New techniques to characterise the vaginal microbiome in pregnancy

Understanding of the vaginal microbiome in health and disease is essential to screen, detect and manage complications in pregnancy. One of the major complications of pregnancy is preterm birth, which is the leading world-wide cause of death and disability in children under five years of age. The aetiology of preterm birth is multifactorial, but a causal link has been established with infection. Despite the importance of understanding the vaginal microbiome in pregnancy in order to evaluate strategies to prevent and manage PTB, currently used culture based techniques provide limited information as not all pathogens are able to be cultured. The implementation of culture-independent high-throughput techniques and bioinformatics tools are advancing our understanding of the vaginal microbiome. New methods employing 16S rRNA and metagenomics analyses make possible a more comprehensive description of the bacteria of the human microbiome. Several studies on the vaginal microbiota of pregnant women have identified a large number of taxa. Studies also suggest reduced diversity of the microbiota in pregnancy compared to non-pregnant women, with a relative enrichment of the overall abundance of Lactobacillus species, and significant differences in the diversity of Lactobacillus spp. A number of advantages and disadvantages of these techniques are discussed briefly. The potential clinical importance of the new techniques is illustrated through recent reports where traditional culture-based techniques failed to identify pathogens in high risk complicated pregnancies whose presence subsequently was established using culture-independent, high-throughput analyses

The Microbial Ecology of Bacterial Vaginosis: A Fine Scale Resolution Metagenomic Analysis

The vaginal microbiota play an important protective role in maintaining the health of women. Disruption of the mutualistic relationship that exists between bacterial communities in the vagina and their hosts can lead to bacterial vaginosis (BV), a condition in which lactic acid producing bacteria are supplanted by a diverse array of strictly anaerobic bacteria. BV has been shown to be an independent risk factor for adverse outcomes including preterm delivery and low infant birth weight, acquisition of sexually transmitted infections and HIV, and development of pelvic inflammatory disease. National surveys indicate the prevalence of BV among U.S. women is 29.2%, and yet, despite considerable effort, the etiology of BV remains unknown. Moreover, there are no broadly effective therapies for the treatment of BV, and reoccurrence is common. In the proposed research we will test the overarching hypothesis that vaginal microbial community dynamics and activities are indicators of risk to BV. To do this, we propose to conduct a high resolution prospective study in which samples collected daily from 200 reproductive-age women over two menstrual cycles are used to capture molecular events that take place before, during, and after the spontaneous remission of BV episodes. We will use modern genomic technologies to obtain the data needed to correlate shifts in vaginal microbial community composition and function, metabolomes, and epidemiological and behavioral metadata with the occurrence of BV to better define the syndrome itself and identify patterns that are predictive of BV. The three specific aims of the research are: (1) Evaluate the association between the dynamics of vaginal microbial communities and risk to BV by characterizing the community composition of vaginal specimens archived from a vaginal douching cessation study in which 33 women self-collected vaginal swabs twice-weekly for 16 weeks; (2) Enroll 135 women in a prospective study in which self-collected vaginal swab samples and secretions are collected daily along with data on the occurrence of BV, vaginal pH, and information on time varying habits and practices; (3) Apply model-based statistical clustering and classification approaches to associate the microbial community composition and function, with metadata and clinical diagnoses of BV. The large body of information generated will facilitate understanding vaginal microbial community dynamics, the etiology of BV, and drive the development of better diagnostic tools for BV. Furthermore, the information will enable a more personalized and effective treatment of BV and ultimately help prevent adverse sequelae associated with this highly prevalent disruption of the vaginal microbiome

Strategy for the identification of micro-organisms producing food and feed products : bacteria producing food enzymes as study case

Recent European regulations require safety assessments of food enzymes (FE) before their commercialization. FE are mainly produced by micro-organisms, whose viable strains nor associated DNA can be present in the final products. Currently, no strategy targeting such impurities exists in enforcement laboratories. Therefore, a generic strategy of first line screening was developed to detect and identify, through PCR amplification and sequencing of the 16S-rRNA gene, the potential presence of FE producing bacteria in FE preparations. First, the specificity was verified using all microbial species reported to produce FE. Second, an in-house database, with 16S reference sequences from bacteria producing FE, was constructed for their fast identification through blast analysis. Third, the sensitivity was assessed on a spiked FE preparation. Finally, the applicability was verified using commercial FE preparations. Using straightforward PCR amplifications, Sanger sequencing and blast analysis, the proposed strategy was demonstrated to be convenient for implementation in enforcement laboratories

Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats : a comparison between bidirectional pyrosequencing and cultivation

The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques. By contrast, cultivation-based approaches allow the retrieval of both common and uncommon bacteria that can grow in the conditions used and provide access to strains for biotechnological applications. We performed bidirectional pyrosequencing of the bacterial 16S rRNA gene diversity in two terrestrial and seven aquatic Antarctic microbial mat samples previously studied by heterotrophic cultivation. While, not unexpectedly, 77.5% of genera recovered by pyrosequencing were not among the isolates, 25.6% of the genera picked up by cultivation were not detected by pyrosequencing. To allow comparison between both techniques, we focused on the five phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Deinococcus-Thermus) recovered by heterotrophic cultivation. Four of these phyla were among the most abundantly recovered by pyrosequencing. Strikingly, there was relatively little overlap between cultivation and the forward and reverse pyrosequencing-based datasets at the genus (17.1–22.2%) and OTU (3.5–3.6%) level (defined on a 97% similarity cut-off level). Comparison of the V1–V2 and V3–V2 datasets of the 16S rRNA gene revealed remarkable differences in number of OTUs and genera recovered. The forward dataset missed 33% of the genera from the reverse dataset despite comprising 50% more OTUs, while the reverse dataset did not contain 40% of the genera of the forward dataset. Similar observations were evident when comparing the forward and reverse cultivation datasets. Our results indicate that the region under consideration can have a large impact on perceived diversity, and should be considered when comparing different datasets. Finally, a high number of OTUs could not be classified using the RDP reference database, suggesting the presence of a large amount of novel diversity

