Application of phylogenetic microarrays to interrogation of human microbiota

Human-associated microbiota is recognized to play vital roles in maintaining host health, and it is implicated in many disease states. While the initial surge in the profiling of these microbial communities was achieved with Sanger and next-generation sequencing, many oligonucleotide microarrays have also been developed recently for this purpose. Containing probes complementary to small ribosomal subunit RNA gene sequences of community members, such phylogenetic arrays provide direct quantitative comparisons of microbiota composition among samples and between sample groups. Some of the developed microarrays including PhyloChip, Microbiota Array, and HITChip can simultaneously measure the presence and abundance of hundreds and thousands of phylotypes in a single sample. This review describes the currently available phylogenetic microarrays that can be used to analyze human microbiota, delineates the approaches for the optimization of microarray use, and provides examples of recent findings based on microarray interrogation of human-associated microbial communities.     

Human-associated microbial signatures: examining their predictive value

Host-associated microbial communities are unique to individuals, affect host health, and correlate with disease states. Although advanced technologies capture detailed snapshots of microbial communities, high within- and between-subject variation hampers discovery of microbial signatures in diagnostic or forensic settings. We suggest turning to machine learning and discuss key directions toward harnessing human-associated microbial signatures.     

Investigating the biological and clinical significance of human dysbioses

Culture-independent microbiological technologies that interrogate complex microbial populations without prior axenic culture, coupled with high-throughput DNA sequencing, have revolutionized the scale, speed and economics of microbial ecological studies. Their application to the medical realm has led to a highly productive merger of clinical, experimental and environmental microbiology. The functional roles played by members of the human microbiota are being actively explored through experimental manipulation of animal model systems and studies of human populations. In concert, these studies have appreciably expanded our understanding of the composition and dynamics of human-associated microbial communities (microbiota). Of note, several human diseases have been linked to alterations in the composition of resident microbial communities, so-called dysbiosis. However, how changes in microbial communities contribute to disease etiology remains poorly defined. Correlation of microbial composition represents integration of only two datasets (phenotype and microbial composition). This article explores strategies for merging the human microbiome data with multiple additional datasets (e.g. host single nucleotide polymorphisms and host gene expression) and for integrating patient-based data with results from experimental animal models to gain deeper understanding of how host-microbe interactions impact disease.     

Lachnospiraceae and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor

The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.     

Improved annotation of antibiotic resistance determinants reveals microbial resistomes cluster by ecology

Antibiotic resistance is a dire clinical problem with important ecological dimensions. While antibiotic resistance in human pathogens continues to rise at alarming rates, the impact of environmental resistance on human health is still unclear. To investigate the relationship between human-associated and environmental resistomes, we analyzed functional metagenomic selections for resistance against 18 clinically relevant antibiotics from soil and human gut microbiota as well as a set of multidrug-resistant cultured soil isolates. These analyses were enabled by Resfams, a new curated database of protein families and associated highly precise and accurate profile hidden Markov models, confirmed for antibiotic resistance function and organized by ontology. We demonstrate that the antibiotic resistance functions that give rise to the resistance profiles observed in environmental and human-associated microbial communities significantly differ between ecologies. Antibiotic resistance functions that most discriminate between ecologies provide resistance to β-lactams and tetracyclines, two of the most widely used classes of antibiotics in the clinic and agriculture. We also analyzed the antibiotic resistance gene composition of over 6000 sequenced microbial genomes, revealing significant enrichment of resistance functions by both ecology and phylogeny. Together, our results indicate that environmental and human-associated microbial communities harbor distinct resistance genes, suggesting that antibiotic resistance functions are largely constrained by ecology.     

In search of an uncultured human-associated TM7 bacterium in the environment

We have identified an environmental bacterium in the Candidate Division TM7 with ≥98.5% 16S rDNA gene homology to a group of TM7 bacteria associated with the human oral cavity and skin. The environmental TM7 bacterium (referred to as TM7a-like) was readily detectable in wastewater with molecular techniques over two years of sampling. We present the first images of TM7a-like cells through FISH technique and the first images of any TM7 as viable cells through the STARFISH technique. In situ quantification showed TM7 concentration in wastewater up to five times greater than in human oral sites. We speculate that upon further characterization of the physiology and genetics of the TM7a-like bacterium from environmental sources and confirmation of its genomic identity to human-associated counterparts it will serve as model organisms to better understand its role in human health. The approach proposed circumvents difficulties imposed by sampling humans, provides an alternative strategy to characterizing some diseases of unknown etiology, and renders a much needed understanding of the ecophysiological role hundreds of unique Bacteria and Archaea strains play in mixed microbial communities.     

