Bacteroides vicugnae sp. nov. isolated from the fecal material of an alpaca.

Two strictly anaerobic, Gram-stain-negative rod-shaped bacterial isolates, A2-P53T and A1-P5, were isolated from an enrichment of fecal material from two alpacas (Vicugna pacos). Based on a comparative 16S rRNA gene sequence analysis, the isolates were assigned to the genus Bacteroides with the highest sequence similarities to Bacteroides koreensis YS-aM39T (A2- P53T 97.7% and A1-P5 97.9%). Additionally, the average nucleotide identity and digital DNA-DNA hybridization values between these isolates and their closest relatives within Bacteroides were less than 92.1% and 49.1%, respectively. The average nucleotide identity between isolates A2-P53T and A1-P5 was 99.9%. The predominant cellular fatty acid for isolates A2-P53T and A1-P5 was C15:0 antesio. The G+C % content of the isolates was 41.7%. Based on biochemical, phylogenetic, genotypic, and chemotaxonomic criteria, these isolates A2-P53T and A1-P5 represent two individual strains of a novel species within the genus Bacteroides for which the name Bacteroides vicugnae sp. nov. is proposed. The type strain of this species is strain A2-P53T (CCUG 77273T=CCM 9377T=NRRL B-65693T).

Clostridium tanneri sp. nov., isolated from the faecal material of an alpaca.

A strictly anaerobic, Gram-stain-negative rod-shaped bacterium, designated A1-XYC3T, was isolated from the faeces of an alpaca (Lama pacos). On the basis of the results of a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Clostridium with the highest sequence similarities to Clostridium magnum DSM 2767T (96.8 %), Clostridium carboxidivorans P7T (96.3 %) and Clostridium aciditolerans JW/YJL-B3T (96.1 %). The average nucleotide identity between A1-XYC3T, C. magnum, C. carboxidivorans and C. aciditolerans was 77.4, 76.1 and 76.6  %, respectively. The predominant components of the cellular fatty acids of A1-XYC3T were C14 : 0, C16 : 0 and summed feature 10, containing C18:0/C17:0 cyclo. The DNA G+C content was 32.4 mol%. On the basis of biochemical, phylogenetic, genotypic and chemotaxonomic criteria, this isolate represents a novel species within Clostridium sensu stricto for which the name Clostridium tanneri sp. nov. is proposed. The type strain of this species is strain A1-XYC3T (=CCM 9376T=NRRL B-65691T).

Methylocystis suflitae sp. nov., a novel type II methanotrophic bacterium isolated from landfill cover soil.

A bacterial strain, designated NLS-7T, was isolated through enrichment of landfill cover soil in methane-oxidizing conditions. Strain NLS-7T is a Gram-stain negative, non-motile rod, approximately 0.8 µm wide by 1.3 µm long. Phylogenetic analysis based on 16S rRNA gene sequencing places it within the genus Methylocystis, with its closest relatives being M. hirsuta, M. silviterrae and M. rosea, with 99.9, 99.7 and 99.6 % sequence similarity respectively. However, average nucleotide identity and average amino acid identity values below the 95 % threshold compared to all the close relatives and digital DNA-DNA hybridization values between 20.9 and 54.1 % demonstrate that strain NLS-7T represents a novel species. Genome sequencing generated 4.31 million reads and genome assembly resulted in the generation of 244 contigs with a total assembly length of 3 820 957 bp (N50, 37 735 bp; L50, 34). Genome completeness is 99.5 % with 3.98 % contamination. It is capable of growth on methane and methanol. It grows optimally at 30 °C between pH 6.5 and 7.0. Strain NLS-7T is capable of atmospheric dinitrogen fixation and can use ammonium (as NH4Cl), l-aspartate, l-arginine, yeast extract, nitrate, l-leucine, l-proline, l-methionine, l-lysine and l-alanine as nitrogen sources. The major fatty acids are C18:1 ω8c and C18:1 ω7c. Based upon this polyphasic taxonomic study, strain NLS-7T represents a novel species of the genus Methylocystis, for which the name Methylocystis suflitae sp. nov. is proposed. The type strain is NLS-7T (=ATCC TSD-256T=DSM 112294T). The 16S rRNA gene and genome sequences of strain NLS-7T have been deposited in GenBank under accession numbers ON715489 and GCA_024448135.1, respectively.

