Cold-chain free nucleic acid preservation using porous super-absorbent polymer (PSAP) beads to facilitate wastewater surveillance.

The instability of viral targets including SARS-CoV-2 in sewage is an important challenge in wastewater monitoring projects. The unrecognized interruptions in the 'cold-chain' transport from the sample collection to RNA quantification in the laboratory may undermine the accurate quantification of the virus. In this study, bovine serum albumin (BSA)-modified porous superabsorbent polymer (PSAP) beads were applied to absorb raw sewage samples as a simple method for viral RNA preservation. The preservation efficiency for SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA were examined during storage for 14 days at 4 °C or room temperature against the control (no beads applied). While a non-significant difference was observed at 4 °C (∼80 % retention for both control and PSAP-treated sewage), the reduction of SARS-CoV-2 RNA concentrations was significantly lower in sewage retrieved from PSAP beads (25-40 % reduction) compared to control (>60 % reduction) at room temperature. On the other hand, the recovery of PMMoV, known for its high persistence in raw sewage, from PSAP beads or controls were consistently above 85 %, regardless of the storage temperature. Our results demonstrate the applicability of PSAP beads to wastewater-based epidemiology (WBE) projects for preservation of SARS-CoV-2 RNA in sewage, especially in remote settings with no refrigeration capabilities.

Metagenomic insights into the impact of litter from poultry Concentrated Animal Feeding Operations (CAFOs) to adjacent soil and water microbial communities.

In recent decades, human food consumption has led to an increased demand for animal-based foods, particularly chicken meat production. The state of Georgia, USA is one of the top broiler chicken producers in the United States, where animals are raised in Concentrated Animal Feeding Operations (CAFOs). Without proper management, CAFOs could negatively impact the environment and become a public health risk as a source of water and air pollution and/or by spreading antimicrobial resistance genes. In this study, we used metagenome sequencing to investigate the impact of the application of the CAFO's litter on adjacent soils and downstream creek waters in terms of microbial diversity and antimicrobial resistance profile changes. Our data indicate that while a few microbial groups increased in abundance within a short period of time after litter application, these populations subsequently decreased to levels similar to those found prior to the litter application or to below the detection limit of our metagenome sequencing effort. Microbial taxonomic composition analyses, relative abundance of Metagenome-Assembled Genomes (MAGs) and detection of Antimicrobial Resistance Genes (ARGs) allow us to conclude that this practice of litter application had a negligible effect on the microbiome or resistome profile of these soils and nearby waterways, likely due to its dilution in the field and/or outcompetition by indigenous microbes, revealing a minimal impact of these poultry facilities on the natural microbial communities.

Metagenomic evaluation of the performance of passive Moore swabs for sewage monitoring relative to composite sampling over time resolved deployments.

Moore swabs have re-emerged as a versatile tool in the field of wastewater-based epidemiology during the COVID-19 pandemic and offer unique advantages for monitoring pathogens in sewer systems, especially at the neighborhood-level. However, whether Moore swabs provide comparable results to more commonly used composite samples remains to be rigorously tested including the optimal duration of Moore swab deployment. This study provides new insights into these issues by comparing the results from Moore swab samples to those of paired composite samples collected from the same sewer lines continuously over six to seventy-two hours post-deployment, during low COVID-19 prevalence periods. Our results show that Moore swabs accumulated approximately 10-fold higher PMMoV concentrations (on a basis of mL of Moore swab squeezed filtrate to mL of composite sewage) and showed comparable trends in terms of bacterial species abundance when compared to composite samples. Moore swabs also generally captured higher SARS-CoV-2 N1/N2 RNA concentrations than composite samples. Moore swabs showed comparable trends in terms of abundance dynamics of the sewage microbiome to composite samples and variable signs of saturation over time that were site and/or microbial population-specific. Based on our dual ddRT-PCR and shotgun metagenomic approach, we find that Moore swabs at our sites were optimally deployed for 6 h at a time at two sites.

A user's guide to the bioinformatic analysis of shotgun metagenomic sequence data for bacterial pathogen detection.

