The macroecology of butyrate-producing bacteria via metagenomic assessment of butyrate production capacity.

Butyrate-producing bacteria are found in many outdoor ecosystems and host organisms, including humans, and are vital to ecosystem functionality and human health. These bacteria ferment organic matter, producing the short-chain fatty acid butyrate. However, the macroecological influences on their biogeographical distribution remain poorly resolved. Here we aimed to characterise their global distribution together with key explanatory climatic, geographical and physicochemical variables. We developed new normalised butyrate production capacity (BPC) indices derived from global metagenomic (n = 13,078) and Australia-wide soil 16S rRNA (n = 1331) data, using Geographic Information System (GIS) and modelling techniques to detail their ecological and biogeographical associations. The highest median BPC scores were found in anoxic and fermentative environments, including the human (BPC = 2.99) and non-human animal gut (BPC = 2.91), and in some plant-soil systems (BPC = 2.33). Within plant-soil systems, roots (BPC = 2.50) and rhizospheres (BPC = 2.34) had the highest median BPC scores. Among soil samples, geographical and climatic variables had the strongest overall effects on BPC scores (variable importance score range = 0.30-0.03), with human population density also making a notable contribution (variable importance score = 0.20). Higher BPC scores were in soils from seasonally productive sandy rangelands, temperate rural residential areas and sites with moderate-to-high soil iron concentrations. Abundances of butyrate-producing bacteria in outdoor soils followed complex ecological patterns influenced by geography, climate, soil chemistry and hydrological fluctuations. These new macroecological insights further our understanding of the ecological patterns of outdoor butyrate-producing bacteria, with implications for emerging microbially focused ecological and human health policies.

Restoring soil biodiversity.

Soil health is crucial for all terrestrial life, supporting, among other processes, food production, water purification and carbon sequestration. Soil biodiversity - the variety of life within soils - is key to these processes and thus key to soil restoration. Human activities that degrade ecosystems threaten soil biodiversity and associated ecosystem processes. Indeed, 75% of the world's soils are affected by degradation - a figure that could rise to 90% by 2050 if deforestation, overgrazing, urbanisation and other harmful practices persist. Restoring soil biodiversity is a prerequisite for planetary health, and it comes with many challenges and opportunities. Soil directly supports around 60% of all species on Earth, and land degradation poses a major problem for this biodiversity and the ecosystem services that sustain human populations. Indeed, 98% of human calories come from soil, and earthworms alone underpin 6.5% of the world's grain production. Moreover, the total carbon in terrestrial ecosystems is around 3,170 gigatons (1 gigaton (Gt) = 1 billion metric tons), of which approximately 80% (2,500 Gt) is found in soil. Therefore, restoring soil biodiversity is not just a human need but an ecological and Earth-system imperative. It is pivotal for maintaining ecosystem resilience, sustaining agricultural productivity and mitigating climate change impacts.

Childcare centre soil microbiomes are influenced by substrate type and surrounding vegetation condition.

Urban development has profoundly reduced human exposure to biodiverse environments, which is linked to a rise in human disease. The 'biodiversity hypothesis' proposes that contact with diverse microbial communities (microbiota) benefits human health, as exposure to microbial diversity promotes immune training and regulates immune function. Soils and sandpits in urban childcare centres may provide exposure to diverse microbiota that support immunoregulation at a critical developmental stage in a child's life. However, the influence of outdoor substrate (i.e., sand vs. soil) and surrounding vegetation on these environmental microbiota in urban childcare centres remains poorly understood. Here, we used 16S rRNA amplicon sequencing to examine the variation in bacterial communities in sandpits and soils across 22 childcare centres in Adelaide, Australia, plus the impact of plant species richness and habitat condition on these bacterial communities. We show that sandpits had distinct bacterial communities and lower alpha diversity than soils. In addition, we found that plant species richness in the centres' yards and habitat condition surrounding the centres influenced the bacterial communities in soils but not sandpits. These results demonstrate that the diversity and composition of childcare centre sandpit and soil bacterial communities are shaped by substrate type, and that the soils are also shaped by the vegetation within and surrounding the centres. Accordingly, there is potential to modulate the exposure of children to health-associated bacterial communities by managing substrates and vegetation in and around childcare centres.

