Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses.

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Soil Properties and Moisture Synergistically Influence Nontuberculous Mycobacterial Prevalence in Natural Environments of Hawai'i.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that cause chronic pulmonary disease (PD). NTM infections are thought to be acquired from the environment; however, the basal environmental factors that drive and sustain NTM prevalence are not well understood. The highest prevalence of NTM PD cases in the United States is reported from Hawai'i, which is unique in its climate and soil composition, providing an opportunity to investigate the environmental drivers of NTM prevalence. We used microbiological sampling and spatial logistic regression complemented with fine-scale soil mineralogy to model the probability of NTM presence across the natural landscape of Hawai'i. Over 7 years, we collected and microbiologically cultured 771 samples from 422 geographic sites in natural areas across the Hawaiian Islands for the presence of NTM. NTM were detected in 210 of these samples (27%), with Mycobacterium abscessus being the most frequently isolated species. The probability of NTM presence was highest in expansive soils (those that swell with water) with a high water balance (>1-m difference between rainfall and evapotranspiration) and rich in Fe-oxides/hydroxides. We observed a positive association between NTM presence and iron in wet soils, supporting past studies, but no such association in dry soils. High soil-water balance may facilitate underground movement of NTM into the aquifer system, potentially compounded by expansive capabilities allowing crack formation under drought conditions, representing further possible avenues for aquifer infiltration. These results suggest both precipitation and soil properties are mechanisms by which surface NTM may reach the human water supply. IMPORTANCE Nontuberculous mycobacteria (NTM) are ubiquitous in the environment, being found commonly in soils and natural bodies of freshwater. However, little is known about the environmental niches of NTM and how they relate to NTM prevalence in homes and other human-dominated areas. To characterize NTM environmental associations, we collected and cultured 771 samples from 422 geographic sites in natural areas across Hawai'i, the U.S. state with the highest prevalence of NTM pulmonary disease. We show that the environmental niches of NTM are most associated with highly expansive, moist soils containing high levels of iron oxides/hydroxides. Understanding the factors associated with NTM presence in the natural environment will be crucial for identifying potential mechanisms and risk factors associated with NTM infiltration into water supplies, which are ultimately piped into homes where most exposure risk is thought to occur.

Comparative survival of environmental and clinical Mycobacterium abscessus isolates in a variety of diverse host cells.

AIMS: Mycobacterium abscessus subsp. abscessus (MABS) is an emerging, opportunistic pathogen found globally in freshwater biofilms and soil. Typically, isolates are treated as a uniform group of organisms and very little is known about their comparative survival in healthy host cells. We posit that environmentally- and clinically derived isolates, show differential infectivity in immune cells and resistance to innate defenses. METHODS AND RESULTS: Six MABS isolates were tested including three water biofilm/soil and three sputum-derived isolates. A clinical MABS type strain and an environmental isolate of Arthrobacter were also included. MABS counts were significantly higher compared to Arthrobacter after co-culture with Acanthamoeba lenticulata, BEAS-2B epithelial cells, alveolar macrophages and the THP-1 macrophage cell line. A rough sputum-derived MABS isolate emerged as an isolate with higher virulence compared to others tested, as both a pellicle and cord former, survivor in the human cell models tested, inducer of high and prolonged production of pro-inflammatory cytokines, and the capacity to evade LL-37. CONCLUSIONS: Findings support intraspecies variation between MABS isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate subversion of host immune defenses by environmental and clinical MABS isolates is nuanced and maybe isolate dependent, providing new information regarding the pathogenesis of NTM infections.

Assessment of Soil Features on the Growth of Environmental Nontuberculous Mycobacterial Isolates from Hawai'i.

