Search

Delftia is a diverse betaproteobacterial genus with many strains having agricultural and industrial relevance, including plant-growth promotion, bioremediation of hydrocarbon-contaminated soils, and heavy metal immobilization. Delftia spp. are broadly distributed in the environment, and have been isolated from plant hosts as well as healthy and diseased animal hosts, yet the genetic basis of this ecological versatility has not been characterized. Here, we present a phylogenomic comparison of published Delftia genomes and show that the genus is divided into two well-supported clades: one ‘ Delftia acidovorans ’ clade with isolates from soils and plant rhizospheres, and a second ‘ Delftia lacustris and Delftia tsuruhatensis ’ clade with isolates from humans and sludge. The pan-genome inferred from 61 Delftia genomes contained over 28 000 genes, of which only 884 were found in all genomes. Analysis of industrially relevant functions highlighted the ecological versatility of Delftia and supported their role as generalists.

Background Probiotics have been demonstrated to ameliorate clinical signs of gastrointestinal diseases in dogs in various studies. However, the effect of probiotics in a healthy population, as well as factors contributing individualized responses, remain largely unknown. This trial examined gut microbiota (GM) and health outcomes in household dogs after synbiotic (SN) supplementation containing probiotics and inulin (a prebiotic). Healthy dogs were randomized to receive SN (50 mg/d inulin and 20 billion total CFU/d of L. reuteri, P. acidilactici, E. faecium, L. acidophilus, B. animalis, L. fermentum, L. rhamnosus) or placebo (PL) for 4 weeks. Owners completed a health survey and collected stool samples for GM profiling (shotgun metagenomic sequencing) at baseline and week 4 in both groups, and at week 6 in the SN group. Results A significant shift (p n = 24), but not PL group (n = 19), at week 4 relative to baseline. Forty-five bacterial species, 43 (96%) of which were Lactobacillales, showed an increase in the relative abundances (≥2 fold change, adjusted p E. coli also decreased at week 4 in the SN group (2.8-fold, adjusted p Proteobacteria and lower Lactobacillales responded more robustly to supplementation in terms of the change in β-diversity. Dogs fed SN tended to have lower diarrhea incidence (0% vs 16%, p = 0.08). Conclusions SN supplement had a short-term impact on the gut microbiota in healthy household dogs as characterized by shotgun metagenomic sequencing. Findings warrant further investigation with longer duration and populations at risk of gastrointestinal diseases. The magnitude of response to the supplement was associated with microbial profile at baseline. To our knowledge, this is the first study documenting such association and may provide a basis for personalized nutrition in companion dogs.

The approximately 1011 viruses and microbial cells per gram of fecal matter (dry weight) in the large intestine are important to human health. The responses of three common gut bacteria species, and one opportunistic pathogen, to 117 commonly consumed foods, chemical additives, and plant extracts were tested. Many compounds, including Stevia rebaudiana and bee propolis extracts, exhibited species-specific growth inhibition by prophage induction. Overall, these results show that various foods may change the abundances of gut bacteria by modulating temperate phage and suggests a novel path for landscaping the human gut microbiome.

