Assessing alignment-based taxonomic classification of ancient microbial DNA

The field of palaeomicrobiology-the study of ancient microorganisms-is rapidly growing due to recent methodological and technological advancements. It is now possible to obtain vast quantities of DNA data from ancient specimens in a high-throughput manner and use this information to investigate the dynamics and evolution of past microbial communities. However, we still know very little about how the characteristics of ancient DNA influence our ability to accurately assign microbial taxonomies (i.e. identify species) within ancient metagenomic samples. Here, we use both simulated and published metagenomic data sets to investigate how ancient DNA characteristics affect alignment-based taxonomic classification. We find that nucleotide-to-nucleotide, rather than nucleotide-to-protein, alignments are preferable when assigning taxonomies to short DNA fragment lengths routinely identified within ancient specimens (<60 bp). We determine that deamination (a form of ancient DNA damage) and random sequence substitutions corresponding to ∼100,000 years of genomic divergence minimally impact alignment-based classification. We also test four different reference databases and find that database choice can significantly bias the results of alignment-based taxonomic classification in ancient metagenomic studies. Finally, we perform a reanalysis of previously published ancient dental calculus data, increasing the number of microbial DNA sequences assigned taxonomically by an average of 64.2-fold and identifying microbial species previously unidentified in the original study. Overall, this study enhances our understanding of how ancient DNA characteristics influence alignment-based taxonomic classification of ancient microorganisms and provides recommendations for future palaeomicrobiological studies.Raphael Eisenhofer and Laura Susan Weyric

Signatures of landscape and captivity in the gut microbiota of Southern Hairy-nosed Wombats (Lasiorhinus latifrons)

Background: Herbivorous mammals co-opt microbes to derive energy and nutrients from diets that are recalcitrant to host enzymes. Recent research has found that captive management—an important conservation tool for many species—can alter the gut microbiota of mammals. Such changes could negatively impact the ability of herbivorous mammals to derive energy from their native diets, and ultimately reduce host fitness. To date, nothing is known of how captivity influences the gut microbiota of the Southern Hairy-nosed Wombat (SHNW), a large herbivorous marsupial that inhabits South Australia. Here, using 16S rRNA gene sequencing, we characterized the faecal microbiota of SHNWs in captivity and from three wild populations, two from degraded habitats and one from an intact native grass habitat. Results: We found that captive SHNWs had gut microbiota that were compositionally different and less diverse compared to wild SHNWs. There were major differences in gut microbiota community membership between captive and wild animals, both in statistically significant changes in relative abundance of microbes, and in the presence/absence of microbes. We also observed differences in microbial composition between wild populations, with the largest difference associated with native vs. degraded habitat. Conclusions: These results suggest that captivity has a major impact on the gut microbiota of SHNWs, and that different wild populations harbour distinct microbial compositions. Such findings warrant further work to determine what impacts these changes have on the fitness of SHNWs, and whether they could be manipulated to improve future management of the species.Raphael Eisenhofer, Kristofer M. Helgen, and David Taggar

Adrenal Hormone Interactions and Metabolism: A Single Sample Multi-Omics Approach

The adrenal gland is important for many physiological and pathophysiological processes, but studies are often restricted by limited availability of sample material. Improved methods for sample preparation are needed to facilitate analyses of multiple classes of adrenal metabolites and macromolecules in a single sample. A procedure was developed for preparation of chromaffin cells, mouse adrenals, and human chromaffin tumors that allows for multi-omics analyses of different metabolites and preservation of native proteins. To evaluate the new procedure, aliquots of samples were also prepared using conventional procedures. Metabolites were analyzed by liquid-chromatography with mass spectrometry or electrochemical detection. Metabolite contents of chromaffin cells and tissues analyzed with the new procedure were similar or even higher than with conventional methods. Catecholamine contents were comparable between both procedures. The TCA cycle metabolites, cis-aconitate, isocitate, and α-ketoglutarate were detected at higher concentrations in cells, while in tumor tissue only isocitrate and potentially fumarate were measured at higher contents. In contrast, in a broad untargeted metabolomics approach, a methanol-based preparation procedure of adrenals led to a 1.3-fold higher number of detected metabolites. The established procedure also allows for simultaneous investigation of adrenal hormones and related enzyme activities as well as proteins within a single sample. This novel multi-omics approach not only minimizes the amount of sample required and overcomes problems associated with tissue heterogeneity, but also provides a more complete picture of adrenal function and intra-adrenal interactions than previously possible