Localized cardiac small molecule trajectories and persistent chemical sequelae in experimental Chagas disease.

Post-infectious conditions present major health burdens but remain poorly understood. In Chagas disease (CD), caused by Trypanosoma cruzi parasites, antiparasitic agents that successfully clear T. cruzi do not always improve clinical outcomes. In this study, we reveal differential small molecule trajectories between cardiac regions during chronic T. cruzi infection, matching with characteristic CD apical aneurysm sites. Incomplete, region-specific, cardiac small molecule restoration is observed in animals treated with the antiparasitic benznidazole. In contrast, superior restoration of the cardiac small molecule profile is observed for a combination treatment of reduced-dose benznidazole plus an immunotherapy, even with less parasite burden reduction. Overall, these results reveal molecular mechanisms of CD treatment based on simultaneous effects on the pathogen and on host small molecule responses, and expand our understanding of clinical treatment failure in CD. This link between infection and subsequent persistent small molecule perturbation broadens our understanding of infectious disease sequelae.

Holtiella tumoricola gen. nov. sp. nov., isolated from a human clinical sample.

The diversity of bacteria associated with biopsy material obtained from patients with colorectal cancer was investigated using culture techniques. A novel bacterium, strain CC70AT, was isolated by diluting a sample of homogenized tissue in anaerobic medium, and then plating to yield a pure culture. Strain CC70AT was a Gram-positive, strictly anaerobic, motile, rod-shaped bacterium. Formate, but not acetate, was a fermentative end-product from growth in peptone-yeast extract and peptone-yeast-glucose broth. The G+C content of DNA from strain CC70AT was 34.9 mol%. 16S rRNA gene sequence analysis revealed that the isolate was part of the phylum Bacillota. The closest described relatives of strain CC70AT were Cellulosilyticum lentocellum (93.3 %) and Cellulosilyticum ruminicola (93.3 and 91.9% sequence similarity across 16S rRNA gene, respectively). According to the data obtained in this work, strain CC70AT represents a novel bacterium belonging to a new genus for which the name Holtiella tumoricola gen. nov., sp. nov. is proposed. The type strain for our described novel species is CC70AT (=DSM 27931T= JCM 30568T).

Evolutionary barriers to horizontal gene transfer in macrophage-associated Salmonella.

Horizontal gene transfer (HGT) is a powerful evolutionary force facilitating bacterial adaptation and emergence of novel phenotypes. Several factors, including environmental ones, are predicted to restrict HGT, but we lack systematic and experimental data supporting these predictions. Here, we address this gap by measuring the relative fitness of 44 genes horizontally transferred from Escherichia coli to Salmonella enterica in infection-relevant environments. We estimated the distribution of fitness effects in each environment and identified that dosage-dependent effects across different environments are a significant barrier to HGT. The majority of genes were found to be deleterious. We also found longer genes had stronger negative fitness consequences than shorter ones, showing that gene length was negatively associated with HGT. Furthermore, fitness effects of transferred genes were found to be environmentally dependent. In summary, a substantial fraction of transferred genes had a significant fitness cost on the recipient, with both gene characteristics and the environment acting as evolutionary barriers to HGT.

Oral metagenomes from Native American Ancestors reveal distinct microbial lineages in the pre-contact era.