Background The gut microbiota (GM) is associated with canine health and can be impacted by diet. Dog owners in the U.S. have increasingly shown an interest in feeding their dogs a mildly cooked (MC) diet. However, its impact on canine GM and health remains largely unknown. Methods Healthy household dogs were tracked upon switching from various brands of extruded to MC diets for four weeks. A health assessment was completed and stool samples were collected by each owner before (day 0) and after the diet transition (day 28). Shotgun metagenomic sequencing was performed at both time points to characterize the GM. Results Dogs completed the study by either completing the health assessments (n = 31) or providing stool samples at both time points (n = 28). All owners reported either better or no change in overall health at the end of the study (61% and 39%, respectively), and none reported worse overall health. Defecation frequency was also reported to be lower (58%) or about the same (35%). Principal coordinate (PCo) analysis showed a significant shift (p = 0.004) in the β-diversity of the GM upon diet transition (34.2% and 10.3% explained by the first two axes). The abundances of 70 species increased after the diet change (adjusted p < 0.05), 67% and 24% of which belonged to the Lactobacillales and the Enterobacterales orders respectively. The abundances of 28 species decreased (adjusted p < 0.05), 46%, 18%, and 11% of which belonged to the Clostridiales, Bacillales, and Bacteroidales orders, respectively. Lower Lactobacillales and Enterobacterales, and higher Bacteroidales at baseline were associated with a greater shift along the PCo1 axis. Protein content of the baseline diet was correlated with the shift along the PCo1 axis (ρ = 0.67, p = 0.006). Conclusion Owners reported either improvement or no change in health in dogs transitioning from extruded kibble to MC diets for 4 weeks, but this report of health perception requires further exploration in a controlled trial. Diet change also led to a significant shift in the GM profile of healthy dogs. The magnitude of shift was associated with baseline GM and dietary protein, and warrants further examination of individualized responses and personalized nutrition in companion dogs. These results also support future investigation of the impact of a MC diet on health maintenance given its increasing popularity.

Crohn’s disease (CD) is characterized by a breakdown of the intestinal mucosal barrier. A genetic polymorphism of the autophagy gene ATG16L1 is associated with increased risk of developing CD, and it impairs the protective and regenerative functions of the intestinal epithelium. Autophagy acts as a regulator of cell death and has been implicated in the protection from both necroptosis and apoptosis in the epithelium. Our work showed that mice specifically deleted for Atg16l1 in intestinal epithelial cells (Atg16l1ΔIEC) had exacerbated T cell-mediated intestinal damage in the model of anti-CD3 enteropathy compared to wild-type mice, which was characterized by crypt epithelial cell death and heightened inflammation. Moreover, Atg16l1 deficiency delayed the recovery of the intestinal epithelium, and Atg16l1-deficient IECs were impaired in their proliferative response. Pathology and mortality were largely driven by microbiota-dependent expression of Ifng, Tnf, and type I IFN response genes in Atg16l1ΔIEC mice. Mechanistically, while survival was rescued by blocking these cytokine pathways independently, only anti-IFNγ treatment abrogated intestinal epithelial cell (IEC) death in Atg16l1ΔIEC mice, thereby decoupling IEC death and survival. In summary, our findings suggest differential roles for IFNγ, TNF, and type I IFN in acute enteropathy and IEC death in the context of autophagy deficiency. Supported by grants from CIHR (MOP-12353, FDN-14333) and NSERC (RGPIN-201).

Background: Probiotics ameliorate gastrointestinal symptoms in dogs. However, the effect of probiotics in a healthy population, as well as factors contributing individualized responses, remains largely unknown. This trial examined gut microbiota (GM) and health outcomes in household dogs after synbiotic (SN) supplementation containing probiotics and inulin. Healthy dogs were randomized to receive SN (50 mg/d inulin and 20 billion total CFU/d of L. reuteri, P. acidilactici, E. faecium, L. acidophilus, B. animalis, L. fermentum, L. rhamnosus) or placebo (PL) for 4 weeks. Owners completed a health survey and collected stool samples for GM profiling (metagenomic sequencing) at baseline and week 4 in both groups, and at week 6 in the SN group.Results: A significant shift (p

