Metagenomic Sequencing of Multiple Soil Horizons and Sites in Close Vicinity Revealed Novel Secondary Metabolite Diversity.

Discovery of novel antibiotics is crucial for combating rapidly spreading antimicrobial resistance and new infectious diseases. Most of the clinically used antibiotics are natural products-secondary metabolites produced by soil microbes that can be cultured in the lab. Rediscovery of these secondary metabolites during discovery expeditions costs both time and resources. Metagenomics approaches can overcome this challenge by capturing both culturable and unculturable hidden microbial diversity. To be effective, such an approach should address questions like the following. Which sequencing method is better at capturing the microbial diversity and biosynthesis potential? What part of the soil should be sampled? Can patterns and correlations from such big-data explorations guide future novel natural product discovery surveys? Here, we address these questions by a paired amplicon and shotgun metagenomic sequencing survey of samples from soil horizons of multiple forest sites very close to each other. Metagenome mining identified numerous novel biosynthetic gene clusters (BGCs) and enzymatic domain sequences. Hybrid assembly of both long reads and short reads improved the metagenomic assembly and resulted in better BGC annotations. A higher percentage of novel domains was recovered from shotgun metagenome data sets than from amplicon data sets. Overall, in addition to revealing the biosynthetic potential of soil microbes, our results suggest the importance of sampling not only different soils but also their horizons to capture microbial and biosynthetic diversity and highlight the merits of metagenome sequencing methods. IMPORTANCE This study helped uncover the biosynthesis potential of forest soils via exploration of shotgun metagenome and amplicon sequencing methods and showed that both methods are needed to expose the full microbial diversity in soil. Based on our metagenome mining results, we suggest revising the historical strategy of sampling soils from far-flung places, as we found a significant number of novel and diverse BGCs and domains even in different soils that are very close to each other. Furthermore, sampling of different soil horizons can reveal the additional diversity that often remains hidden and is mainly caused by differences in environmental key parameters such as soil pH and nutrient content. This paired metagenomic survey identified diversity patterns and correlations, a step toward developing a rational approach for future natural product discovery surveys.

Comparative genomics reveals phylogenetic distribution patterns of secondary metabolites in Amycolatopsis species.

BACKGROUND: Genome mining tools have enabled us to predict biosynthetic gene clusters that might encode compounds with valuable functions for industrial and medical applications. With the continuously increasing number of genomes sequenced, we are confronted with an overwhelming number of predicted clusters. In order to guide the effective prioritization of biosynthetic gene clusters towards finding the most promising compounds, knowledge about diversity, phylogenetic relationships and distribution patterns of biosynthetic gene clusters is necessary. RESULTS: Here, we provide a comprehensive analysis of the model actinobacterial genus Amycolatopsis and its potential for the production of secondary metabolites. A phylogenetic characterization, together with a pan-genome analysis showed that within this highly diverse genus, four major lineages could be distinguished which differed in their potential to produce secondary metabolites. Furthermore, we were able to distinguish gene cluster families whose distribution correlated with phylogeny, indicating that vertical gene transfer plays a major role in the evolution of secondary metabolite gene clusters. Still, the vast majority of the diverse biosynthetic gene clusters were derived from clusters unique to the genus, and also unique in comparison to a database of known compounds. Our study on the locations of biosynthetic gene clusters in the genomes of Amycolatopsis' strains showed that clusters acquired by horizontal gene transfer tend to be incorporated into non-conserved regions of the genome thereby allowing us to distinguish core and hypervariable regions in Amycolatopsis genomes. CONCLUSIONS: Using a comparative genomics approach, it was possible to determine the potential of the genus Amycolatopsis to produce a huge diversity of secondary metabolites. Furthermore, the analysis demonstrates that horizontal and vertical gene transfer play an important role in the acquisition and maintenance of valuable secondary metabolites. Our results cast light on the interconnections between secondary metabolite gene clusters and provide a way to prioritize biosynthetic pathways in the search and discovery of novel compounds.

Quantification of waterborne pathogens and associated health risks in urban water.

