeze and atopy incidence exhibited the largest number of shared bacterial taxa with their environment. CONCLUSION: Nasal microbiota development over the course of early childhood and composition at age 3 years are associated with longitudinal respiratory phenotypes. These data provide evidence supporting an early-life window of airway microbiota development that is influenced by environmental microbial exposures in infancy and associates with wheeze- and atopy-associated respiratory phenotypes through age 7 years.

Seasonal airway microbiome and transcriptome interactions promote childhood asthma exacerbations.

BACKGROUND: Seasonal variation in respiratory illnesses and exacerbations in pediatric populations with asthma is well described, though whether upper airway microbes play season-specific roles in these events is unknown. OBJECTIVE: We hypothesized that nasal microbiota composition is seasonally dynamic and that discrete microbe-host interactions modify risk of asthma exacerbation in a season-specific manner. METHODS: Repeated nasal samples from children with exacerbation-prone asthma collected during periods of respiratory health (baseline; n = 181 samples) or first captured respiratory illness (n = 97) across all seasons, underwent bacterial (16S ribosomal RNA gene) and fungal (internal transcribed spacer region 2) biomarker sequencing. Virus detection was performed by multiplex PCR. Paired nasal transcriptome data were examined for seasonal dynamics and integrative analyses. RESULTS: Upper airway bacterial and fungal microbiota and rhinovirus detection exhibited significant seasonal dynamics. In seasonally adjusted analysis, variation in both baseline and respiratory illness microbiota related to subsequent exacerbation. Specifically, in the fall, when respiratory illness and exacerbation events were most frequent, several Moraxella and Haemophilus members were enriched both in virus-positive respiratory illnesses and those that progressed to exacerbations. The abundance of 2 discrete bacterial networks, characteristically comprising either Streptococcus or Staphylococcus, exhibited opposing interactions with an exacerbation-associated SMAD3 nasal epithelial transcriptional module to significantly increase the odds of subsequent exacerbation (odds ratio = 14.7, 95% confidence interval = 1.50-144, P = .02; odds ratio = 39.17, 95% confidence interval = 2.44-626, P = .008, respectively). CONCLUSIONS: Upper airway microbiomes covary with season and with seasonal trends in respiratory illnesses and asthma exacerbations. Seasonally adjusted analyses reveal specific bacteria-host interactions that significantly increase risk of asthma exacerbation in these children.

Longitudinal Phenotypes of Respiratory Health in a High-Risk Urban Birth Cohort.

RATIONALE: Characterization of patterns of wheezing and allergic sensitization in early life may allow for identification of specific environmental exposures impacting asthma development. OBJECTIVES: To define respiratory phenotypes in inner-city children and their associations with early-life environmental exposures. METHODS: Data were collected prospectively from 442 children in the URECA (Urban Environment and Childhood Asthma) birth cohort through age 7 years, reflecting symptoms (wheezing), aeroallergen sensitization, pulmonary function, and body mass index. Latent class mixed models identified trajectories of wheezing, allergic sensitization, and pulmonary function. Cluster analysis defined nonoverlapping groups (termed phenotypes). Potential associations between phenotypes and early-life environmental exposures were examined. MEASUREMENTS AND MAIN RESULTS: Five phenotypes were identified and mainly differentiated by patterns of wheezing and allergic sensitization (low wheeze/low atopy; low wheeze/high atopy; transient wheeze/low atopy; high wheeze/low atopy; high wheeze/high atopy). Asthma was most often present in the high-wheeze phenotypes, with greatest respiratory morbidity among children with frequent wheezing and allergic sensitization. These phenotypes differentially related to early-life exposures, including maternal stress and depression, antenatal environmental tobacco smoke, house dust microbiome, and allergen content (all P < 0.05). Prenatal smoke exposure, maternal stress, and depression were highest in the high-wheeze/low-atopy phenotype. The high-wheeze/high-atopy phenotype was associated with low household microbial richness and diversity. Early-life aeroallergen exposure was low in high-wheeze phenotypes. CONCLUSIONS: Patterns of wheezing, allergic sensitization, and lung function identified five respiratory phenotypes among inner-city children. Early-life environmental exposure to stress, depression, tobacco smoke, and indoor allergens and microbes differentially associate with specific phenotypes.

Distinct nasal airway bacterial microbiotas differentially relate to exacerbation in pediatric patients with asthma.

