Taxonomic Features and Comparison of the Gut Microbiome from Two Edible Fungus-Farming Termites (Macrotermes falciger, M. natalensis) Harvested in the Vhembe District of Limpopo, South Africa

Background Termites are an important food resource for many human populations around the world, and are a good supply of nutrients. The fungus-farming ‘higher’ termite members of Macrotermitinae are also consumed by modern great apes and are implicated as critical dietary resources for early hominins. While the chemical nutritional composition of edible termites is well known, their microbiomes are unexplored in the context of human health. Here we sequenced the V4 region of the 16S rRNA gene of gut microbiota extracted from the whole intestinal tract of two Macrotermes sp. soldiers collected from the Limpopo region of South Africa. Results Major and minor soldier subcastes of M. falciger exhibit consistent differences in taxonomic representation, and are variable in microbial presence and abundance patterns when compared to another edible but less preferred species, M. natalensis. Subcaste differences include alternate patterns in sulfate-reducing bacteria and methanogenic Euryarchaeota abundance, and differences in abundance between Alistipes and Ruminococcaceae. M. falciger minor soldiers and M. natalensissoldiers have similar microbial profiles, likely from close proximity to the termite worker castes, particularly during foraging and fungus garden cultivation. Compared with previously published termite and cockroach gut microbiome data, the taxonomic representation was generally split between termites that directly digest lignocellulose and humic substrates and those that consume a more distilled form of nutrition as with the omnivorous cockroaches and fungus-farming termites. Lastly, to determine if edible termites may point to a shared reservoir for rare bacterial taxa found in the gut microbiome of humans, we focused on the genus Treponema. The majority of Treponemasequences from edible termite gut microbiota most closely relate to species recovered from other termites or from environmental samples, except for one novel OTU strain, which clustered separately with Treponema found in hunter-gatherer human groups. Conclusions Macrotermes consumed by humans display special gut microbial arrangements that are atypical for a lignocellulose digesting invertebrate, but are instead suited to the simplified nutrition in the fungus-farmer diet. Our work brings to light the particular termite microbiome features that should be explored further as avenues in human health, agricultural sustainability, and evolutionary research

Biogeographic Study of Human Gut-Associated CrAssphage Suggests Impacts From Industrialization and Recent Expansion

CrAssphage (cross-assembly phage) is a bacteriophage that was first discovered in human gut metagenomic data. CrAssphage belongs to a diverse family of crAss-like bacteriophages thought to infect gut commensal bacteria belonging to Bacteroides species. However, not much is known about the biogeography of crAssphage and whether certain strains are associated with specific human populations. In this study, we screened publicly available human gut metagenomic data from 3,341 samples for the presence of crAssphage sensu stricto (NC_024711.1). We found that crAssphage prevalence is low in traditional, hunter-gatherer populations, such as the Hadza from Tanzania and Matses from Peru, as compared to industrialized, urban populations. Statistical comparisons showed no association of crAssphage prevalence with variables such as age, sex, body mass index, and health status of individuals. Phylogenetic analyses show that crAssphage strains reconstructed from the same individual over multiple time-points, cluster together. CrAssphage strains from individuals from the same study population do not always cluster together. Some evidence of clustering is seen at the level of broadly defined geographic regions, however, the relative positions of these clusters within the crAssphage phylogeny are not well-supported. We hypothesize that this lack of strong biogeographic structuring is suggestive of an expansion event within crAssphage. Using a Bayesian dating approach, we estimate that this expansion has occurred fairly recently. Overall, we determine that crAssphage presence is associated with an industrialized lifestyle and the absence of strong biogeographic structuring within global crAssphage strains is likely due to a recent population expansion within this bacteriophage.This study was supported by a grant from the National Institutes of Health (https://www.nih.gov/), NIH R01 GM089886, awarded to C.M.L., C.W., and K.S. Open Access fees paid for in whole or in part by the University of Oklahoma Libraries.Ye

Analysis of global human gut metagenomes shows that metabolic resilience potential for short-chain fatty acid production is strongly influenced by lifestyle