From molecules to dynamic biological communities

Microbial ecology is flourishing, and in the process, is making contributions to how the ecology and biology of large organisms is understood. Ongoing advances in sequencing technology and computational methods have enabled the collection and analysis of vast amounts of molecular data from diverse biological communities. While early studies focused on cataloguing microbial biodiversity in environments ranging from simple marine ecosystems to complex soil ecologies, more recent research is concerned with community functions and their dynamics over time. Models and concepts from traditional ecology have been used to generate new insight into microbial communities, and novel system-level models developed to explain and predict microbial interactions. The process of moving from molecular inventories to functional understanding is complex and challenging, and never more so than when many thousands of dynamic interactions are the phenomena of interest. We outline the process of how epistemic transitions are made from producing catalogues of molecules to achieving functional and predictive insight, and show how those insights not only revolutionize what is known about biological systems but also about how to do biology itself. Examples will be drawn primarily from analyses of different human microbiota, which are the microbial consortia found in and on areas of the human body, and their associated microbiomes (the genes of those communities). Molecular knowledge of these microbiomes is transforming microbiological knowledge, as well as broader aspects of human biology, health and disease.     

Comparative analysis of human gut microbiota by barcoded pyrosequencing

Humans host complex microbial communities believed to contribute to health maintenance and, when in imbalance, to the development of diseases. Determining the microbial composition in patients and healthy controls may thus provide novel therapeutic targets. For this purpose, high-throughput, cost-effective methods for microbiota characterization are needed. We have employed 454-pyrosequencing of a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences which enables parallel in-depth analysis of hundreds of samples with limited sample processing. In silico modeling demonstrated that the method correctly describes microbial communities down to phylotypes below the genus level. Here we applied the technique to analyze microbial communities in throat, stomach and fecal samples. Our results demonstrate the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology.     

A cross-species interaction with a symbiotic commensal enables cell-density-dependent growth and in vivo virulence of an oral pathogen

Recent studies describe in detail the shifts in composition of human-associated polymicrobial communities from health to disease. However, the specific processes that drive the colonization and overgrowth of pathogens within these communities remain incompletely understood. We used in vitro culture systems and a disease-relevant mouse model to show that population size, which determines the availability of an endogenous diffusible small molecule, limits the growth, colonization, and in vivo virulence of the human oral pathogen Porphyromonas gingivalis. This bacterial pathogen overcomes the requirement for an endogenous cue by utilizing a cell-density dependent, growth-promoting, soluble molecule provided by the symbiotic early colonizer Veillonella parvula, but not produced by other commensals tested. Our work shows that exchange of cell-density-dependent diffusible cues between specific early and late colonizing species in a polymicrobial community drives microbial successions, pathogen colonization and disease development, representing a target process for manipulation of the microbiome towards the healthy state.Subject terms: Microbiome, Microbial ecology     

A new era in palaeomicrobiology: prospects for ancient dental calculus as a long-term record of the human oral microbiome

The field of palaeomicrobiology is dramatically expanding thanks to recent advances in high-throughput biomolecular sequencing, which allows unprecedented access to the evolutionary history and ecology of human-associated and environmental microbes. Recently, human dental calculus has been shown to be an abundant, nearly ubiquitous, and long-term reservoir of the ancient oral microbiome, preserving not only microbial and host biomolecules but also dietary and environmental debris. Modern investigations of native human microbiota have demonstrated that the human microbiome plays a central role in health and chronic disease, raising questions about changes in microbial ecology, diversity and function through time. This paper explores the current state of ancient oral microbiome research and discusses successful applications, methodological challenges and future possibilities in elucidating the intimate evolutionary relationship between humans and their microbes.     

Microbial biogeography of public restroom surfaces

We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla: Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with hands. On toilet surfaces, gut-associated taxa were more prevalent, suggesting fecal contamination of these surfaces. Floor surfaces were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated bacteria, especially the Propionibacteriaceae, dominated surfaces routinely touched with our hands. Certain taxa were more common in female than in male restrooms as vagina-associated Lactobacillaceae were widely distributed in female restrooms, likely from urine contamination. Use of the SourceTracker algorithm confirmed many of our taxonomic observations as human skin was the primary source of bacteria on restroom surfaces. Overall, these results demonstrate that restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clear linkages between communities on or in different body sites and those communities found on restroom surfaces. More generally, this work is relevant to the public health field as we show that human-associated microbes are commonly found on restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching of surfaces. Furthermore, we demonstrate that we can use high-throughput analyses of bacterial communities to determine sources of bacteria on indoor surfaces, an approach which could be used to track pathogen transmission and test the efficacy of hygiene practices.     

A direct PCR approach to accelerate analyses of human-associated microbial communities

Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.     

Host lifestyle affects human microbiota on daily timescales

Background

Disturbance to human microbiota may underlie several pathologies. Yet, we lack a comprehensive understanding of how lifestyle affects the dynamics of human-associated microbial communities.

Results

Here, we link over 10,000 longitudinal measurements of human wellness and action to the daily gut and salivary microbiota dynamics of two individuals over the course of one year. These time series show overall microbial communities to be stable for months. However, rare events in each subjects’ life rapidly and broadly impacted microbiota dynamics. Travel from the developed to the developing world in one subject led to a nearly two-fold increase in the Bacteroidetes to Firmicutes ratio, which reversed upon return. Enteric infection in the other subject resulted in the permanent decline of most gut bacterial taxa, which were replaced by genetically similar species. Still, even during periods of overall community stability, the dynamics of select microbial taxa could be associated with specific host behaviors. Most prominently, changes in host fiber intake positively correlated with next-day abundance changes among 15% of gut microbiota members.