Short-term passive greenspace exposures have little effect on nasal microbiomes: A cross-over exposure study of a Maori cohort.

Indigenous health interventions have emerged in New Zealand aimed at increasing people's interactions with and exposure to macro and microbial diversity. Urban greenspaces provide opportunities for people to gain such exposures. However, the dynamics and pathways of microbial transfer from natural environments onto a person remain poorly understood. Here, we analysed bacterial 16S rRNA amplicons in air samples (n = 7) and pre- and post-exposure nasal samples (n = 238) from 35 participants who had 30-min exposures in an outdoor park. The participants were organised into two groups: over eight days each group had two outdoor park exposures and two indoor office exposures, with a cross-over study design and washout days between exposure days. We investigated the effects of participant group, location (outdoor park vs. indoor office), and exposures (pre vs. post) on the nasal bacterial community composition and three key suspected health-associated bacterial indicators (alpha diversity, generic diversity of Gammaproteobacteria, and read abundances of butyrate-producing bacteria). The participants had distinct nasal bacterial communities, but these communities did not display notable shifts in composition following exposures. The community composition and key health bacterial indicators were stable throughout the trial period, with no clear or consistent effects of group, location, or exposure. We conclude that 30-min exposure periods to urban greenspaces are unlikely to create notable changes in the nasal microbiome of visitors, which contrasts with previous research. Our results suggest that longer exposures or activities that involves closer interaction with microbial rich ecological components (e.g., soil) are required for greenspace exposures to result in noteworthy changes in the nasal microbiome.

Light-dark cycles may influence in situ soil bacterial networks and diurnally-sensitive taxa.

Soil bacterial taxa have important functional roles in ecosystems (e.g. nutrient cycling, soil formation, plant health). Many factors influence their assembly and regulation, with land cover types (e.g. open woodlands, grasslands), land use types (e.g. nature reserves, urban green space) and plant-soil feedbacks being well-studied factors. However, changes in soil bacterial communities in situ over light-dark cycles have received little attention, despite many plants and some bacteria having endogenous circadian rhythms that could influence soil bacterial communities. We sampled surface soils in situ across 24-h light-dark cycles (at 00:00, 06:00, 12:00, 18:00) at two land cover types (remnant vegetation vs. cleared, grassy areas) and applied 16S rRNA amplicon sequencing to investigate changes in bacterial communities. We show that land cover type strongly affected soil bacterial diversity, with soils under native vegetation expressing 15.4%-16.4% lower alpha diversity but 4.9%-10.6% greater heterogeneity than soils under cleared vegetation. In addition, we report time-dependent and site-specific changes in bacterial network complexity and between 598-922 ASVs showing significant changes in relative abundance across times. Native site node degree (bacterial interactions) at the phylum level was 16.0% higher in the early morning than in the afternoon/evening. Our results demonstrate for the first time that light-dark cycles have subtle yet important effects on soil bacterial communities in situ and that land cover influences these dynamics. We provide a new view of soil microbial ecology and suggest that future studies should consider the time of day when sampling soil bacteria.

Opportunities and challenges for microbiomics in ecosystem restoration.

Microbiomics is the science of characterizing microbial community structure, function, and dynamics. It has great potential to advance our understanding of plant-soil-microbe processes and interaction networks which can be applied to improve ecosystem restoration. However, microbiomics may be perceived as complex and the technology is not accessible to all. The opportunities of microbiomics in restoration ecology are considerable, but so are the practical challenges. Applying microbiomics in restoration must move beyond compositional assessments to incorporate tools to study the complexity of ecosystem recovery. Advances in metaomic tools provide unprecedented possibilities to aid restoration interventions. Moreover, complementary non-omic applications, such as microbial inoculants and biopriming, have the potential to improve restoration objectives by enhancing the establishment and health of vegetation communities.

Environmental DNA as an innovative technique to identify the origins of falsified antimalarial tablets-a pilot study of the pharmabiome.