The phylum Caldiserica was identified from the hot spring 16S rRNA gene lineage 'OP5' and named for the sole isolate Caldisericum exile, a hot spring sulfur-reducing chemoheterotroph. Here we characterize 7 Caldiserica metagenome-assembled genomes (MAGs) from a thawing permafrost site in Stordalen Mire, Arctic Sweden. By 16S rRNA and marker gene phylogenies, and average nucleotide and amino acid identities, these Stordalen Mire Caldiserica (SMC) MAGs form part of a divergent clade from C. exile. Genome and meta-transcriptome and proteome analyses suggest that unlike Caldisericum, the SMCs (i) are carbohydrate- and possibly amino acid fermenters that can use labile plant compounds and peptides, and (ii) encode adaptations to low temperature. The SMC clade rose to community dominance within permafrost, with a peak metagenome-based relative abundance of ∼60%. It was also physiologically active in the upper seasonally-thawed soil. Beyond Stordalen Mire, analysis of 16S rRNA gene surveys indicated a global distribution of this clade, predominantly in anaerobic, carbon-rich and cold environments. These findings establish the SMCs as four novel phenotypically and ecologically distinct species within a single novel genus, distinct from C. exile clade at the phylum level. The SMCs are thus part of a novel cold-habitat phylum for an understudied, globally-distributed superphylum encompassing the Caldiserica. We propose the names Candidatus Cryosericota phylum nov., Ca. Cryosericia class nov., Ca. Cryosericales ord. nov., Ca. Cryosericaceae fam. nov., Ca. Cryosericum gen. nov., Ca. Cryosericum septentrionale sp. nov., Ca. C. hinesii sp. nov., Ca. C. odellii sp. nov., and Ca. C. terrychapinii sp. nov.

Previous papers in this series described the “evolutionary genetic species concept” which is based on population genetic theory and should be applicable to any organism. Also described was a species criterion, the K/θ ratio, that delimits independently evolving evolutionary species based on single-gene sequences, and its application to sexual and asexual eukaryotes was illustrated. Here, we show how the evolutionary genetic species concept and the K/θ ratio can be applied to bacteria, using the sequences from some genes of the core genome that are rarely, if ever, transferred horizontally between different species. This completes the demonstration that K/θ is a general method for species delimitation, applicable to all organisms. Also, it adds to the evidence that bacteria have species in the most general sense, even though they have the ability to exchange genes across species boundaries. Finally, we show that a published critique of the use of K/θ ≥ 4 as a criterion for independently evolving species rests on two errors in the application of population/evolutionary genetic theory.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ(+)CD8(+) T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα(+)IFNγ(+)CD8(+) T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

The human oral cavity is colonized by one of the densest bacterial communities on the planet, with consistent microbial composition throughout life and in response to perturbations such as tooth cleaning. The oral microbiome has important associations with health in the context of gingivitis and even heart disease and preterm birth. In this chapter, we will provide an overview of what is known about the human oral microbiome, starting from the first glimpse of microbes when van Leeuwenhoek looked at his own oral microbes with the first microscope, to recent expansion of knowledge with both high-throughput sequencing and microscopy. We will describe what is known about healthy oral microbial communities, including how they develop in childhood and become shared within families and others who cohabitate, and how the biogeographic and biophysical forces drive construction of largely anaerobic microbial communities unique to each tooth. Finally, we touch on what is known about health-and disease-associated human oral microbial communities and briefly discuss the potential for using saliva samples to monitor health.

Proper management of polymicrobial infections in patients with cystic fibrosis (CF) has extended their life span. Information about the composition and dynamics of each patient’s microbial community aids in the selection of appropriate treatment of pulmonary exacerbations. We propose the cystic fibrosis rapid response (CFRR) as a fast approach to determine viral and microbial community composition and activity during CF pulmonary exacerbations. The CFRR potential is illustrated with a case study in which a cystic fibrosis fatal exacerbation was characterized by the presence of shigatoxigenic Escherichia coli. The incorporation of the CFRR within the CF clinic could increase the life span and quality of life of CF patients., Pulmonary exacerbations are the leading cause of death in cystic fibrosis (CF) patients. To track microbial dynamics during acute exacerbations, a CF rapid response (CFRR) strategy was developed. The CFRR relies on viromics, metagenomics, metatranscriptomics, and metabolomics data to rapidly monitor active members of the viral and microbial community during acute CF exacerbations. To highlight CFRR, a case study of a CF patient is presented, in which an abrupt decline in lung function characterized a fatal exacerbation. The microbial community in the patient’s lungs was closely monitored through the multi-omics strategy, which led to the identification of pathogenic shigatoxigenic Escherichia coli (STEC) expressing Shiga toxin. This case study illustrates the potential for the CFRR to deconstruct complicated disease dynamics and provide clinicians with alternative treatments to improve the outcomes of pulmonary exacerbations and expand the life spans of individuals with CF.

