Edible flora in pre-Columbian Caribbean coprolites: Expected and unexpected data.

Coprolites, or mummified feces, are valuable sources of information on ancient cultures as they contain ancient DNA (aDNA). In this study, we analyzed ancient plant DNA isolated from coprolites belonging to two pre-Columbian cultures (Huecoid and Saladoid) from Vieques, Puerto Rico, using shotgun metagenomic sequencing to reconstruct diet and lifestyles. We also analyzed DNA sequences of putative phytopathogenic fungi, likely ingested during food consumption, to further support dietary habits. Our findings show that pre-Columbian Caribbean cultures had a diverse diet consisting of maize (Zea mays), sweet potato (Ipomoea batatas), chili peppers (Capsicum annuum), peanuts (Arachis spp.), papaya (Carica papaya), tomato (Solanum lycopersicum) and, very surprisingly cotton (Gossypium barbadense) and tobacco (Nicotiana sylvestris). Modelling of putative phytopathogenic fungi and plant interactions confirmed the potential consumption of these plants as well as edible fungi, particularly Ustilago spp., which suggest the consumption of maize and huitlacoche. These findings suggest that a variety of dietary, medicinal, and hallucinogenic plants likely played an important role in ancient human subsistence and societal customs. We compared our results with coprolites found in Mexico and the United States, as well as present-day faeces from Mexico, Peru, and the United States. The results suggest that the diet of pre-Columbian cultures resembled that of present-day hunter-gatherers, while agriculturalists exhibited a transitional state in dietary lifestyles between the pre-Columbian cultures and larger scale farmers and United States individuals. Our study highlights differences in dietary patterns related to human lifestyles and provides insight into the flora present in the pre-Columbian Caribbean area. Importantly, data from ancient fecal specimens demonstrate the importance of ancient DNA studies to better understand pre-Columbian populations.

Accelerating Gut Microbiome Research with Robust Sample Collection.

Background: Inferior quality of biological material compromises data, slows discovery, and wastes research funds. The gut microbiome plays a critical role in human health and disease, yet little attention has been given to optimizing collection and processing methods of human stool. Methods: We collected the entire bowel movement from 2 healthy volunteers: one to examine stool sample heterogeneity and one to test stool sample handling parameters. Sequencing and bioinformatic analyses were used to examine the microbiome composition. Results: The microbiome profile varied depending on where the subsample was obtained from the stool. The exterior cortex of the stool was rich in specific phyla and deficient in others while the interior core of the stool revealed opposite microbiome profiles. Sample processing also resulted in varying microbiome profiles. Homogenization and stabilization at 4°C gave superior microbial diversity profiles compared to the fresh or frozen subsamples of the same stool sample. Bacterial proliferation continued in the fresh subsample when processed at ambient temperature. Bacteroidetes proliferated and Firmicutes diminished during the 30-minute processing of fresh sample. The frozen sample had good overall diversity but Proteobacteria diminished likely because of the freeze/thaw. Conclusion: The microbiome profile is specific to the section of the stool being sampled. Stool sample collection, homogenization and stabilization at 4°C for 24 hours provides a neat, high-quality sample of sufficient quantity that can be banked into aliquots with nearly identical microbial diversity profiles. This collection pipeline is essential to accelerate our understanding of the gut microbiome in health and disease.

A mobile lab for ancient DNA extraction in Perug.

We report the use of a mobile laboratory set up to extract ancient DNA (aDNA) from 34 human coprolites (fossilized faeces) samples. Our approach enabled the rapid genetic characterization of 5,000 years old archeological samples. It is useful for the on-site screening of museums and freshly excavated samples for DNA. This approach is accessible to other investigators as the mobile laboratory was set up using commercially available instruments.

Commensal and Pathogenic Members of the Dental Calculus Microbiome of Badia Pozzeveri Individuals from the 11th to 19th Centuries.

The concept of the human oral microbiome was applied to understand health and disease, lifestyles, and dietary habits throughout part of human history. In the present study, we augment the understanding of ancient oral microbiomes by characterizing human dental calculus samples recovered from the ancient Abbey of Badia Pozzeveri (central Italy), with differences in socioeconomic status, time period, burial type, and sex. Samples dating from the Middle Ages (11th century) to the Industrial Revolution era (19th century) were characterized using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. Consistent with previous studies, individuals from Badia Pozzeveri possessed commensal oral bacteria that resembled modern oral microbiomes. These results suggest that members of the oral microbiome are ubiquitous despite differences in geographical regions, time period, sex, and socioeconomic status. The presence of fecal bacteria could be in agreement with poor hygiene practices, consistent with the time period. Respiratory tract, nosocomial, and other rare pathogens detected in the dental calculus samples are intriguing and could suggest subject-specific comorbidities that could be reflected in the oral microbiome.