Investigating the demographic history of Japan using ancient oral microbiota

While microbial communities in the human body (microbiota) are now commonly associated with health and disease in industrialised populations, we know very little about how these communities co-evolved and changed with humans throughout history and deep prehistory. We can now examine these communities by sequencing ancient DNA preserved within calcified dental plaque (calculus), providing insights into the origins of disease and their links to human history. Here, we examine ancient DNA preserved within dental calculus samples and their associations with two major cultural periods in Japan: the Jomon period hunter–gatherers approximately 3000 years before present (BP) and the Edo period agriculturalists 400–150 BP. We investigate how human oral microbiomes have changed in Japan through time and explore the presence of microorganisms associated with oral diseases (e.g. periodontal disease, dental caries) in ancient Japanese populations. Finally, we explore oral microbial strain diversity and its potential links to ancient demography in ancient Japan by performing phylogenomic analysis of a widely conserved oral species—Anaerolineaceae oral taxon 439. This research represents, to our knowledge, the first study of ancient oral microbiomes from Japan and demonstrates that the analysis of ancient dental calculus can provide key information about the origin of non-infectious disease and its deep roots with human demography. This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.Raphael Eisenhofer, Hideaki Kanzawa-Kiriyama, Ken-ichi Shinoda and Laura S. Weyric

Contamination in low microbial biomass microbiome studies: issues and recommendations

Next-generation sequencing approaches in microbiome research have allowed surveys of microbial communities, their genomes, and their functions with higher sensitivity than ever before. However, this sensitivity is a double-edged sword because these tools also efficiently detect contaminant DNA and cross-contamination, which can confound the interpretation of microbiome data. Therefore, there is an urgent need to integrate key controls into microbiome research to improve the integrity of microbiome studies. Here, we review how contaminant DNA and cross-contamination arise within microbiome studies and discuss their negative impacts, especially during the analysis of low microbial biomass samples. We then identify several key measures that researchers can implement to reduce the impact of contaminant DNA and cross-contamination during microbiome research. We put forward a set of minimal experimental criteria, the 'RIDE' checklist, to improve the validity of future low microbial biomass research.Raphael Eisenhofer, Jeremiah J. Minich, Clarisse Marotz, Alan Cooper, Rob Knight, and Laura S. Weyric

Microbial biogeography of the wombat gastrointestinal tract

Most herbivorous mammals have symbiotic microbes living in their gastrointestinal tracts that help with harvesting energy from recalcitrant plant fibre. The bulk of research into these microorganisms has focused on samples collected from faeces, representing the distal region of the gastrointestinal (GI) tract. However, the GI tract in herbivorous mammals is typically long and complex, containing different regions with distinct physico-chemical properties that can structure resident microbial communities. Little work has been done to document GI microbial communities of herbivorous animals at these sites. In this study, we use 16S rRNA gene sequencing to characterize the microbial biogeography along the GI tract in two species of wombats. Specifically, we survey the microbes along four major gut regions (stomach, small intestine, proximal colon, distal colon) in a single bare-nosed wombat (Vombatus ursinus) and a single southern hairy-nosed wombat (Lasiorhinus latifrons). Our preliminary results show that GI microbial communities of wombats are structured by GI region. For both wombat individuals, we observed a trend of increasing microbial diversity from stomach to distal colon. The microbial composition in the first proximal colon region was more similar between wombat species than the corresponding distal colon region in the same species. We found several microbial genera that were differentially abundant between the first proximal colon (putative site for primary plant fermentation) and distal colon regions (which resemble faecal samples). Surprisingly, only 10.6% (98) and 18.8% (206) of amplicon sequence variants (ASVs) were shared between the first proximal colon region and the distal colon region for the bare-nosed and southern hairy-nosed wombat, respectively. These results suggest that microbial communities in the first proximal colon region—the putative site of primary plant fermentation in wombats—are distinct from the distal colon, and that faecal samples may have limitations in capturing the diversity of these communities. While faeces are still a valuable and effective means of characterising the distal colon microbiota, future work seeking to better understand how GI microbiota impact the energy economy of wombats (and potentially other hindgut-fermenting mammals) may need to take gut biogeography into account

Host reproductive cycle influences the pouch microbiota of wild southern hairy-nosed wombats (Lasiorhinus latifrons)

