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.

Draft genome sequence of Lactobacillus helveticus OSU-BDGOAK2 and Lactobacillus kefiranofaciens OSU-BDGOA1, kefir grain isolates with potential antibacterial activity.

We present the draft genome sequence and assembly of Lactobacillus helveticus OSU-BDGOAK2 and Lactobacillus kefiranofaciens OSU-BDGOA1 isolated from kefir grains that exhibited in vitro antibacterial activity against Escherichia coli ATCC 25922, Listeria innocua ATCC 51742, and Staphylococcus epidermidis ATCC 1222. Genome analysis of both strains revealed gene clusters encoding bacteriocins.

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.

The Skin Microbiome: Current Techniques, Challenges, and Future Directions.

Skin acts as a barrier that promotes the colonization of bacteria, fungi, archaea, and viruses whose membership and function may differ depending on the various specialized niches or micro-environments of the skin. The group of microorganisms inhabiting the skin, also known as the skin microbiome, offers protection against pathogens while actively interacting with the host's immune system. Some members of the skin microbiome can also act as opportunistic pathogens. The skin microbiome is influenced by factors such as skin site, birth mode, genetics, environment, skin products, and skin conditions. The association(s) of the skin microbiome with health and disease has (have) been identified and characterized via culture-dependent and culture-independent methods. Culture-independent methods (such as high-throughput sequencing), in particular, have expanded our understanding of the skin microbiome's role in maintaining health or promoting disease. However, the intrinsic challenges associated with the low microbial biomass and high host content of skin microbiome samples have hindered advancements in the field. In addition, the limitations of current collection and extraction methods and biases derived from sample preparation and analysis have significantly influenced the results and conclusions of many skin microbiome studies. Therefore, the present review discusses the technical challenges associated with the collection and processing of skin microbiome samples, the advantages and disadvantages of current sequencing approaches, and potential future areas of focus for the field.

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.

Viral Metagenomics as a Tool to Track Sources of Fecal Contamination: A One Health Approach.

The One Health framework recognizes that human, animal, and environmental health are linked and highly interdependent. Fecal contamination of water, soil, foodstuff, and air may impact many aspects of One Health, and culture, PCR-based, and sequencing methods are utilized in the detection of fecal contamination to determine source, load, and risk to inform targeted mitigation strategies. Viruses, particularly, have been considered as fecal contamination indicators given the narrow host range many exhibit and their association with other biological contaminants. Culture- and molecular-based methods are considered the gold-standards for virus detection and for determining specific sources of fecal contamination via viral indicators. However, viral metagenomics is also being considered as a tool for tracking sources of fecal contamination. In the present review, studies tracking potential sources of fecal contamination in freshwaters, marine waters, foodstuff, soil, and air using viral metagenomics are discussed to highlight the potential of viral metagenomics for optimizing fecal source tracking. Limitations of the use of viral metagenomics to track fecal contamination sources, including sample processing, nucleic acid recovery, sequencing depth, and bioinformatics are also discussed. Finally, the present review discusses the potential of viral metagenomics as part of the toolbox of methods in a One Health approach.

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 [...].

Unraveling the viral dark matter through viral metagenomics.

Viruses are part of the microbiome and have essential roles in immunology, evolution, biogeochemical cycles, health, and disease progression. Viruses influence a wide variety of systems and processes, and the continued discovery of novel viruses is anticipated to reveal new mechanisms influencing the biology of diverse environments. While the identity and roles of viruses continue to be discovered and understood through viral metagenomics, most of the sequences in virome datasets cannot be attributed to known viruses or may be only distantly related to species already described in public sequence databases, at best. Such viruses are known as the viral dark matter. Ongoing discoveries from the viral dark matter have provided insights into novel viruses from a variety of environments, as well as their potential in immunological processes, virus evolution, health, disease, therapeutics, and surveillance. Increased understanding of the viral dark matter will continue with a combination of cultivation, microscopy, sequencing, and bioinformatic efforts, which are discussed in the present review.

Fresh Food Consumption Increases Microbiome Diversity and Promotes Changes in Bacteria Composition on the Skin of Pet Dogs Compared to Dry Foods.

The skin is the first barrier the body has to protect itself from the environment. There are several bacteria that populate the skin, and their composition may change throughout the dog's life due to several factors, such as environmental changes and diseases. The objective of this research was to determine the skin microbiome changes due to a change in diet on healthy pet dogs. Healthy client-owned dogs (8) were fed a fresh diet for 30 days then dry foods for another 30 days after a 4-day transition period. Skin bacterial population samples were collected after each 30-day feeding period and compared to determine microbiome diversity. Alpha diversity was higher when dogs were fed the fresh diet compared to the dry foods. Additionally, feeding fresh food to dogs increased the proportion of Staphylococcus and decreased Porphyromonas and Corynebacterium. In conclusion, changing from fresh diet to dry foods promoted a relative decrease in skin microbiome in healthy pet dogs.

Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

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 [...].

Multi 'omic data integration: A review of concepts, considerations, and approaches.

The application of 'omic techniques including, but not limited to genomics/metagenomics, transcriptomics/meta-transcriptomics, proteomics/meta-proteomics, and metabolomics to generate multiple datasets from a single sample have facilitated hypothesis generation leading to the identification of biological, molecular and ecological functions and mechanisms, as well as associations and correlations. Despite their power and promise, a variety of challenges must be considered in the successful design and execution of a multi-omics study. In this review, various 'omic technologies applicable to single- and meta-organisms (i.e., host + microbiome) are described, and considerations for sample collection, storage and processing prior to data generation and analysis, as well as approaches to data storage, dissemination and analysis are discussed. Finally, case studies are included as examples of multi-omic applications providing novel insights and a more holistic understanding of biological processes.

Consumption of High-Fructose Corn Syrup Compared with Sucrose Promotes Adiposity and Increased Triglyceridemia but Comparable NAFLD Severity in Juvenile Iberian Pigs.

BACKGROUND: Fructose consumption has been linked to nonalcoholic fatty liver disease (NAFLD) in children. However, the effect of high-fructose corn syrup (HFCS) compared with sucrose in pediatric NAFLD has not been investigated. OBJECTIVES: We tested whether the isocaloric substitution of dietary sucrose by HFCS would increase the severity of NAFLD in juvenile pigs, and whether this effect would be associated with changes in gut histology, SCFA production, and microbial diversity. METHODS: Iberian pigs, 53-d-old and pair-housed in pens balanced for weight and sex, were randomly assigned to receive a mash diet top-dressed with increasing amounts of sucrose (SUC; n = 3 pens; 281.6-486.8 g/kg diet) or HFCS (n = 4; 444.3-724.8 g/kg diet) during 16 wk. Diets exceeded the animal's energy requirements by providing sugars in excess, but met the requirements for all other nutrients. Animals were killed at 165 d of age after blood sampling, and liver, muscle, and gut were collected for histology, metabolome, and microbiome analyses. Data were analyzed by multivariate and univariate statistics. RESULTS: Compared with SUC, HFCS increased subcutaneous fat, triacylglycerides in plasma, and butyrate in colon (P ≤ 0.05). In addition, HFCS decreased UMP and short-chain acyl carnitines in liver, and urea nitrogen and creatinine in serum (P ≤ 0.05). Microbiome analysis showed a 24.8% average dissimilarity between HFCS and SUC associated with changes in SCFA-producing bacteria. Body weight gain, intramuscular fat, histological and serum markers of liver injury, and circulating hormones, glucose, and proinflammatory cytokines did not differ between diets. CONCLUSIONS: Fructose consumption derived from HFCS promoted butyrate synthesis, triglyceridemia, and subcutaneous lipid deposition in juvenile Iberian pigs, but did not increase serum and histological markers of NAFLD compared with a sucrose-enriched diet. Longer studies could be needed to observe differences in liver injury among sugar types.

Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.

OBJECTIVE: Altered bacterial composition is associated with disease progression in cirrhosis but the role of virome, especially phages, is unclear. DESIGN: Cross-sectional and pre/post rifaximin cohorts were enrolled. Cross-sectional: controls and cirrhotic outpatients (compensated, on lactulose (Cirr-L), on rifaximin (Cirr-LR)) were included and followed for 90-day hospitalisations. Pre/post: compensated cirrhotics underwent stool collection pre/post 8 weeks of rifaximin. Stool metagenomics for bacteria and phages and their correlation networks were analysed in controls versus cirrhosis, within cirrhotics, hospitalised/not and pre/post rifaximin. RESULTS: Cross-sectional: 40 controls and 163 cirrhotics (63 compensated, 43 Cirr-L, 57 Cirr-LR) were enrolled. Cirr-L/LR groups were similar on model for end-stage liver disease (MELD) score but Cirr-L developed greater hospitalisations versus Cirr-LR (56% vs 30%, p=0.008). Bacterial alpha/beta diversity worsened from controls through Cirr-LR. While phage alpha diversity was similar, beta diversity was different between groups. Autochthonous bacteria linked negatively, pathobionts linked positively with MELD but only modest phage-MELD correlations were seen. Phage-bacterial correlation network complexity was highest in controls, lowest in Cirr-L and increased in Cirr-LR. Microviridae and Faecalibacterium phages were linked with autochthonous bacteria in Cirr-LR, but not Cirr-L hospitalised patients had greater pathobionts, lower commensal bacteria and phages focused on Streptococcus, Lactococcus and Myoviridae. Pre/post: No changes in alpha/beta diversity of phages or bacteria were seen postrifaximin. Phage-bacterial linkages centred around urease-producing Streptococcus species collapsed postrifaximin. CONCLUSION: Unlike bacteria, faecal phages are sparsely linked with cirrhosis characteristics and 90-day outcomes. Phage and bacterial linkages centred on urease-producing, ammonia-generating Streptococcus species were affected by disease progression and rifaximin therapy and were altered in patients who experienced 90-day hospitalisations.