Corals and humans represent two extremely disparate metazoan lineages and are therefore useful for comparative evolutionary studies. Two lipid-based molecules that are central to human immunity, platelet-activating factor (PAF) and Lyso-PAF were recently identified in scleractinian corals. To identify processes in corals that involve these molecules, PAF and Lyso-PAF biosynthesis was quantified in conditions known to stimulate PAF production in mammals (tissue growth and exposure to elevated levels of ultraviolet light) and in conditions unique to corals (competing with neighbouring colonies over benthic space). Similar to observations in mammals, PAF production was higher in regions of active tissue growth and increased when corals were exposed to elevated levels of ultraviolet light. PAF production also increased when corals were attacked by the stinging cells of a neighbouring colony, though only the attacked coral exhibited an increase in PAF. This reaction was observed in adjacent areas of the colony, indicating that this response is coordinated across multiple polyps including those not directly subject to the stress. PAF and Lyso-PAF are involved in coral stress responses that are both shared with mammals and unique to the ecology of cnidarians.

Five batch cultures of Bacillus subtilis were subjected to evolution in the laboratory for 6,000 generations under conditions repressing sporulation in complex liquid medium containing glucose. Between generations 1,000 and 2,000, variants with a distinct small-colony morphology arose and swept through four of the five populations that had been previously noted for their loss of sporulation (H. Maughan et al., Genetics 177:937-948, 2007). To better understand the nature of adaptation in these variants, individual strains were isolated from one population before (WN715) and after (WN716) the sweep. In addition to colony morphology, strains WN715 and WN716 differed in their motility, aerotaxis, and cell morphology. Competition experiments showed that strain WN716 had evolved a distinct fitness advantage over the ancestral strain and strain WN715 during growth and the transition to the postexponential growth phase, which was more pronounced when WN715 was present in the coculture. Microarray analyses revealed candidate genes in which mutations may have produced some of the observed phenotypes. For example, loss of motility in WN716 was accompanied by decreased transcription of all flagellar, motility, and chemotaxis genes on the microarray. Transcription of alsS and alsD was also lower in strain WN716, and the predicted loss of acetoin production and enhanced acetate production was confirmed by high-performance liquid chromatography (HPLC) analysis. The results suggested that the derived colony morphology of strain WN716 was associated with increased fitness, the alteration of several metabolic pathways, and the loss of a typical postexponential-phase response.

We used microarrays to identify the causes of sporulation deficiencies in Bacillus subtilis after 6,000 generations of evolution. We found that sporulation loss did not result from large-scale deletions; therefore, it must have resulted from smaller indels and/or substitutions. Transcription patterns of one strain versus its ancestor showed that sporulation was not initiated and suggested that sporulation loss may be part of an overall decline in plasticity.

Phenotypic loss is an important evolutionary force in nature but the mechanism(s) responsible for loss remains unclear. We used both simulation and multiple-regression approaches to analyze data on the loss of sporulation, a complex bacterial developmental process, during experimental evolution of Bacillus subtilis. Neutral processes of mutational degradation alone were sufficient to explain loss-of-sporulation ability in four of five populations, while evidence that selection facilitated mutational loss was found for only one population. These results are discussed in the context of the evolution of sporulation in particular and phenotypic loss in general.

Rates of molecular evolution are known to vary considerably among lineages, partially due to differences in life-history traits such as generation time. The generation-time effect has been well documented in some eukaryotes, but its prevalence in prokaryotes is unknown. "Because many species of Firmicute bacteria spend long periods of time as metabolically dormant spores, which could result in fewer DNA substitutions per unit time, they present an excellent system for testing predictions of the molecular clock hypothesis." To test whether spore-forming bacteria evolve more slowly than their non-spore-forming relatives, I used phylogenetic methods to determine if there were differences in rates of amino acid substitution between spore-forming and non-spore-forming lineages of Firmicute bacteria. Although rates of evolution do vary among lineages, I find no evidence for an effect of spore-formation on evolutionary rate and, furthermore, evolutionary rates are similar to those calculated for enteric bacteria. These results support the notion that variation in generation time does not affect evolutionary rates in bacterial lineages.