General Anesthesia Alters the Diversity and Composition of the Intestinal Microbiota in Mice.

Dysbiosis of the intestinal microbiota has been shown to result in altered immune responses and increased susceptibility to infection; as such, the state of the intestinal microbiome may have profound implications in the perioperative setting. In this first-in-class study, we used 16s ribosomal RNA sequencing and analysis in a mouse model of general anesthesia to investigate the effects of volatile anesthetics on the diversity and composition of the intestinal microbiome. After 4-hour exposure to isoflurane, we observed a decrease in bacterial diversity. Taxonomic alterations included depletion of several commensal bacteria including Clostridiales. These data identify volatile anesthetics as potential contributors to microbial dysbiosis in the postoperative patient.

Definitions and Historical Perspectives in Environmental Forensics.

Environmental forensics is a tool that uses chemical, physical, and statistical techniques to investigate contaminants in the environment as a means to determine attribution for legal purposes. Environmental microbiology is a branch of science that has benefited from the use of metagenomics. The term microbial forensics, which includes nucleic acid sequencing methods, is now used to investigate the sources of microorganisms for attribution purposes as well. Environmental microbial forensics can fully address the questions that must be answered for attribution of causation and subsequent remedial actions within a reasonably short time frame. Although sensu stricto forensics refers to obtaining scientific evidence to be presented during legal proceedings, the term sensu lato is used as a description of the procedures used to reconstruct previous events, such as contamination. The term microbial forensics was first used to describe a forensic science approach for attribution purposes, specifically for bioterror as a purposeful release of pathogen microorganisms, but it also especially refers to investigations on the inadvertent or accidental release of pathogenic agents. However, microbial forensics can be used to determine the source of a microorganism or a group of microorganisms, regardless of whether they are pathogenic or not. Microbial forensics has limitations, but it should be used as part of a toolbox of methods to be relied upon when doing forensic studies. Environmental microbial forensics can only benefit from the development of new methods, and we already are experiencing a paradigm change in terms of approaches to the forensic sciences.

Future Technologies.

Microbiome analysis of environmental samples may represent the next frontier in environmental microbial forensics. Next-generation sequencing technologies significantly increased the available genetic data that could be used as evidentiary material. It is not clear, however, whether the microbiome can scale across institutions using forensic-based evidence due to the data resource requirements and the associated costs of maintaining these databases. A successful microbiome study is impacted by the quality of the information gathered and the steps in sample processing and data analysis. To ascertain the validity of methods and the results obtained, there needs to be a stringent procedure to validate the methods and ensure that the results are comparable and reproducible, not only within the laboratory but also between laboratories conducting similar research. Of primary importance for meaningful microbiome studies is an experimental design that leads to carefully executed, controlled, and reproducible studies. The microbiome literature contains a fair share of anecdotal descriptions of microbial community composition and "diagnostic" relative abundance of the taxa therein. These studies are now being supplemented by experimental designs that feature repeated measurements, error estimates, correlations of microbiota with covariates, and increasingly sophisticated statistical tests that enhance the robustness of data analysis and study conclusions. It is imperative to be careful, especially when carrying out attribution studies, to be fully aware of the possible biases included in a specific sample being analyzed.

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

Correction: Insights of the dental calculi microbiome of pre-Columbian inhabitants from Puerto Rico.

[This corrects the article DOI: 10.7717/peerj.3277.].

Insights of the dental calculi microbiome of pre-Columbian inhabitants from Puerto Rico.

