The secret microbiome

Auto-immune disease, obesity and even cancer… the microbiome is clearly involved in much of human health. No surprise really, we are as much microbe as we are human. What is a surprise, says Alexandre Almeida, is that we don’t know the half of it yet…

It was thought that our bodies were more bacterial cells than human cells – while this may be fun to quote in the pub, it turns out not to be true. However, we are still about as much microbe as we are human… and there are still many different microbiome species to be discovered.

Researchers have been looking for ways to manipulate the human microbiota for health, but new studies reveal that there are still thousands of gut bacteria we do not know much about…

Up until a few years ago, there was a common misconception that the number of microbial cells outnumbered that of human origin by a ratio of over 10:1. However, studies now estimate that we are about as much microbe as we are human¹.

This is still hugely significant and illustrates how almost every aspect of human biology needs to take into account the effect or role that the microbiota – the complex community of microorganisms living with us – may play.

Many years of research show, with varying degrees of confidence, that the microbiota – specifically that colonising our intestinal tract – has an impact on human physiology and diseases² such as infection, obesity, auto-immune disorders and cancer. One of the most prominent and successful cases of microbiota intervention for improving health has been demonstrated in the treatment of diarrheal infections caused by the bacterium Clostridium difficile. Because this disease is characterised by the absence of protective, commensal bacteria in the gut, fecal microbiota transplantation (FMT) of stool from a healthy individual to one affected by recurrent C. difficile infection was shown to resolve up to 90% of cases³.

Studies now estimate that we are about as much microbe as we are human  

Researchers are striving to use similar approaches for the treatment of other diseases associated with the microbiota, but many of the results and conclusions so far are based on data correlations alone; it is unclear whether certain species linked to disease are causal or happen to be found in affected individuals either by chance or as a result (or effect) of the disease. Therefore, there is a need to go beyond these simple observations to determine whether particular species, or combinations of species, have clinical relevance. This requires access to the individual microbes of interest in order to perform additional experiments and gain new mechanistic insights, meaning we as researchers need to be able to grow and culture these species under laboratory conditions. However, a phenomenon that has historically been termed “The Great Plate Count Anomaly”⁴ has revealed that many of the existing microbial species remain uncultured.

Several research teams have dedicated their efforts to solving this issue, which has led to two recent papers describing comprehensive collections of cultured intestinal bacterial^5,6. In spite of these ground-breaking contributions, which have now cultured and sequenced around 500-600 gut species, it has remained unclear how much of the human gut microbiota it has been possible to capture. This is an important question to tackle, as many associations between the microbiota and specific human phenotypes could be hidden behind incomplete reference databases.

What’s still to be discovered?

In our work, recently published in Nature, we utilised computational methods to determine how much of the human gut microbiota is left to be characterised. Metagenomics – the analysis of genetic material directly from the environment, circumventing the need for culturing – allows us to investigate the microbial diversity present in each sequenced sample. By aligning and grouping sequences that appear to be from the same organism (a process called assembly and binning) it is possible to reconstruct almost complete genomes from species that have never been seen before.

At EMBL’s European Bioinformatics Institute, we used these methods in the analysis of thousands of publicly available samples from individuals worldwide (mostly from North America and Europe) and found almost 2,000 bacterial species that have not yet been cultured. This number was much higher than we anticipated given that the most recent culture collections have obtained fewer than 600 species each. However, our results showed that many of the uncultured species are presently at a low abundance in the intestinal tract of typical Western populations, so would therefore be much more difficult to find.

Looking at the genetic diversity of all the bacterial species, we found that many of the uncultured genomes were very distinct from the cultured species, as several hundred of them could not even be assigned to known bacterial families or genera. Another interesting observation was that, by comparing the genomes of the cultured and uncultured species, we found that the uncultured group lacked many genes related to resistance to reactive oxygen species. Given the low oxygen concentration in the gut, most of the intestinal microbiota consists of anaerobic species. Our results suggest that, beyond being strict anaerobes, these unknown species may be even more sensitive to oxygen exposure when compared to known, cultured bacteria.

Beyond Europe and North America

Most of the reference cultured genomes available have been sampled from both European and North American populations, revealing a clear bias in current microbiome studies. Our research further highlighted this by showing that?some of the uncultured species we found were particularly prevalent and abundant in underrepresented populations (e.g. from Africa and South America). This means that, with just a few samples from these regions, we were able to find lots of bacterial diversity missing from current reference databases.

Concurrent to our study, another work published in Cell⁷ investigated the human microbiota diversity in thousands of samples across multiple body-sites. They similarly found a high abundance of uncultured species in the same underrepresented populations. An initial comparison with our dataset suggests that at least 80% of the species we found were also detected in their work, which highlights the reproducibility of the methods used in both studies and increases confidence in the generated datasets.

Both of these works have important implications for the field. By making the genomes of all the unknown bacteria available to the community, other researchers will be able to use our data to look for these species in their own datasets and identify those of particular interest much more accurately. Based on the functions predicted from the genomic data, new methods for sampling and culturing could be developed to isolate candidate species for further experimental testing. Having a more comprehensive database of intestinal bacteria will also allow researchers to perform higher resolution analyses to investigate the strain/population diversity within each species and how it may relate to specific functions in the human gut.

However, more extensive sampling efforts beyond Europe and North America are needed to move the field forward and better understand the role of the microbiota across populations with different lifestyles, cultures and host genetics.

References:

1.Sender, R., Fuchs, S. & Milo, R. Revised estimates for the number of human and bacteria cells in the body. PLOS Biol. 14, e1002533 (2016).

2.Duvallet, C., Gibbons, S. M., Gurry, T., Irizarry, R. A. & Alm, E. J. Meta-analysis of gut microbiome studies identifies disease-specific and shared responses. Nat. Commun. 8, 1784 (2017).

3.Brandt, L. J. et al. Long-term follow-up of colonoscopic fecal microbiota transplant for recurrent Clostridium difficile infection. Am. J. Gastroenterol. 107, 1079–1087 (2012).

4.Staley, J. T. & Konopka, A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu. Rev. Microbiol. 39, 321–346 (1985).

5.Zou, Y. et al. 1,520 reference genomes from cultivated human gut bacteria enable functional microbiome analyses. Nat. Biotechnol. 37, 179–185 (2019).

6.Forster, S. C. et al. A human gut bacterial genome and culture collection for improved metagenomic analyses. Nat. Biotechnol. 37, 186–192 (2019).

7.Pasolli, E. et al. Extensive unexplored human microbiome diversity revealed by over 150,000 genomes from metagenomes spanning age, geography, and lifestyle. Cell 176, 649–662.e20 (2019).

Author:

Alexandre Almeida is a Postdoctoral Fellow at?EMBL’s European Bioinformatics Institute?and the?Wellcome Sanger Institute