Metagenomics, i.e., shotgun sequencing of the total microbial community DNA from a sample, has become a mature technique but its application to pathogen detection in clinical, environmental, and food samples is far from common or standardized. In this review, we summarize ongoing developments in metagenomic sequence analysis that facilitate its wider application to pathogen detection. We examine theoretical frameworks for estimating the limit of detection for a particular level of sequencing effort, current approaches for achieving species and strain analytical resolution, and discuss some relevant modern tools for these tasks. While these recent advances are significant and establish metagenomics as a powerful tool to provide insights not easily attained by culture-based approaches, metagenomics is unlikely to emerge as a widespread, routine monitoring tool in the near future due to its inherently high detection limits, cost, and inability to easily distinguish between viable and non-viable cells. Instead, metagenomics seems best poised for applications involving special circumstances otherwise challenging for culture-based and molecular (e.g., PCR-based) approaches such as the de novo detection of novel pathogens, cases of co-infection by more than one pathogen, and situations where it is important to assess the genomic composition of the pathogenic population(s) and/or its impact on the indigenous microbiome.

Fecal microbiota transplantation promotes reduction of antimicrobial resistance by strain replacement.

Multidrug-resistant organism (MDRO) colonization is a fundamental challenge in antimicrobial resistance. Limited studies have shown that fecal microbiota transplantation (FMT) can reduce MDRO colonization, but its mechanisms are poorly understood. We conducted a randomized, controlled trial of FMT for MDRO decolonization in renal transplant recipients called PREMIX (NCT02922816). Eleven participants were enrolled and randomized 1:1 to FMT or an observation period followed by delayed FMT if stool cultures were MDRO positive at day 36. Participants who were MDRO positive after one FMT were treated with a second FMT. At last visit, eight of nine patients who completed all treatments were MDRO culture negative. FMT-treated participants had longer time to recurrent MDRO infection versus PREMIX-eligible controls who were not treated with FMT. Key taxa (Akkermansia muciniphila, Alistipes putredinis, Phocaeicola dorei, Phascolarctobacterium faecium, Alistipes species, Mesosutterella massiliensis, Barnesiella intestinihominis, and Faecalibacterium prausnitzii) from the single feces donor used in the study that engrafted in recipients and metabolites such as short-chain fatty acids and bile acids in FMT-responding participants uncovered leads for rational microbiome therapeutic and diagnostic development. Metagenomic analyses revealed a previously unobserved mechanism of MDRO eradication by conspecific strain competition in an FMT-treated subset. Susceptible Enterobacterales strains that replaced baseline extended-spectrum ß-lactamase-producing strains were not detectable in donor microbiota manufactured as FMT doses but in one case were detectable in the recipient before FMT. These data suggest that FMT may provide a path to exploit strain competition to reduce MDRO colonization.

Distribution of Escherichia coli Pathotypes along an Urban-Rural Gradient in Ecuador.

Diarrheal diseases are a leading cause of mortality and morbidity in low- and middle-income countries. Diarrhea is associated with a wide array of etiological agents including bacterial, viral, and parasitic enteropathogens. Previous studies have captured between- but not within-country heterogeneities in enteropathogen prevalence and severity. We conducted a case-control study of diarrhea to understand how rates and outcomes of infection with diarrheagenic pathotypes of Escherichia coli vary across an urban-rural gradient in four sites in Ecuador. We found variability by site in enteropathogen prevalence and infection outcomes. Any pathogenic E. coli infection, coinfections, diffuse adherent E. coli (DAEC), enteroinvasive E. coli (EIEC), and rotavirus were significantly associated with acute diarrhea. DAEC was the most common pathotype overall and was more frequently associated with disease in urban areas. Enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) were more common in rural areas. ETEC was only associated with diarrhea in one site. Phylogenetic analysis revealed that associations with disease were not driven by any single clonal complex. Higher levels of antibiotic resistance were detected in rural areas. Enteropathogen prevalence, virulence, and antibiotic resistance patterns vary substantially by site within Ecuador. The variations in E. coli pathotype prevalence and virulence in this study have important implications for control strategies by context and demonstrate the importance of capturing within-country differences in enteropathogen disease dynamics.