Microbialization refers to the observed shift in ecosystem trophic structure towards higher microbial biomass and energy use. On coral reefs, the proximal causes of microbialization are overfishing and eutrophication, both of which facilitate enhanced growth of fleshy algae, conferring a competitive advantage over calcifying corals and coralline algae. The proposed mechanism for this competitive advantage is the DDAM positive feedback loop (dissolved organic carbon (DOC), disease, algae, microorganism), where DOC released by ungrazed fleshy algae supports copiotrophic, potentially pathogenic bacterial communities, ultimately harming corals and maintaining algal competitive dominance. Using an unprecedented data set of >400 samples from 60 coral reef sites, we show that the central DDAM predictions are consistent across three ocean basins. Reef algal cover is positively correlated with lower concentrations of DOC and higher microbial abundances. On turf and fleshy macroalgal-rich reefs, higher relative abundances of copiotrophic microbial taxa were identified. These microbial communities shift their metabolic potential for carbohydrate degradation from the more energy efficient Embden-Meyerhof-Parnas pathway on coral-dominated reefs to the less efficient Entner-Doudoroff and pentose phosphate pathways on algal-dominated reefs. This 'yield-to-power' switch by microorganism directly threatens reefs via increased hypoxia and greater CO2 release from the microbial respiration of DOC.

Although many examples of trait loss exist in nature, the underlying population genetic mechanism responsible for the loss is usually unknown. Selective or neutral processes can result in the deterioration of a trait, and often one of these is inferred based on indirect evidence. Furthermore, selective pressures that are unique to particular environments and the effect these might have on the population genetic cause of trait loss are not well understood. Here we describe an experimental evolution system where two different environments were used for addressing the population genetic cause of trait loss throughout evolutionary time. We found that growth in minimal medium (i.e., prototrophy) was lost in all populations regardless of the experimental environment and that the pattern of trait loss in one environment was due to selection, whereas in the other environment the cause remains inconclusive.

Previously, spontaneous rifampin resistance mutations were isolated in cluster I of the rpoB gene, resulting in amino acid replacements (Q469R, H482R, H482Y, or S487L) in the Bacillus subtilis RNA polymerase β subunit (W. L. Nicholson and H. Maughan, J. Bacteriol. 184: 4936-4940, 2002). In this study, each amino acid change in the β subunit was observed to result in its own unique spectrum of effects on growth and various developmental events, including sporulation, germination, and competence for transformation. The results thus establish the important role played by the RNA polymerase β subunit, not only in the catalytic aspect of transcription, but also in the regulation of major developmental events in B. subtilis .

Phenotypic plasticity is ubiquitous and generally regarded as a key mechanism for enabling organisms to survive in the face of environmental change. Because no organism is infinitely or ideally plastic, theory suggests that there must be limits (for example, the lack of ability to produce an optimal trait) to the evolution of phenotypic plasticity, or that plasticity may have inherent significant costs. Yet numerous experimental studies have not detected widespread costs. Explicitly differentiating plasticity costs from phenotype costs, we re-evaluate fundamental questions of the limits to the evolution of plasticity and of generalists vs specialists. We advocate for the view that relaxed selection and variable selection intensities are likely more important constraints to the evolution of plasticity than the costs of plasticity. Some forms of plasticity, such as learning, may be inherently costly. In addition, we examine opportunities to offset costs of phenotypes through ontogeny, amelioration of phenotypic costs across environments, and the condition-dependent hypothesis. We propose avenues of further inquiry in the limits of plasticity using new and classic methods of ecological parameterization, phylogenetics and omics in the context of answering questions on the constraints of plasticity. Given plasticity's key role in coping with environmental change, approaches spanning the spectrum from applied to basic will greatly enrich our understanding of the evolution of plasticity and resolve our understanding of limits.

Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the β subunit of RNA polymerase. In this study, we demonstrated that spores of B. subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells. In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB , H482R, from vegetative cells. Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L. The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis. The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.