Environmental nontuberculous mycobacteria (NTM), with the potential to cause opportunistic lung infections, can reside in soil. This might be particularly relevant in Hawai'i, a geographic hot spot for NTM infections and whose soil composition differs from many other areas of the world. Soil components are likely to contribute to NTM prevalence in certain niches as food sources or attachment scaffolds, but the particular types of soils, clays, and minerals that impact NTM growth are not well-defined. Hawai'i soil and chemically weathered rock (saprolite) samples were examined to characterize the microbiome and quantify 11 mineralogical features as well as soil pH. Machine learning methods were applied to identify important soil features influencing the presence of NTM. Next, these features were directly tested in vitro by incubating synthetic clays and minerals in the presence of Mycobacteroides abscessus and Mycobacterium chimaera isolates recovered from the Hawai'i environment, and changes in bacterial growth were determined. Of the components examined, synthetic gibbsite, a mineral form of aluminum hydroxide, inhibited the growth of both M. abscessus and M. chimaera, while other minerals tested showed differential effects on each species. For example, M. abscessus (but not M. chimaera) growth was significantly higher in the presence of hematite, an iron oxide mineral. In contrast, M. chimaera (but not M. abscessus) counts were significantly reduced in the presence of birnessite, a manganese-containing mineral. These studies shed new light on the mineralogic features that promote or inhibit the presence of Hawai'i NTM in Hawai'i soil.IMPORTANCE Globally and in the United States, the prevalence of NTM pulmonary disease-a potentially life-threatening but underdiagnosed chronic illness-is prominently rising. While NTM are ubiquitous in the environment, including in soil, the specific soil components that promote or inhibit NTM growth have not been elucidated. We hypothesized that NTM culture-positive soil contains minerals that promote NTM growth in vitro Because Hawai'i is a hot spot for NTM and a unique geographic archipelago, we examined the composition of Hawai'i soil and identified individual clay, iron, and manganese minerals associated with NTM. Next, individual components were evaluated for their ability to directly modulate NTM growth in culture. In general, gibbsite and some manganese oxides were shown to decrease NTM, whereas iron-containing minerals were associated with higher NTM counts. These data provide new information to guide future analyses of soil-associated factors impacting persistence of these soil bacteria.

Hawaiian Volcanic Ash, an Airborne Fomite for Nontuberculous Mycobacteria.

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that can cause chronic lung disease. Within the U.S., Hawai'i shows the highest prevalence rates of NTM lung infections. Here, we investigated a potential role for active volcanism at the Kilauea Volcano located on Hawai'i Island in promoting NTM growth and diversity. We recovered NTM that are known to cause lung disease from plumbing biofilms and soils collected from the Kilauea environment. We also discovered viable Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium intracellulare subsp. chimaera on volcanic ash collected during the 2018 Kilauea eruption. Analysis of soil samples showed that NTM prevalence is positively associated with bulk content of phosphorus, sulfur, and total organic carbon. In growth assays, we showed that phosphorus utilization is essential for proliferation of Kilauea-derived NTM, and demonstrate that NTM cultured with volcanic ash adhere to ash surfaces and remain viable. Ambient dust collected on O'ahu concurrent with the 2018 eruption contained abundant fresh volcanic glass, suggestive of inter-island ash transport. Phylogenomic analyses using whole genome sequencing revealed that Kilauea-derived NTM are genetically similar to respiratory isolates identified on other Hawaiian Islands. Consequently, we posit that volcanic eruptions could redistribute environmental microorganisms over large scales. While additional studies are needed to confirm a direct role of ash in NTM dispersal, our results suggest that volcanic particulates harbor and can redistribute NTM and should therefore be studied as a fomite for these burgeoning, environmentally acquired respiratory infections.

Genomic and microbiological analyses of iron acquisition pathways among respiratory and environmental nontuberculous mycobacteria from Hawai'i.