Parallel deployment of passive and composite samplers for surveillance and variant profiling of SARS-CoV-2 in sewage.

Wastewater-based epidemiology during the COVID-19 pandemic has proven useful for public health decision-making but is often hampered by sampling methodology constraints, particularly at the building- or neighborhood-level. Time-weighted composite samples are commonly used; however, autosamplers are expensive and can be affected by intermittent flows in sub-sewershed contexts. In this study, we compared time-weighted composite, grab, and passive sampling via Moore swabs, at four locations across a college campus to understand the utility of passive sampling. After optimizing the methods for sample handling and processing for viral RNA extraction, we quantified SARS-CoV-2 N1 and N2, as well as a fecal strength indicator, PMMoV, by ddRT-PCR and applied tiled amplicon sequencing of the SARS-CoV-2 genome. Passive samples compared favorably with composite samples in our study area: for samples collected concurrently, 42 % of the samples agreed between Moore swab and composite samples and 58 % of the samples were positive for SARS-CoV-2 using Moore swabs while composite samples were below the limit of detection. Variant profiles from Moore swabs showed a shift from variant BA.1 to BA.2, consistent with in-person saliva samples. These data have implications for the broader implementation of sewage surveillance without advanced sampling technologies and for the utilization of passive sampling approaches for other emerging pathogens.

Quinone Moieties Link the Microbial Respiration of Natural Organic Matter to the Chemical Reduction of Diverse Nitroaromatic Compounds.

Insensitive munitions compounds (IMCs) are emerging nitroaromatic contaminants developed by the military as safer-to-handle alternatives to conventional explosives. Biotransformation of nitroaromatics via microbial respiration has only been reported for a limited number of substrates. Important soil microorganisms can respire natural organic matter (NOM) by reducing its quinone moieties to hydroquinones. Thus, we investigated the NOM respiration combined with the abiotic reduction of nitroaromatics by the hydroquinones formed. First, we established nitroaromatic concentration ranges that were nontoxic to the quinone respiration. Then, an enrichment culture dominated by Geobacter anodireducens could indirectly reduce a broad array of nitroaromatics by first respiring NOM components or the NOM surrogate anthraquinone-2,6-disulfonate (AQDS). Without quinones, no nitroaromatic tested was reduced except for the IMC 3-nitro-1,2,4-triazol-5-one (NTO). Thus, the quinone respiration expanded the spectrum of nitroaromatics susceptible to transformation. The system functioned with very low quinone concentrations because NOM was recycled by the nitroaromatic reduction. A metatranscriptomic analysis demonstrated that the microorganisms obtained energy from quinone or NTO reduction since respiratory genes were upregulated when AQDS or NTO was the electron acceptor. The results indicated microbial NOM respiration sustained by the nitroaromatic-dependent cycling of quinones. This process can be applied as a nitroaromatic remediation strategy, provided that a quinone pool is available for microorganisms.

Metagenomics indicate that public health risk may be higher from flooding following dry versus rainy periods.

Urban floodwater could lead to significant risk for public and environmental health from mobilization of microbial pathogens and overflow of wastewater treatment systems. Here, we attempted to assess this risk by obtaining metagenomic profiles of antibiotic resistance genes (ARGs), virulence factors (VFs) and pathogens present in floodwater samples collected in urban Atlanta, GA that were categorized in two distinct groups: floods that occurred after periods of drought and those after regular (seasonal) rain events. Even though no major (known) pathogens were present at the limit of detection of our sequencing effort (~3 Gbp/sample), we observed that floodwaters after drought showed a 2.5-fold higher abundance of both ARGs and VFs compared to floodwater after rainy days. These differences were mainly derived by several novel species of the Pseudomonas genus, which were more dominant in the former versus the latter samples and carried several genes to cope with osmotic stress in addition to ARGs and VFs. These results revealed that there are previously undescribed species that become mobilized after flooding events in the Southeast US urban settings and could represent an increased public health risk, especially after periods of drought, which warrants further attention.

