Environmental risk of nontuberculous mycobacterial infection: Strategies for advancing methodology.

The National Institute of Allergy and Infectious Diseases organized a symposium in June 2022, to facilitate discussion of the environmental risks for nontuberculous mycobacteria exposure and disease. The expert researchers presented recent studies and identified numerous research gaps. This report summarizes the discussion and identifies six major areas of future research related to culture-based and culture independent laboratory methods, alternate culture media and culturing conditions, frameworks for standardized laboratory methods, improved environmental sampling strategies, validation of exposure measures, and availability of high-quality spatiotemporal data.

Methods of isolation and identification of nontuberculous mycobacteria from environmental samples: A scoping review.

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment. Some species of NTM are pathogenic and cause lung disease in susceptible persons. Epidemiologic studies of environmental NTM infection risk rely on both culture-dependent and culture-independent techniques for NTM isolation and identification. In this review, we summarized current methods used to isolate and identify NTM from the environment. We searched PubMed, Embase, Scopus, Web of Science: Core Collection, and Global Health (CAB Direct) for peer-reviewed studies from the last 12 years. We identified 1685 unique citations and 110 studies met our inclusion and exclusion criteria. Approximately half (55%) of the studies identified in this review used a combination of culture-independent and culture-dependent methods. The most common environmental substrate analyzed was water (n = 90). Identification of current, common methods for the isolation and identification of NTM from environmental samples may contribute to the development of standard methodological practices in the future. The choice of isolation method is based on the research question, environment, and species. A summary of common methods may contribute to the development of standard practices for isolation and identification of NTM from environmental samples, which may lead to more robust and comparable results.

A Metagenomic Investigation of Spatial and Temporal Changes in Sewage Microbiomes across a University Campus.

Wastewater microbial communities are not static and can vary significantly across time and space, but this variation and the factors driving the observed spatiotemporal variation often remain undetermined. We used a shotgun metagenomic approach to investigate changes in wastewater microbial communities across 17 locations in a sewer network, with samples collected from each location over a 3-week period. Fecal material-derived bacteria constituted a relatively small fraction of the taxa found in the collected samples, highlighting the importance of environmental sources to the sewage microbiome. The prokaryotic communities were highly variable in composition depending on the location within the sampling network, and this spatial variation was most strongly associated with location-specific differences in sewage pH. However, we also observed substantial temporal variation in the composition of the prokaryotic communities at individual locations. This temporal variation was asynchronous across sampling locations, emphasizing the importance of independently considering both spatial and temporal variation when assessing the wastewater microbiome. The spatiotemporal patterns in viral community composition closely tracked those of the prokaryotic communities, allowing us to putatively identify the bacterial hosts of some of the dominant viruses in these systems. Finally, we found that antibiotic resistance gene profiles also exhibit a high degree of spatiotemporal variability, with most of these genes unlikely to be derived from fecal bacteria. Together, these results emphasize the dynamic nature of the wastewater microbiome, the challenges associated with studying these systems, and the utility of metagenomic approaches for building a multifaceted understanding of these microbial communities and their functional attributes. IMPORTANCE Sewage systems harbor extensive microbial diversity, including microbes derived from both human and environmental sources. Studies of the sewage microbiome are useful for monitoring public health and the health of our infrastructure, but the sewage microbiome can be highly variable in ways that are often unresolved. We sequenced DNA recovered from wastewater samples collected over a 3-week period at 17 locations in a single sewer system to determine how these communities vary across time and space. Most of the wastewater bacteria, and the antibiotic resistance genes they harbor, were not derived from human feces, but human usage patterns did impact how the amounts and types of bacteria and bacterial genes we found in these systems varied over time. Likewise, the wastewater communities, including both bacteria and their viruses, varied depending on location within the sewage network, highlighting the challenges and opportunities in efforts to monitor and understand the sewage microbiome.

Structure and Functional Attributes of Bacterial Communities in Premise Plumbing Across the United States.