Conclusions

Our findings suggest that although human-associated microbial communities are generally stable, they can be quickly and profoundly altered by common human actions and experiences.

Electronic supplementary material

The online version of this article (doi:10.1186/gb-2014-15-7-r89) contains supplementary material, which is available to authorized users.     

Metaproteomic strategies and applications for gut microbial research

The human intestine hosts various complex microbial communities that are closely associated with multiple health and disease processes. Determining the composition and function of these microbial communities is critical to unveil disease mechanisms and promote human health. Recently, meta-omic strategies have been developed that use high-throughput techniques to provide a wealth of information, thus accelerating the study of gut microbes. Metaproteomics is a newly emerged analytical approach that aims to identify proteins on a large scale in complex environmental microbial communities (e.g., the gut microbiota). This review introduces the recent analytical strategies and applications of metaproteomics, with a focus on advances in gut microbiota research, including a discussion of the limitations and challenges of these approaches.

     

Subgingival Microbial Communities in Leukocyte Adhesion Deficiency and Their Relationship with Local Immunopathology

Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of β2 integrins which result in defective transmigration of neutrophils into the tissues. Affected patients suffer from recurrent life threatening infections and severe oral disease (periodontitis). Microbial communities in the local environment (subgingival plaque) are thought to be the triggers for inflammatory periodontitis, yet little is known regarding the microbial communities associated with LAD-I periodontitis. Here we present the first comprehensive characterization of the subgingival communities in LAD-I, using a 16S rRNA gene-based microarray, and investigate the relationship of this tooth adherent microbiome to the local immunopathology of periodontitis. We show that the LAD subgingival microbiome is distinct from that of health and Localized Aggressive Periodontitits. Select periodontitis-associated species in the LAD microbiome included Parvimonas micra, Porphyromonas endodontalis, Eubacterium brachy and Treponema species. Pseudomonas aeruginosa, a bacterium not typically found in subgingival plaque is detected in LAD-I. We suggest that microbial products from LAD-associated communities may have a role in stimulating the local inflammatory response. We demonstrate that bacterial LPS translocates into the lesions of LAD-periodontitis potentially triggering immunopathology. We also show in in vitro assays with human macrophages and in vivo in animal models that microbial products from LAD-associated subgingival plaque trigger IL-23-related immune responses, which have been shown to dominate in patient lesions. In conclusion, our current study characterizes the subgingival microbial communities in LAD-periodontitis and supports their role as triggers of disease pathogenesis.     

The potential for probiotic manipulation of the gastrointestinal microbiome

Multiple internal and external sites of the healthy human body are colonized by a diversity of symbiotic microbes. The microbial assemblages found in the intestine represent some of the most dense and diverse of these human-associated ecosystems. Unsurprisingly, the enteric microbiome, that is the totality of microbes, their combined genomes, and their interactions with the human body, has a profound impact on physiological aspects of mammalian function, not least, host immune response. Lack of early-life exposure to certain microbes, or shifts in the composition of the gastrointestinal microbiome have been linked to the development and progression of several intestinal and extra-intestinal diseases, including childhood asthma development and inflammatory bowel disease. Modulating microbial exposure through probiotic supplementation represents a long-held strategy towards ameliorating disease via intestinal microbial community restructuring. This field has experienced somewhat of a resurgence over the past few years, primarily due to the exponential increase in human microbiome studies and a growing appreciation of our dependence on resident microbiota to modulate human health. This review aims to review recent regulatory aspects related to probiotics in food. It also summarizes what is known to date with respect to human gastrointestinal microbiota - the niche which has been most extensively studied in the human system - and the evidence for probiotic supplementation as a viable therapeutic strategy for modulating this consortium.     

The role of competition versus cooperation in microbial community coalescence

New microbial communities often arise through the mixing of two or more separately assembled parent communities, a phenomenon that has been termed “community coalescence”. Understanding how the interaction structures of complex parent communities determine the outcomes of coalescence events is an important challenge. While recent work has begun to elucidate the role of competition in coalescence, that of cooperation, a key interaction type commonly seen in microbial communities, is still largely unknown. Here, using a general consumer-resource model, we study the combined effects of competitive and cooperative interactions on the outcomes of coalescence events. To do so, we simulate coalescence events between pairs of communities with different degrees of competition for shared carbon resources and cooperation through cross-feeding on leaked metabolic by-products (facilitation). We also study how structural and functional properties of post-coalescence communities evolve when they are subjected to repeated coalescence events. We find that in coalescence events, the less competitive and more cooperative parent communities contribute a higher proportion of species to the new community because of their superior ability to deplete resources and resist invasions. Consequently, when a community is subjected to repeated coalescence events, it gradually evolves towards being less competitive and more cooperative, as well as more speciose, robust and efficient in resource use. Encounters between microbial communities are becoming increasingly frequent as a result of anthropogenic environmental change, and there is great interest in how the coalescence of microbial communities affects environmental and human health. Our study provides new insights into the mechanisms behind microbial community coalescence, and a framework to predict outcomes based on the interaction structures of parent communities.