Toward shotgun metagenomic approaches for microbial source tracking sewage spills based on laboratory mesocosms.

Little is known about the genomic diversity of the microbial communities associated with raw municipal wastewater (sewage), including whether microbial populations specific to sewage exist and how such populations could be used to improve source attribution and apportioning in contaminated waters. Herein, we used the influent of three wastewater treatment plants in Atlanta, Georgia (USA) to perturb laboratory freshwater mesocosms, simulating sewage contamination events, and followed these mesocosms with shotgun metagenomics over a 7-day observational period. We describe 15 abundant non-redundant bacterial metagenome-assembled genomes (MAGs) ubiquitous within all sewage inocula yet absent from the unperturbed freshwater control at our analytical limit of detection. Tracking the dynamics of the populations represented by these MAGs revealed varied decay kinetics, depending on (inferred) phenotypes, e.g., anaerobes decayed faster than aerobes under the well-aerated incubation conditions. Notably, a portion of these populations showed decay patterns similar to those of common markers, Enterococcus and HF183. Despite the apparent decay of these populations, the abundance of ß-lactamase encoding genes remained high throughout incubation relative to the control. Lastly, we constructed genomic libraries representing several different fecal sources and outline a bioinformatic approach which leverages these libraries for identifying and apportioning contamination signal among multiple probable sources using shotgun metagenomic data.

Higher pathogen load in children from Mozambique vs. USA revealed by comparative fecal microbiome profiling.

The infant gut microbiome has lifelong implications on health and immunity but there is still limited understanding of the microbiome differences and similarities between children in low- and middle-income countries (LMICs) vs. high-income countries (HICs). Here, we describe and compare the microbiome profile of children aged under 48 months in two urban areas: Maputo, Mozambique and Atlanta, USA using shotgun metagenomics. The gut microbiome of American children showed distinct development, characterized by higher alpha diversity after infancy, compared to the same age group of African children, and the microbiomes clustered separately based on geographic location or age. The abundances of antibiotic resistance genes (ARGs) and virulence factors (VFs) were significantly higher in Maputo children, driven primarily by several primary and opportunistic pathogens. Most notably, about 50% of Maputo children under the age of two were positive for enterotoxigenic (ETEC) and typical enteropathogenic (EPEC) Escherichia coli diagnostic genes while none of the Atlanta age-matched children showed such a positive signal. In contrast, commensal species such as Phocaeicola vulgatus and Bacteroides caccae were more abundant in Atlanta, potentially reflecting diets rich in animal protein and susceptibility to inflammatory diseases. Overall, our results suggest that the different environments characterizing the two cities have significant, distinctive signatures on the microbiota of children and its development over time. Lack of safe water, sanitation, and hygiene (WASH) conditions and/or unsafe food sources may explain the higher enteric pathogen load among children in Maputo.

Toward quantifying the adaptive role of bacterial pangenomes during environmental perturbations.

Metagenomic surveys have revealed that natural microbial communities are predominantly composed of sequence-discrete, species-like populations but the genetic and/or ecological processes that maintain such populations remain speculative, limiting our understanding of population speciation and adaptation to perturbations. To address this knowledge gap, we sequenced 112 Salinibacter ruber isolates and 12 companion metagenomes from four adjacent saltern ponds in Mallorca, Spain that were experimentally manipulated to dramatically alter salinity and light intensity, the two major drivers of this ecosystem. Our analyses showed that the pangenome of the local Sal. ruber population is open and similar in size (~15,000 genes) to that of randomly sampled Escherichia coli genomes. While most of the accessory (noncore) genes were isolate-specific and showed low in situ abundances based on the metagenomes compared to the core genes, indicating that they were functionally unimportant and/or transient, 3.5% of them became abundant when salinity (but not light) conditions changed and encoded for functions related to osmoregulation. Nonetheless, the ecological advantage of these genes, while significant, was apparently not strong enough to purge diversity within the population. Collectively, our results provide an explanation for how this immense intrapopulation gene diversity is maintained, which has implications for the prokaryotic species concept.