BACKGROUND: In infants, distinct nasopharyngeal bacterial microbiotas differentially associate with the incidence and severity of acute respiratory tract infection and childhood asthma development. OBJECTIVE: We hypothesized that distinct nasal airway microbiota structures also exist in children with asthma and relate to clinical outcomes. METHODS: Nasal secretion samples (n = 3122) collected after randomization during the fall season from children with asthma (6-17 years, n = 413) enrolled in a trial of omalizumab (anti-IgE) underwent 16S rRNA profiling. Statistical analyses with exacerbation as the primary outcome and rhinovirus infection and respiratory illnesses as secondary outcomes were performed. Using A549 epithelial cells, we assessed nasal isolates of Moraxella, Staphylococcus, and Corynebacterium species for their capacity to induce epithelial damage and inflammatory responses. RESULTS: Six nasal airway microbiota assemblages, each dominated by Moraxella, Staphylococcus, Corynebacterium, Streptococcus, Alloiococcus, or Haemophilus species, were observed. Moraxella and Staphylococcus species-dominated microbiotas were most frequently detected and exhibited temporal stability. Nasal microbiotas dominated by Moraxella species were associated with increased exacerbation risk and eosinophil activation. Staphylococcus or Corynebacterium species-dominated microbiotas were associated with reduced respiratory illness and exacerbation events, whereas Streptococcus species-dominated assemblages increased the risk of rhinovirus infection. Nasal microbiota composition remained relatively stable despite viral infection or exacerbation; only a few taxa belonging to the dominant genera exhibited relative abundance fluctuations during these events. In vitro, Moraxella catarrhalis induced significantly greater epithelial damage and inflammatory cytokine expression (IL-33 and IL-8) compared with other dominant nasal bacterial isolates tested. CONCLUSION: Distinct nasal airway microbiotas of children with asthma relate to the likelihood of exacerbation, rhinovirus infection, and respiratory illnesses during the fall season.

Gut microbiota in early pediatric multiple sclerosis: a case-control study.

BACKGROUND AND PURPOSE: Alterations in the gut microbial community composition may be influential in neurological disease. Microbial community profiles were compared between early onset pediatric multiple sclerosis (MS) and control children similar for age and sex. METHODS: Children ≤18 years old within 2 years of MS onset or controls without autoimmune disorders attending a University of California, San Francisco, USA, pediatric clinic were examined for fecal bacterial community composition and predicted function by 16S ribosomal RNA sequencing and phylogenetic reconstruction of unobserved states (PICRUSt) analysis. Associations between subject characteristics and the microbiota, including beta diversity and taxa abundance, were identified using non-parametric tests, permutational multivariate analysis of variance and negative binomial regression. RESULTS: Eighteen relapsing-remitting MS cases and 17 controls (mean age 13 years; range 4-18) were studied. Cases had a short disease duration (mean 11 months; range 2-24) and half were immunomodulatory drug (IMD) naïve. Whilst overall gut bacterial beta diversity was not significantly related to MS status, IMD exposure was (Canberra, P < 0.02). However, relative to controls, MS cases had a significant enrichment in relative abundance for members of the Desulfovibrionaceae (Bilophila, Desulfovibrio and Christensenellaceae) and depletion in Lachnospiraceae and Ruminococcaceae (all P and q < 0.000005). Microbial genes predicted as enriched in MS versus controls included those involved in glutathione metabolism (Mann-Whitney, P = 0.017), findings that were consistent regardless of IMD exposure. CONCLUSIONS: In recent onset pediatric MS, perturbations in the gut microbiome composition were observed, in parallel with predicted enrichment of metabolic pathways associated with neurodegeneration. Findings were suggestive of a pro-inflammatory milieu.

Associations between the gut microbiota and host immune markers in pediatric multiple sclerosis and controls.

