Claire L. Thompson

The immediate environment during postnatal development has long-term impact on gut community structure in pigs

The gastrointestinal tract of mammals contains a complex microbial community that influences numerous aspects of health and development. It is postulated that establishment of this community during early life has long-term consequences on the health status of adults. Potential influences on colonization are expected to include environmental microbes, diet and the developmental changes of the host. Denaturing gradient gel electrophoresis was used to follow the individual community dynamics of 24 piglets over the period of 3–36 days after birth. The community of piglets older than 31 days was inferred to show high stability relative to the first 28 days post birth. The stable day 36 community showed significant correlation between cohabiting piglets, but not between siblings. This cohabitation effect was not observable in 1- or 2-week-old piglets but was strongest at either 3 or 4 weeks post birth. The onset of this change after 2 weeks is predicted to be after the development of key induction elements of the immune system and before significant levels of piglet sIgA were observable (4 weeks). The outcome is altered community dynamics that result in significant similarity between the stable communities that develop in cohabiting pigs. We conclude that for a finite period in their development, the outcome of gut colonization in piglets is greatly influenced by the immediate environment. The implication is that mammals have a developmental window, in which the developing host–gut microbiota interaction will be simultaneously more amenable to engineering and more susceptible to disturbance.

The Bacterial Community in the Gut of the Cockroach Shelfordella lateralis Reflects the Close Evolutionary Relatedness of Cockroaches and Termites

ABSTRACT Termites and cockroaches are closely related, with molecular phylogenetic analyses even placing termites within the radiation of cockroaches. The intestinal tract of wood-feeding termites harbors a remarkably diverse microbial community that is essential for the digestion of lignocellulose. However, surprisingly little is known about the gut microbiota of their closest relatives, the omnivorous cockroaches. Here, we present a combined characterization of physiological parameters, metabolic activities, and bacterial microbiota in the gut of Shelfordella lateralis, a representative of the cockroach family Blattidae, the sister group of termites. We compared the bacterial communities within each gut compartment using terminal-restriction fragment length polymorphism (T-RFLP) analysis and made a 16S rRNA gene clone library of the microbiota in the colon—the dilated part of the hindgut with the highest density and diversity of bacteria. The colonic community was dominated by members of the Bacteroidetes, Firmicutes (mainly Clostridia), and some Deltaproteobacteria. Spirochaetes and Fibrobacteres, which are abundant members of termite gut communities, were conspicuously absent. Nevertheless, detailed phylogenetic analysis revealed that many of the clones from the cockroach colon clustered with sequences previously obtained from the termite gut, which indicated that the composition of the bacterial community reflects at least in part the phylogeny of the host.

Community dynamics in the mouse gut microbiota: a possible role for IRF9-regulated genes in community homeostasis.

Background Gut microbial communities of mammals are thought to show stable differences between individuals. This means that the properties imparted by the gut microbiota become a unique and constant characteristic of the host. Manipulation of the microbiota has been proposed as a useful tool in health care, but a greater understanding of mechanisms which lead to community stability is required. Here we have examined the impact of host immunoregulatory phenotype on community dynamics. Methods and Findings Denaturing gradient gel electrophoresis was used to analyse the faecal bacterial community of BALB/c and C57BL/6 mice and C57BL/6 mice deficient for either type I interferon (IFN) signalling (IRF9 KO mice) or type I and type II IFN signalling (STAT1 KO mice). Temporal variation was found in all mouse strains. A measure of the ability for a community structure characteristic of the host to be maintained over time, the individuality index, varied between mouse strains and available data from pigs and human models. IRF9 KO mice had significantly higher temporal variation, and lower individuality, than other mouse strains. Examination of the intestinal mucosa of the IRF9 KO mice revealed an increased presence of T-cells and neutrophils in the absence of inflammation. Significance The high temporal variation observed in the gut microbiota of inbred laboratory mice has implications for their use as experimental models for the human gut microbiota. The distinct IRF9 and STAT1 phenotypes suggest a role for IRF9 in immune regulation within the gut mucosa and that further study of interferon responsive genes is necessary to understand host-gut microbe relationships.