Microbial diversity in a military impacted lagoon (Vieques, Puerto Rico) and description of "Candidatus Biekeibacterium resiliens" gen. nov., sp. nov. comprising a new bacterial family.

The Anones Lagoon, located in the Island Municipality of Vieques, Puerto Rico (PR), received extensive bombing by the US Navy during military exercises for decades until 2003 when military activities ceased. Here, we employed shotgun metagenomic sequencing to investigate how microbial communities responded to pollution by heavy metals and explosives at this lagoon. Sediment samples (0-5 cm) from Anones were collected in 2005 and 2014 and compared to samples from two reference lagoons, i.e., Guaniquilla, Cabo Rojo (a natural reserve) and Condado, San Juan (PR's capital city). Consistent with low anthropogenic inputs, Guaniquilla exhibited the highest degree of diversity with a lower frequency of genes related to xenobiotics metabolism between the three lagoons. Notably, a clear shift was observed in Anones, with Euryarchaeota becoming enriched (9% of total) and a concomitant increase in community diversity, by about one order of magnitude, after almost 10 years without bombing activities. In contrast, genes associated with explosives biodegradation and heavy metal transformation significantly decreased in abundance in Anones 2014 (by 91.5%). Five unique metagenome-assembled genomes (MAGs) were recovered from the Anones 2005 sample that encoded genetic determinants implicated in biodegradation of contaminants, and we propose to name one of them as "Candidatus Biekeibacterium resiliens" gen. nov., sp. nov. within the Gammaproteobacteria class. Collectively, these results provide new insights into the natural attenuation of explosive contaminants by the benthic microbial communities of the Anones lagoon and provide a reference point for assessing other similarly impacted sites and associated bioremediation efforts.

A novel, divergent alkane monooxygenase (alkB) clade involved in crude oil biodegradation.

Alkanes are ubiquitous in marine ecosystems and originate from diverse sources ranging from natural oil seeps to anthropogenic inputs and biogenic production by cyanobacteria. Enzymes that degrade cyanobacterial alkanes (typically C15-C17 compounds) such as the alkane monooxygenase (AlkB) are widespread, but it remains unclear whether or not AlkB variants exist that specialize in degradation of crude oil from natural or accidental spills, a much more complex mixture of long-chain hydrocarbons. In the present study, large-scale analysis of available metagenomic and genomic data from the Gulf of Mexico (GoM) oil spill revealed a novel, divergent AlkB clade recovered from genomes with no cultured representatives that was dramatically increased in abundance in crude-oil impacted ecosystems. In contrast, the AlkB clades associated with biotransformation of cyanobacterial alkanes belonged to 'canonical' or hydrocarbonoclastic clades, and based on metatranscriptomics data and compared to the novel clade, were much more weakly expressed during crude oil biodegradation in laboratory mesocosms. The absence of this divergent AlkB clade in metagenomes of uncontaminated samples from the global ocean survey but not from the GoM as well as its frequent horizontal gene transfer indicated a priming effect of the Gulf for crude oil biodegradation likely driven by natural oil seeps.

Transcriptomic and rRNA:rDNA Signatures of Environmental versus Enteric Enterococcus faecalis Isolates under Oligotrophic Freshwater Conditions.

