An Outlook for the Acquisition of Marine Sedimentary Ancient DNA (sedaDNA) From North Atlantic Ocean Archive Material

Studies incorporating sedimentary ancient DNA (sedaDNA) analyses to investigate paleo-environments have increased considerably over the last few years, and the possibility of utilizing archived sediment cores from previous field campaigns could unlock an immense resource of sampling material for such paleo-investigations. However, sedaDNA research is at a high risk of contamination by modern environmental DNA, as sub-optimal sediment storage conditions may allow for contaminants (e.g., fungi) to grow and become dominant over preserved sedaDNA in the sample. Here, we test the feasibility of sedaDNA analysis applied to archive sediment material from five sites in the North Atlantic, collected between 1994 and 2013. We analyzed two samples (one younger and one older) per site using a metagenomic shotgun approach and were able to recover eukaryotic sedaDNA from all samples. We characterized the authenticity of each sample through sedaDNA fragment size and damage analyses, which allowed us to disentangle sedaDNA and contaminant DNA. Although we determined that contaminant sequences originated mainly from Ascomycota (fungi), most samples were dominated by Emiliania huxleyi, a haptophyte species that commonly blooms in the study region. We attribute the presence of contaminants to non-ideal sampling and sample storage conditions of the investigated samples. Therefore, while we demonstrate that sedaDNA analysis of archival North Atlantic seafloor sediment samples are generally achievable, we stress the importance of best-practice ship-board sampling techniques and storage conditions to minimize contamination. We highly recommend the application of robust bioinformatic tools that help distinguish ancient genetic signals from modern contaminants, especially when working with archive material.Caitlin A. Selway, Linda Armbrecht, and David Thornalle

Variable preterm oral microbiome stabilizes and reflects a full-term infant profile within three months.

OnlinePublBACKGROUND: Preterm infants suffer higher morbidity and mortality rates compared to full-term infants, but little is known about how changes to oral and respiratory tract microbiota may impact disease development. METHODS: Here, very preterm neonates (n = 50) were selected to study oral and respiratory microbiota development during the first few months post-birth, where 26 individuals were diagnosed with BPD and/or sepsis. These infants were compared to 14 healthy full-term infants and 16 adults. Microbiota diversity, composition, and species abundances were calculated from 16S ribosomal RNA gene sequences in buccal swabs and tracheal aspirates at two time points (within a week and 1-3 months post-birth). RESULTS: Collection time point was the biggest factor to significantly influence the preterm oral microbial diversity and composition. In addition, BPD and sepsis were linked to distinct preterm oral microbiota diversity and composition, and opportunistic pathogens previously associated with these diseases were identified in the initial sample for both healthy preterm neonates and those with the disease. Compared to the full-term infant and adult dataset, preterm infant diversity and composition was initially significantly different, but resembled full-term infant diversity and composition over time. CONCLUSION: Overall, consequences of microbiota development need further examination in preterm infant infections and later development. IMPACT: Non-gut microbiota research on preterm infants is limited. At one week post-birth, preterm infants harbor distinct oral microbiota that are not shared with full-term children or adults, eventually becoming similar to full-term infants at 36 weeks postmenstrual age. DNA from potential opportunistic pathogens was observed in the mouth and lungs of preterm infants within a week of birth, and microbes associated with BPD were identified in the lungs. Oral microbiota in preterm infants over the first 2-3 months is unique and may be connected to short- and long-term health outcomes in these children.Caitlin A. Selway, Carmel T. Collins, Maria Makrides, Thomas R. Sullivan, N3RO Steering Committee, and Laura S. Weyric

Transfer of environmental microbes to the skin and respiratory tract of humans after urban green space exposure

Available online 22 September 2020BACKGROUND: In industrialized countries, non-communicable diseases have been increasing in prevalence since the middle of the 20th century. While the causal mechanisms remain poorly understood, increased population density, pollution, sedentary behavior, smoking, changes in diet, and limited outdoor exposure have all been proposed as significant contributors. Several hypotheses (e.g. Hygiene, Old Friends, and Biodiversity Hypotheses) also suggest that limited environmental microbial exposures may underpin part of this rise in non-communicable diseases. In response, the Microbiome Rewilding Hypothesis proposes that adequate environmental microbial exposures could be achieved by restoring urban green spaces and could potentially decrease the prevalence of non-communicable diseases. However, the microbial interactions between humans and their surrounding environment and the passaging of microbes between both entities remains poorly understood, especially within an urban context. RESULTS: Here, we survey human skin (n = 90 swabs) and nasal (n = 90 swabs) microbiota of three subjects that were exposed to air (n = 15), soil (n = 15), and leaves (n = 15) from different urban green space environments in three different cities across different continents (Adelaide, Australia; Bournemouth, United Kingdom; New Delhi, India). Using 16S ribosomal RNA metabarcoding, we examined baseline controls (pre-exposure) of both skin (n = 16) and nasal (n = 16) swabs and tracked microbiota transfer from the environment to the human body after exposure events. Microbial richness and phylogenetic diversity increased after urban green space exposure in skin and nasal samples collected in two of the three locations. The microbial composition of skin samples also became more similar to soil microbiota after exposure, while nasal samples became more similar to air samples. Nasal samples were more variable between sites and individuals than skin samples. CONCLUSIONS: We show that exposure to urban green spaces can increase skin and nasal microbial diversity and alter human microbiota composition. Our study improves our understanding of human-environmental microbial interactions and suggests that increased exposure to diverse outdoor environments may increase the microbial diversity, which could lead to positive health outcomes for non-communicable diseases.Caitlin A. Selway, Jacob G. Mills, Philip Weinstein, Chris Skelly, Sudesh Yadav, Andrew Lowe, Martin F. Breed, Laura S. Weyric

