High-throughput isolation and sorting of gut microbes reduce biases of traditional cultivation strategies

Abstract

Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and often yield biased sampling of taxa due to ecological and evolutionary factors. New strategies are needed to enable ample representation of rare taxa and slow-growers that are outcompeted by fast-growing organisms. We developed a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts droplets based on colony density. We applied our strategy to mouse and human gut microbiomes and used 16S ribosomal RNA gene amplicons to characterize taxonomic composition of cells grown using different media. We found up to 4-fold increase in richness and larger representation of rare taxa among cells grown in droplets compared to conventional culture plates. Automated sorting of droplets for slow-growing colonies further enhanced the relative abundance of rare populations. Our method improves the cultivation and analysis of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.

Interindividual Variation in Dietary Carbohydrate Metabolism by Gut Bacteria Revealed with Droplet Microfluidic Culture

“…1C). This finding is consistent with the hypothesis that droplet isolation enables slowgrowing microbes to be sheltered from competition with fast-growing bacteria (18,24,31).…”

“…Because droplets are not limited by the need to microfabricate physical wells or channels, millions of distinct culture volumes can be created in minutes. So far, droplet techniques have been used to isolate uncultured microbes from seawater, soil, and gut communities (18,(22)(23)(24); assess microbial cross-feeding (25); track population dynamics of individual bacteria (26); and examine antibiotic sensitivity and commensal-pathogen interactions of human gut and oral microbiota (27,28). Still, existing droplet microfluidic approaches for assaying bacteria have required combining complex emulsion techniques (water-oil-water) with the use of flow cytometers or custom on-chip droplet sorting devices.…”

Highly parallelized microfluidic droplet cultivation and prioritization on antibiotic producers from complex natural microbial communities

“…While we used dynamic droplet incubation to continuously supply the droplet population with oxygen, the perflourinated oil carrying the dissolved gases could also be charged with nitrogen or other gases to enable a microaerophilic or even anaerobic incubation. It was recently shown that the anaerobic incubation of human microbiomes in droplets increases the species richness dramatically 21,22 . Besides encapsulating single cells, several cells could be confined per droplet creating simplified multispecies communities, which have the potential to either support growth of dependent species 44 or to exert a change in natural product biosynthesis 45,46 .…”

“…A complementary trend is the miniaturization of the culture vessels 12,14,[18][19][20][21][22][23][24][25][26][27][28][29][30] to save material and time while increasing the throughput and thereby the chance to find non-dormant, culturable variants of naturally occurring species. These microscale culture techniques have the idea in common to partition complex bacterial communities on the single cell level, allowing the various species to grow at their own speed, since they do not have to compete neither for space nor for nutrients.…”

“…At the same time, the compartments that confine single cells are small and densely packed to establish a highly parallelized cultivation. The reported microscale approaches can be divided into methods that arrange the compartments into arrays 18,20,[23][24][25][26][27][28][29][30] and techniques that keep the compartments not fixed to positions but rather mobile within the population 12,14,19,21,22 . The clear advantage of arrays is the possibility to address the same culture several times, since the compartments are identified by their coordinates.…”