The use of enterococci as a fecal indicator bacterial group for public health risk assessment has been brought into question by recent studies showing that "naturalized" populations of Enterococcus faecalis exist in the extraenteric environment. The extent to which these naturalized E. faecalis organisms can confound water quality monitoring is unclear. To determine if strains isolated from different habitats display different survival strategies and responses, we compared the decay patterns of three E. faecalis isolates from the natural environment (environmental strains) against three human gut isolates (enteric strains) in laboratory mesocosms that simulate an oligotrophic, aerobic freshwater environment. Our results showed similar overall decay rates between enteric and environmental isolates based on viable plate and quantitative PCR (qPCR) counts. However, the enteric isolates exhibited a spike in copy number ratios of 16S rRNA gene transcripts to 16S rRNA gene DNA copies (rRNA:rDNA ratios) between days 1 and 3 of the mesocosm incubations that was not observed in environmental isolates, which could indicate a different stress response. Nevertheless, there was no strong evidence of differential gene expression between environmental and enteric isolates related to habitat adaptation in the accompanying mesocosm metatranscriptomes. Overall, our results provide novel information on how rRNA levels may vary over different growth conditions (e.g., standard lab versus oligotrophic) for this important indicator bacteria. We also observed some evidence for habitat adaptation in E. faecalis; however, this adaptation may not be substantial or consistent enough for integration in water quality monitoring. IMPORTANCE Enterococci are commonly used worldwide to monitor environmental fecal contamination and public health risk for waterborne diseases. However, closely related enterococci strains adapted to living in the extraenteric environment may represent a lower public health risk and confound water quality estimates. We developed an rRNA:rDNA viability assay for E. faecalis (a predominant species within this fecal group) and tested it against both enteric and environmental isolates in freshwater mesocosms to assess whether this approach can serve as a more sensitive water quality monitoring tool. We were unable to reliably distinguish the different isolate types using this assay under the conditions tested; thus, environmental strains should continue to be counted during routine water monitoring. However, this assay could be useful for distinguishing more recent (i.e., higher-risk) fecal pollution because rRNA levels significantly decreased after 1 week in all isolates.

The influence of alfalfa-switchgrass intercropping on microbial community structure and function.

The use of nitrogen fertilizer on bioenergy crops such as switchgrass results in increased costs, nitrogen leaching and emissions of N2 O, a potent greenhouse gas. Intercropping with nitrogen-fixing alfalfa has been proposed as an environmentally sustainable alternative, but the effects of synthetic fertilizer versus intercropping on soil microbial community functionality remain uncharacterized. We analysed 24 metagenomes from the upper soil layer of agricultural fields from Prosser, WA over two growing seasons and representing three agricultural practices: unfertilized switchgrass (control), fertilized switchgrass and switchgrass intercropped with alfalfa. The synthetic fertilization and intercropping did not result in major shifts of microbial community taxonomic and functional composition compared with the control plots, but a few significant changes were noted. Most notably, mycorrhizal fungi, ammonia-oxidizing archaea and bacteria increased in abundance with intercropping and fertilization. However, only betaproteobacterial ammonia-oxidizing bacteria abundance in fertilized plots significantly correlated to N2 O emission and companion qPCR data. Collectively, a short period of intercropping elicits minor but significant changes in the soil microbial community toward nitrogen preservation and that intercropping may be a viable alternative to synthetic fertilization.

Beach sand oil spills select for generalist microbial populations.

The specialization-disturbance hypothesis predicts that, in the event of a disturbance, generalists are favored, while specialists are selected against. This hypothesis has not been rigorously tested in microbial systems and it remains unclear to what extent it could explain microbial community succession patterns following perturbations. Previous field observations of Pensacola Beach sands that were impacted by the Deepwater Horizon (DWH) oil spill provided evidence in support of the specialization-disturbance hypothesis. However, ecological drift as well as uncounted environmental fluctuations (e.g., storms) could not be ruled out as confounding factors driving these field results. In this study, the specialization-disturbance hypothesis was tested on beach sands, disturbed by DWH crude oil, ex situ in closed laboratory advective-flow chambers that mimic in situ conditions in saturated beach sediments. The chambers were inoculated with weathered DWH oil and unamended chambers served as controls. The time series of shotgun metagenomic and 16S rRNA gene amplicon sequence data from a two-month long incubation showed that functional diversity significantly increased while taxonomic diversity significantly declined, indicating a decrease in specialist taxa. Thus, results from this laboratory study corroborate field observations, providing verification that the specialization-disturbance hypothesis can explain microbial succession patterns in crude oil impacted beach sands.

Gut Microbiome Changes with Acute Diarrheal Disease in Urban Versus Rural Settings in Northern Ecuador.