High taxonomic diversity in non-industrial human gut microbiomes is often interpreted as beneficial; however, it is unclear if taxonomic diversity engenders ecological resilience (i.e. community stability and metabolic continuity). We estimate resilience through genus and species-level richness, phylogenetic diversity, and evenness in short-chain fatty acid (SCFA) production among a global gut metagenome panel of 12 populations (n = 451) representing industrial and non-industrial lifestyles, including novel metagenomic data from Burkina Faso (n = 90). We observe significantly higher genus-level resilience in non-industrial populations, while SCFA production in industrial populations is driven by a few phylogenetically closely related species (belonging to Bacteroides and Clostridium), meaning industrial microbiomes have low resilience potential. Additionally, database bias obfuscates resilience estimates, as we were 2–5 times more likely to identify SCFA-encoding species in industrial microbiomes compared to non-industrial. Overall, we find high phylogenetic diversity, richness, and evenness of bacteria encoding SCFAs in non-industrial gut microbiomes, signaling high potential for resilience in SCFA production, despite database biases that limit metagenomic analysis of non-industrial populations

Biogeographic Study of Human Gut Associated CrAssphage Suggests Impacts from Industrialization and Recent Expansion

CrAssphage (cross-assembly phage) is a bacteriophage that was first discovered in human gut metagenomic data. CrAssphage belongs to a diverse family of crAss-like bacteriophages thought to infect gut commensal bacteria belonging to Bacteroides species. However, not much is known about the biogeography of crAssphage and whether certain strains are associated with specific human populations. In this study, we screened publicly available human gut metagenomic data from 3,341 samples for the presence of crAssphage sensu stricto (NC_024711.1). We found that crAssphage prevalence is low in traditional, hunter-gatherer populations, such as the Hadza from Tanzania and Matses from Peru, as compared to industrialized, urban populations. Statistical comparisons showed no association of crAssphage prevalence with variables such as age, sex, body mass index, and health status of individuals. Phylogenetic analyses show that crAssphage strains reconstructed from the same individual over multiple time-points, cluster together. CrAssphage strains from individuals from the same study population do not always cluster together. Some evidence of clustering is seen at the level of broadly defined geographic regions, however, the relative positions of these clusters within the crAssphage phylogeny are not well-supported. We hypothesize that this lack of strong biogeographic structuring is suggestive of a recent expansion event within crAssphage. Using a Bayesian dating approach, we estimate this expansion has occurred within the past 200 years. Overall, we determine that crAssphage presence is associated with an industrialized lifestyle. The absence of strong biogeographic structuring within global crAssphage strains is likely due to a recent population expansion within this bacteriophage

Comparison of aDNA Yields from Calculus and Tooth Roots in Pre-Columbian Skeletal Remains

In recent years, dental calculus has emerged as an important source of ancient genetic material. However, calculus has not been extensively utilized as a source of endogenous host DNA when working with human skeletal remains. In this study we compare endogenous DNA yields obtained from extractions performed from both dental calculus and dental tooth roots for three pre-Columbian individuals, originating from three different archaeological sites of the island of Puerto Rico. Furthermore, in order to assess the effects of physical decontamination procedures on recovery of endogenous DNA, tooth root samples were further subdivided into two groups: one group was treated by removal of the cementum and the second group was left untreated. Extractions were then performed in three replicates for each individual, one from calculus, one from treated tooth roots and one from untreated tooth roots. DNA extracts were quantified, transformed into sequencing libraries, and enriched for the complete mitochondrial genome through in-solution hybridization capture. Preliminary results indicate that out of nine extracts obtained (three replicates for each individual) only seven were successfully built into libraries. Sequence data suggest that libraries made from treated tooth root extracts contain, on average, more sequence reads mapping to the reference and higher coverage than libraries built from untreated tooth root or calculus extracts. These results suggest that although calculus is a viable source of endogenous DNA, treated tooth root extractions result in higher overall endogenous DNA yields and a reduced presence of contaminant DNA molecules in these samples

Mycobacterium leprae genomes from naturally infected nonhuman primates.

Leprosy is caused by the bacterial pathogens Mycobacterium leprae and Mycobacterium lepromatosis. Apart from humans, animals such as nine-banded armadillos in the Americas and red squirrels in the British Isles are naturally infected with M. leprae. Natural leprosy has also been reported in certain nonhuman primates, but it is not known whether these occurrences are due to incidental infections by human M. leprae strains or by M. leprae strains specific to nonhuman primates. In this study, complete M. leprae genomes from three naturally infected nonhuman primates (a chimpanzee from Sierra Leone, a sooty mangabey from West Africa, and a cynomolgus macaque from The Philippines) were sequenced. Phylogenetic analyses showed that the cynomolgus macaque M. leprae strain is most closely related to a human M. leprae strain from New Caledonia, whereas the chimpanzee and sooty mangabey M. leprae strains belong to a human M. leprae lineage commonly found in West Africa. Additionally, samples from ring-tailed lemurs from the Bezà Mahafaly Special Reserve, Madagascar, and chimpanzees from Ngogo, Kibale National Park, Uganda, were screened using quantitative PCR assays, to assess the prevalence of M. leprae in wild nonhuman primates. However, these samples did not show evidence of M. leprae infection. Overall, this study adds genomic data for nonhuman primate M. leprae strains to the existing M. leprae literature and finds that this pathogen can be transmitted from humans to nonhuman primates as well as between nonhuman primate species. While the prevalence of natural leprosy in nonhuman primates is likely low, nevertheless, future studies should continue to explore the prevalence of leprosy-causing pathogens in the wild

