Functional Metagenomics To Discover Antibiotic Resistance Genes In Natural Environments

In this book, the latest tools available for functional metagenomics research are described.This research enables scientists to directly access the genomes from diverse microbial genomes at one time and study these “metagenomes”. Using the modern tools of genome sequencing and cloning, researchers have now been able to harness this astounding metagenomic diversity to understand and exploit the diverse functions of microorganisms. Leading scientists from around the world demonstrate how these approaches have been applied in many different settings, including aquatic and terrestrial habitats, microbiomes, and many more environments. This is a highly informative and carefully presented book, providing microbiologists with a summary of the latest functional metagenomics literature on all specific habitats.

The majority of microbes in many environments are considered “as yet uncultured” and were traditionally considered inaccessible for study through the microbiological gold standard of pure culture. The emergence of metagenomic approaches has allowed researchers to access and study these microbes in a culture-independent manner through DNA sequencing and functional expression of metagenomic DNA in a heterologous host. Metagenomics has revealed an extraordinary degree of diversity and novelty, not only among microbial communities themselves, but also within the genomes of these microbes. This Research Topic aims to showcase the utility of metagenomics to gain insights on the microbial and genomic diversity in different environments by revealing the breadth of novelty that was in the past, largely untapped.

Functional Metagenomics: Microbial Diversity, Interaction, and Application in Bioremediation uses a 20-chapter, easy-to-understand format to centralize the practical application ideas for functional metagenomics. This important resource not only includes chapters on next-generation sequencing technologies to study important biogeochemical cycles, degradation pathways and detoxification, but also gives insight into several tools that have been developed to integrate metadata and sequence data, allowing downstream comparative analyses of different datasets using several ecological indices.Content further explains the newly developed techniques for sequencing DNA; generating shorter fragments than Sanger sequencing techniques to quickly read larger sequences in a shorter amount of time. The content finally explores the role of metagenomics in studying microbial diversity, interaction and application in bioremediation studies and gives the reader overall detailed information regarding metagenomics, its application as well as techniques. Provides comprehensive information to readers on state-of-the-art applications in metagenomics Summarizes our current knowledge of the use of metagenomics and gives a novel and powerful insight into the already existing bioremediation process Gives an overall picture of metagenomics, its application, processes, and future prospects in the field of bioremediation

Although we can't usually see them, microbes are essential for every part of human life-indeed all life on Earth. The emerging field of metagenomics offers a new way of exploring the microbial world that will transform modern microbiology and lead to practical applications in medicine, agriculture, alternative energy, environmental remediation, and many others areas. Metagenomics allows researchers to look at the genomes of all of the microbes in an environment at once, providing a "meta" view of the whole microbial community and the complex interactions within it. It's a quantum leap beyond traditional research techniques that rely on studying-one at a time-the few microbes that can be grown in the laboratory. At the request of the National Science Foundation, five Institutes of the National Institutes of Health, and the Department of Energy, the National Research Council organized a committee to address the current state of metagenomics and identify obstacles current researchers are facing in order to determine how to best support the field and encourage its success. The New Science of Metagenomics recommends the establishment of a "Global Metagenomics Initiative" comprising a small number of large-scale metagenomics projects as well as many medium- and small-scale projects to advance the technology and develop the standard practices needed to advance the field. The report also addresses database needs, methodological challenges, and the importance of interdisciplinary collaboration in supporting this new field.

This book gives an overview of gene transfer and stability in those aquatic and terrestrial environments where bacteria and fungi can survive and interact genetically. It examines the role played by sex between microbes in the evolution of populations and their survival. Special emphasis is placed on methodology, including the analysis by novel techniques of genetic material extracted directly from soils, rivers and lakes. The natural spread of antibiotic resistance and the safe use of genetically manipulated microbes are matters of considerable scientific, medical and public concern upon which the investigations presented here have direct bearing. This unique collection will be of value to specialist researchers in applied microbiology, ecology and biotechnology as well as biomedical scientists interested in the environmental risks of genetic engineering.