As environmental opportunistic pathogens, nontuberculous mycobacteria (NTM) can cause severe and difficult to treat pulmonary disease. In the United States, Hawai'i has the highest prevalence of infection. Rapid growing mycobacteria (RGM) such as Mycobacterium abscessus and M. porcinum and the slow growing mycobacteria (SGM) including M. intracellulare subspecies chimaera are common environmental NTM species and subspecies in Hawai'i. Although iron acquisition is an essential process of many microorganisms, iron acquisition via siderophores among the NTM is not well-characterized. In this study, we apply genomic and microbiological methodologies to better understand iron acquisition via siderophores for environmental and respiratory isolates of M. abscessus, M. porcinum, and M. intracellulare subspecies chimaera from Hawai'i. Siderophore synthesis and transport genes, including mycobactin (mbt), mmpL/S, and esx-3 were compared among 47 reference isolates, 29 respiratory isolates, and 23 environmental Hawai'i isolates. Among all reference isolates examined, respiratory isolates showed significantly more siderophore pertinent genes compared to environmental isolates. Among the Hawai'i isolates, RGM M. abscessus and M. porcinum had significantly less esx-3 and mbt genes compared to SGM M. chimaera when stratified by growth classification. However, no significant differences were observed between the species when grown on low iron culture agar or siderophore production by the chrome azurol S (CAS) assay in vitro. These results indicate the complex mechanisms involved in iron sequestration and siderophore activity among diverse NTM species.

Immunological and metabolic characterization of environmental Mycobacterium chimaera infection in a murine model.

Mycobacterium chimaera causes pulmonary disease, but little is known of gradations in isolate virulence. Previously, 17 M. chimaera isolates were screened for survival in THP1 macrophages. "M. chimaera 1" was categorized as "more virulent" because it showed the greatest survival in macrophages, whereas "M. chimaera 2" was categorized as "less virulent" with reduced survival. Herein, we infected C3HeB/FeJ mice to compare the in vivo immune responses to M. chimaera 1 and 2. Unlike macrophages, significantly lower M. chimaera 1 counts were recovered from mouse lung tissue and BAL cells with less lung histopathologic changes compared to M. chimaera 2. Compared to M. chimaera 2, significantly more IL-1ß, IL-6, and TNFα was produced early after M. chimaera 1 infection. LC-MS metabolomics analyses of BAL fluid revealed divergence in sphingolipid, phospholipid metabolism between M. chimaera 1 versus M. chimaera 2 mice. From pan-GWAS analyses, virulence and organizing DNA/molecular structure genes were associated with more virulent M. chimaera isolates. Vigorous lung-specific immune responses to M. chimaera 1 may influence effective bacterial control, but for a different isolate M. chimaera 2, subvert immune control. Continued studies of the gradations in virulence among the same NTM species will advance our understanding of NTM pathogenesis.

Complete genome assembly of Hawai'i environmental nontuberculous mycobacteria reveals unexpected co-isolation with methylobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous environmental opportunistic pathogens that can cause chronic lung disease. Within the United States, Hawai'i has the highest incidence of NTM lung disease, though the precise reasons are yet to be fully elucidated. One possibility is the high prevalence of NTM in the Hawai'i environment acting as a potential reservoir for opportunistic NTM infections. Through our previous initiatives to collect and characterize NTM in Hawai'i, community scientists of Hawai'i have collected thousands of environmental samples for sequencing. Here, these community scientists were invited for the first time into a high school lab in O'ahu for a genomic sequencing workshop, where participants sequenced four of the collected isolate genomic samples using the Oxford Nanopore Technologies MinION sequencer. Participants generated high quality long read data that when combined with short read Illumina data yielded complete bacterial genomic assemblies suitable for in-depth analysis. The gene annotation analysis identified a suite of genes that might help NTM thrive in the Hawai'i environment. Further, we found evidence of co-occurring methylobacteria, revealed from the sequencing data, suggesting that in some cases methylobacteria and NTM may coexist in the same niche, challenging previously accepted paradigms. The sequencing efforts presented here generated novel insights regarding the potential survival strategies and microbial interactions of NTM in the geographic hot spot of Hawai'i. We highlight the contributions of community scientists and present an activity that can be reimplemented as a workshop or classroom activity by other research groups to engage their local communities.