Microbes that thrive in premise plumbing can have potentially important effects on human health. Yet, how and why plumbing-associated microbial communities vary across broad spatial scales remain undetermined. We characterized the bacterial communities in 496 showerheads collected from across the continental United States. The overall community structure, determined by 16S rRNA gene amplicon sequencing, revealed high levels of bacterial diversity. Although a large fraction of the observed variation in community composition could not be explained, differences in bacterial community composition were associated with water supply (private well water vs public municipal water), water source (groundwater vs surface water), and associated differences in water chemistry (pH and chlorine). Most notably, showerheads in homes supplied with public water had higher abundances of Blastomonas, Mycobacterium, and Porphyrobacter, while Pseudorhodoplanes, Novosphingobium, and Nitrospira were more abundant in those receiving private well water. We conducted shotgun metagenomic analyses on 92 of these samples to assess differences in genomic attributes. Public water-sourced showerheads had communities enriched in genes related to lipid and xenobiotic metabolisms, virulence factors, and antibiotic resistance. In contrast, genes associated with oxidative stress and membrane transporters were over-represented in communities from private well water-sourced showerheads compared to those supplied by public water systems. These results highlight the broad diversity of bacteria found in premise plumbing across the United States and the role of the water source and treatment in shaping the microbial community structure and functional potential.

Global Patterns and Climatic Controls of Dust-Associated Microbial Communities.

The ubiquity and long-range transport of the microorganisms inhabiting dust can pose a serious risk to human, animal, and plant health. The well-recognized importance of dust-associated microorganisms contrasts starkly with our limited understanding of the factors determining the variation in the composition of these communities at the global scale. Here, we provide the first insight into the global determinants of dust-associated microorganisms by quantifying the environmental factors shaping bacterial and fungal community composition in 467 outdoor settled dust samples collected from 33 countries and 6 continents. Our results show that the global variation in dust-associated bacterial and fungal community composition was, to some degree, predictable from mean annual precipitation and temperature. Notably, our results show that the fungal genera Alternaria and Aspergillus, which contain many species that can serve as triggers of allergenic disease in humans and as plant pathogens, were more abundant in drier regions. Collectively, these results highlight the key influence of climate on the global distribution of dust-associated microorganisms and provide the baseline information needed to build a more comprehensive understanding of how microbial exposures vary across the globe and in response to climate change. IMPORTANCE A broad diversity of microorganisms can be found in dust, with some of these microorganisms capable of causing allergenic disease in human via inhalation or affecting plant health by acting as plant pathogens. However, the spatial variation in dust microbiomes and the environmental factors associated with this variation have not been comprehensively assessed at the global scale. Here, we investigated the bacteria and fungi found in outdoor settled dust samples spanning 33 countries and 6 continents. Our results show that dust-associated bacteria and fungi exhibit climate-driven variability in community composition at the global scale. Our results call for the development of strategies to predict the geographic distribution of dust-associated microorganisms and to identify the potential associations between microbial exposures and the health of humans, animals, and plants.

Structure of Chimpanzee Gut Microbiomes across Tropical Africa.

Understanding variation in host-associated microbial communities is important given the relevance of microbiomes to host physiology and health. Using 560 fecal samples collected from wild chimpanzees (Pan troglodytes) across their range, we assessed how geography, genetics, climate, vegetation, and diet relate to gut microbial community structure (prokaryotes, eukaryotic parasites) at multiple spatial scales. We observed a high degree of regional specificity in the microbiome composition, which was associated with host genetics, available plant foods, and potentially with cultural differences in tool use, which affect diet. Genetic differences drove community composition at large scales, while vegetation and potentially tool use drove within-region differences, likely due to their influence on diet. Unlike industrialized human populations in the United States, where regional differences in the gut microbiome are undetectable, chimpanzee gut microbiomes are far more variable across space, suggesting that technological developments have decoupled humans from their local environments, obscuring regional differences that could have been important during human evolution. IMPORTANCE Gut microbial communities are drivers of primate physiology and health, but the factors that influence the gut microbiome in wild primate populations remain largely undetermined. We report data from a continent-wide survey of wild chimpanzee gut microbiota and highlight the effects of genetics, vegetation, and potentially even tool use at different spatial scales on the chimpanzee gut microbiome, including bacteria, archaea, and eukaryotic parasites. Microbial community dissimilarity was strongly correlated with chimpanzee population genetic dissimilarity, and vegetation composition and consumption of algae, honey, nuts, and termites were potentially associated with additional divergence in microbial communities between sampling sites. Our results suggest that host genetics, geography, and climate play a far stronger role in structuring the gut microbiome in chimpanzees than in humans.

Comparing the effects of two different strains of mycobacteria, Mycobacterium vaccae NCTC 11659 and M. vaccae ATCC 15483, on stress-resilient behaviors and lipid-immune signaling in rats.

