The Magnificent Microbiome: Where have we been and where are we going? (Part 1)

In this blog, we will attempt to cover some of the many ways our microbiomes impact our health and wider lives, and how they might shape our future. Of course, to do the field of microbiome research justice, we’d have to devote a lot more words to it than we have here – just look at the number of papers that have been published in the last few years (Figure 1). But we need to start somewhere – so let’s start with ourselves.

Figure 1: Overview of Microbiome Research. The interactive bar chart shows the number of research papers published since 2000 with either “microbiome” or “microbiota” in their title/abstract.

More microbe than man

Where are you right now? Are you sat in your home office? Perhaps you’re on the commute into work? Maybe you’re in your garden, or at a local park? No matter where you are, I can assure you of one thing: you are not alone.

You might not be able to see them (unless you happen to have a microscope to hand), but present on just about every surface around you is a complex ecosystem of microscopic organisms. That’s right – you may, in your very human way, be under the (false) impression that hominin species have for millions of years been the sole masters of the world. But you would be wrong.

The true rulers of the world are the microscopic organisms that have inhabited every known environment we have ever explored on this planet (and even those we haven’t) for billions of years. And we humans are but a speck on the timeline of their long rule. Trillions of these microbes (and their genes) live in and on the human body, and it is now widely accepted that humans must be considered as a “superorganism” that is mostly comprised of microbes. The Human Genome Project concluded that we are made up of around 20,000 genes – approximately the same number as mice. This, though a breakthrough at the time, would turn out to be but the beginning. Now, researchers believe the genome of our microbiome far outweighs our own, contributing more than 300 times the number of genes as our own genomes (to get an idea of this scale, look at Figure 2 below).

**Figure 2: Ratio of microbial to human cells and genes in human body.** Source: P Wischmeyer at al., “Role of the microbiome, probiotics, and ‘dysbiosis therapy’ in critical illness,” Current Opinion in Critical Care, 22, 347 (2016). DOI: 10.1097/MCC.0000000000000321

The truth is, we’ve only really begun to appreciate these microbes in the last few decades. Since then, our eyes have been opened to the many ways they influence our lives, from helping us to digest certain foods to producing their own antibiotics to ward off invading pathogens. Within this landscape, the blossoming field of microbiome research has emerged – one which has advanced from a fledgling area of research to a multi-billion-dollar industry in the past decade alone. But we still have a lot to learn.

First, to appreciate where the field is heading, we must look at where it has been…

A brief history of microbiome research

Though we might not have always been able to see our ever-present microscopic pals (or have any real understanding of the processes underlying some of our scientific observations), our fascination with our microbiomes arose many moons ago – initially, in the form of our bowels.

The early days

For millennia, humans have treated varying conditions with things such as specific diets, fasting, spring waters and purgatives. The use of faecal microbiota transplants (FMT) to treat food poisoning and severe diarrhoea is noted as far back as the fourth century in China, and animal excrement was used to treat certain conditions up until the 18^th century in Europe.

It was around this time that Antonie van Leeuwenhoek, microscope in hand, would sample some of his own stool and observe “more than 1000 living animalcules,” as he termed microorganisms at the time. Despite his intriguing observations, research on these “animalcules” would only really take off in the mid-nineteenth century.

Seeing is believing

One of the first descriptions of microbes present in the body would come from Scottish Surgeon, John Goodsir, who discovered Sarcina centriculi in the ejected stomach fluid of his 19-year-old patient. This would ignite a debate around why such microbes were present and whether they were harmful or innocuous residents of the internal organs. In 1853, Joseph Leidy famously published ‘A Flora and Fauna Within Living Animals’ – widely considered to be the origin of microbiota research. A few years later, Louis Pasteur and Robert Koch would build on Leidy’s work and bring about the acceptance of the germ theory of disease that linked microbes or “germs” to certain conditions. Though moving in the right direction, this would lay the groundwork for a long-held belief that microbes were mostly pathogenic and harmful to their human hosts.

Building on our understanding of host-microorganism interactions, Theodore Escherich (more widely known for his discovery of “Bacterium coli commun” or Escherichia coli) published two papers detailing the intestinal bacteria of infants, how this transformed over time, and the role of these bacteria in certain processes in the gut. In the early twentieth century, Henry Tissier discovered that most of the bacteria in the infant gut was in fact Bacillus bifidus communis and decided to try using “good bacteria” as a therapy in children with gastrointestinal disease. He found that the children all but recovered once their gut flora was converted to that of a breastfeeding child, and the probiotic was born.

During WWI, medical microbiologist Alfred Nissle discovered that a particular E. coli strain from one soldier was “antagonistically strong” against the pathogenic enterobacteria behind the deadly dysentery epidemic. He cultured this strain (later named Nissle 1917 – a reference strain for genetically engineering bacteria) in the lab and encapsulated it inside gelatine pills. The result – Mutaflor – is still used to this day to treat chronic constipation, diarrhoea, and other conditions such as ulcerative colitis.

FMT and anaerobic culture

Despite the translational potential of these breakthroughs, microbiome research would only really pick up again in the mid-twentieth century when Robert E. Hungate discovered how to anaerobically culture microbes, leading to the first isolation of human-associated anaerobes. This allowed researchers to study the human microbiota outside of a lab setting and helped advance the field dramatically. In 1958, the first use of FMT in western medicine was published by Ben Eiseman and colleagues, who used a faecal enema to successfully treat pseudomembranous enterocolitis (caused by Clostridium difficile). Seven years later, researchers compared germ-free and colonised mice and realised that gut microbiota transfer could be used to reconstitute some of their physiology – an essential step in studying the effects of gut microbiota on the host.