Falsified medicines are a major threat to global health. Antimalarial drugs have been particularly targeted by criminals. As DNA analysis has revolutionized forensic criminology, we hypothesized that these techniques could also be used to investigate the origins of falsified medicines. Medicines may contain diverse adventitious biological contamination, and the sealed nature of blister-packages may capture and preserve genetic signals from the manufacturing processes allowing identification of production source(s). We conducted a blinded pilot study to determine if such environmental DNA (eDNA) could be detected in eleven samples of falsified and genuine artesunate antimalarial tablets, collected in SE Asia, which could be indicative of origin. Massively Parallel Sequencing (MPS) was used to characterize microbial and eukaryote diversity. Two mitochondrial DNA analysis approaches were explored to detect the presence of human DNA. Trace eDNA from these low biomass samples demonstrated sample specific signals using two target markers. Significant differences in bacterial and eukaryote DNA community structures were observed between genuine and falsified tablets and between different packaging types of falsified artesunate. Human DNA, which was indicative of likely east Asian ancestry, was found in falsified tablets. This pilot study of the 'pharmabiome' shows the potential of environmental DNA as a powerful forensic tool to assist with the identification of the environments, and hence location and timing, of the source and manufacture of falsified medicines, establish links between seizures and complement existing tools to build a more complete picture of criminal trade routes. The finding of human DNA in tablets raises important ethical issues that need to be addressed.

Ecosystem restoration is integral to humanity's recovery from COVID-19.

COVID-19 has devastated global communities and economies. The pandemic has exposed socioeconomic disparities and weaknesses in health systems worldwide. Long-term health effects and economic recovery are major concerns. Ecosystem restoration-ie, the repair of ecosystems that have been degraded-relates directly to tackling the health and socioeconomic burdens of COVID-19, because stable and resilient ecosystems are fundamental determinants of health and socioeconomic stability. Here, we use COVID-19 as a case study, showing how ecosystem restoration can reduce the risk of infection and adverse sequelae and have an integral role in humanity's recovery from COVID-19. The next decade will be crucial for humanity's recovery from COVID-19 and for ecosystem repair. Indeed, in the absence of effective, large-scale restoration, 95% of the Earth's land could be degraded by 2050. The UN Decade on Ecosystem Restoration (2021-30) declaration reflects the growing urgency and scale at which we should repair ecosystems. Importantly, ecosystem restoration could also help to combat the health and socioeconomic issues that are associated with COVID-19, yet it is poorly integrated into current responses to the disease. Ecosystem restoration can be a core public health intervention and assist in COVID-19 recovery if it is closely integrated with socioeconomic, health, and environmental policies.

Gut microbiota composition does not associate with toxoplasma infection in rats.

Toxoplasma infection in intermediate host species closely associates with inflammation. This association has led to suggestions that the behavioural changes associated with infection may be indirectly driven by the resulting sustained inflammation rather than a direct behavioural manipulation by the parasite. If this is correct, sustained inflammation in chronically infected rodents should present as widespread differences in the gastrointestinal microbiota due to the dependency between the composition of these microbiota and sustained inflammation. We conducted a randomized controlled experiment in rats that were assigned to a Toxoplasma-treatment, placebo-treatment or negative control group. We euthanised rats during the chronic phase of infection, collected their caecal stool samples and sequenced the V3-V4 region of the 16S rRNA gene to characterize the bacterial community in these samples. Toxoplasma infection did not induce widespread differences in the bacterial community composition of the gastrointestinal tract of rats. Rather, we found sex differences in the bacterial community composition of rats. We conclude that it is unlikely that sustained inflammation is the mechanism driving the highly specific behavioural changes observed in Toxoplasma-positive rats.

Next generation restoration metrics: Using soil eDNA bacterial community data to measure trajectories towards rehabilitation targets.

