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Culturing the uncultured: isolating human microbial ‘dark matter’

A large fraction of the microorganisms that constitute the human microbiome have remained uncultured. Some of the uncultured microbes have close relatives that have been cultured. However, there are also whole groups of microbes that don’t have relatives in culture.

Some of these uncultured microbes have been associated with certain diseases such as periodontal disease. Generally, they’re not very dominant or in high abundance, but this doesn’t mean they aren’t significant. They might interact with other microbes that are linked to disease.

For example, in the human mouth, there are several microbes that are classified as the so-called candidate phyla bacteria. For several decades there have only been known based on molecular signatures. Although these types of bacteria occur in other environments they haven’t been cultured from any other environment, human or otherwise. What they look like and how they behave has remained a complete mystery.

If we look at a broader level at the human microbiome there are many strains and many species that are still to be cultured. Furthermore, most of the work done so far has been on western populations. More recently people have started to analyse microbial gut sequencing from indigenous populations in remote areas of the world, who are isolated or have a distinctive form of diet. There’s a lot more diversity out there than the diversity we find in Western populations.

There have been a number of studies showing that once people become urbanized and adapt to a more Western diet, they start losing some of the microbes associated with more traditional lifestyles. Of course, there are many microbes that potentially could be beneficial, and people are looking at options for developing new probiotics.

Why is it important to cultivate these microbes?

Cultivation is important for studying pathogenicity mechanisms, assessing potential beneficial effects, and understanding how microbes interact with each other and with their human host. 

It’s vital to understand the role of different microbial types in human evolution, health, and disease. Whether from a prophylactic or treatment perspective, it’s important to understand as much as we can about the role and the potential of individual microbes. If we start losing some of these microbes because of changing habits and nutrition, then we could potentially be losing them forever. The role of cultivating unknown microbes for understanding and using the human microbiome is very important.

There have been a number of approaches, some of which have been high throughput. Sometimes referred to as ‘culturomics’ they are a nontargeted, agnostic way to get the diversity of microbes into samples. It is very powerful and provides a lot of new strains and potentially new species.

However, there are very few ways to target specific strains or species if one wants to isolate a particular one that appears to be correlated with some disease or another phenotype.

How can these microbes be isolated, cultivated and studied?

We designed an approach based on genomic information generated by culture-independent studies to selectively target, isolate and culture-specific organisms from the human microbiome. 

Of course, isolating a microbe does not guarantee that you’re actually going to be able to culture it. However, the first step is physically separating it from other microbes.

We took sequencing data from either a single-cell genome of from shotgun metagenomes. Using the assembled genomes and genetic information, we were able to decide which organism to target because either it was very novel or because it had specific associations.

We searched for proteins we predicted had exposed domains, and then took sequence information from those particular proteins. We generally narrowed it down to one to three target proteins. Because we looked at their homologs in other bacteria, we could be relatively confident that they were likely to be expressed on the surface of the cell. 

Then we essentially generated an antibody against the surface domains of those proteins: we selected epitopes or entire protein domains to raise antibodies. Once we had the antibodies, we labelled them with a fluorescent molecule before applying them to biological samples where the relevant organisms occur. This is a non-destructive process where we can label cells whilst keeping them alive.

With the target cells labelled, we used flow cytometry to isolate and sort the individual cells. Those cells could then be deposited in different types of culture media and under different conditions. Conditions can then be screened to get the cells to divide, propagate and grow. Eventually, we could then culture and purify them so that they can be studied.

What is the benefit of a targeted response?

Such targeted, genomics-driven strategies for cultivation will help to identify microbial health-disease determinants and preserving human microbiome diversity.

We’re losing a lot of the microbial diversity across the human population because of Westernization. We may not have the time to apply classical methods to isolate some from samples that are becoming rarer. Using a targeted approach could allow us to isolate desired types of organisms in order to better understand the human microbiome.

Mircea Podar is a Distinguished Scientist and Group Leader in Systems Genetics at Oak Ridge National Laboratory.

The 7th Microbiome and Probiotics R&D and Business Collaboration Forum will explore the latest developments in microbiome research and applications. Find out more here.

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