Many other scientific developments toward the end of the 20^th century helped push microbiome research along, such as the discovery of DNA and the development of sequencing-based technology to study cultivation-independent microorganisms. In 1996, the bacteria in a faecal sample were analysed using 16s ribosomal RNA sequencing. Two years later, the same technology was used to uncover that each adult had their own unique microbial community.

The era of the microbiome

At this point, microbiome research hits a new stride. In 2006, the Gordon group showed that human phenotypes could be transferred to mice via FMT – namely, the microbiome of an obese human would cause the mice to gain weight, and vice versa. Two years later, the Human Microbiome Project would launch, catapulting the filed into a new era. In 2013, the clinical relevance of the microbiome would be proven once and for all after a randomized control trial to treat Clostridium difficile infection (CDI) patients was stopped prematurely due to the outstanding results obtained by FMT versus antibiotic treatment – it simply did not seem ethical to continue treating some selected patients with the antibiotic.

Since then, research into how to alter our microbiomes to treat disease has blossomed and many new applications have opened up. Alongside this, the development of multi-omics technologies – including metatranscriptome, metaproteome, and metabolome approaches – has provided unprecedented detail on the metabolic activities of these microbes and launched us into a new era of microbiome research.

The Human Microbiome Project

In 2008, The National Institutes of Health launched the Human Microbiome Project (HMP) with $170 million in funding dedicated to characterizing the genomic makeup of all the microbes inhabiting the body. The main aims are around creating resources for the research community, such as a “healthy cohort” reference database of human metagenomic sequences, computational tools for analysing these sequences, and clinical protocols for sampling the microbiome.

The “healthy cohort” project is based on sampling the microbiome from five key sites: the nasal passages, oral cavities, skin, gastrointestinal tract, and urogenital tract. In total, 300 “disease-free” adults were recruited and sampled up to three times over a two-year period. Data on the taxonomy of the microbes present (16S rRNA data) was collected alongside sequencing data on entire microbial communities (metagenomics).

The second phase of the project, known as the Integrative Microbiome Project (iHMP), worked to establish links between the microbiome and health or disease using multi-omics. The three key focuses areas were Type 2 diabetes, pregnancy and preterm birth, and IBD. In 2019, the results from this research phase were published, elucidating the mechanisms of host-microbiome interactions and providing a unique data resource (hosted by the HMP Data Coordination Center).

**Figure 3:** **The first and second phases of the NIH Human Microbiome Project.** The Integrative HMP (iHMP) Research Network Consortium. The Integrative Human Microbiome Project. *Nature* 569, 641–648 (2019). DOI: 10.1038/s41586-019-1238-8

The impact of the HMP can perhaps best be seen in the more than 650 peer-reviewed publications listed on the HMP website from June 2009 to the end of 2017, which had been cited over 70,000 times. Together, the results of the HMP provide a rich multi-omic data resource that can be mined in future research.

The dawn of omics

In the last decade, the emergence and continued development of omics technologies has helped push microbiome research to new heights. With the combined power of multiple omics technologies, we are now able to gain unprecedented insights into host-microbiome interactions and make the transition from purely analysing which microbes are present in a particular environment to exploring their functional potential – in other words, looking at the molecules they produce.

There are a number of available methods to study the intricacies of the microbiome, including (but not limited to) culturomics (the high-throughput isolation and cultivation of microbes), metabarcoding (taxonomically classifying a community), and the various methods for analysing the metabolic output of a microbial community (metaproteomics, metabolomics, and metatranscriptomics). An overview of these approaches, and how they link together, can be seen in Figure 4.

**Figure 4: Methods for assessing microbial function.** Complex microbiome studies cover various areas, starting from the level of complete microbial cells (microscopy, culturomics), followed by the DNA (single cell genomics, metabarcoding, metagenomics), RNA (metatranscriptomics), protein (metaproteomics), and metabolites (metabolomics). In that order, the focus of the studies shifts from the microbial potential (learning about available microbiota in the given habitat) over the metabolic potential (deciphering available genetic material) towards microbial functioning (e.g., the discovery of the active metabolic pathways). Microbiome 8, 103 (2020). DOI: 10.1186/s40168-020-00875-0

Initially, metagenomics allowed researchers to characterise the genetic material present in a given microbial population in silico. This approach overcomes the limitations around isolating and culturing different bacterial species from a mixture and is useful for obtaining a broader picture of the human microbiome. On the other hand, single-cell genomics has emerged as a promising method for recovering genome sequences from individual cells, allowing the finer details of closely related species to be characterised.

Despite these advances, 40-70% of the annotated genes in fully sequenced microbial genomes still have no known or predicted function. Here, metabolomics is making headway, allowing us to not only characterise the genomes of microbes, but also the 10s of thousands of potentially active biomolecules they secrete.

Crucially, metabolomics will have a significant impact on the realisation of personalised medicine. Genes are just one part of the gene-diet-microbe system that must be fully understood before true “precision” healthcare comes to fruition. As Jeremy Nicholson, an internationally renowned pioneer in metabolic phenotyping and systems medicine, said “The microbiome represents yet another entire level of genetic connectivity. The challenge for the future is to think about layers of networks on top of networks. This is really quite a tough problem, probably the toughest problem in 21st-century biology.”

In part 2 of this blog, we will take a closer look at how researchers are solving this problem by exploring the complex and shifting nature of our microbiomes – including how they evolve alongside us, both throughout history and during our lifetimes. We will also discover the many ways our increased knowledge of the microbiome is being used in cutting-edge applications, such as precision medicine.

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