The family Bacillaceae constitutes a phenotypically diverse and globally ubiquitous assemblage of bacteria. Investigation into how evolution has shaped, and continues to shape, this family has relied on several widely ranging approaches from classical taxonomy, ecological field studies, and evolution in soil microcosms to genomic-scale phylogenetics, laboratory, and directed evolution experiments. One unifying characteristic of the Bacillaceae , the endospore, poses unique challenges to answering questions regarding both the calculation of evolutionary rates and claims of extreme longevity in ancient environmental samples.

A continuously mixed series of microbial communities inhabits various points of the respiratory tract, with community composition determined by distance from colonization sources, colonization rates, and extinction rates. Ecology and evolution theory developed in the context of biogeography is relevant to clinical microbiology and could reframe the interpretation of recent studies comparing communities from lung explant samples, sputum samples, and oropharyngeal swabs. We propose an island biogeography model of the microbial communities inhabiting different niches in human airways. Island biogeography as applied to communities separated by time and space is a useful parallel for exploring microbial colonization of healthy and diseased lungs, with the potential to inform our understanding of microbial community dynamics and the relevance of microbes detected in different sample types. In this perspective, we focus on the intermixed microbial communities inhabiting different regions of the airways of patients with cystic fibrosis.

As DNA sequencing becomes faster and cheaper, genomics-based approaches are being explored for their use in personalized diagnoses and treatments. Here, we provide a proof of principle for disease monitoring using personal metagenomic sequencing and traditional clinical microbiology by focusing on three adults with cystic fibrosis (CF). The CF lung is a dynamic environment that hosts a complex ecosystem composed of bacteria, viruses, and fungi that can vary in space and time. Not surprisingly, the microbiome data from the induced sputum samples we collected revealed a significant amount of species diversity not seen in routine clinical laboratory cultures. The relative abundances of several species changed as clinical treatment was altered, enabling the identification of the climax and attack communities that were proposed in an earlier work. All patient microbiomes encoded a diversity of mechanisms to resist antibiotics, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in CF patients. The metabolic potentials of these communities differed by the health status and recovery route of each patient. Thus, this pilot study provides an example of how metagenomic data might be used with clinical assessments for the development of treatments tailored to individual patients.

It has recently been proposed that phenotypic variation in clonal populations of bacterial species results from intracellular “noise,” i.e., random fluctuations in levels of cellular molecules, which would be predicted to be insensitive to selective pressure. To test this notion, we propagated five populations of Bacillus subtilis for 5,000 generations with selection for one phenotype: the decision to sporulate. In support of the noise hypothesis, we report that none of the populations responded to selection by improving their efficiency of sporulation, indicating that intracellular noise is independent of heritable genotype.

The impaired mucociliary clearance in individuals with Cystic Fibrosis (CF) enables opportunistic pathogens to colonize CF lungs. Here we show that Rothia mucilaginosa is a common CF opportunist that was present in 83% of our patient cohort, almost as prevalent as Pseudomonas aeruginosa (89%). Sequencing of lung microbial metagenomes identified unique R. mucilaginosa strains in each patient, presumably due to evolution within the lung. The de novo assembly of a near-complete R. mucilaginosa (CF1E) genome illuminated a number of potential physiological adaptations to the CF lung, including antibiotic resistance, utilization of extracellular lactate, and modification of the type I restriction-modification system. Metabolic characteristics predicted from the metagenomes suggested R. mucilaginosa have adapted to live within the microaerophilic surface of the mucus layer in CF lungs. The results also highlight the remarkable evolutionary and ecological similarities of many CF pathogens; further examination of these similarities has the potential to guide patient care and treatment.

Background Inverse associations between allergic disease and having pets or siblings are commonly attributed to the hygiene hypothesis. As an extension, one could posit that a less diverse gut microbiome in the infant, also linked with the development of allergic disease, would be a function of fewer microbes in the home environment. Piglet studies, however, indicate that greater microbe diversity in the environment actually leads to reduced diversity of the gut microbiota. In this study, we characterize the infant gut microbiota in relation to environmental factors traditionally associated with the hygiene hypothesis.

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.