Laboratory contamination over time during low-biomass sample analysis

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (e.g., skin swabs, tissue biopsies, ice, water, degraded forensic samples or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high-throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no-template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over 5 years to characterize long-term contaminant diversity. We additionally compared the contaminant content within a home-made silica-based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human-associated taxa in comparison to the home-made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low-biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high-throughput metagenomic study.Laura S. Weyrich, Andrew G. Farrer, Raphael Eisenhofer, Luis A. Arriola Jennifer Young, Caitlin A. Selway, Matilda Handsley‐Davis, Christina J. Adler James Breen, Alan Coope

Early markers of periodontal disease and altered oral microbiota are associated with glycemic control in children with type 1 diabetes

Objectives: To determine the relationship between periodontal disease and glycemic control in children with type 1 diabetes and to characterize the diversity and composition of their oral microbiota. Methods: Cross-sectional study including children with type 1 diabetes recruited from clinics at the Women's and Children's Hospital (Australia). Participants had a comprehensive dental assessment, periodontal examination, and buccal and gingival samples collected for 16S rRNA sequencing. Results: Seventy-seven participants (age 13.3 ± 2.6 years, 38 males, BMI z-score 0.81 ± 0.75) had a diabetes duration of 5.6 ± 3.9 years and median HbA1c of 8.5% (range 5.8–13.3), 69.4 mmol/mol (range 39.9–121.9). Thirty-eight (49%) had early markers of periodontal disease. HbA1c was positively correlated with plaque index (Rho = 0.34, P = 0.002), gingival index (Rho = 0.30, P = 0.009), bleeding on probing (Rho = 0.44, P = 0.0001) and periodontal pocket depth >3 mm (Rho = 0.21, P = 0.06). A 1% increase in HbA1c was independently associated with an average increase in bleeding on probing of 25% (P = 0.002) and with an increase in the rate of sites with pocket depth >3 mm of 54% (P = 0.003). Higher HbA1c was independently related to increased phylogenetic alpha diversity (P = 0.008) and increased compositional variation (beta diversity P = 0.02) in gingival, but not buccal, microbiota. Brushing frequency, plaque index, and gingival index had a significant effect on microbiota composition, independent of HbA1c. Conclusions: Children with type 1 diabetes showed a continuous relationship between less favorable glycemic control and increased early markers of periodontal disease. Glycemic control was also related to the complexity and richness of the plaque microbiota, with diversity increasing as HbA1c levels increase.Emilija D. Jensen, Caitlin A. Selway, Gabrielle Allen, Jana Bednarz, Laura S. Weyrich, Sam Gue, Alexia S. Peña, Jennifer Coupe

Revegetation of urban green space rewilds soil microbiotas with implications for human health and urban design

Many noncommunicable diseases are linked to degraded diversity in the human and environmental microbiota and are rising globally in epidemic proportions in industrialized urban populations. Reducing this disease burden may be aided by the ecological restoration of microbiota and their habitat in urban green spaces—a process termed microbiome rewilding. Microbiome rewilding could serve as a mechanism to increase urban exposure to biodiversity; biodiversity could introduce microbiota species or functional diversity to improve immune training and regulation in urban populations. As a first step in examining this hypothesis, we explored the microbial diversity and composition of a variety of urban green space vegetation types relative to urban revegetated woodlands of varying levels of vegetation diversity, including lawns, vacant lots, parklands, and remnant woodlands. We generated amplicon sequence variant community profiles from bacterial and archaeal 16S rRNA, fungal ITS1 region, and eukaryotic 18S rRNA marker genes. We also made trophic‐mode predictions of the fungal amplicon sequence variants. Across sites, soil microbiotas in revegetated urban green spaces were similar to remnant woodland microbiotas and differed greatly from lawns and vacant lots. There were several differentially abundant genera likely driving these differences that had strong correlations to plant species richness, soil pH, and conductivity. We provide the first evidence, as far as we know, that revegetation can improve urban soil microbiota diversity toward a more natural, biodiverse state by creating more wild habitat conditions. This evidence supports initiating further studies within the growing field of microbiome rewilding.Jacob G. Mills, Andrew Bissett, Nicholas J.C. Gellie, Andrew J. Lowe, Caitlin A. Selway, Torsten Thomas, Philip Weinstein, Laura S. Weyrich, Martin F. Bree