OBJECTIVES: Limited studies have focused on how European contact and colonialism impacted Native American oral microbiomes, specifically, the diversity of commensal or opportunistically pathogenic oral microbes, which may be associated with oral diseases. Here, we studied the oral microbiomes of pre-contact Wichita Ancestors, in partnership with the Descendant community, The Wichita and Affiliated Tribes, Oklahoma, USA. MATERIALS AND METHODS: Skeletal remains of 28 Wichita Ancestors from 20 archeological sites (dating approximately to 1250-1450 CE) were paleopathologically assessed for presence of dental calculus and oral disease. DNA was extracted from calculus, and partial uracil deglycosylase-treated double-stranded DNA libraries were shotgun-sequenced using Illumina technology. DNA preservation was assessed, the microbial community was taxonomically profiled, and phylogenomic analyzes were conducted. RESULTS: Paleopathological analysis revealed signs of oral diseases such as caries and periodontitis. Calculus samples from 26 Ancestors yielded oral microbiomes with minimal extraneous contamination. Anaerolineaceae bacterium oral taxon 439 was found to be the most abundant bacterial species. Several Ancestors showed high abundance of bacteria typically associated with periodontitis such as Tannerella forsythia and Treponema denticola. Phylogenomic analyzes of Anaerolineaceae bacterium oral taxon 439 and T. forsythia revealed biogeographic structuring; strains present in the Wichita Ancestors clustered with strains from other pre-contact Native Americans and were distinct from European and/or post-contact American strains. DISCUSSION: We present the largest oral metagenome dataset from a pre-contact Native American population and demonstrate the presence of distinct lineages of oral microbes specific to the pre-contact Americas.

Reclassification of Clostridium cocleatum, Clostridium ramosum, Clostridium spiroforme and Clostridium saccharogumia as Thomasclavelia cocleata gen. nov., comb. nov., Thomasclavelia ramosa comb. nov., gen. nov., Thomasclavelia spiroformis comb. nov. and Thomasclavelia saccharogumia comb. nov.

The genus Clostridium is phenotypically and genotypically diverse, with many species phylogenetically located outside Clostridium sensu stricto. One such group consists of the species Clostridium cocleatum, Clostridium ramosum, Clostridium spiroforme and Clostridium saccharogumia (formally clostridial rRNA cluster XVIII) [1]. Sequencing of the 16S rRNA and, more recently, the results of genomic analyses have demonstrated that these species represent a coherent cluster separated from other closely related genera located in the family Coprobacillaceae within the order Erysipelotrichales [2]. In addition to phenotypic, phylogenetic and genomic comparisons, chemotaxonomic features were consistent between all four species, the predominant fatty acids were C16 : 0 and C18 : 1ω9c, while glucose and ribose were the whole cell sugars present in the cell walls. Furthermore, he results of peptidoglycan analysis indicated that meso-2,6-diaminopimelic acid was present as the diagnostic diamino acid in all four species. Biochemical profiles were also concordant with them being closely related species. Therefore, on the basis of phylogenetic, genomic, phenotypic and chemotaxonomic information, a novel genus, Thomasclavelia gen. nov., is proposed. It is suggested that Clostridium cocleatum, Clostridium ramosum, Clostridium spiroforme and Clostridium saccharogumia be transferred to this genus as Thomasclavelia cocleata comb. nov., Thomasclavelia ramosa comb. nov., Thomasclavelia saccharogumia comb. nov. and Thomasclavelia spiroformis comb. nov. The type species of the genus is Thomasclavelia ramosa CCUG 24038T (=ATCC 25582T=DSM 1402T).

Untargeted Fecal Metabolomic Analyses across an Industrialization Gradient Reveal Shared Metabolites and Impact of Industrialization on Fecal Microbiome-Metabolome Interactions.

