Erin A. McKenney

Alteration of the rat cecal microbiome during colonization with the helminth Hymenolepis diminuta

The microbiome is now widely recognized as being important in health and disease, and makes up a substantial subset of the biome within the ecosystem of the vertebrate body. At the same time, multicellular, eukaryotic organisms such as helminths are being recognized as an important component of the biome that shaped the evolution of our genes. The absence of these macroscopic organisms during the early development and life of humans in Western culture probably leads to a wide range of human immunological diseases. However, the interaction between the microbiome and macroscopic components of the biome remains poorly characterized. In this study, the microbiome of the cecum in rats colonized for 2 generations with the small intestinal helminth Hymenolepis diminuta was evaluated. The introduction of this benign helminth, which is of considerable therapeutic interest, led to several changes in the cecal microbiome. Most of the changes were within the Firmicutes phylum, involved about 20% of the total bacteria, and generally entailed a shift from Bacilli to Clostridia species in the presence of the helminth. The results point toward ecological relationships between various components of the biome, with the observed shifts in the microbiome suggesting potential mechanisms by which this helminth might exert therapeutic effects.

Patterns of gut bacterial colonization in three primate species.

Host fitness is impacted by trillions of bacteria in the gastrointestinal tract that facilitate development and are inextricably tied to life history. During development, microbial colonization primes the gut metabolism and physiology, thereby setting the stage for adult nutrition and health. However, the ecological rules governing microbial succession are poorly understood. In this study, we examined the relationship between host lineage, captive diet, and life stage and gut microbiota characteristics in three primate species (infraorder, Lemuriformes). Fecal samples were collected from captive lemur mothers and their infants, from birth to weaning. Microbial DNA was extracted and the v4 region of 16S rDNA was sequenced on the Illumina platform using protocols from the Earth Microbiome Project. Here, we show that colonization proceeds along different successional trajectories in developing infants from species with differing dietary regimes and ecological profiles: frugivorous (fruit-eating) Varecia variegata, generalist Lemur catta, and folivorous (leaf-eating) Propithecus coquereli. Our analyses reveal community membership and succession patterns consistent with previous studies of human infants, suggesting that lemurs may serve as a useful model of microbial ecology in the primate gut. Each lemur species exhibits distinct species-specific bacterial diversity signatures correlating to life stages and life history traits, implying that gut microbial community assembly primes developing infants at species-specific rates for their respective adult feeding strategies.

Binturong (Arctictis binturong) and Kinkajou (Potos flavus) digestive strategy: implications for interpreting frugivory in Carnivora and primates.

Exclusive frugivory is rare. As a food resource, fruit is temporally and spatially patchy, low in protein, and variable in terms of energy yield from different carbohydrate types. Here, we evaluate the digestive physiology of two frugivorous Carnivora species (Potos flavus, Arctictis binturong) that converge with primates in a diversity of ecological and anatomical traits related to fruit consumption. We conducted feeding trials to determine mean digestive retention times (MRT) on captive animals at the Carnivore Preservation Trust (now Carolina Tiger Rescue), Pittsboro, NC. Fecal samples were collected on study subjects for in vitro analysis to determine methane, pH, and short chain fatty acid profiles; fiber was assayed using standard neutral detergent (NDF) and acid detergent (ADF) fiber methods. Results indicate that both carnivoran species have rapid digestive passage for mammals that consume a predominantly plant-based diet: A. binturong MRT = 6.5 hrs (0.3); P. flavus MRT = 2.5 hrs (1.6). In vitro experiments revealed no fermentation of structural polysaccharides – methane levels did not shift from 0 h to either 24 or 48 hours and no short chain fatty acids were detected. In both species, however, pH declined from one incubation period to another suggesting acidification and bacterial activity of microbes using soluble carbohydrates. A comparison with primates indicates that the study species are most similar in digestive retention times to Ateles – the most frugivorous anthropoid primate taxon.

