Researchers from the University of Oxford have developed a droplet-based 3D printing method capable of customizing bacterial genotypes at the micron-scale, which could drive major shifts in ecology.
Using droplet printing, the researchers were able to print strains of the gut bacterium Escherichia coli, also known as E. coli, and alter its spacing to create customized communities of the bacteria in order to see how strains react to each other when placed side-by-side in specific patterns.
Being able to manipulate the arrangement of such bacteria at the micron-scale enables greater observation and understanding of its behavior, and could even be “critical for ecological outcomes”, the researchers claim.
Manipulating bacteria and what it tells us
The position of different strains and species of bacteria in space is thought to be critical for the ecology of bacterial “communities”, and in turn how they affect us. The way in which different strains of bacteria are arranged when a community first develops is also thought to be a key predictor of which strains dominate and, more generally, whether that community will thrive or perish.
When strains grow apart in distinct patches, this is expected to limit the impacts of competitive mechanisms, essentially where different species of bacteria fight each other for dominance, and promote coexistence. This is still a hypothesis, however, and until now there have been barriers to the direct testing of this theory.
As the researchers point out, a variety of 3D printing technologies have been developed that are capable of positioning and patterning microbes, however typically this work has focused more on the production of biomaterials, such as bacterial sensors. How bacteria grow and interact as a community has received less attention, although 3D printing has previously facilitated research in this field.
In November 2020, physicists from Leiden University 3D printed micron-scale “microswimmer” structures to help them better study water bacteria, while elsewhere researchers from Purdue University developed a 3D printed capsule capable of sampling bacteria inside the gastrointestinal tract of humans.
Printing patterned bacterial communities
To combat the barriers to arranging bacterium structures at the micrometer scale, the Oxford researchers developed a high-resolution droplet printing technique to enable them to position interacting microbes in specific sub-milimeter patterns. As a result, they were able to manipulate and study the ecology of microbial communities through changing their arrangements across very fine scales.
To prove their method, the researchers printed tailored bioinks containing E. coli cells in 110µm diameter droplets which were deposited into patterns by line-by-line printing. The droplets were initially surrounded by monolayers of phospholipid – a class of lipid containing two fatty acids, a phosphate group, and a glycerol molecule – to create a stabilized, support-free structure.
The resulting structure was then gelled and the lipid bilayers removed by adding oil in order to create the final printed community of bacteria. Within its printed community, the E. coli bacterium grew from single-cell dispersions into 3D microcolonies. The researchers printed two strains of E. coli in different fluorescent colours in order to demonstrate their ability to print specific patterns and vary the amount of genetic mixing. Through this, the researchers were able to clearly observe how the two strains grew and interacted with each other depending on their spatial arrangement.
Impacting microbial communities
At the end of their experiments, the researchers found that varying the spatial structure of bacteria at the micrometer scale did impact the outcome of interference and competition between the E. Coli strains. Significantly, they observed that altering the structure of the bacterium at such a fine scale could shift the outcome of competition from one strain dominating another, and this patterning could be the difference between one strain thriving and mutual destruction.
A key finding of the study confirmed that spatial segregation was often protective and limited the effects of one strain on the other. In the future, the researchers will look to further explore factors that provide ‘refuge’ for one bacterial strain from another, such as the length of time that strains grow together, the turnover in nutrients for the bacterium, and the initial frequencies of strains.
For now, they believe their work could have great importance across many species and ecological interactions, and that it has significant potential for both understanding and controlling microbial communities.
Further information on the study can be found in the paper titled “Droplet printing reveals the importance of micron-scale structure for bacterial ecology”, published in the Nature journal. The paper is co-authored by R. Kumar, T. Meiller-Legrand, A. Alcinesio, D. Gonzalez, D. Mavridou, O. Meacock, W. Smith, L. Zhou, W. Kim, G. Pulcu, H. Bayley, and K. Foster.
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Featured image shows droplet printing generates viable bacterial communities with defined micron-scale patterning. Image via Nature Communications.