Pyrotag sequencing of the gut microbiota of the cockroach Shelfordella lateralis reveals a highly dynamic core but only limited effects of diet on community structure.

Although blattid cockroaches and termites share a common ancestor, their diets are distinctly different. While termites consume a highly specialized diet of lignocellulose, cockroaches are omnivorous and opportunistic feeders. The role of the termite gut microbiota has been studied intensively, but little is known about the cockroach gut microbiota and its function in digestion and nutrition, particularly the adaptation to different diets. Our analyses of the bacterial gut microbiota of the blattid cockroach Shelfordella lateralis combining terminal restriction fragment length polymorphism of their 16S rRNA genes with physiological parameters (microbial metabolites, hydrogen and methane emission) indicated substantial variation between individuals but failed to identify any diet-related response. Subsequent deep-sequencing of the 16S rRNA genes of the colonic gut microbiota of S. lateralis fed either a high- or a low-fiber diet confirmed the absence of bacterial taxa that responded to diet. Instead, we found a small number of abundant phylotypes that were consistently present in all samples and made up half of the community in both diet groups. They varied strongly in abundance between individual samples at the genus but not at the family level. The remaining phylotypes were inconsistently present among replicate batches. Our findings suggest that S. lateralis harbors a highly dynamic core gut microbiota that is maintained even after fundamental dietary shifts, and that any dietary effects on the gut community are likely to be masked by strong individual variations.

Metagenomic analysis of the microbiota in the highly compartmented hindguts of six wood- or soil-feeding higher termites

BackgroundTermites are important contributors to carbon and nitrogen cycling in tropical ecosystems. Higher termites digest lignocellulose in various stages of humification with the help of an entirely prokaryotic microbiota housed in their compartmented intestinal tract. Previous studies revealed fundamental differences in community structure between compartments, but the functional roles of individual lineages in symbiotic digestion are mostly unknown.ResultsHere, we conducted a highly resolved analysis of the gut microbiota in six species of higher termites that feed on plant material at different levels of humification. Combining amplicon sequencing and metagenomics, we assessed similarities in community structure and functional potential between the major hindgut compartments (P1, P3, and P4). Cluster analysis of the relative abundances of orthologous gene clusters (COGs) revealed high similarities among wood- and litter-feeding termites and strong differences to humivorous species. However, abundance estimates of bacterial phyla based on 16S rRNA genes greatly differed from those based on protein-coding genes.ConclusionCommunity structure and functional potential of the microbiota in individual gut compartments are clearly driven by the digestive strategy of the host. The metagenomics libraries obtained in this study provide the basis for future studies that elucidate the fundamental differences in the symbiont-mediated breakdown of lignocellulose and humus by termites of different feeding groups. The high proportion of uncultured bacterial lineages in all samples calls for a reference-independent approach for the correct taxonomic assignment of protein-coding genes.

Deterministic Assembly of Complex Bacterial Communities in Guts of Germ-Free Cockroaches.

ABSTRACT The gut microbiota of termites plays important roles in the symbiotic digestion of lignocellulose. However, the factors shaping the microbial community structure remain poorly understood. Because termites cannot be raised under axenic conditions, we established the closely related cockroach Shelfordella lateralis as a germ-free model to study microbial community assembly and host-microbe interactions. In this study, we determined the composition of the bacterial assemblages in cockroaches inoculated with the gut microbiota of termites and mice using pyrosequencing analysis of their 16S rRNA genes. Although the composition of the xenobiotic communities was influenced by the lineages present in the foreign inocula, their structure resembled that of conventional cockroaches. Bacterial taxa abundant in conventional cockroaches but rare in the foreign inocula, such as Dysgonomonas and Parabacteroides spp., were selectively enriched in the xenobiotic communities. Donor-specific taxa, such as endomicrobia or spirochete lineages restricted to the gut microbiota of termites, however, either were unable to colonize germ-free cockroaches or formed only small populations. The exposure of xenobiotic cockroaches to conventional adults restored their normal microbiota, which indicated that autochthonous lineages outcompete foreign ones. Our results provide experimental proof that the assembly of a complex gut microbiota in insects is deterministic.