BACKGROUND: As little is known of association(s) between gut microbiota profiles and host immunological markers, we explored these in children with and without multiple sclerosis (MS). METHODS: Children ≤18 years provided stool and blood. MS cases were within 2-years of onset. Fecal 16S rRNA gene profiles were generated on an Illumina Miseq platform. Peripheral blood mononuclear cells were isolated, and Treg (CD4+CD25hiCD127lowFoxP3+) frequency and CD4+ T-cell intracellular cytokine production evaluated by flow cytometry. Associations between microbiota diversity, phylum-level abundances and immune markers were explored using Pearson's correlation and adjusted linear regression. RESULTS: Twenty-four children (15 relapsing-remitting, nine controls), averaging 12.6 years were included. Seven were on a disease-modifying drug (DMD) at sample collection. Although immune markers (e.g. Th2, Th17, Tregs) did not differ between cases and controls (p > 0.05), divergent gut microbiota associations occurred; richness correlated positively with Th17 for cases (r = +0.665, p = 0.018), not controls (r = -0.644, p = 0.061). Bacteroidetes inversely associated with Th17 for cases (r = -0.719, p = 0.008), not controls (r = +0.320, p = 0.401). Fusobacteria correlated with Tregs for controls (r = +0.829, p = 0.006), not cases (r = -0.069, p = 0.808). CONCLUSIONS: Our observations motivate further exploration to understand disruption of the microbiota-immune balance so early in the MS course.

Heritable vaginal bacteria influence immune tolerance and relate to early-life markers of allergic sensitization in infancy.

Maternal asthma status, prenatal exposures, and infant gut microbiota perturbation are associated with heightened risk of atopy and asthma risk in childhood, observations hypothetically linked by intergenerational microbial transmission. Using maternal vaginal (n = 184) and paired infant stool (n = 172) samples, we identify four compositionally and functionally distinct Lactobacillus-dominated vaginal microbiota clusters (VCs) that relate to prenatal maternal health and exposures and infant serum immunoglobulin E (IgE) status at 1 year. Variance in bacteria shared between mother and infant pairs relate to VCs, maternal allergy/asthma status, and infant IgE levels. Heritable bacterial gene pathways associated with infant IgE include fatty acid synthesis and histamine and tryptophan degradation. In vitro, vertically transmitted Lactobacillus jensenii strains induce immunosuppressive phenotypes on human antigen-presenting cells. Murine supplementation with L. jensenii reduces lung eosinophils, neutrophilic expansion, and the proportion of interleukin-4 (IL-4)+ CD4+ T cells. Thus, bacterial and atopy heritability are intimately linked, suggesting a microbial component of intergenerational disease transmission.

Moraxella-dominated pediatric nasopharyngeal microbiota associate with upper respiratory infection and sinusitis.

BACKGROUND: Distinct bacterial upper airway microbiota structures have been described in pediatric populations, and relate to risk of respiratory viral infection and, exacerbations of asthma. We hypothesized that distinct nasopharyngeal (NP) microbiota structures exist in pediatric populations, relate to environmental exposures and modify risk of acute sinusitis or upper respiratory infection (URI) in children. METHODS: Bacterial 16S rRNA profiles from nasopharyngeal swabs (n = 354) collected longitudinally over a one-year period from 58 children, aged four to seven years, were analyzed and correlated with environmental variables, URI, and sinusitis outcomes. RESULTS: Variance in nasopharyngeal microbiota composition significantly related to clinical outcomes, participant characteristics and environmental exposures including dominant bacterial genus, season, daycare attendance and tobacco exposure. Four distinct nasopharyngeal microbiota structures (Cluster I-IV) were evident and differed with respect to URI and sinusitis outcomes. These clusters were characteristically either dominated by Moraxella with sparse underlying taxa (Cluster I), comprised of a non-dominated, diverse microbiota (Cluster II), dominated by Alloiococcus/Corynebacterium (Cluster III), or by Haemophilus (Cluster IV). Cluster I was associated with increased risk of URI and sinusitis (RR = 1.18, p = 0.046; RR = 1.25, p = 0.009, respectively) in the population studied. CONCLUSION: In a pediatric population, URI and sinusitis associate with the presence of Moraxella-dominated NP microbiota.

Hydroxyapatite-mediated separation of double-stranded DNA, single-stranded DNA, and RNA genomes from natural viral assemblages.

Metagenomics can be used to determine the diversity of complex, often unculturable, viral communities with various nucleic acid compositions. Here, we report the use of hydroxyapatite chromatography to efficiently fractionate double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), dsRNA, and ssRNA genomes from known bacteriophages. Linker-amplified shotgun libraries were constructed to generate sequencing reads from each hydroxyapatite fraction. Greater than 90% of the reads displayed significant similarity to the expected genomes at the nucleotide level. These methods were applied to marine viruses collected from the Chesapeake Bay and the Dry Tortugas National Park. Isolated nucleic acids were fractionated using hydroxyapatite chromatography followed by linker-amplified shotgun library construction and sequencing. Taxonomic analysis demonstrated that the majority of environmental sequences, regardless of their source nucleic acid, were most similar to dsDNA viruses, reflecting the bias of viral metagenomic sequence databases.