BACKGROUND: The study of ancient microorganisms in mineralized dental plaque or calculi is providing insights into microbial evolution, as well as lifestyles and disease states of extinct cultures; yet, little is still known about the oral microbial community structure and function of pre-Columbian Caribbean cultures. In the present study, we investigated the dental calculi microbiome and predicted function of one of these cultures, known as the Saladoid. The Saladoids were horticulturalists that emphasized root-crop production. Fruits, as well as small marine and terrestrial animals were also part of the Saladoid diet. METHODS: Dental calculi samples were recovered from the archaeological site of Sorcé, in the municipal island of Vieques, Puerto Rico, characterized using 16S rRNA gene high-throughput sequencing, and compared to the microbiome of previously characterized coprolites of the same culture, as well modern plaque, saliva and stool microbiomes available from the Human Microbiome Project. RESULTS: Actinobacteria, Proteobacteria and Firmicutes comprised the majority of the Saladoid dental calculi microbiome. The Saladoid dental calculi microbiome was distinct when compared to those of modern saliva and dental plaque, but showed the presence of common inhabitants of modern oral cavities including Streptococcus sp., Veillonella dispar and Rothia mucilaginosa. Cell motility, signal transduction and biosynthesis of other secondary metabolites may be unique features of the Saladoid microbiome. DISCUSSION: Results suggest that the Saladoid dental calculi microbiome structure and function may possibly reflect a horticulturalist lifestyle and distinct dietary habits. Results also open the opportunity to further elucidate oral disease states in extinct Caribbean cultures and extinct indigenous cultures with similar lifestyles.

Soil Microbial Forensics.

Soil microbial forensics can be defined as the study of how microorganisms can be applied to forensic investigations. The field of soil microbial forensics is of increasing interest and applies techniques commonly used in diverse disciplines in order to identify microbes and determine their abundances, complexities, and interactions with soil and surrounding objects. Emerging new techniques are also providing insights into the complexity of microbes in soil. Soil may harbor unique microbes that may reflect specific physical and chemical characteristics indicating site specificity. While applications of some of these techniques in the field of soil microbial forensics are still in early stages, we are still gaining insight into how microorganisms may be more robustly used in forensic investigations.

Approved Glycopeptide Antibacterial Drugs: Mechanism of Action and Resistance.

The glycopeptide antimicrobials are a group of natural product and semisynthetic glycosylated peptides that show antibacterial activity against Gram-positive organisms through inhibition of cell-wall synthesis. This is achieved primarily through binding to the d-alanyl-d-alanine terminus of the lipid II bacterial cell-wall precursor, preventing cross-linking of the peptidoglycan layer. Vancomycin is the foundational member of the class, showing both clinical longevity and a still preferential role in the therapy of methicillin-resistant Staphylococcus aureus and of susceptible Enterococcus spp. Newer lipoglycopeptide derivatives (telavancin, dalbavancin, and oritavancin) were designed in a targeted fashion to increase antibacterial activity, in some cases through secondary mechanisms of action. Resistance to the glycopeptides emerged in delayed fashion and occurs via a spectrum of chromosome- and plasmid-associated elements that lead to structural alteration of the bacterial cell-wall precursor substrates.

Taxonomic and predicted metabolic profiles of the human gut microbiome in pre-Columbian mummies.

Characterization of naturally mummified human gut remains could potentially provide insights into the preservation and evolution of commensal and pathogenic microorganisms, and metabolic profiles. We characterized the gut microbiome of two pre-Columbian Andean mummies dating to the 10-15th centuries using 16S rRNA gene high-throughput sequencing and metagenomics, and compared them to a previously characterized gut microbiome of an 11th century AD pre-Columbian Andean mummy. Our previous study showed that the Clostridiales represented the majority of the bacterial communities in the mummified gut remains, but that other microbial communities were also preserved during the process of natural mummification, as shown with the metagenomics analyses. The gut microbiome of the other two mummies were mainly comprised by Clostridiales or Bacillales, as demonstrated with 16S rRNA gene amplicon sequencing, many of which are facultative anaerobes, possibly consistent with the process of natural mummification requiring low oxygen levels. Metagenome analyses showed the presence of other microbial groups that were positively or negatively correlated with specific metabolic profiles. The presence of sequences similar to both Trypanosoma cruzi and Leishmania donovani could suggest that these pathogens were prevalent in pre-Columbian individuals. Taxonomic and functional profiling of mummified human gut remains will aid in the understanding of the microbial ecology of the process of natural mummification.

Natural mummification of the human gut preserves bacteriophage DNA.