Down for the count: Cryptosporidium infection depletes the gut microbiome in Coquerel’s sifakas

ABSTRACT Background: The gut microbiome (GMB) is the first line of defense against enteric pathogens, which are a leading cause of disease and mortality worldwide. One such pathogen, the protozoan Cryptosporidium, causes a variety of digestive disorders that can be devastating and even lethal. The Coquerel’s sifaka (Propithecus coquereli) – an endangered, folivorous primate endemic to Madagascar – is precariously susceptible to cryptosporidiosis under captive conditions. If left untreated, infection can rapidly advance to morbidity and death. Objective: To gain a richer understanding of the pathophysiology of this pathogen while also improving captive management of endangered species, we examine the impact of cryptosporidiosis on the GMB of a flagship species known to experience a debilitating disease state upon infection. Design: Using 16S sequencing of DNA extracted from sifaka fecal samples, we compared the microbial communities of healthy sifakas to those of infected individuals, across infection and recovery periods. Results: Over the course of infection, we found that the sifaka GMB responds with decreased microbial diversity and increased community dissimilarity. Compared to the GMB of unaffected individuals, as well as during pre-infection and recovery periods, the GMB during active infection was enriched for microbial taxa associated with dysbiosis and rapid transit time. Time to recovery was inversely related to age, with young animals being slowest to recover GMB diversity and full community membership. Antimicrobial treatment during infection caused a significant depletion in GMB diversity. Conclusions: Although individual sifakas show unique trajectories of microbial loss and recolonization in response to infection, recovering sifakas exhibit remarkably consistent patterns, similar to initial community assembly of the GMB in infants. This observation, in particular, provides biological insight into the rules by which the GMB recovers from the disease state. Fecal transfaunation may prove effective in restoring a healthy GMB in animals with specialized diets.

Bamboo Specialists from Two Mammalian Orders (Primates, Carnivora) Share a High Number of Low-Abundance Gut Microbes

Bamboo specialization is one of the most extreme examples of convergent herbivory, yet it is unclear how this specific high-fiber diet might selectively shape the composition of the gut microbiome compared to host phylogeny. To address these questions, we used deep sequencing to investigate the nature and comparative impact of phylogenetic and dietary selection for specific gut microbial membership in three bamboo specialists—the bamboo lemur (Hapalemur griseus, Primates: Lemuridae), giant panda (Ailuropoda melanoleuca, Carnivora: Ursidae), and red panda (Ailurus fulgens, Carnivora: Musteloideadae), as well as two phylogenetic controls—the ringtail lemur (Lemur catta) and the Asian black bear (Ursus thibetanus). We detected significantly higher Shannon diversity in the bamboo lemur (10.029) compared to both the giant panda (8.256; p = 0.0001936) and the red panda (6.484; p = 0.0000029). We also detected significantly enriched bacterial taxa that distinguished each species. Our results complement previous work in finding that phylogeny predominantly governs high-level microbiome community structure. However, we also find that 48 low-abundance OTUs are shared among bamboo specialists, compared to only 8 OTUs shared by the bamboo lemur and its sister species, the ringtail lemur (Lemur catta, a generalist). Our results suggest that deep sequencing is necessary to detect low-abundance bacterial OTUs, which may be specifically adapted to a high-fiber diet. These findings provide a more comprehensive framework for understanding the evolution and ecology of the microbiome as well as the host.

Feeding strategy shapes gut metagenomic enrichment and functional specialization in captive lemurs.

ABSTRACT Many studies have demonstrated the effects of host diet on gut microbial membership, metagenomics, and fermentation individually; but few have attempted to interpret the relationship among these biological phenomena with respect to host features (e.g. gut morphology). We quantitatively compare the fecal microbial communities, metabolic pathways, and fermentation products associated with the nutritional intake of frugivorous (fruit-eating) and folivorous (leaf-eating) lemurs. Our results provide a uniquely multidimensional and comparative perspective on the adaptive dynamics between host and microbiome. Shotgun metagenomic sequencing revealed significant differential taxonomic and metabolic pathway enrichment, tailored to digest and detoxify different diets. Frugivorous metagenomes feature pathways to degrade simple carbohydrates and host-derived glycosaminoglycans, while folivorous metagenomes are equipped to break down phytic acid and other phytochemical compounds in an anaerobic environment. We used nuclear magnetic resonance based metabolic profiling of fecal samples to link metabolic pathways to fermentation products, confirming that the dissimilar substrates provided in each diet select for specific microbial functions. Fecal samples from frugivorous lemurs contained significantly different profiles of short chain fatty acids, alcohol fermentation products, amino acids, glucose, and glycerol compared to folivorous lemurs. We present the relationships between these datasets as an integrated visual framework, which we refer to as microbial geometry. We use microbial geometry to compare empirical gut microbial profiles across different feeding strategies, and suggest additional utility as a tool for hypothesis-generation.