Distinct lung microbiota associate with HIV-associated chronic lung disease in children.

Chronic lung disease (CLD) is a common co-morbidity for HIV-positive children and adolescents on antiretroviral therapy (ART) in sub-Saharan Africa. In this population, distinct airway microbiota may differentially confer risk of CLD. In a cross-sectional study of 202 HIV-infected children aged 6-16 years in Harare, Zimbabwe, we determined the association of sputum microbiota composition (using 16S ribosomal RNA V4 gene region sequencing) with CLD defined using clinical, spirometric, or radiographic criteria. Forty-two percent of children were determined to have CLD according to our definition. Dirichlet multinomial mixtures identified four compositionally distinct sputum microbiota structures. Patients whose sputum microbiota was dominated by Haemophilus, Moraxella or Neisseria (HMN) were at 1.5 times higher risk of CLD than those with Streptococcus or Prevotella (SP)-dominated microbiota (RR = 1.48, p = 0.035). Cell-free products of HMN sputum microbiota induced features of epithelial disruption and inflammatory gene expression in vitro, indicating enhanced pathogenic potential of these CLD-associated microbiota. Thus, HIV-positive children harbor distinct sputum microbiota, with those dominated by Haemophilus, Moraxella or Neisseria associated with enhanced pathogenesis in vitro and clinical CLD.

Gut microbiota in HIV-pneumonia patients is related to peripheral CD4 counts, lung microbiota, and in vitro macrophage dysfunction.

Pneumonia is common and frequently fatal in HIV-infected patients, due to rampant, systemic inflammation and failure to control microbial infection. While airway microbiota composition is related to local inflammatory response, gut microbiota has been shown to correlate with the degree of peripheral immune activation (IL6 and IP10 expression) in HIV-infected patients. We thus hypothesized that both airway and gut microbiota are perturbed in HIV-infected pneumonia patients, that the gut microbiota is related to peripheral CD4+ cell counts, and that its associated products differentially program immune cell populations necessary for controlling microbial infection in CD4-high and CD4-low patients. To assess these relationships, paired bronchoalveolar lavage and stool microbiota (bacterial and fungal) from a large cohort of Ugandan, HIV-infected patients with pneumonia were examined, and in vitro tests of the effect of gut microbiome products on macrophage effector phenotypes performed. While lower airway microbiota stratified into three compositionally distinct microbiota as previously described, these were not related to peripheral CD4 cell count. In contrast, variation in gut microbiota composition significantly related to CD4 cell count, lung microbiota composition, and patient mortality. Compared with patients with high CD4+ cell counts, those with low counts possessed more compositionally similar airway and gut microbiota, evidence of microbial translocation, and their associated gut microbiome products reduced macrophage activation and IL-10 expression and increased IL-1ß expression in vitro. These findings suggest that the gut microbiome is related to CD4 status and plays a key role in modulating macrophage function, critical to microbial control in HIV-infected patients with pneumonia.

Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus.

BACKGROUND: Bracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome. RESULTS: Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses. CONCLUSION: Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to large differences in the predicted gene composition of BV genomes, sequences that appear to mediate processes of viral segment formation, such as proviral segment excision and circularization, appear to be highly conserved, supporting the hypothesis of a single origin for BVs.

Characterizing the gut microbiome in trauma: significant changes in microbial diversity occur early after severe injury.