In post-mining rehabilitation, successful mine closure planning requires specific, measurable, achievable, relevant and time-bound (SMART) completion criteria, such as returning ecological communities to match a target level of similarity to reference sites. Soil microbiota are fundamentally linked to the restoration of degraded ecosystems, helping to underpin ecological functions and plant communities. High-throughput sequencing of soil eDNA to characterise these communities offers promise to help monitor and predict ecological progress towards reference states. Here we demonstrate a novel methodology for monitoring and evaluating ecological restoration using three long-term (>25 year) case study post-mining rehabilitation soil eDNA-based bacterial community datasets. Specifically, we developed rehabilitation trajectory assessments based on similarity to reference data from restoration chronosequence datasets. Recognising that numerous alternative options for microbiota data processing have potential to influence these assessments, we comprehensively examined the influence of standard versus compositional data analyses, different ecological distance measures, sequence grouping approaches, eliminating rare taxa, and the potential for excessive spatial autocorrelation to impact on results. Our approach reduces the complexity of information that often overwhelms ecologically-relevant patterns in microbiota studies, and enables prediction of recovery time, with explicit inclusion of uncertainty in assessments. We offer a step change in the development of quantitative microbiota-based SMART metrics for measuring rehabilitation success. Our approach may also have wider applications where restorative processes facilitate the shift of microbiota towards reference states.

Exposure to airborne bacteria depends upon vertical stratification and vegetation complexity.

Exposure to biodiverse aerobiomes supports human health, but it is unclear which ecological factors influence exposure. Few studies have investigated near-surface green space aerobiome dynamics, and no studies have reported aerobiome vertical stratification in different urban green spaces. We used columnar sampling and next generation sequencing of the bacterial 16S rRNA gene, combined with geospatial and network analyses to investigate urban green space aerobiome spatio-compositional dynamics. We show a strong effect of habitat on bacterial diversity and network complexity. We observed aerobiome vertical stratification and network complexity that was contingent on habitat type. Tree density, closer proximity, and canopy coverage associated with greater aerobiome alpha diversity. Grassland aerobiomes exhibited greater proportions of putative pathogens compared to scrub, and also stratified vertically. We provide novel insights into the urban ecosystem with potential importance for public health, whereby the possibility of differential aerobiome exposures appears to depend on habitat type and height in the airspace. This has important implications for managing urban landscapes for the regulation of aerobiome exposure.

Outdoor artificial light at night: A forgotten factor in green space and health research.

Exposure to green space has been associated with a range of human health benefits, largely in spatial epidemiological studies. Green space and artificial light at night are both associated with breast and prostate cancer, depressive symptoms, obesity, and sleep problems, and are both influenced by the built environment and urbanisation. We identified a negative correlation between green space diversity and outdoor artificial light at night for Australian major cities. Outdoor artificial light at night is therefore a potential, but overlooked, confounder in spatial epidemiological studies of green space exposure and human health, and should be incorporated into future models.

The potential of outdoor environments to supply beneficial butyrate-producing bacteria to humans.

Butyrate is an important mediator of human health and disease. The mechanisms of action of butyrate are becoming increasingly well-known. Many commensal bacteria that inhabit the human gut can synthesise butyrate, which is then absorbed into the human host. Simultaneously, several immune- and inflammatory-mediated diseases are being linked to insufficient exposure to beneficial microbes from our environment, including butyrate-producing bacteria. However, the role of outdoor environmental exposure to butyrate-producing bacteria remains poorly understood. Here we review the literature on the human exposure pathways to butyrate-producing bacteria, with a particular focus on outdoor environmental sources (e.g. associated with plants, plant-based residues, and soil), and the health implications of exposure to them. Emerging evidence suggests that environmental butyrate-producers may help supplement the human gut microbiota and represent an important component of the Biodiversity and Old Friends hypotheses. Improving our understanding of potential sources, precursors, and exposure pathways of environmental butyrate-producers that influence the gut microbiota and butyrate production offers promise to advance multiple disciplines of health and environmental science. We outline research priorities to address knowledge gaps in the outdoor environment-butyrate-health nexus and build knowledge of the potential pathways to help optimise exposure to human-beneficial butyrate-producing bacteria from the outdoor environment during childhood and adulthood.

Increased plant species richness associates with greater soil bacterial diversity in urban green spaces.

The vegetation and soil microbiome within urban green spaces is increasingly managed to help conserve biodiversity and improve human health concurrently. However, the effects of green space management on urban soil ecosystems is poorly understood, despite their importance. Across 40 urban green spaces in metropolitan Adelaide, South Australia, we show that soil bacterial communities are strongly affected by urban green space type (incl. sport fields, community gardens, parklands and revegetated areas), and that plant species richness is positively associated with soil bacterial diversity. Importantly, these microbiome trends were not affected by geographic proximity of sample sites. Our results provide early evidence that urban green space management can have predictable effects on the soil microbiome, at least from a diversity perspective, which could prove important to inform policy development if urban green spaces are to be managed to optimise population health benefits.