Metagenomic Information Recovery from Human Stool Samples Is Influenced by Sequencing Depth and Profiling Method.

Sequencing of the 16S rRNA gene (16S) has long been a go-to method for microbiome characterization due to its accessibility and lower cost compared to shotgun metagenomic sequencing (SMS). However, 16S sequencing rarely provides species-level resolution and cannot provide direct assessment of other taxa (e.g., viruses and fungi) or functional gene content. Shallow shotgun metagenomic sequencing (SSMS) has emerged as an approach to bridge the gap between 16S sequencing and deep metagenomic sequencing. SSMS is cost-competitive with 16S sequencing, while also providing species-level resolution and functional gene content insights. In the present study, we evaluated the effects of sequencing depth on marker gene-mapping- and alignment-based annotation of bacteria in healthy human stool samples. The number of identified taxa decreased with lower sequencing depths, particularly with the marker gene-mapping-based approach. Other annotations, including viruses and pathways, also showed a depth-dependent effect on feature recovery. These results refine the understanding of the suitability and shortcomings of SSMS, as well as annotation tools for metagenomic analyses in human stool samples. Results may also translate to other sample types and may open the opportunity to explore the effect of sequencing depth and annotation method.

Potential Applications of Human Viral Metagenomics and Reference Materials: Considerations for Current and Future Viruses.

Viruses are ubiquitous particles comprising genetic material that can infect bacteria, archaea, and fungi, as well as human and other animal cells. Given that determining virus composition and function in association with states of human health and disease is of increasing interest, we anticipate that the field of viral metagenomics will continue to expand and be applied in a variety of areas ranging from surveillance to discovery and will rely heavily upon the continued development of reference materials and databases. Information regarding viral composition and function readily translates into biological and clinical applications, including the rapid sequence identification of pathogenic viruses in various sample types. However, viral metagenomic approaches often lack appropriate standards and reference materials to enable cross-study comparisons and assess potential biases which can be introduced at the various stages of collection, storage, processing, and sequence analysis. In addition, implementation of appropriate viral reference materials can aid in the benchmarking of current and development of novel assays for virus identification, discovery, and surveillance. As the field of viral metagenomics expands and standardizes, results will continue to translate into diverse applications.

Dysregulated FXR-FGF19 signaling and choline metabolism are associated with gut dysbiosis and hyperplasia in a novel pig model of pediatric NASH.

To investigate the role of bile acids (BAs) in the pathogenesis of diet-induced nonalcoholic steatohepatitis (NASH), we fed a "Western-style diet" [high fructose, high fat (HFF)] enriched with fructose, cholesterol, and saturated fat for 10 wk to juvenile Iberian pigs. We also supplemented probiotics with in vitro BA deconjugating activity to evaluate their potential therapeutic effect in NASH. Liver lipid and function, cytokines, and hormones were analyzed using commercially available kits. Metabolites, BAs, and fatty acids were measured by liquid chromatography-mass spectrometry. Histology and gene and protein expression analyses were performed using standard protocols. HFF-fed pigs developed NASH, cholestasis, and impaired enterohepatic Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling in the absence of obesity and insulin resistance. Choline depletion in HFF livers was associated with decreased lipoprotein and cholesterol in serum and an increase of choline-containing phospholipids in colon contents and trimethylamine-N-oxide in the liver. Additionally, gut dysbiosis and hyperplasia increased with the severity of NASH, and were correlated with increased colonic levels of choline metabolites and secondary BAs. Supplementation of probiotics in the HFF diet enhanced NASH, inhibited hepatic autophagy, increased excretion of taurine and choline, and decreased gut microbial diversity. In conclusion, dysregulation of BA homeostasis was associated with injury and choline depletion in the liver, as well as increased biliary secretion, gut metabolism and excretion of choline-based phospholipids. Choline depletion limited lipoprotein synthesis, resulting in hepatic steatosis, whereas secondary BAs and choline-containing phospholipids in colon may have promoted dysbiosis, hyperplasia, and trimethylamine synthesis, causing further damage to the liver.NEW & NOTEWORTHY Impaired Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling and cholestasis has been described in nonalcoholic fatty liver disease (NAFLD) patients. However, therapeutic interventions with FXR agonists have produced contradictory results. In a swine model of pediatric nonalcoholic steatohepatitis (NASH), we show that the uncoupling of intestinal FXR-FGF19 signaling and a decrease in FGF19 levels are associated with a choline-deficient phenotype of NASH and increased choline excretion in the gut, with the subsequent dysbiosis, colonic hyperplasia, and accumulation of trimethylamine-N-oxide in the liver.