The complex interplay between routine antibiotic use and zoonotic pathogen presence makes livestock farming environments unique nexuses for the potential emergence of zoonotic diseases and/or antibiotic resistant bacteria and their resistance genes. Livestock can further facilitate transmission and emergence by serving as intermediary or amplifying hosts in which pathogens and antibiotic resistant bacteria and their genes can evolve and spill over into humans. As such, we were interested in understanding differences in the dairy worker microbiota that may arise due to exposure to livestock farming environments to evaluate potential risks of these environments in facilitating global dissemination of zoonotic disease, antibiotic resistant bacteria and antibiotic resistance genes. We used culture independent methods that go beyond the traditional single pathogen approach to conduct comparisons between the gut microbiota and resistome of dairy workers and of community controls as well as to interrogate functional differences in commensal genomes recovered from both groups. To enable our study of functional differences, we first addressed methodological limitations with novel statistical methods for pangenomics. We developed happi, a statistical method for modeling gene presence that accounts for differential genome quality factors (e.g., mean coverage). We evaluated happi's performance using simulated and shotgun sequencing data and found that happi is accurate and robust even in scenarios when genome quality is correlated with the main covariate of interest. happi can furthermore be broadly applied to functional comparisons of genomes of other microorganisms beyond bacteria, and used in functional comparisons of metagenomes to adjust for differential quality (e.g., sequencing depths) of metagenomes. Using happi to facilitate our functional comparisons, we conducted a metagenomics and pangenomics investigation of the effects of occupational exposure to dairy farm environments on metagenome differences in taxonomy, diversity and gene presence (i.e., co-abundant gene groups (CAGs), antibiotic resistance genes (ARGs), and virulence factors) and on functional differences of gut commensal bacteria genomes in dairy workers and community controls. A major strength of our study was the multi-level interrogation of dairy worker and community control microbiomes. Our cross-sectional study examining differences in microbial genes and genomes from dairy workers and community controls observed several patterns for further investigation including greater abundance of tetracycline resistance genes and higher occurrence of cephamycin resistance genes in dairy workers' metagenomes; evidence of commensal organism association with plasmid-mediated tetracycline resistance genes found in both dairy workers and community controls; and lower average gene and genome diversity in dairy workers' metagenomes compared to community controls. These findings point towards possible avenues for future research to better understand the impact of exposure to zoonotic pathogens, antibiotic resistant organisms, and ARGs on the microbiome and resistome of livestock workers and others with close animal contact.

Antibiotic resistance genes from soil are ancient, diverse, and represent an evolutionary origin for resistance seen in the clinic. In my thesis, I demonstrate that the lateral exchange of antibiotic resistance genes has occurred between non-pathogenic soil bacteria and diverse human pathogens on recent timescales, indicating that the vast soil resistome may serve as a reservoir of resistance available to the clinic. Despite the apparent overlap between soil and clinical resistomes, factors that influence resistance gene composition in soil and their movement between genomes and habitats are largely unknown. To investigate these relationships, I performed functional metagenomic selections for antibiotic resistance genes from 18 grassland and agricultural soils, identifying 2895 resistance genes with sequences predominantly unlike those in public databases. I was able to identify factors that drive resistome composition across these soils, detecting strong correlations between resistance genes and (i) soil type, (ii) the amount of added nitrogenous fertilizer, and (iii) bacterial community composition. I also describe a novel family of tetracycline-inactivating enzymes, widespread in soil and with ties to pathogenic Legionella, but previously unrecognizable as antibiotic resistance genes. Because tetracycline inactivation is scarcely observed in hospitals, these enzymes may fill an empty niche in pathogenic organisms and should therefore be monitored for their dissemination potential into the clinic. Taken together, my data indicate that soil resistomes are largely structured by bacterial community composition, but are capable of exchanging resistance genes with clinical pathogens, including previously unseen resistance phenotypes. Further work to identify the ecological, taxonomic, and genetic risk factors associated with this resistance gene flow is warranted.

Strikingly the genes identified using culture dependent methods are highly similar to genes harbored by human pathogens whereas genes identified using culture independent methods are distantly related to resistance genes of pathogens. This highlights a reservoir of previously unknown resistance genes harbored by the uncultured fraction of the human microbiome and demonstrates that exchange of resistance genes between microbiome isolates and pathogens has occurred. We also identify 170 antibiotic resistance genes from bacteria capable of subsisting on antibiotics and show that 10% of these are identical at the nucleotide level to resistance genes harbored by pathogens demonstrating recent evolutionary exchange of resistance genes between these bacteria. Finally we apply metagenomic functional selections to identify novel genetic parts from the soil microbiome, that confer tolerance to important biomass inhibitors currently limiting sustainable biofuel production, providing proof of concept of how to make available Nature's diverse inventory to synthetic biology.