Vertical Stratification in Urban Green Space Aerobiomes.

BACKGROUND: Exposure to a diverse environmental microbiome is thought to play an important role in "educating" the immune system and facilitating competitive exclusion of pathogens to maintain human health. Vegetation and soil are key sources of airborne microbiota--the aerobiome. A limited number of studies have attempted to characterize the dynamics of near surface green space aerobiomes, and no studies to date have investigated these dynamics from a vertical perspective. Vertical stratification in the aerobiome could have important implications for public health and for the design, engineering, and management of urban green spaces. OBJECTIVES: The primary objectives of this study were to: a) assess whether significant vertical stratification in bacterial species richness and evenness (alpha diversity) of the aerobiome occurred in a parkland habitat in Adelaide, South Australia; b) assess whether significant compositional differences (beta diversity) between sampling heights occurred; and c) to preliminarily assess whether there were significant altitudinal differences in potentially pathogenic and beneficial bacterial taxa. METHODS: We combined an innovative columnar sampling method at soil level, 0.0, 0.5, 1.0, and 2.0m, using passive petri dish sampling to collect airborne bacteria. We used a geographic information system (GIS) to select study sites, and we used high-throughput sequencing of the bacterial 16S rRNA gene to assess whether significant vertical stratification of the aerobiome occurred. RESULTS: Our results provide evidence of vertical stratification in both alpha and beta (compositional) diversity of airborne bacterial communities, with diversity decreasing roughly with height. We also found significant vertical stratification in potentially pathogenic and beneficial bacterial taxa. DISCUSSION: Although additional research is needed, our preliminary findings point to potentially different exposure attributes that may be contingent on human height and activity type. Our results lay the foundations for further research into the vertical characteristics of urban green space aerobiomes and their implications for public health and urban planning. https://doi.org/10.1289/EHP7807.

Naturally-diverse airborne environmental microbial exposures modulate the gut microbiome and may provide anxiolytic benefits in mice.

Growing epidemiological evidence links natural green space exposure with a range of health benefits, including for mental health. Conversely, greater urbanisation associates with increased risk of mental health disorders. Microbiomes are proposed as an important but understudied link that may help explain many green space-human health associations. However, there remains a lack of controlled experimental evidence testing possible beneficial effects from passive exposure to natural biodiversity via airborne microbiota. Previous mouse model studies have used unrealistic environmental microbial exposures-including excessive soil and organic matter contact, feed supplements and injections-to demonstrate host microbiota, immune biomarker, and behavioural changes. Here, in a randomised controlled experiment, we demonstrate that realistic exposures to trace-level dust from a high biodiversity soil can change mouse gut microbiota, in comparison to dust from low biodiversity soil or no soil (control) (n = 54 total mice, comprising 3 treatments × 18 mice, with 9 females + 9 males per group). Furthermore, we found a nominal soil-derived anaerobic spore-forming butyrate-producer, Kineothrix alysoides, was supplemented to a greater extent in the gut microbiomes of high biodiversity treatment mice. Also, increasing relative abundance of this rare organism correlated with reduced anxiety-like behaviour in the most anxious mice. Our results point to an intriguing new hypothesis: that biodiverse soils may represent an important supplementary source of butyrate-producing bacteria capable of resupplying the mammalian gut microbiome, with potential for gut health and mental health benefits. Our findings have potential to inform cost-effective population health interventions through microbiome-conscious green space design and, ultimately, the mainstreaming of biodiversity into health care.

Can bacterial indicators of a grassy woodland restoration inform ecosystem assessment and microbiota-mediated human health?