Evolution of the hygiene hypothesis into biota alteration theory: what are the paradigms and where are the clinical applications?

For thousands of years, changes in human cultures have altered the biota associated with the human body, and those alterations have strongly influenced human health. The hygiene hypothesis has evolved over the past 30 years into a nuanced biota alteration theory, but modern medical priorities and regulatory policies have resulted in tragic underutilization of the acquired knowledge.

The critical role of dietary foliage in maintaining the gut microbiome and metabolome of folivorous sifakas

The gut microbiome (GMB) of folivores metabolizes dietary fiber into nutrients, including short-chain fatty acids (SCFAs); however, experiments probing the consequences of foliage quality on host GMBs are lacking. We therefore examined GMB structure and function via amplicon sequencing and Nuclear Magnetic Resonance spectroscopy in 31 captive sifakas (Propithecus coquereli) during dietary manipulations associated with husbandry. Supplementing standard diets with diverse foliage blends, versus with a single plant species, promoted more diverse GMBs, enriched for taxa implicated in plant-fiber metabolism, but depleted in taxa implicated in starch metabolism and bile tolerance. The consumption of diverse blends was associated with greater concentrations of colonic SCFAs. Abundant foliage, via forest access, promoted compositionally distinct and more stable GMBs, but reduced concentrations of SCFAs, possibly reflecting selection of high-quality leaves. In 11 subjects denied forest access, we examined the temporal pace of microbial shifts when supplemental foliage was abruptly switched between diverse blends and single species. The sifaka GMB responded within days, with community diversity and composition closely tracking foliage diversity. By providing experimental evidence that the folivore GMB is sensitive to minor changes in dietary foliage, we reveal the fragility of specialist GMBs, with implications for managing the wellbeing of endangered wildlife.

Symbiosis in the Soil: Citizen Microbiology in Middle and High School Classrooms

Microorganisms are vital to environmental health, yet their association with disease often overshadows these benefits. Building citizen-science activities around the positive role of microorganisms and an understanding of their ubiquity can begin to dispel misconceptions while simultaneously engaging the public in research. Here, we describe a citizen-science microbiology project geared toward implementation in middle and high school classrooms. Students culture environmental microorganisms and document microbial diversity of plant root systems compared with adjacent bulk soil. Results contribute data toward research on microbiome recruitment of weeds and other successful plants while addressing core topics in science education.

30. Biome reconstitution as a novel mechanism of preventing neonatal infection-induced cognitive dysfunction

The incidence of autoimmune and inflammatory diseases has risen in post-industrial societies. “Biome depletion” – loss of factors interacting with the immune system – may contribute to these increases. Helminths are commensalist organisms capable of modulating the immune system that have been lost in post-industrial society. Here, we investigated their therapeutic effects in a neonatal infection model known to cause cognitive impairments in adult rats. Breeders were given rat tapeworms (Hymenolepsis diminuta) or vehicle. The male offspring were injected with PBS or live Escherichia coli on postnatal day (PD) 4. At PD21, offspring were inoculated with tapeworms or vehicle, matching their mothers’ treatment. At PD65–70, rats were tested in a fear conditioning task, in which they received an injection of saline or LPS following context exploration/learning. When tested for freezing behavior/memory, the “two hit” rats – those receiving E. coli on PD4 and LPS after learning – froze less, evidence of a significant memory deficit. However, helminth-colonized “two hit” rats demonstrated no cognitive impairment. Additionally, neonatally-infected pups sacrificed on PD5 had increased IL-1 mRNA within the hippocampus compared to PBS controls, whereas maternal helminth inoculation prevented this increase. The worms had no impact on adaptive immune cell subsets, whereas exaggerated innate inflammatory responses within cultured splenocytes were prevented. Together these data indicate that biome reconstitution is a viable preventative treatment for inflammation-induced cognitive impairments.