BACKGROUND: Recent studies have demonstrated the vital influence of commensal microbial communities on human health. The central role of the gut in the response to injury is well described; however, no prior studies have used culture-independent profiling techniques to characterize the gut microbiome after severe trauma. We hypothesized that in critically injured patients, the gut microbiome would undergo significant compositional changes in the first 72 hours after injury. METHODS: Trauma stool samples were prospectively collected via digital rectal examination at the time of presentation (0 hour). Patients admitted to the intensive care unit (n=12) had additional stool samples collected at 24 hours and/or 72 hours. Uninjured patients served as controls (n=10). DNA was extracted from stool samples and 16S rRNA-targeted PCR amplification was performed; amplicons were sequenced and binned into operational taxonomic units (OTUs; 97% sequence similarity). Diversity was analyzed using principle coordinates analyses, and negative binomial regression was used to determine significantly enriched OTUs. RESULTS: Critically injured patients had a median Injury Severity Score of 27 and suffered polytrauma. At baseline (0 hour), there were no detectable differences in gut microbial community diversity between injured and uninjured patients. Injured patients developed changes in gut microbiome composition within 72 hours, characterized by significant alterations in phylogenetic composition and taxon relative abundance. Members of the bacterial orders Bacteroidales, Fusobacteriales and Verrucomicrobiales were depleted during 72 hours, whereas Clostridiales and Enterococcus members enriched significantly. DISCUSSION: In this initial study of the gut microbiome after trauma, we demonstrate that significant changes in phylogenetic composition and relative abundance occur in the first 72 hours after injury. This rapid change in intestinal microbiota represents a critical phenomenon that may influence outcomes after severe trauma. A better understanding of the nature of these postinjury changes may lead to the ability to intervene in otherwise pathological clinical trajectories. LEVEL OF EVIDENCE: III. STUDY TYPE: Prognostic/epidemiological.

Gut microbiota composition and relapse risk in pediatric MS: A pilot study.

We explored the association between baseline gut microbiota (16S rRNA biomarker sequencing of stool samples) in 17 relapsing-remitting pediatric MS cases and risk of relapse over a mean 19.8 months follow-up. From the Kaplan-Meier curve, 25% relapsed within an estimated 166 days from baseline. A shorter time to relapse was associated with Fusobacteria depletion (p=0.001 log-rank test), expansion of the Firmicutes (p=0.003), and presence of the Archaea Euryarchaeota (p=0.037). After covariate adjustments for age and immunomodulatory drug exposure, only absence (vs. presence) of Fusobacteria was associated with relapse risk (hazard ratio=3.2 (95% CI: 1.2-9.0), p=0.024). Further investigation is warranted. Findings could offer new targets to alter the MS disease course.

Gut-Resident Lactobacillus Abundance Associates with IDO1 Inhibition and Th17 Dynamics in SIV-Infected Macaques.

Gut microbes can profoundly modulate mucosal barrier-promoting Th17 cells in mammals. A salient feature of HIV/simian immunodeficiency virus (SIV) immunopathogenesis is the loss of Th17 cells, which has been linked to increased activity of the immunomodulatory enzyme, indoleamine 2,3-dioxygenase 1 (IDO 1). The role of gut microbes in this system remains unknown, and the SIV-infected rhesus macaque provides a well-described model for HIV-associated Th17 loss and mucosal immune disruption. We observed a specific depletion of gut-resident Lactobacillus during acute and chronic SIV infection of rhesus macaques, which was also seen in early HIV-infected humans. This depletion in rhesus macaques correlated with increased IDO1 activity and Th17 loss. Macaques supplemented with a Lactobacillus-containing probiotic exhibited decreased IDO1 activity during chronic SIV infection. We propose that Lactobacillus species inhibit mammalian IDO1 and thus may help to preserve Th17 cells during pathogenic SIV infection, providing support for Lactobacillus species as modulators of mucosal immune homeostasis.

An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform.

BACKGROUND: To take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality. RESULTS: Here we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp "heterogeneity spacer" to the index sequence that allows an equal proportion of samples to be sequenced out of phase. CONCLUSIONS: Our approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.

The vaginal microbiota of pregnant women who subsequently have spontaneous preterm labor and delivery and those with a normal delivery at term.

BACKGROUND: This study was undertaken to determine whether the vaginal microbiota of pregnant women who subsequently had a spontaneous preterm delivery is different from that of women who had a term delivery. RESULTS: This was a nested case-control study of pregnant women who had a term delivery (controls) and those who had a spontaneous preterm delivery before 34 weeks of gestation (cases). Samples of vaginal fluid were collected longitudinally and stored at -70°C until assayed. A microbial survey using pyrosequencing of V1-V3 regions of 16S rRNA genes was performed. We tested the hypothesis of whether the relative abundance of individual microbial species (phylotypes) was different between women who had a term versus preterm delivery. A suite of bioinformatic and statistical tools, including linear mixed effects models and generalized estimating equations, was used. We show that: 1) the composition of the vaginal microbiota during normal pregnancy changed as a function of gestational age, with an increase in the relative abundance of four Lactobacillus spp., and decreased in anaerobe or strict-anaerobe microbial species as pregnancy progressed; 2) no bacterial taxa differed in relative abundance between women who had a spontaneous preterm delivery and those who delivered at term; and 3) no differences in the frequency of the vaginal community state types (CST I, III, IV-B) between women who delivered at term and those who delivered preterm were detected. CONCLUSIONS: The bacterial taxa composition and abundance of vaginal microbial communities, characterized with 16S rRNA gene sequence-based techniques, were not different in pregnant women who subsequently delivered a preterm neonate versus those who delivered at term.