The natural mummification process of the human gut represents a unique opportunity to study the resulting microbial community structure and composition. While results are providing insights into the preservation of bacteria, fungi, pathogenic eukaryotes and eukaryotic viruses, no studies have demonstrated that the process of natural mummification also results in the preservation of bacteriophage DNA. We characterized the gut microbiome of three pre-Columbian Andean mummies, namely FI3, FI9 and FI12, and found sequences homologous to viruses. From the sequences attributable to viruses, 50.4% (mummy FI3), 1.0% (mummy FI9) and 84.4% (mummy FI12) were homologous to bacteriophages. Sequences corresponding to the Siphoviridae, Myoviridae, Podoviridae and Microviridae families were identified. Predicted putative bacterial hosts corresponded mainly to the Firmicutes and Proteobacteria, and included Bacillus, Staphylococcus, Clostridium, Escherichia, Vibrio, Klebsiella, Pseudomonas and Yersinia. Predicted functional categories associated with bacteriophages showed a representation of structural, replication, integration and entry and lysis genes. The present study suggests that the natural mummification of the human gut results in the preservation of bacteriophage DNA, representing an opportunity to elucidate the ancient phageome and to hypothesize possible mechanisms of preservation.

Retroviral DNA Sequences as a Means for Determining Ancient Diets.

For ages, specialists from varying fields have studied the diets of the primeval inhabitants of our planet, detecting diet remains in archaeological specimens using a range of morphological and biochemical methods. As of recent, metagenomic ancient DNA studies have allowed for the comparison of the fecal and gut microbiomes associated to archaeological specimens from various regions of the world; however the complex dynamics represented in those microbial communities still remain unclear. Theoretically, similar to eukaryote DNA the presence of genes from key microbes or enzymes, as well as the presence of DNA from viruses specific to key organisms, may suggest the ingestion of specific diet components. In this study we demonstrate that ancient virus DNA obtained from coprolites also provides information reconstructing the host's diet, as inferred from sequences obtained from pre-Columbian coprolites. This depicts a novel and reliable approach to determine new components as well as validate the previously suggested diets of extinct cultures and animals. Furthermore, to our knowledge this represents the first description of the eukaryotic viral diversity found in paleofaeces belonging to pre-Columbian cultures.

Gut Microbiome of an 11th Century A.D. Pre-Columbian Andean Mummy.

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.

Paleomicrobiology: revealing fecal microbiomes of ancient indigenous cultures.

Coprolites are fossilized feces that can be used to provide information on the composition of the intestinal microbiota and, as we show, possibly on diet. We analyzed human coprolites from the Huecoid and Saladoid cultures from a settlement on Vieques Island, Puerto Rico. While more is known about the Saladoid culture, it is believed that both societies co-existed on this island approximately from 5 to 1170 AD. By extracting DNA from the coprolites, followed by metagenomic characterization, we show that both cultures can be distinguished from each other on the basis of their bacterial and fungal gut microbiomes. In addition, we show that parasite loads were heavy and also culturally distinct. Huecoid coprolites were characterized by maize and Basidiomycetes sequences, suggesting that these were important components of their diet. Saladoid coprolite samples harbored sequences associated with fish parasites, suggesting that raw fish was a substantial component of their diet. The present study shows that ancient DNA is not entirely degraded in humid, tropical environments, and that dietary and/or host genetic differences in ancient populations may be reflected in the composition of their gut microbiome. This further supports the hypothesis that the two ancient cultures studied were distinct, and that they retained distinct technological/cultural differences during an extended period of close proximity and peaceful co-existence. The two populations seemed to form the later-day Taínos, the Amerindians present at the point of Columbian contact. Importantly, our data suggest that paleomicrobiomics can be a powerful tool to assess cultural differences between ancient populations.

luxS in bacteria isolated from 25- to 40-million-year-old amber.

Interspecies bacterial communication is mediated by autoinducer-2, whose synthesis depends on luxS. Due to the apparent universality of luxS (present in more than 40 bacterial species), it may have an ancient origin; however, no direct evidence is currently available. We amplified luxS in bacteria isolated from 25- to 40-million-year-old amber. The phylogenies and molecular clocks of luxS and the 16S rRNA gene from ancient and extant bacteria were determined as well. Luminescence assays using Vibrio harveyi BB170 aimed to determine the activity of luxS. While the phylogeny of luxS was very similar to that of extant Bacillus spp., amber isolates exhibited unique 16S rRNA gene phylogenies. This suggests that luxS may have been acquired by horizontal transfer millions of years ago. Molecular clocks of luxS suggest slow evolutionary rates, similar to those of the 16S rRNA gene and consistent with a conserved gene. Dendograms of the 16S rRNA gene and luxS show two separate clusters for the extant and ancient bacteria, confirming the uniqueness of the latter group.