Gut microbiome composition in the Hispanic Community Health Study/Study of Latinos is shaped by geographic relocation, environmental factors, and obesity.

Background: Hispanics living in the USA may have unrecognized potential birthplace and lifestyle influences on the gut microbiome. We report a cross-sectional analysis of 1674 participants from four centers of the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), aged 18 to 74 years old at recruitment.Results: Amplicon sequencing of 16S rRNA gene V4 and fungal ITS1 fragments from self-collected stool samples indicate that the host microbiome is determined by sociodemographic and migration-related variables. Those who relocate from Latin America to the USA at an early age have reductions in Prevotella to Bacteroides ratios that persist across the life course. Shannon index of alpha diversity in fungi and bacteria is low in those who relocate to the USA in early life. In contrast, those who relocate to the USA during adulthood, over 45 years old, have high bacterial and fungal diversity and high Prevotella to Bacteroides ratios, compared to USA-born and childhood arrivals. Low bacterial diversity is associated in turn with obesity. Contrasting with prior studies, our study of the Latino population shows increasing Prevotella to Bacteroides ratio with greater obesity. Taxa within Acidaminococcus, Megasphaera, Ruminococcaceae, Coriobacteriaceae, Clostridiales, Christensenellaceae, YS2 (Cyanobacteria), and Victivallaceae are significantly associated with both obesity and earlier exposure to the USA, while Oscillospira and Anaerotruncus show paradoxical associations with both obesity and late-life introduction to the USA.Conclusions: Our analysis of the gut microbiome of Latinos demonstrates unique features that might be responsible for health disparities affecting Hispanics living in the USA

Author Correction: Gut microbiome composition in the Hispanic Community Health Study/Study of Latinos is shaped by geographic relocation, environmental factors, and obesity.

Following publication of the original paper [1], an error was reported in the third paragraph in the section "Analysis of GMB composition and its correlates" (page 3 of the PDF). The first sentence of the text should refer to Table 2, but mistakenly refers to Table 1

Comparison of Fecal Collection Methods for Microbiome and Metabolomics Studies

Background: Integrated microbiome and metabolomics analyses hold the potential to reveal interactions between host and microbiota in relation to disease risks. However, there are few studies evaluating how field methods influence fecal microbiome characterization and metabolomics profiling.Methods: Five fecal collection methods [immediate freezing at −20°C without preservative, OMNIgene GUT, 95% ethanol, RNAlater, and Flinders Technology Associates (FTA) cards] were used to collect 40 fecal samples from eight healthy volunteers. We performed gut microbiota 16S rRNA sequencing, untargeted metabolomics profiling, and targeted metabolomics focusing on short chained fatty acids (SCFAs). Metrics included α-diversity and β-diversity as well as distributions of predominant phyla. To evaluate the concordance with the “gold standard” immediate freezing, the intraclass correlation coefficients (ICCs) for alternate fecal collection systems were calculated. Correlations between SCFAs and gut microbiota were also examined.Results: The FTA cards had the highest ICCs compared to the immediate freezing method for α-diversity indices (ICCs = 0.96, 0.96, 0.76 for Shannon index, Simpson's Index, Chao-1 Index, respectively), followed by OMNIgene GUT, RNAlater, and 95% ethanol. High ICCs (all >0.88) were observed for all methods for the β-diversity metric. For untargeted metabolomics, in comparison to immediate freezing which detected 621 metabolites at ≥75% detectability level, 95% ethanol showed the largest overlapping set of metabolites (n = 430; 69.2%), followed by FTA cards (n = 330; 53.1%) and OMNIgene GUT (n = 213; 34.3%). Both OMNIgene GUT (ICCs = 0.82, 0.93, 0.64) and FTA cards (ICCs = 0.87, 0.85, 0.54) had acceptable ICCs for the top three predominant SCFAs (butyric acid, propionic acid and acetic acid). Nominally significant correlations between bacterial genera and SCFAs (P < 0.05) were observed in fecal samples collected by different methods. Of note, a high correlation between the genus Blautia (known butyrate producer) and butyric acid was observed for both immediate freezing (r = 0.83) and FTA cards (r = 0.74).Conclusions: Four alternative fecal collection methods are generally comparable with immediate freezing, but there are differences in certain measures of the gut microbiome and fecal metabolome across methods. Choice of method depends on the research interests, simplicity of fecal collection procedures and ease of transportation to the lab, especially for large epidemiological studies

Evaluation of Oral Cavity DNA Extraction Methods on Bacterial and Fungal Microbiota

Abstract The objective of this study was to evaluate the most effective method of DNA extraction of oral mouthwash samples for use in microbiome studies that utilize next generation sequencing (NGS). Eight enzymatic and mechanical DNA extraction methods were tested. Extracted DNA was amplified using barcoded primers targeting the V6 variable region of the bacterial 16S rRNA gene and the ITS1 region of the fungal ribosomal gene cluster and sequenced using the Illumina NGS platform. Sequenced reads were analyzed using QIIME and R. The eight methods yielded significantly different quantities of DNA (p < 0.001), with the phenol-chloroform extraction method producing the highest total yield. There were no significant differences in observed bacterial or fungal Shannon diversity (p = 0.64, p = 0.93 respectively) by extraction method. Bray-Curtis beta-diversity did not demonstrate statistically significant differences between the eight extraction methods based on bacterial (R2 = 0.086, p = 1.00) and fungal (R2 = 0.039, p = 1.00) assays. No differences were seen between methods with or without bead-beating. These data indicate that choice of DNA extraction method affect total DNA recovery without significantly affecting the observed microbiome

Novel ITS1 Fungal Primers for Characterization of the Mycobiome

ABSTRACT Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states

Staphylococcus Aureus Nasal Carriage and Microbiome Composition Among Medical Students from Colombia: a Cross-Sectional Study

Background: The anterior nares are the main ecological niche for Staphylococcus aureus, an important commensal and opportunistic pathogen. Medical students are frequently colonized by a variety of pathogens. Microbial interactions in the human nose can prevent or favor colonization by pathogens, and individuals colonized by pathogens have increased risk of infection and are the source of transmission to other community members or susceptible individuals. According to recent studies, the microbiome from several anatomic areas of healthy individuals varies across different ethnicities. Although previous studies analyzed the nasal microbiome in association with S. aureus carriage, those studies did not provide information regarding ethnicity of participants. Our aim was to assess S. aureus nasal carriage patterns and prevalence among medical students from Colombia, a country of Hispanic origin, and to investigate possible associations of colonization and nasal microbiome composition (bacterial and fungal) in a subgroup of students with known S. aureus carriage patterns. Methods: Nasal swabs from second-year medical students were used to determine prevalence and patterns of S. aureus nasal carriage. Based on microbiological results, we assigned participants into one of three patterns of S. aureus colonization: persistent, intermittent, and non-carrier. Then, we evaluated the composition of nasal microbial communities (bacterial and fungal) in 5 individuals from each carriage category using 16S rRNA and Internal-Transcribed-Spacer sequencing. Results: Prevalence of S. aureus nasal carriage among medical students was 28%. Carriage of methicillin-resistant strains was 8.4% and of methicillin-sensitive strains was 19.6%. We identified 19.6% persistent carriers, 17.5% intermittent carriers, and 62.9% non-carriers. Conclusions: Analysis of nasal microbiome found that bacterial and fungal diversity was higher in individuals colonized by S. aureus than in non-carriers; however, the difference among the three groups was non-significant. We confirmed that fungi were present within the healthy anterior nares at substantial biomass and richness