Understanding how microbial communities change with environmental degradation and restoration may offer new insights into the understudied ecology that connects humans, microbiota, and the natural world. Immunomodulatory microbial diversity and 'Old Friends' are thought to be supplemented from biodiverse natural environments, yet deficient in anthropogenically disturbed or degraded environments. However, few studies have compared the microbiomes of natural vs. human-altered environments and there is little knowledge of which microbial taxa are representative of ecological restoration-i.e. the assisted recovery of degraded ecosystems typically towards a more natural, biodiverse state. Here we use novel bootstrap-style resampling of site-level soil bacterial 16S rRNA gene environmental DNA data to identify genus-level indicators of restoration from a 10-year grassy eucalypt woodland restoration chronosequence at Mt Bold, South Australia. We found two key indicator groups emerged: 'opportunistic taxa' that decreased in relative abundance with restoration and more stable and specialist, 'niche-adapted taxa' that increased. We validated these results, finding seven of the top ten opportunists and eight of the top ten niche-adapted taxa displayed consistent differential abundance patterns between human-altered vs. natural samples elsewhere across Australia. Extending this, we propose a two-dimensional mapping for ecosystem condition based on the proportions of these divergent indicator groups. We also show that restoring a more biodiverse ecosystem at Mt Bold has increased the potentially immune-boosting environmental microbial diversity. Furthermore, environmental opportunists including the pathogen-containing genera Bacillus, Clostridium, Enterobacter, Legionella and Pseudomonas associated with disturbed ecosystems. Our approach is generalizable with potential to inform DNA-based methods for ecosystem assessment and help target environmental interventions that may promote microbiota-mediated human health gains.

Relating Urban Biodiversity to Human Health With the 'Holobiont' Concept.

A relatively unaccounted ecosystem service from biodiversity is the benefit to human health via symbiotic microbiota from our environment. This benefit occurs because humans evolved alongside microbes and have been constantly exposed to diverse microbiota. Plants and animals, including humans, are organised as a host with symbiotic microbiota, whose collective genome and life history form a single holobiont. As such, there are interdependencies between biodiversity, holobionts, and public health which lead us to argue that human health outcomes could be improved by increasing contact with biodiversity in an urban context. We propose that humans, like all holobionts, likely require a diverse microbial habitat to appropriate resources for living healthy, long lives. We discuss how industrial urbanisation likely disrupts the symbiosis between microbiota and their hosts, leading to negative health outcomes. The industrialised urban habitat is low in macro and microbial biodiversity and discourages contact with beneficial environmental microbiota. These habitat factors, alongside diet, antibiotics, and others, are associated with the epidemic of non-communicable diseases in these societies. We suggest that restoration of urban microbial biodiversity and micro-ecological processes through microbiome rewilding can benefit holobiont health and aid in treating the urban non-communicable disease epidemic. Further, we identify research gaps and some solutions to economic and strategic hurdles in applying microbiome rewilding into daily urban life.

Ambient soil cation exchange capacity inversely associates with infectious and parasitic disease risk in regional Australia.

Human contact with soil may be important for building and maintaining normal healthy immune defence mechanisms, however this idea remains untested at the population-level. In this continent-wide, cross-sectional study we examine the possible public health benefit of ambient exposures to soil of high cation exchange capacity (CEC), a surrogate for potential immunomodulatory soil microbial diversity. We compare distributions of normalized mean 2011/12-2012/13 age-standardized public hospital admission rates (cumulative incidence) for infectious and parasitic diseases across regional Australia (representing an average of 29,516 patients/year in 228 local government areas), within tertiles of socioeconomic status and soil exposure. To test the significance of soil CEC, we use probabilistic individual-level environmental exposure data (with or without soil), and group-level variables, in robust non-parametric multilevel modelling to predict disease rates in unseen groups. Our results show that in socioeconomically-deprived areas with high CEC soils, rates of infectious and parasitic disease are significantly lower than areas with low CEC soils. Also, health inequality (relative risk) due to socioeconomic status is significantly lower in areas with high CEC soils compared to low CEC soils (Δ relative risk = 0.47; 95% CI: 0.13, 0.82). Including soil exposure when modelling rates of infectious and parasitic disease significantly improves prediction performance, explaining an additional 7.5% (Δ r2 = 0.075; 95% CI: 0.05, 0.10) of variation in disease risk, in local government areas that were not used for model building. Our findings suggest that exposure to high CEC soils (typically high soil biodiversity) associates with reduced risk of infectious and parasitic diseases, particularly in lower socioeconomic areas.