A window of environmental dependence is evident in multiple phylogenetically distinct subgroups in the faecal community of piglets

Microbial colonization in neonates has a profound impact on host development. In pigs, we have observed that a window of environmental dependence occurs during neonatal development. This was evident by the sudden onset of faecal community similarity in cohoused neonatal piglets at 3 weeks of age. This effect is postulated to represent a general change in gut community structure. Here, three phylogenetic groups (Clostridium leptum subgroup, Bacteroides and enterobacteria) that were predicted to have distinct ecological roles were monitored using nested denaturing gradient gel electrophoresis to determine the extent to which this window of environmental dependence was exerted throughout the gut community. Colonization trends were found to be similar for all subgroups despite predicted differences in the functional role and niche, and increased similarity between cohabiting piglets occurred in multiple phylogenetic subgroups. This supports the hypothesis that a distinct phase in neonatal development commences after 2 weeks whereby multiple subcommunities of the gut are strongly influenced by the environment.

Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model

ABSTRACT The gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroach Shelfordella lateralis as a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches. Fluorescence microscopy revealed that both strains specifically colonized the germfree hindgut. In diassociated cockroaches, the facultatively anaerobic strain EbSL (a new species of Enterobacteriaceae) always outnumbered the obligately anaerobic strain FuSL (a close relative of Fusobacterium varium), irrespective of the sequence of inoculation, which showed that precolonization by facultatively anaerobic bacteria does not necessarily favor colonization by obligate anaerobes. Comparison of the fermentation products of the cultures formed in vitro with those accumulated in situ indicated that the gut environment strongly affected the metabolic activities of both strains. The pure cultures formed the typical products of mixed-acid or butyrate fermentation, whereas the guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Similar shifts toward more-oxidized products were observed when the pure cultures were exposed to oxygen, which corroborated the strong effects of oxygen on the metabolic fluxes previously observed in termite guts. Oxygen microsensor profiles of the guts of germfree, gnotobiotic, and conventional cockroaches indicated that both gut tissue and microbiota contribute to oxygen consumption and suggest that the oxygen status influences the colonization success.

Analysis of Community Dynamics in Environmental Samples Using Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) is a culture-independent fingerprinting technique that allows for rapid comparative analysis of changes to microbial communities. 16S rRNA genes amplified from environmental samples can be separated based on their melting behavior in a denaturing gradient of urea and formamide. A fingerprint of the microbial community is generated with each band on the gel assumed to correspond to a different bacterial species. Community dynamics can then be assessed through statistical analysis of DGGE profiles and the sequencing of excised bands.

High-resolution phylogenetic analysis of Endomicrobia reveals multiple acquisitions of endosymbiotic lineages by termite gut flagellates

Bacteria of the class Endomicrobia form a deep-branching clade in the Elusimicrobia phylum. They are found almost exclusively in the intestinal tract of animals and are particularly abundant in many termites, where they reside as intracellular symbionts in the cellulolytic gut flagellates. Although small populations of putatively free-living lineages have been detected in faunated and flagellate-free hosts, the evolutionary origin of the endosymbionts is obscured by the limited amount of phylogenetic information provided by the 16S rRNA gene fragment amplified with Endomicrobia-specific primers. Here, we present a robust phylogenetic framework based on the near-full-length 16S-23S rRNA gene region of a diverse set of Endomicrobia from termites and cockroaches, which also allowed us to classify the shorter reads from previous studies. Our data revealed that endosymbionts arose independently at least four times from different free-living lineages, which were already present in ancestral cockroaches but became associated with their respective hosts long after the digestive symbiosis between termites and flagellates had been established. Pyrotag sequencing revealed that the proportion of putatively free-living lineages increased, when all flagellates and their symbionts were removed from the gut of lower termites by starvation, starch feeding or hyperbaric oxygen, but results varied between different methods.