Molecular biological methods for studying the gut microbiota : the EU human gut flora project

Seven European laboratories co-operated in a joint project (FAIR CT97-3035) to develop, refine and apply molecular methods towards facilitating elucidation of the complex composition of the human intestinal microflora and to devise robust methodologies for monitoring the gut flora in response to diet. An extensive database of 16S rRNA sequences for tracking intestinal bacteria was generated by sequencing the 16S rRNA genes of new faecal isolates and of clones obtained by amplification with polymerase chain reaction (PCR) on faecal DNA from subjects belonging to different age groups. The analyses indicated that the number of different species (diversity) present in the human gut increased with age. The sequence information generated, provided the basis for design of 16S rRNA-directed oligonucleotide probes to specifically detect bacteria at various levels of phylogenetic hierarchy. The probes were tested for their specificity and used in whole-cell and dot-blot hybridisations. The applicability of the developed methods was demonstrated in several studies and the major outcomes are described

Ecological and Genomic Attributes of Novel Bacterial Taxa That Thrive in Subsurface Soil Horizons.

While most bacterial and archaeal taxa living in surface soils remain undescribed, this problem is exacerbated in deeper soils, owing to the unique oligotrophic conditions found in the subsurface. Additionally, previous studies of soil microbiomes have focused almost exclusively on surface soils, even though the microbes living in deeper soils also play critical roles in a wide range of biogeochemical processes. We examined soils collected from 20 distinct profiles across the United States to characterize the bacterial and archaeal communities that live in subsurface soils and to determine whether there are consistent changes in soil microbial communities with depth across a wide range of soil and environmental conditions. We found that bacterial and archaeal diversity generally decreased with depth, as did the degree of similarity of microbial communities to those found in surface horizons. We observed five phyla that consistently increased in relative abundance with depth across our soil profiles: Chloroflexi, Nitrospirae, Euryarchaeota, and candidate phyla GAL15 and Dormibacteraeota (formerly AD3). Leveraging the unusually high abundance of Dormibacteraeota at depth, we assembled genomes representative of this candidate phylum and identified traits that are likely to be beneficial in low-nutrient environments, including the synthesis and storage of carbohydrates, the potential to use carbon monoxide (CO) as a supplemental energy source, and the ability to form spores. Together these attributes likely allow members of the candidate phylum Dormibacteraeota to flourish in deeper soils and provide insight into the survival and growth strategies employed by the microbes that thrive in oligotrophic soil environments.IMPORTANCE Soil profiles are rarely homogeneous. Resource availability and microbial abundances typically decrease with soil depth, but microbes found in deeper horizons are still important components of terrestrial ecosystems. By studying 20 soil profiles across the United States, we documented consistent changes in soil bacterial and archaeal communities with depth. Deeper soils harbored communities distinct from those of the more commonly studied surface horizons. Most notably, we found that the candidate phylum Dormibacteraeota (formerly AD3) was often dominant in subsurface soils, and we used genomes from uncultivated members of this group to identify why these taxa are able to thrive in such resource-limited environments. Simply digging deeper into soil can reveal a surprising number of novel microbes with unique adaptations to oligotrophic subsurface conditions

Estuarine Nitrifiers: New Players, Patterns and Processes

Ever since the first descriptions of ammonia-oxidizing Bacteria by Winogradsky in the late 1800s, the metabolic capability of aerobic ammonia oxidation has been restricted to a phylogenetically narrow group of bacteria. However, the recent discovery of ammonia-oxidizing Archaea has forced microbiologists and ecologists to re-evaluate long-held paradigms and the role of niche partitioning between bacterial and archaeal ammonia oxidizers. Much of the current research has been conducted in open ocean or terrestrial systems, where community patterns of archaeal and bacterial ammonia oxidizers are highly congruent. Studies of archaeal and bacterial ammonia oxidizers in estuarine systems, however, present a very different picture, with highly variable patterns of archaeal and bacterial ammonia oxidizer abundances. Although salinity is often identified as an important factor regulating abundance, distribution, and diversity of both archaeal and bacterial ammonia oxidizers, the data suggest that the variability in the observed patterns is likely not due to a simple salinity effect. Here we review current knowledge of ammonia oxidizers in estuaries and propose that because of their steep physico-chemical gradients, estuaries may serve as important natural laboratories in which to investigate the relationships between archaeal and bacterial ammonia oxidizers

Long-term impact of fecal transplantation in healthy volunteers

Fecal microbiota transplantation (FMT) has been recently approved by FDA for the treatment of refractory recurrent clostridial colitis (rCDI). Success of FTM in treatment of rCDI led to a number of studies investigating the effectiveness of its application in the other gastrointestinal diseases. However, in the majority of studies the effects of FMT were evaluated on the patients with initially altered microbiota. The aim of our study was to estimate effects of FMT on the gut microbiota composition in healthy volunteers and to monitor its long-term outcomes.Peer ReviewedPostprint (published version