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.

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. This communication includes a descriptive analysis of zoonotic parasites infecting Caribbean pre-Columbian cultures in Vieques and network modeling of parasite-host interaction. Our conclusions were based on microscopy and DNA reads extracted from approximately 1,400 to 540 year-old coprolites (un-calibrated years before present). Age and exposure to taphonomic conditions most likely caused the degradation of a portion of the organic material preserved in the coprolites, including parasite eggs, cysts and genetic material. In this study, singleton parasite reads were detected in the metagenomic data. This was likely the outcome of various factors. For instance, non-targeted metagenomic sequencing mainly obtains reads from abundant organisms within the sample, and in the case of fecal metagenomic datasets, these are commonly overpowered by high numbers of bacterial cells. In addition to small amounts of detectable parasite DNA in the feces, ancient DNA damage causes difficulty during sequencing and subsequent alignment of reads to an extant reference sequence. This along with limited representation of parasite genomes in curated reference databases limits the prediction of parasite homologue reads [1]. Furthermore, although contamination and sequencing errors are also a possibility, we believe that this is not the case as precautions were taken to avoid contamination with extant DNA. To overcome these challenges, we performed phylogenetic inference of predicted amino acid homologues to obtain as close as possible to an accurate taxonomic identification. To complement the inherent limitations of the analyses, some of the conclusions presented in this article include intellectual assumptions by the authors based on established knowledge of the field after analyzing and discussing the data.

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.

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.

Given that microbial interactions in nature are very complex, we propose that quorum-sensing, as well as quorum-quenching, phenazine and secondary metabolite production, resistance and toxin-antitoxin systems within a microbial community should all comprise the battery of processes involving the study of what we would define as the “sociomicrobiome”. In the present study the genes/molecules, subsystems and taxonomic breakup of the mentioned processes were identified in decaying tropical wood from the El Yunque rainforest in Puerto Rico, and soil using a shotgun metagenomic approach. The rapid decomposition of wood and litter in tropical regions suggests that processes in these settings are governed by unexplored microbes with the potential of being further studied and exploited for various purposes. Both ecosystems were characterized by the presence of specific genes/molecules, subsystems and microbes associated with the mentioned processes, although the average abundances for specific processes differed. Of the sociomicrobiomes studied, that from El Yunque was found to be the most complex. The approach considered in the present study could also be applied to study the sociomicrobiome of other ecosystems.