Stress-related disorders, such as posttraumatic stress disorder (PTSD), are highly prevalent and often difficult to treat. In rodents, stress-related, anxiety-like defensive behavioral responses may be characterized by social avoidance, exacerbated inflammation, and altered metabolic states. We have previously shown that, in rodents, subcutaneous injections of a heat-killed preparation of the soil-derived bacterium Mycobacterium vaccae NCTC 11659 promotes stress resilience effects that are associated with immunoregulatory signaling in the periphery and the brain. In the current study, we sought to determine whether treatment with a heat-killed preparation of the closely related M. vaccae type strain, M. vaccae ATCC 15483, would also promote stress-resilience in adult male rats, likely due to biologically similar characteristics of the two strains. Here we show that immunization with either M. vaccae NCTC 11659 or M. vaccae ATCC 15483 prevents stress-induced increases in hippocampal interleukin 6 mRNA expression, consistent with previous studies showing that M. vaccae NCTC 11659 prevents stress-induced increases in peripheral IL-6 secretion, and prevents exaggeration of anxiety-like defensive behavioral responses assessed 24 h after exposure to inescapable tail shock stress (IS) in adult male rats. Analysis of mRNA expression, protein abundance, and flow cytometry data demonstrate overlapping but also unique effects of treatment with the two M. vaccae strains on immunological and metabolic signaling in the host. These data support the hypothesis that treatment with different M. vaccae strains may immunize the host against stress-induced dysregulation of physiology and behavior.

A Global Survey of Mycobacterial Diversity in Soil.

Mycobacteria are a diverse bacterial group ubiquitous in many soil and aquatic environments. Members of this group have been associated with human and other animal diseases, including the nontuberculous mycobacteria (NTM), which are of growing relevance to public health worldwide. Although soils are often considered an important source of environmentally acquired NTM infections, the biodiversity and ecological preferences of soil mycobacteria remain largely unexplored across contrasting climates and ecosystem types. Using a culture-independent approach by combining 16S rRNA marker gene sequencing with mycobacterium-specific hsp65 gene sequencing, we analyzed the diversity, distributions, and environmental preferences of soil-dwelling mycobacteria in 143 soil samples collected from a broad range of ecosystem types. The surveyed soils harbored highly diverse mycobacterial communities that span the full extent of the known mycobacterial phylogeny, with most soil mycobacteria (97% of mycobacterial clades) belonging to previously undescribed lineages. While mycobacteria tended to have higher relative abundances in cool, wet, and acidic soil environments, several individual mycobacterial clades had contrasting environmental preferences. We identified the environmental preferences of many mycobacterial clades, including the clinically relevant Mycobacterium avium complex that was more commonly detected in wet and acidic soils. However, most of the soil mycobacteria detected were not closely related to known pathogens, calling into question previous assumptions about the general importance of soil as a source of NTM infections. Together, this work provides novel insights into the diversity, distributions, and ecological preferences of soil mycobacteria and lays the foundation for future efforts to link mycobacterial phenotypes to their distributions.IMPORTANCE Mycobacteria are common inhabitants of soil, and while most members of this bacterial group are innocuous, some mycobacteria can cause environmentally acquired infections of humans and other animals. Human infections from nontuberculous mycobacteria (NTM) are increasingly prevalent worldwide, and some areas appear to be "hotspots" for NTM disease. While exposure to soil is frequently implicated as an important mode of NTM transmission, the diversity, distributions, and ecological preferences of soil mycobacteria remain poorly understood. We analyzed 143 soils from a range of ecosystems and found that mycobacteria and lineages within the group often exhibited predictable preferences for specific environmental conditions. Soils harbor large amounts of previously undescribed mycobacterial diversity, and lineages that include known pathogens were rarely detected in soil. Together, these findings suggest that soil is an unlikely source of many mycobacterial infections. The biogeographical patterns we documented lend insight into the ecology of this important group of soil-dwelling bacteria.

Ecological Analyses of Mycobacteria in Showerhead Biofilms and Their Relevance to Human Health.