Exposure Pathways of Nontuberculous Mycobacteria Through Soil, Streams, and Groundwater, Hawai'i, USA.

Although uncommon, nontuberculous mycobacterial (NTM) pulmonary infection in the Hawaiian Islands has a relatively high incidence and mortality compared to the mainland U.S. As a result, this study examines the possible geological and hydrological pathways by which NTM patients may become infected, including the environmental conditions that may favor growth and transport. Previously suggested infection routes include the inhalation of NTM attached to micro-droplets from infected home plumbing systems and aerosolized dust from garden soil. In this study, we evaluate the possible routes NTM may take from riparian environments, into groundwater, into public water supplies and then into homes. Because NTM are notoriously hydrophobic and prone to attach to surfaces, mineralogy, and surface chemistry of suspended sediment in streams, soils, and rock scrapings suggest that NTM may especially attach to Fe-oxides/hydroxides, and be transported as particles from losing streams to the aquifer on time-scales of minutes to days. Within the aquifer, flow models indicate that water may be drawn into production wells on time scales (months) that permit NTM to survive and enter domestic water supplies. These processes depend on the presence of interconnected fracture networks with sufficient aperture to preclude complete autofiltration. The common occurrence of NTM in and around streams, in addition to wells, implies that the natural and built environments are capable of introducing a source of NTM into domestic water supplies via groundwater withdrawals. This may produce a persistent source of NTM infection to individuals through the presence of NTM-laden biofilms in home plumbing.

Lower Recovery of Nontuberculous Mycobacteria from Outdoor Hawai'i Environmental Water Biofilms Compared to Indoor Samples.

Nontuberculous mycobacteria (NTM) are environmental organisms that can cause opportunistic pulmonary disease with species diversity showing significant regional variation. In the United States, Hawai'i shows the highest rate of NTM pulmonary disease. The need for improved understanding of NTM reservoirs led us to identify NTM from patient respiratory specimens and compare NTM diversity between outdoor and indoor locations in Hawai'i. A total of 545 water biofilm samples were collected from 357 unique locations across Kaua'i (n = 51), O'ahu (n = 202), Maui (n = 159), and Hawai'i Island (n = 133) and divided into outdoor (n = 179) or indoor (n = 366) categories. rpoB sequence analysis was used to determine NTM species and predictive modeling applied to develop NTM risk maps based on geographic characteristics between environments. M. chimaera was frequently identified from respiratory and environmental samples followed by M. chelonae and M. abscessus; yet significantly less NTM were consistently recovered from outdoor compared to indoor biofilms, as exemplified by showerhead biofilm samples. While the frequency of M. chimaera recovery was comparable between outdoor and indoor showerhead biofilms, phylogenetic analyses demonstrate similar rpoB gene sequences between all showerhead and respiratory M. chimaera isolates, supporting outdoor and indoor environments as possible sources for pulmonary M. chimaera infections.

Brought to you courtesy of the red, white, and blue-pigments of nontuberculous mycobacteria.

Pigments are chromophores naturally synthesized by animals, plants, and microorganisms, as well as produced synthetically for a wide variety of industries such as food, pharmaceuticals, and textiles. Bacteria produce various pigments including melanin, pyocyanin, bacteriochlorophyll, violacein, prodigiosin, and carotenoids that exert diverse biological activities as antioxidants and demonstrate anti-inflammatory, anti-cancer, and antimicrobial properties. Nontuberculous mycobacteria (NTM) include over 200 environmental and acid-fast species; some of which can cause opportunistic disease in humans. Early in the study of mycobacteriology, the vast majority of mycobacteria were not known to synthesize pigments, particularly NTM isolates of clinical significance such as the Mycobacterium avium complex (MAC) species. This paper reviews the overall understanding of microbial pigments, their applications, as well as highlights what is currently known about pigments produced by NTM, the circumstances that trigger their production, and their potential roles in NTM survival and virulence.