The metabolome is a central determinant of human phenotypes and includes the plethora of small molecules produced by host and microbiome or taken up from exogenous sources. However, studies of the metabolome have so far focused predominantly on urban, industrialized populations. Through an untargeted metabolomic analysis of 90 fecal samples from human individuals from Africa and the Americas-the birthplace and the last continental expansion of our species, respectively-we characterized a shared human fecal metabolome. The majority of detected metabolite features were ubiquitous across populations, despite any geographic, dietary, or behavioral differences. Such shared metabolite features included hyocholic acid and cholesterol. However, any characterization of the shared human fecal metabolome is insufficient without exploring the influence of industrialization. Here, we show chemical differences along an industrialization gradient, where the degree of industrialization correlates with metabolomic changes. We identified differential metabolite features such as amino acid-conjugated bile acids and urobilin as major metabolic correlates of these behavioral shifts. Additionally, coanalyses with over 5,000 publicly available human fecal samples and cooccurrence probability analyses with the gut microbiome highlight connections between the human fecal metabolome and gut microbiome. Our results indicate that industrialization significantly influences the human fecal metabolome, but diverse human lifestyles and behavior still maintain a shared human fecal metabolome. This study represents the first characterization of the shared human fecal metabolome through untargeted analyses of populations along an industrialization gradient. IMPORTANCE As the world becomes increasingly industrialized, understanding the biological consequences of these lifestyle shifts and what it means for past, present, and future human health is critical. Indeed, industrialization is associated with rises in allergic and autoimmune health conditions and reduced microbial diversity. Exploring these health effects on a chemical level requires consideration of human lifestyle diversity, but understanding the significance of any differences also requires knowledge of what molecular components are shared between human groups. Our study reveals the key chemistry of the human gut as defined by varied industrialization-based differences and ubiquitous shared features. Ultimately, these novel findings extend our knowledge of human molecular biology, especially as it is influenced by lifestyle and behavior, and provide steps toward understanding how human biology has changed over our species' history.

Remembering St. Louis individual-structural violence and acute bacterial infections in a historical anatomical collection.

Incomplete documentary evidence, variable biomolecular preservation, and limited skeletal responses have hindered assessment of acute infections in the past. This study was initially developed to explore the diagnostic potential of dental calculus to identify infectious diseases, however, the breadth and depth of information gained from a particular individual, St. Louis Individual (St.LI), enabled an individualized assessment and demanded broader disciplinary introspection of ethical research conduct. Here, we document the embodiment of structural violence in a 23-year-old Black and/or African American male, who died of lobar pneumonia in 1930s St. Louis, Missouri. St.LI exhibits evidence of systemic poor health, including chronic oral infections and a probable tuberculosis infection. Metagenomic sequencing of dental calculus recovered three pre-antibiotic era pathogen genomes, which likely contributed to the lobar pneumonia cause of death (CoD): Klebsiella pneumoniae (13.8X); Acinetobacter nosocomialis (28.4X); and Acinetobacter junii (30.1X). Ante- and perimortem evidence of St.LI's lived experiences chronicle the poverty, systemic racism, and race-based structural violence experienced by marginalized communities in St. Louis, which contributed to St.LI's poor health, CoD, anatomization, and inclusion in the Robert J. Terry Anatomical Collection. These same embodied inequalities continue to manifest as health disparities affecting many contemporary communities in the United States.

Shifts in gut and vaginal microbiomes are associated with cancer recurrence time in women with ovarian cancer.

Many studies investigating the human microbiome-cancer interface have focused on the gut microbiome and gastrointestinal cancers. Outside of human papillomavirus driving cervical cancer, little is known about the relationship between the vaginal microbiome and other gynecological cancers, such as ovarian cancer. In this retrospective study, we investigated the relationship between ovarian cancer, platinum-free interval (PFI) length, and vaginal and gut microbiomes. We observed that Lactobacillus-dominated vaginal communities were less common in women with ovarian cancer, as compared to existing datasets of similarly aged women without cancer. Primary platinum-resistance (PPR) disease is strongly associated with survivability under one year, and we found over one-third of patients with PPR (PFI < 6 months, n = 17) to have a vaginal microbiome dominated by Escherichia (>20% relative abundance), while only one platinum super-sensitive (PFI > 24 months, n = 23) patient had an Escherichia-dominated microbiome. Additionally, L. iners was associated with little, or no, gross residual disease, while other Lactobacillus species were dominant in women with >1 cm gross residual disease. In the gut microbiome, we found patients with PPR disease to have lower phylogenetic diversity than platinum-sensitive patients. The trends we observe in women with ovarian cancer and PPR disease, such as the absence of Lactobacillus and presence of Escherichia in the vaginal microbiome as well as low gut microbiome phylogenetic diversity have all been linked to other diseases and/or pro-inflammatory states, including bacterial vaginosis and autoimmune disorders. Future prospective studies are necessary to explore the translational potential and underlying mechanisms driving these associations.