Citizens are exposed to microbial hazards in urban waters. To quantify health risks associated with this exposure, pathogen concentrations in an urban river, lake, rainwater sedimentation pond, a pond in a park, and a wadi, were assessed. E. coli concentrations were variable in all locations, with mean values ranging between 1.2 × 10(2) (lake) and 1.7 × 10(4) (sedimentation pond) cfu (colony forming units)/100 mL. High concentrations of Campylobacter were found, being the lowest in the lake (4.2 × 10(1) gc (genomic copies)/L) and the highest in the wadi (1.7 × 10(4) gc/L). Cryptosporidium was not found in any sample. Low levels of adenovirus 40/41 were found in some samples in the river (1.8 × 10(1) gc/L) and lake (7.2 × 10° gc/L), indicating human fecal contamination. Legionella pneumophila was found in the sedimentation pond, with higher concentrations after rain events (1.3 × 10(2) gc/L). Cyanochlorophyll-a was found in the lake (7.0 × 10(-1) µg/L), the sedimentation pond (1.1 × 10° µg/L), and the pond in the park (2.9 × 10(1) µg/L), where low levels of microcystin were found (2.1 × 10° µg/L). Campylobacter data were used to estimate gastrointestinal risks from recreational exposure. This revealed risks above the annual disease incidence of campylobacteriosis in The Netherlands, being highest in the wadi and river. Measures are proposed to reduce the health risks.

Microbial health risks associated with exposure to stormwater in a water plaza.

Climate change scenarios predict an increase of intense rainfall events in summer in Western Europe. Current urban drainage systems cannot cope with such intense precipitation events. Cities are constructing stormwater storage facilities to prevent pluvial flooding. Combining storage with other functions, such as recreation, may lead to exposure to contaminants. This study assessed the microbial quality of rainwater collected in a water plaza and the health risks associated with recreational exposure. The water plaza collects street run-off, diverges first flush to the sewer system and stores the rest of the run-off in the plaza as open water. Campylobacter, Cryptosporidium and Legionella pneumophila were the pathogens investigated. Microbial source tracking tools were used to determine the origin (human, animal) of the intestinal pathogens. Cryptosporidium was not found in any sample. Campylobacter was found in all samples, with higher concentrations in samples containing human Bacteroides than in samples with zoonotic contamination (15 vs 3.7 gc (genomic copies)/100 mL). In both cases, the estimated disease risk associated with Campylobacter and recreational exposure was higher than the Dutch national incidence. This indicates that the health risk associated with recreational exposure to the water plaza is significant. L. pneumophila was found only in two out of ten pond samples. Legionnaire's disease risks were lower than the Dutch national incidence. Presence of human Bacteroides indicates possible cross-connections with the CSS that should be identified and removed.

Screening-level microbial risk assessment of urban water locations: a tool for prioritization.

People in urban areas are exposed to microbial hazards in urban waters. In this study, various hazards, diseases, and water systems, where different recreation activities take place, are compared in an integrated quantitative microbial risk assessment (QMRA). The event and annual probability of gastrointestinal illness (GI) and Legionnaires'disease (LD) were analyzed in QMRA models using selected literature data. Highest mean event probabilities of GI were found for playing in pluvial flood from a combined sewer overflow (34%), swimming (18%), and rowing (13%) in the river, swimming (8.7%) and rowing (4.5%) in the lake, and playing in a water playground (3.7%) and in the pluvial flood from stormwater sewers (4.7%). At these locations, the GI probability was above the EU Bathing Water Directive threshold for excellent water quality (3%). All the annual risk medians were below the national incidence of legionellosis of 0.002%. The illness probability was most sensitive to the pathogens concentration (particularly Campylobacter, Norovirus, and Legionella) and exposure frequency. Therefore, site-specific pathogen data collection is the best next step to strengthen the certainty of the risk estimates. This study created an evidence-base that was used by water authorities to understand the health risks and set priorities for risk management.

Screening-level risk assessment of Coxiella burnetii (Q fever) transmission via aeration of drinking water.

A screening-level risk assessment of Q fever transmission through drinking water produced from groundwater in the vicinity of infected goat barnyards that employed aeration of the water was performed. Quantitative data from scientific literature were collected and a Quantitative Microbial Risk Assessment approach was followed. An exposure model was developed to calculate the dose to which consumers of aerated groundwater are exposed through aerosols inhalation during showering. The exposure assessment and hazard characterization were integrated in a screening-level risk characterization using a dose-response model for inhalation to determine the risk of Q fever through tap water. A nominal range sensitivity analysis was performed. The estimated risk of disease was lower than 10(-4) per person per year (pppy), hence the risk of transmission of C. burnetii through inhalation of drinking water aerosols is very low. The sensitivity analysis shows that the most uncertain parameters are the aeration process, the transport of C. burnetii in bioaerosols via the air, the aerosolization of C. burnetii in the shower, and the air filtration efficiency. The risk was compared to direct airborne exposure of persons in the vicinity of infected goat farms; the relative risk of exposure through inhalation of drinking water aerosols was 0.002%.