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.

Coliphages as indicators of primary wastewater treatment efficiency by constructed wetlands.

Constructed wetlands are an efficient and cost-effective system for the treatment of wastewater that can be reused for diverse purposes, including irrigation; however, few studies have determined the efficiency of microbial removal by constructed wetlands in tropical regions. Therefore, the present study aimed to determine the microbial quality of the influent and effluent of a constructed wetland in Puerto Rico, using traditional bacterial indicators (i.e., thermotolerant coliforms and enterococci), as well as somatic and male-specific (F+) coliphages. Results showed that over 99.9 and 97.7% of thermotolerant coliforms and enterococci were removed after treatment by constructed wetlands, respectively. Notably, approximately 84.0% of male-specific (F+) coliphages were removed, while somatic and total coliphages exhibited differing removal percentages at different steps during treatment by constructed wetlands. The potential risk of the presence of enteric viruses in treated wastewater by constructed wetlands may increase when considering traditional bacterial indicators exclusively. The present study may aid in the efforts to determine public health concerns associated with the exposure of bioaerosols resulting from wastewater treatment by constructed wetlands.

Deciphering Diets and Lifestyles of Prehistoric Humans through Paleoparasitology: A Review.

Parasites have affected and coevolved with humans and animals throughout history. Evidence of ancient parasitic infections, particularly, reside in archeological remains originating from different sources dating to various periods of times. The study of ancient parasites preserved in archaeological remains is known as paleoparasitology, and it initially intended to interpret migration, evolution, and dispersion patterns of ancient parasites, along with their hosts. Recently, paleoparasitology has been used to better understand dietary habits and lifestyles of ancient human societies. Paleoparasitology is increasingly being recognized as an interdisciplinary field within paleopathology that integrates areas such as palynology, archaeobotany, and zooarchaeology. Paleoparasitology also incorporates techniques such as microscopy, immunoassays, PCR, targeted sequencing, and more recently, high-throughput sequencing or shotgun metagenomics to understand ancient parasitic infections and thus interpret migration and evolution patterns, as well as dietary habits and lifestyles. The present review covers the original theories developed in the field of paleoparasitology, as well as the biology of some parasites identified in pre-Columbian cultures. Conclusions, as well as assumptions made during the discovery of the parasites in ancient samples, and how their identification may aid in better understanding part of human history, ancient diet, and lifestyles are discussed.

MULTI-OMICS as Invaluable Tools for the Elucidation of Host-Microbe-Microbiota Interactions.

"Omics" is becoming an increasingly recognizable term, even to the general public, as it is used more and more often in everyday scientific research [...].

Mycobiome-Host Coevolution? The Mycobiome of Ancestral Human Populations Seems to Be Different and Less Diverse Than Those of Extant Native and Urban-Industrialized Populations.

Few data exist on the human gut mycobiome in relation to lifestyle, ethnicity, and dietary habits. To understand the effect of these factors on the structure of the human gut mycobiome, we analyzed sequences belonging to two extinct pre-Columbian cultures inhabiting Puerto Rico (the Huecoid and Saladoid) and compared them to coprolite samples found in Mexico and Ötzi, the Iceman's large intestine. Stool mycobiome samples from extant populations in Peru and urban cultures from the United States were also included. The ancient Puerto Rican cultures exhibited a lower fungal diversity in comparison to the extant populations. Dissimilarity distances showed that the Huecoid gut mycobiome resembled that from ancient Mexico. Fungal genera including Aspergillus spp., Penicillium spp., Rasamsonia spp., Byssochlamys spp., Talaromyces spp., Blastomyces spp., Monascus spp., and Penicilliopsis spp. were differentially abundant in the ancient and extant populations. Despite cultural differences, certain fungal taxa were present in all samples. These results suggest that culture and diet may impact the gut mycobiome and emphasize that modern lifestyles could be associated with the alteration of gut mycobiome diversity. The present study presents data on ancient and extant human gut mycobiomes in terms of lifestyle, ethnicity, and diet in the Americas.

Multiomics and Health: A Holistic Approach to Better Understand the Role of the Microbiome.

The present Special Issue focuses on the latest approaches to health and public health microbiology using multiomics [...].

Pre-Columbian zoonotic enteric parasites: An insight into Puerto Rican indigenous culture diets and life styles.

The pre-Columbian Huecoid and Saladoid cultures were agricultural ethnic groups that supplemented their diets by fishing, hunting and scavenging. Archaeological deposits associated to these cultures contained a variety of faunal osseous remains that hinted at the cultures' diets. The present study identified zoonotic parasites that may have infected these two cultures as a result of their diets. We used metagenomic sequencing and microscopy data from 540-1,400 year old coprolites as well as the zooarchaeological data to recreate the possible interactions between zoonotic parasites and their hosts. Microscopy revealed Diphyllobothrium spp. and Dipylidium caninum eggs along with unidentified cestode and trematode eggs. DNA sequencing together with functional prediction and phylogenetic inference identified reads of Cryptosporidium spp., Giardia intestinalis and Schistosoma spp. The complimentary nature of the molecular, microscopy and zooarchaeology data provided additional insight into the detected zoonotic parasites' potential host range. Network modeling revealed that rodents and canids living in close proximity to these cultures were most likely the main source of these zoonotic parasite infections.