Reclassification of Facklamia ignava, Facklamia sourekii and Facklamia tabacinasalis as Falseniella ignava gen. nov., comb. nov., Hutsoniella sourekii gen. nov., comb. nov., and Ruoffia tabacinasalis gen. nov., comb. nov., and description of Ruoffia halotolerans sp. nov., isolated from hypersaline Inland Sea of Qatar.

A Gram-stain-positive, non-pigmented, coccus-shaped, facultatively anaerobic and α-hemolytic bacterium designated as INB8T was isolated from a hypersaline marine water sample collected at the Inland Sea of Qatar. The isolate was able to grow at 25-40 °C (optimum, 30 °C), at pH 5-11 and with 2-8% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain INB8T was placed within the family Aerococcaceae with the highest sequence similarity to Facklamia tabacinasalis CCUG 30090T (99.5%), followed by Facklamia hominis CCUG 36813T (93.9%), Facklamia sourekii Y17312T (93.8%), Facklamia ignava CCUG 37419T (93.6%), Facklamia miroungae CCUG 42728T (93.5%), Suicoccus acidiformans ZY16052T (93.5%), Facklamia languida CCUG 37842T (93.2%), Ignavigranum ruoffiae (93.1%), and Dolosicoccus paucivorans DSM 15742T (90.8%). Average nucleotide identity and digital DNA-DNA hybridization values between strain INB8T and F. tabacinasalis CCUG 30090T were determined to be 94.5% and 58.9% respectively, confirming strain INB8T represents a novel species. The major fatty acids were C14:0, C16:0, C18:0 and C18:1 ω9c. The G + C content of strain INB8T determined from the genome was 36.3 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic information, it is proposed that Facklamia tabacinasalis should be reclassified as Ruoffia tabacinasalis, Facklamia ignava be reclassified as Falseniella ignava, and Facklamia sourekii be reclassified Hutsoniella sourekii. It is further proposed that strain INB8T should be classified as a species of the genus Ruoffia for which the name Ruoffia halotolerans sp. nov. is proposed. The type strain is INB8T (= LMG 30291T = CCUG 70701T = QCC/B60/17T).

The evolution and changing ecology of the African hominid oral microbiome.

The oral microbiome plays key roles in human biology, health, and disease, but little is known about the global diversity, variation, or evolution of this microbial community. To better understand the evolution and changing ecology of the human oral microbiome, we analyzed 124 dental biofilm metagenomes from humans, including Neanderthals and Late Pleistocene to present-day modern humans, chimpanzees, and gorillas, as well as New World howler monkeys for comparison. We find that a core microbiome of primarily biofilm structural taxa has been maintained throughout African hominid evolution, and these microbial groups are also shared with howler monkeys, suggesting that they have been important oral members since before the catarrhine-platyrrhine split ca. 40 Mya. However, community structure and individual microbial phylogenies do not closely reflect host relationships, and the dental biofilms of Homo and chimpanzees are distinguished by major taxonomic and functional differences. Reconstructing oral metagenomes from up to 100 thousand years ago, we show that the microbial profiles of both Neanderthals and modern humans are highly similar, sharing functional adaptations in nutrient metabolism. These include an apparent Homo-specific acquisition of salivary amylase-binding capability by oral streptococci, suggesting microbial coadaptation with host diet. We additionally find evidence of shared genetic diversity in the oral bacteria of Neanderthal and Upper Paleolithic modern humans that is not observed in later modern human populations. Differences in the oral microbiomes of African hominids provide insights into human evolution, the ancestral state of the human microbiome, and a temporal framework for understanding microbial health and disease.