Bacteria within the genus Mycobacterium can be abundant in showerheads, and the inhalation of aerosolized mycobacteria while showering has been implicated as a mode of transmission in nontuberculous mycobacterial (NTM) lung infections. Despite their importance, the diversity, distributions, and environmental predictors of showerhead-associated mycobacteria remain largely unresolved. To address these knowledge gaps, we worked with citizen scientists to collect showerhead biofilm samples and associated water chemistry data from 656 households located across the United States and Europe. Our cultivation-independent analyses revealed that the genus Mycobacterium was consistently the most abundant genus of bacteria detected in residential showerheads, and yet mycobacterial diversity and abundances were highly variable. Mycobacteria were far more abundant, on average, in showerheads receiving municipal water than in those receiving well water and in U.S. households than in European households, patterns that are likely driven by differences in the use of chlorine disinfectants. Moreover, we found that water source, water chemistry, and household location also influenced the prevalence of specific mycobacterial lineages detected in showerheads. We identified geographic regions within the United States where showerheads have particularly high abundances of potentially pathogenic lineages of mycobacteria, and these "hot spots" generally overlapped those regions where NTM lung disease is most prevalent. Together, these results emphasize the public health relevance of mycobacteria in showerhead biofilms. They further demonstrate that mycobacterial distributions in showerhead biofilms are often predictable from household location and water chemistry, knowledge that advances our understanding of NTM transmission dynamics and the development of strategies to reduce exposures to these emerging pathogens.IMPORTANCE Bacteria thrive in showerheads and throughout household water distribution systems. While most of these bacteria are innocuous, some are potential pathogens, including members of the genus Mycobacterium that can cause nontuberculous mycobacterial (NTM) lung infection, an increasing threat to public health. We found that showerheads in households across the United States and Europe often harbor abundant mycobacterial communities that vary in composition depending on geographic location, water chemistry, and water source, with households receiving water treated with chlorine disinfectants having particularly high abundances of certain mycobacteria. The regions in the United States where NTM lung infections are most common were the same regions where pathogenic mycobacteria were most prevalent in showerheads, highlighting the important role of showerheads in the transmission of NTM infections.

Microbial community assembly and metabolic function during mammalian corpse decomposition.

Vertebrate corpse decomposition provides an important stage in nutrient cycling in most terrestrial habitats, yet microbially mediated processes are poorly understood. Here we combine deep microbial community characterization, community-level metabolic reconstruction, and soil biogeochemical assessment to understand the principles governing microbial community assembly during decomposition of mouse and human corpses on different soil substrates. We find a suite of bacterial and fungal groups that contribute to nitrogen cycling and a reproducible network of decomposers that emerge on predictable time scales. Our results show that this decomposer community is derived primarily from bulk soil, but key decomposers are ubiquitous in low abundance. Soil type was not a dominant factor driving community development, and the process of decomposition is sufficiently reproducible to offer new opportunities for forensic investigations.

Antigen recognition in the islets changes with progression of autoimmune islet infiltration.

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention because immune infiltration of the islets is well established at diagnosis. Therefore, understanding the events that underlie the continued progression of the autoimmune response and islet destruction is critical. Islet infiltration and destruction is an asynchronous process, making it important to analyze the disease process on a single islet basis. To understand how T cell stimulation evolves through the process of islet infiltration, we analyzed the dynamics of T cell movement and interactions within individual islets of spontaneously autoimmune NOD mice. Using both intravital and explanted two-photon islet imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was Ag dependent and due, at least in part, to Ag recognition through sustained interactions with CD11c(+) APCs. As islet infiltration progressed, T cell motility became Ag independent, with a loss of T cell arrest and sustained interactions with CD11c(+) APCs. These studies suggest that the autoimmune T cell response in the islets may be temporarily dampened during the course of islet infiltration and disease progression.

A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system.

Establishing the time since death is critical in every death investigation, yet existing techniques are susceptible to a range of errors and biases. For example, forensic entomology is widely used to assess the postmortem interval (PMI), but errors can range from days to months. Microbes may provide a novel method for estimating PMI that avoids many of these limitations. Here we show that postmortem microbial community changes are dramatic, measurable, and repeatable in a mouse model system, allowing PMI to be estimated within approximately 3 days over 48 days. Our results provide a detailed understanding of bacterial and microbial eukaryotic ecology within a decomposing corpse system and suggest that microbial community data can be developed into a forensic tool for estimating PMI. DOI:http://dx.doi.org/10.7554/eLife.01104.001.

Alterations in the gut microbiota associated with HIV-1 infection.

Understanding gut microbiota alterations associated with HIV infection and factors that drive these alterations may help explain gut-linked diseases prevalent with HIV. 16S rRNA sequencing of feces from HIV-infected individuals revealed that HIV infection is associated with highly characteristic gut community changes, and antiretroviral therapy does not consistently restore the microbiota to an HIV-negative state. Despite the chronic gut inflammation characteristic of HIV infection, the associated microbiota showed limited similarity with other inflammatory states and instead showed increased, rather than decreased, diversity. Meta-analysis revealed that the microbiota of HIV-infected individuals in the U.S. was most similar to a Prevotella-rich community composition typically observed in healthy individuals in agrarian cultures of Malawi and Venezuela and related to that of U.S. individuals with carbohydrate-rich, protein- and fat-poor diets. By evaluating innate and adaptive immune responses to lysates from bacteria that differ with HIV, we explore the functional drivers of these compositional differences.