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Culture-free strain-level population genomics to identify disappearing human-associated microbes in the westernized world

Periodic Reporting for period 3 - MetaPG (Culture-free strain-level population genomics to identify disappearing human-associated microbes in the westernized world)

Reporting period: 2020-04-01 to 2021-09-30

The microbes in one healthy human body (the human microbiome) outnumber the total human beings on Earth one million to one and the human cells in the same body ten to one. Even considering only the lower gastrointestinal tract, thousands of different species live in symbiosis with our body and are crucial for a number of immunological and metabolic functions. The gut microbiome is shaped by several lifestyle factors. MetaPG aims at identifying and characterize the microbial organisms that were part of our microbiome but have now disappeared or are disappearing from the microbiome as a consequence of the changed lifestyles in our post-industrialization Westernized population. Pre-MetaPG research found that the composition of the intestinal microbiome between urbanized typically high-fat diet populations and rural low-fat diet populations are different, with some bacterial species and subspecies that are dramatically and consistently underrepresented in westernized population. However, a large fraction of the gut microbiome remains uncharacterized especially in non-Westernized population greatly limiting our understanding of the microbiome differences in different populations. Moreover, the ecological and life-style associated causes of the disappearing diversity in the gut of westernized population remain unclear. Unraveling the microbial ecology dynamics together with the investigation of their effects is of crucial relevance in the effort of characterizing our ancestral microbiome and defending the biodiversity that co-evolved with our body during our evolution.

The identification, validation, and characterization of the disappearing intestinal microbes in the westernized population will be the basis for multiple fullow-up lines of research. First of all, the catalogue of such disappearing microbes will crucially increase the awareness in the scientific and non-scientific community of substantial modifications of the gut microbiome as a consequence of the Westernization process. Given the limited timescale on which this occurred and the non-modulability of the human genome, this should raise several concerns about the consequences of disrupting the host/microbiome coevolution. The catalogue will also promote efforts to sample and appropriately conserve this microbial diversity in microbiological facilities, and to better study the biology of the specific organisms undergoing extinction. This could lead to new crucial insights into host-microbe and microbe-microbe interaction dynamics and, potentially, also to consider these strains as probiotic products in the future.

MetaPG defines the foundation for studying the human microbiome using metagenomics (cultivation-free) at a resolution previously attainable only via sequencing of microbes in pure cultures. Technically called "strain-level metagenomics", MetaPG is developing a new computational framework that, coupled with newly sampled Westernized and non-Westernized populations, is enabling finding hundreds of under-investigated microbes, and compiling a catalogue of strains undergoing or at risk of primary, secondary, or ecological extinction in Westernized populations. The advances in computational methods to study members of the human microbiome via metagenomics will also play a crucial role for other investigations of the human microbiome, including those focusing on human diseases.

The project consists of three main aims. Aim1: development of highly innovative computational methods that will allow a high resolution characterization of the human microbiome. Aim2: characterization of hundreds of unknown microbes living in the human body using the newly developed methods. Aim3: we will complement the available data with new targeted cohorts of both westernized and non-westernized populations, to identify those microbes that are currently disappearing in westernized populations as a consequence of urbanization, industrialization, high-fat diets.
We developed highly innovative computational methods to study microbial communities at unprecedented depth and resolution, based on the growing availability of microbiome data being scalable to many thousands of samples and thus considered "big data". The computational tools were published on scientific journals, presented at scientific conferences, taught in classes and courses, and are open source and publicly available with guides and tutorials.

With the new tools developed, we analyzed thousands of microbiome samples from multiple existing studies that have been curated and integrated within MetaPG. This generated massive information about the population genomics of hundreds of species that were previously completely or almost completely neglected. We investigated association between specific strains/subspecies with conditions of interest and produced several large comparative meta-analysis. In total we processed more than 10,000 samples and over 150,000 reconstructed microbial genomes. Among the investigations, we analysed metagenomes from various countries, body sites, ages, and lifestyles, identifying thousands of microbial genomes from yet-to-be-named species and expanding the knowledge of the human-associated microbes. In another meta-analysis of the gut microbiome in patients with colorectal cancer, we identified reproducible microbiome biomarkers and accurate disease-predictive models that can form the basis for clinical prognostic tests. We also performed a cross-continent meta-analysis exploiting >6,500 metagenomes specifically on Prevotella copri, which is a common human gut microbes particularly affected by lifestyle conditions.

By the large scale comparative meta-analysis performed in Aim2, Aim3 is identifying and characterizing microbes that are undergoing extinction in the westernized world using newly sequenced non-Westerninzed cohorts and publicly available data. Specifically, three three main findings we revealed so far are: i) Prevotella copri, previously considered just one microbial species, is a complex composed of four distinct groups and was found more prevalent in non-Westernized populations probably due to diet\lifestyle; ii) thousands of new human microbiome species were uncovered by a large-scale analysis, most of which are associated to non-Westernized populations; iii) the study on Blastocystis, a commonly colonizer of the gut, detected different subtypes and extended previous observations on its high prevalence in the population of non-westernized individuals; iv) an investigation on Eubacterium rectale, one of the most prevalent human gut bacteria, identified possible association of its distribution and human migration along time.

The most relevant biological aspects of this project is the identification, validation, and characterization of the disappearing intestinal microbes in the westernized population as a consequence of factors such as antibiotic use/abuse, high-fat diet, industrialization, pollution, increased hygiene. We feel confident of the results obtained so far that will lead to new crucial insights into host-microbe and microbe-microbe interaction dynamics and, potentially, also to consider these strains as probiotic products in the future.
Despite comparative analyses between metagenomes can be performed on multiple levels, MetaPG is enabling for the first time the possibility to exploit large-scale metagenome databases at high resolution scale. We, thus, developed several computational publicly available tools that can be applied to characterize the genetic structure of thousands of strains in different populations across the globe. These tools are being widely adopted by the research community.

Thanks to these achievements, we were able to perform detailed microbial comparative genomics investigation, unravelling patterns and association between microbial features and diseases/conditions to compile a catalogue of disappearing strains in westernized countries. We obtained interesting results on the large scale human microbiome diversity, and more specific on three common human gut bacteria, Prevotella copri complex found mostly in populations with non-Westernized lifestyles, Blastocystis study and Eubacterium rectale distribution affected by human migration.
MetaPG overview