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.

Ningiella ruwaisensis gen. nov., sp. nov., a member of the family Alteromonadaceae isolated from marine water of the Arabian Gulf.

Strain B66T was isolated from a marine water sample collected at Al Ruwais, located on the northern tip of Qatar. Cells were Gram-stain-negative, strictly aerobic and short- rod-shaped with a polar flagellum. The isolate was able to grow at 15-45 °C (optimum, 30 °C), at pH 5-11 (optimum, pH 6.5-8) and with 0-6 % NaCl. 16S rRNA gene sequence analysis revealed that strain B66T was affiliated with the family Alteromonadaceae, sharing the highest sequence similarities to the genera Alteromonas (93.7-95.4 %), Aestuariibacter (94.0-95.1 %), Agaribacter (93.3-93.7 %), Glaciecola (92.0-93.7 %), Marisendiminitalea (93.2-93.3 %) and Planctobacterium (92.9 %). In the phylogenetic trees, strain B66T demonstrated the novel organism formed a distinct lineage closely associated with Aestuariibacter and Planctobacterium. Major fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c/iso-C15 : 0 2-OH and iso-C15 : 0 3-OH. The major respiratory quinone was ubiquinone-8 and the major polar lipids are phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content derived from the genome was 43.2 mol%. Based on the phenotypic, chemotaxonomic, phylogenetic and genomic data, strain B66T is considered to represent a novel species and genus for which the name Ningiella ruwaisensis gen. nov., sp. nov., is proposed. The type strain is B66T (=QCC B003/17T=LMG 30288 T=CCUG 70703T).

The earliest farmers of northwest China exploited grain-fed pheasants not chickens.

Though chickens (Gallus gallus domesticus) are globally ubiquitous today, the timing, location, and manner of their domestication is contentious. Until recently, archaeologists placed the origin of the domestic chicken in northern China, perhaps as early as 8,000 years ago. Such evidence however complicates our understanding of how the chicken was domesticated because its wild progenitor - the red jungle fowl (Gallus gallus) - lives in tropical ecosystems and does not exist in northern China today or in the recent past. Increasingly, multiple lines of evidence suggest that many of the archaeological bird remains underlying this northern origins hypothesis have been misidentified. Here we analyze the mitochondrial DNA of some of the earliest purported chickens from the Dadiwan site in northern China and conclude that they are pheasants (Phasianus colchicus). Curiously, stable isotope values from the same birds reveal that their diet was heavy in agricultural products (namely millet), meaning that they lived adjacent to or among some of the earliest farming communities in East Asia. We suggest that the exploitation of these baited birds was an important adaptation for early farmers in China's arid north, and that management practices like these likely played a role in the domestication of animals - including the chicken - in similar contexts throughout the region.

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.

Comparison of extraction methods for recovering ancient microbial DNA from paleofeces.

OBJECTIVES: Paleofeces are valuable to archeologists and evolutionary biologists for their potential to yield health, dietary, and host information. As a rich source of preserved biomolecules from host-associated microorganisms, they can also provide insights into the recent evolution and changing ecology of the gut microbiome. However, there is currently no standard method for DNA extraction from paleofeces, which combine the dual challenges of complex biological composition and degraded DNA. Due to the scarcity and relatively poor preservation of paleofeces when compared with other archeological remains, it is important to use efficient methods that maximize ancient DNA (aDNA) recovery while also minimizing downstream taxonomic biases. METHODS: In this study, we use shotgun metagenomics to systematically compare the performance of five DNA extraction methods on a set of well-preserved human and dog paleofeces from Mexico (~1,300 BP). RESULTS: Our results show that all tested DNA extraction methods yield a consistent microbial taxonomic profile, but that methods optimized for ancient samples recover significantly more DNA. CONCLUSIONS: These results show promise for future studies that seek to explore the evolution of the human gut microbiome by comparing aDNA data with those generated in modern studies.