Background: Marsupials are born much earlier than placental mammals, with most crawling from the birth canal to the protective marsupium (pouch) to further their development. However, little is known about the microbiology of the pouch and how it changes throughout a marsupial's reproductive cycle. Here, using stringent controls, we characterized the microbial composition of multiple body sites from 26 wild Southern Hairy-nosed Wombats (SHNWs), including pouch samples from animals at different reproductive stages. Results: Using qPCR of the 16S rRNA gene we detected a microbial community in the SHNW pouch. We observed significant differences in microbial composition and diversity between the body sites tested, as well as between pouch samples from different reproductive stages. The pouches of reproductively active females had drastically lower microbial diversity (mean ASV richness 19 ± 8) compared to reproductively inactive females (mean ASV richness 941 ± 393) and were dominated by gram positive bacteria from the Actinobacteriota phylum (81.7-90.6%), with the dominant families classified as Brevibacteriaceae, Corynebacteriaceae, Microbacteriaceae, and Dietziaceae. Three of the five most abundant sequences identified in reproductively active pouches had closest matches to microbes previously isolated from tammar wallaby pouches. Conclusions: This study represents the first contamination-controlled investigation into the marsupial pouch microbiota, and sets a rigorous framework for future pouch microbiota studies. Our results indicate that SHNW pouches contain communities of microorganisms that are substantially altered by the host reproductive cycle. We recommend further investigation into the roles that pouch microorganisms may play in marsupial reproductive health and joey survival.Sesilje Weiss, David Taggart, Ian Smith, Kristofer M. Helgen and Raphael Eisenhofe

Sarcoptic mange changes bacterial and fungal microbiota of bare-nosed wombats (Vombatus ursinus)

Published online: 13 September 2022Background: Sarcoptes scabiei is globally distributed and one of the most impactful mammalian ectoparasites. Sarcoptic mange, caused by infection with S. scabiei, causes disruption of the epidermis and its bacterial microbiota, but its effects on host fungal microbiota and on the microbiota of marsupials in general have not been studied. Here, we (i) examine bacterial and fungal microbiota changes associated with mange in wild bare-nosed wombats (BNWs) and (ii) evaluate whether opportunistic pathogens are potentiated by S. scabiei infection in this species. Methods: Using Amplicon Sequencing of the 16S rRNA and ITS2 rDNA genes, we detected skin microbiota changes of the bare-nosed wombat (Vombatus ursinus). We compared the alpha and beta diversity among healthy, moderate, and severe disease states using ANOVA and PERMANOVA with nesting. Lastly, we identified taxa that differed between disease states using analysis of composition of microbes (ANCOM) testing. Results: We detected significant changes in the microbial communities and diversity with mange in BNWs. Severely affected BNWs had lower amplicon sequence variant (ASV) richness compared to that of healthy individuals, and the microbial communities were significantly different between disease states with higher relative abundance of potentially pathogenic microbial taxa in mange-affected BNWs including Staphylococcus sciuri, Corynebacterium spp., Brevibacterium spp., Brachybacterium spp., and Pseudogymnascus spp. and Debaryomyces spp. Conclusion: This study represents the first investigation of microbial changes in association with sarcoptic mange in a marsupial host, as well as the first investigation of fungal microbial changes on the skin of any host suffering from sarcoptic mange. Our results are broadly consistent with bacterial microbiota changes observed in humans, pigs, canids, and Iberian ibex, suggesting the epidermal microbial impacts of mange may be generalisable across host species. We recommend that future studies investigating skin microbiota changes include both bacterial and fungal data to gain a more complete picture of the effects of sarcoptic mange.Christina Næsborg, Nielsen, Raphael Eisenhofer, Tamieka A. Fraser, Vicky Wilkinson, Christopher P. Burridge and Scott Carve

A systematic review of the literature examining the diagnostic efficacy of measurement of fractionated plasma free metanephrines in the biochemical diagnosis of pheochromocytoma

BACKGROUND: Fractionated plasma metanephrine measurements are commonly used in biochemical testing in search of pheochromocytoma. METHODS: We aimed to critically appraise the diagnostic efficacy of fractionated plasma free metanephrine measurements in detecting pheochromocytoma. Nine electronic databases, meeting abstracts, and the Science Citation Index were searched and supplemented with previously unpublished data. Methodologic and reporting quality was independently assessed by two endocrinologists using a checklist developed by the Standards for Reporting of Diagnostic Studies Accuracy Group and data were independently abstracted. RESULTS: Limitations in methodologic quality were noted in all studies. In all subjects (including those with genetic predisposition): the sensitivities for detection of pheochromocytoma were 96%–100% (95% CI ranged from 82% to 100%), whereas the specificities were 85%–100% (95% CI ranged from 78% to 100%). Statistical heterogeneity was noted upon pooling positive likelihood ratios when those with predisposition to disease were included (p < 0.001). However, upon pooling the positive or negative likelihood ratios for patients with sporadic pheochromocytoma (n = 191) or those at risk for sporadic pheochromocytoma (n = 718), no statistical heterogeneity was noted (p = 0.4). For sporadic subjects, the pooled positive likelihood ratio was 5.77 (95% CI = 4.90, 6.81) and the pooled negative likelihood ratio was 0.02 (95% CI = 0.01, 0.07). CONCLUSION: Negative plasma fractionated free metanephrine measurements are effective in ruling out pheochromocytoma. However, a positive test result only moderately increases suspicion of disease, particularly when screening for sporadic pheochromocytoma