Microbiota dispersion in the Uyuni salt flat (Bolivia) as determined by community structure analyses

Soil microbial communities are an important component of biological diversity and terrestrial ecosystems which is responsible for processes such as decomposition, mineralization of nutrients, and accumulation of organic matter. One of the factors that provide information on the mechanisms regulating biodiversity is spatial scaling. We characterized the microbial communities using 16S rRNA gene sequences from DNA isolated from halite at various locations and correlated these to geographic distance in the Uyuni salt flat (Bolivia). Sequences from each site were analyzed to determine any spatial patterns of diversity, as well as to describe the microbial communities. Results suggest that different taxa are able to disperse over Uyuni's surface crust regardless of distance. As expected, ubiquitous taxa included members of Halobacteriaceae such as Haloarcula, Halorubrum, Halorhabdus, Halolamina, and halophilic bacteria Salinibacter, Halorhodospira, and unclassified members of the Gammaproteobacteria. Archaeal communities were homogeneous across the salt flat. In contrast, bacterial communities present strong local variations which could be attributed to external factors. Likely sources for these variations are the Rio Grande river influent in the south shore and the Tunupa volcano influencing the northern area

No disponible

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.

Microbiota dispersion in the Uyuni salt flat (Bolivia) as determined by community structure analyses.

Soil microbial communities are an important component of biological diversity and terrestrial ecosystems which is responsible for processes such as decomposition, mineralization of nutrients, and accumulation of organic matter. One of the factors that provide information on the mechanisms regulating biodiversity is spatial scaling. We characterized the microbial communities using 16S rRNA gene sequences from DNA isolated from halite at various locations and correlated these to geographic distance in the Uyuni salt flat (Bolivia). Sequences from each site were analyzed to determine any spatial patterns of diversity, as well as to describe the microbial communities. Results suggest that different taxa are able to disperse over Uyuni's surface crust regardless of distance. As expected, ubiquitous taxa included members of Halobacteriaceae such as Haloarcula, Halorubrum, Halorhabdus, Halolamina, and halophilic bacteria Salinibacter, Halorhodospira, and unclassified members of the Gammaproteobacteria. Archaeal communities were homogeneous across the salt flat. In contrast, bacterial communities present strong local variations which could be attributed to external factors. Likely sources for these variations are the Rio Grande river influent in the south shore and the Tunupa volcano influencing the northern area.

On Controls in Ancient Microbiome Studies, and Microbial Resilience in Ancient Samples.

In the following comment, we reply to Eisenhofer and Weyrich's letter "Proper authentication of ancient DNA is still essential" responding to the article "Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies" by Santiago-Rodriguez et al. One of the concerns raised was the possibility that the patterns noted in the gut microbiome of pre-Inca/Inca and Italian nobility mummies were due to contamination of the blank control. When examining the blank controls and filtering the operational taxonomic units (OTUs) present in the blank controls, and further performing in-silico contamination analyses, we noticed very similar patterns as those previously reported. We also discuss controls in ancient microbiome studies, and aspects of microbial resilience in ancient samples.

Proteus vulgaris - Pt electrode system for urea to nitrogen conversion in synthetic urine.

One of the most challenging problems when trying to recycle urine for different purposes is the removal of urea. In this project we studied an ureolysis system using the bacterium Proteus vulgaris for the transformation of urea to ammonia and its subsequent oxidation to nitrogen at a Pt working electrode. Our system was tested under different pH, microbial reaction times, and urea and bacteria concentrations. Our results indicate that a pH8 is optimal for the combined Proteus vulgaris urease activity and the ammonia oxidation reaction at a Pt electrode. The reaction time and concentration dependence on the ammonia oxidation reaction current densities was also studied. Results showed limited ammonia oxidation under high urea concentrations in ~2.5×109cfu/mL Proteus vulgaris in synthetic urine.

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.

Forensic Approaches to Detect Possible Agents of Bioterror.

Many biological agents have been strategic pathogenic agents throughout history. Some have even changed history as a consequence of early discoveries of their use as weapons of war. Many of these bioagents can be easily isolated from the environment, and some have recently been genetically manipulated to become more pathogenic for biowarfare. However, it is difficult to determine accidental outbreaks of disease from intentional exposures. In this review, we examine how molecular tools have been used in combination with forensic research to resolve cases of unusual outbreaks and trace the source of the biocrime. New technologies are also discussed in terms of their crucial role impacting forensic science. The anthrax event of 2001 serves as an example of the real threat of bioterrorism and the employment of bioagents as weapons against a population. The Amerithrax investigation has given us lessons of the highest resolution possible with new technologies capable of distinguishing isolates at the base-pair level of sensitivity. In addition, we discuss the implications of proper sanitation to avoid waterborne diseases. The use of new methods in forensic science and health-related surveillance will be invaluable in determining the source of any new disease outbreak, and these data will allow for a quick response to any type of public health threat, whether accidental or purposely initiated.

From Evolutionary Advantage to Disease Agents: Forensic Reevaluation of Host-Microbe Interactions and Pathogenicity.

As the "human microbiome era" continues, there is an increasing awareness of our resident microbiota and its indispensable role in our fitness as holobionts. However, the host-microbe relationship is not so clearly defined for some human symbionts. Here we discuss examples of "accidental pathogens," meaning previously nonpathogenic and/or environmental microbes thought to have inadvertently experienced an evolutionary shift toward pathogenicity. For instance, symbionts such as Helicobacter pylori and JC polyomavirus have been shown to have accompanied humans since prehistoric times and are still abundant in extant populations as part of the microbiome. And yet, the relationship between a subgroup of these microbes and their human hosts seems to have changed with time, and they have recently gained notoriety as gastrointestinal and neuropathogens, respectively. On the other hand, environmental microbes such as Legionella spp. have recently experienced a shift in host range and are now a major problem in industrialized countries as a result of artificial ecosystems. Other variables involved in this accidental phenomenon could be the apparent change or reduction in the diversity of human-associated microbiota because of modern medicine and lifestyles. All of this could result in an increased prevalence of accidental pathogens in the form of emerging pathogens.