Previous studies have reported lower fecal bacterial diversity in urban populations compared with those living in rural settings. However, most of these studies compare geographically distant populations from different countries and even continents. The extent of differences in the gut microbiome in adjacent rural versus urban populations, and the role of such differences, if any, during enteric infections remain poorly understood. To provide new insights into these issues, we sampled the gut microbiome of young children with and without acute diarrheal disease (ADD) living in rural and urban areas in northern Ecuador. Shotgun metagenomic analyses of non-ADD samples revealed small but significant differences in the abundance of microbial taxa, including a greater abundance of Prevotella and a lower abundance of Bacteroides and Alistipes in rural populations. Greater and more significant shifts in taxon abundance, metabolic pathway abundance, and diversity were observed between ADD and non-ADD status when comparing urban to rural sites (Welch's t-test, P < 0.05). Collectively our data show substantial functional, diversity, and taxonomic shifts in the gut microbiome of urban populations with ADD, supporting the idea that the microbiome of rural populations may be more resilient to ADD episodes.

Metagenomic Characterization of Soil Microbial Communities in the Luquillo Experimental Forest (Puerto Rico) and Implications for Nitrogen Cycling.

The phylogenetic and functional diversities of microbial communities in tropical rainforests and how these differ from those of temperate communities remain poorly described but are directly related to the increased fluxes of greenhouse gases such as nitrous oxide (N2O) from the tropics. Toward closing these knowledge gaps, we analyzed replicated shotgun metagenomes representing distinct life zones and an elevation gradient from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico. These soils had a distinct microbial community composition and lower species diversity compared to those of temperate grasslands or agricultural soils. In contrast to the overall distinct community composition, the relative abundances and nucleotide sequences of N2O reductases (nosZ) were highly similar between tropical forest and temperate soils. However, respiratory NO reductase (norB) was 2-fold more abundant in the tropical soils, which might be relatable to their greater N2O emissions. Nitrogen fixation (nifH) also showed higher relative abundance in rainforest than in temperate soils, i.e., 20% versus 0.1 to 0.3% of bacterial genomes in each soil type harbored the gene, respectively. Finally, unlike temperate soils, LEF soils showed little stratification with depth in the first 0 to 30 cm, with ∼45% of community composition differences explained solely by location. Collectively, these results advance our understanding of spatial diversity and metabolic repertoire of tropical rainforest soil communities and should facilitate future ecological studies of these ecosystems. IMPORTANCE Tropical rainforests are the largest terrestrial sinks of atmospheric CO2 and the largest natural source of N2O emissions, two greenhouse gases that are critical for the climate. The microbial communities of rainforest soils that directly or indirectly, through affecting plant growth, contribute to these fluxes remain poorly described by cultured-independent methods. To close this knowledge gap, the present study applied shotgun metagenomics to samples selected from three distinct life zones within the Puerto Rico rainforest. The results advance our understanding of microbial community diversity in rainforest soils and should facilitate future studies of natural or manipulated perturbations of these critical ecosystems.

Bacteria Make a Living Breathing the Nitroheterocyclic Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO).

The nitroheterocyclic 3-nitro-1,2,4-triazol-5-one (NTO) is an ingredient of insensitive explosives increasingly used by the military, becoming an emergent environmental pollutant. Cometabolic biotransformation of NTO occurs in mixed microbial cultures in soils and sludges with excess electron-donating substrates. Herein, we present the unusual energy-yielding metabolic process of NTO respiration, in which the NTO reduction to 3-amino-1,2,4-triazol-5-one (ATO) is linked to the anoxic acetate oxidation to CO2 by a culture enriched from municipal anaerobic digester sludge. Cell growth was observed simultaneously with NTO reduction, whereas the culture was unable to grow in the presence of acetate only. Extremely low concentrations (0.06 mg L-1) of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited NTO reduction, indicating that the process was linked to respiration. The ultimate evidence of NTO respiration was adenosine triphosphate production due to simultaneous exposure to NTO and acetate. Metagenome sequencing revealed that the main microorganisms (and relative abundances) were Geobacter anodireducens (89.3%) and Thauera sp. (5.5%). This study is the first description of a nitroheterocyclic compound being reduced by anaerobic respiration, shedding light on creative microbial processes that enable bacteria to make a living reducing NTO.