The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women.

BACKGROUND: This study was undertaken to characterize the vaginal microbiota throughout normal human pregnancy using sequence-based techniques. We compared the vaginal microbial composition of non-pregnant patients with a group of pregnant women who delivered at term. RESULTS: A retrospective case-control longitudinal study was designed and included non-pregnant women (n = 32) and pregnant women who delivered at term (38 to 42 weeks) without complications (n = 22). Serial samples of vaginal fluid were collected from both non-pregnant and pregnant patients. A 16S rRNA gene sequence-based survey was conducted using pyrosequencing to characterize the structure and stability of the vaginal microbiota. Linear mixed effects models and generalized estimating equations were used to identify the phylotypes whose relative abundance was different between the two study groups. The vaginal microbiota of normal pregnant women was different from that of non-pregnant women (higher abundance of Lactobacillus vaginalis, L. crispatus, L. gasseri and L. jensenii and lower abundance of 22 other phylotypes in pregnant women). Bacterial community state type (CST) IV-B or CST IV-A characterized by high relative abundance of species of genus Atopobium as well as the presence of Prevotella, Sneathia, Gardnerella, Ruminococcaceae, Parvimonas, Mobiluncus and other taxa previously shown to be associated with bacterial vaginosis were less frequent in normal pregnancy. The stability of the vaginal microbiota of pregnant women was higher than that of non-pregnant women; however, during normal pregnancy, bacterial communities shift almost exclusively from one CST dominated by Lactobacillus spp. to another CST dominated by Lactobacillus spp. CONCLUSION: We report the first longitudinal study of the vaginal microbiota in normal pregnancy. Differences in the composition and stability of the microbial community between pregnant and non-pregnant women were observed. Lactobacillus spp. were the predominant members of the microbial community in normal pregnancy. These results can serve as the basis to study the relationship between the vaginal microbiome and adverse pregnancy outcomes.

Daily temporal dynamics of vaginal microbiota before, during and after episodes of bacterial vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common gynecologic diagnosis characterized by dysbiosis of the vaginal microbiota. It is often accompanied by vaginal symptoms such as odor and discharge, but can be asymptomatic. Despite over 50 years of research, the etiology of BV is not well understood, which is a major impediment to treatment and prevention of BV. RESULTS: Here we report on the temporal dynamics of 25 vaginal communities over a 10 week period using samples collected daily from women who were diagnosed with symptomatic BV (15 women), asymptomatic BV (6 women), and women who did not have BV (4 women). CONCLUSION: This unique resource of samples and data will contribute to a better understanding of the role that the vaginal microbes have in the natural history of BV and lead to improved diagnosis and treatment.

Metagenomic investigation of microbes and viruses in patients with jaw osteonecrosis associated with bisphosphonate therapy.

OBJECTIVE: The goal of this preliminary study was to use metagenomic approaches to investigate the taxonomic diversity of microorganisms in patients with bisphosphonate-related osteonecrosis of the jaw (BRONJ). STUDY DESIGN: Samples of saliva for planktonic microbial analysis and biofilm cultivation were collected from 10 patients (5 with BRONJ and 5 non-BRONJ control subjects) who met all ascertainment criteria. Prophage induction experiments-16S rRNA polymerase chain reaction and 454 pyrosequencing-and epifluorescent microscopy were performed for characterization and enumeration of microbes and viruses. RESULTS: Three phyla of microbes-Proteobacteria (70%), Firmicutes (26.9%), and Actinobacteria (1.95%)-dominated all BRONJ samples and accounted for almost 99% of the total data. Viral abundance was ∼1 order of magnitude greater than microbial cell abundance and comprised mainly phage viruses. CONCLUSIONS: Individuals with jaw osteonecrosis harbored different microbial assemblages than nonaffected patients, and in general viral abundance and prophage induction increased with biofilm formation, suggesting that biofilm formation encouraged lysogenic interactions between viruses and microbial hosts and may contribute to pathogenicity.