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus

OBJECTIVES: Archaeological dental calculus is a rich source of host-associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. MATERIALS AND METHODS: Extracted DNA from six individuals at the 700-year-old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in-solution capture techniques, followed by Illumina high-throughput sequencing. RESULTS: Full mitogenomes (7-34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92-100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. DISCUSSION: Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains

Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution

Mycobacterium lepraemurium is the causative agent of murine leprosy, a chronic, granulomatous disease similar to human leprosy. Due to the similar clinical manifestations of human and murine leprosy and the difficulty of growing both bacilli axenically, Mycobacterium leprae and M. lepraemurium were once thought to be closely related, although it was later suggested that M. lepraemurium might be related to Mycobacterium avium. In this study, the complete genome of M. lepraemurium was sequenced using a combination of PacBio and Illumina sequencing. Phylogenomic analyses confirmed that M. lepraemurium is a distinct species within the M. avium complex (MAC). The M. lepraemurium genome is 4.05 Mb in length, which is considerably smaller than other MAC genomes, and it comprises 2,682 functional genes and 1,139 pseudogenes, which indicates that M. lepraemurium has undergone genome reduction. An error-prone repair homologue of the DNA polymerase III alpha-subunit was found to be nonfunctional in M. lepraemurium, which might contribute to pseudogene formation due to the accumulation of mutations in nonessential genes. M. lepraemurium has retained the functionality of several genes thought to influence virulence among members of the MAC. IMPORTANCE Mycobacterium lepraemurium seems to be evolving toward a minimal set of genes required for an obligatory intracellular lifestyle within its host, a niche seldom adopted by most mycobacteria, as they are free-living. M. lepraemurium could be used as a model to elucidate functions of genes shared with other members of the MAC. Its reduced gene set can be exploited for studying the essentiality of genes in related pathogenic species, which might lead to discovery of common virulence factors or clarify host-pathogen interactions. M. lepraemurium can be cultivated in vitro only under specific conditions and even then with difficulty. Elucidating the metabolic (in) capabilities of M. lepraemurium will help develop suitable axenic media and facilitate genetic studies

Ancient Dental Calculus as a Reservoir of Whole Human Mitogenomes

Until recently, one challenge of ancient DNA research has been the necessary destruction of skeletal material in order to extract human genetic information. Calcified dental plaque (dental calculus) can be removed from dentition without damaging the underlying teeth or surrounding bone and it has been shown to be an excellent reservoir of microbial and dietary biomolecules and microfossils. Here, we examine the preservation of human mitochondrial DNA in dental calculus by performing high-throughput mitogenome sequencing of material from six individuals from Norris Farms #36, a Mississippian period Oneota cemetery in Illinois dating back to 700BP. DNA was extracted in a dedicated ancient DNA facility at the University of Oklahoma and built into shotgun libraries. Following library amplification, each sample underwent a mitochondrial DNA capture and enrichment protocol at Arizona State University and was sequenced on an Illumina MiSeq platform. Medium coverage (7-34x) whole mitogenomes were successfully recovered from all six samples from Norris Farms #36 including three which were previously unsuccessful for HVRI sequencing using traditional PCR-based methods. In one calculus sample, the proportion of endogenous mtDNA increased from \u3c0.0001% in unenriched shotgun to 0.34% using mitochondrial capture and enrichment. This research demonstrates that calculus, an abundant and ubiquitous archaeological substrate, contains sufficient host information for whole mitogenome reconstruction. These findings have important implications for the use of dental calculus as an alternative material in archaeological studies of maternal ancestry

Maximum clade credibility tree of <i>M</i>. <i>leprae</i> strains.

<p>The five <i>M</i>. <i>leprae</i> branches are highlighted. Nodes are labeled with median divergence times in years before present, with the 95% HPD given in brackets. Posterior probabilities for each branch are shown next to the branches. The nonhuman primate <i>M</i>. <i>leprae</i> genomes sequenced in this study are marked in red.</p