High-throughput isolation and culture of human gut bacteria with droplet microfluidics

Abstract

30Isolation and culture of gut bacteria enable testing for microbial roles in disease and 31 may also lead to novel therapeutics. However, the diversity of human gut microbial 32 communities (microbiota) impedes comprehensive experimental studies of individual 33 bacterial taxa. Here, we combine advances in droplet microfluidics and high-throughput 34 DNA sequencing to develop a platform for isolating and assaying microbiota members 35 in picoliter droplets (MicDrop). MicDrop can be used to create millions of distinct 36 bacterial colonies in a single experiment while using off-the-shelf parts compact enough 37 to fit in an anaerobic chamber. In proof-of-concept experiments, we used the platform to 38 characterize inter-individual metabolic variation among hundreds of polysaccharide-39 degrading gut bacteria from nine stool donors. We also used MicDrop to test the 40 hypothesis that growth kinetics of individual gut bacterial taxa are associated with long-41 term community dynamics in an artificial gut. These demonstrations suggest the 42 MicDrop platform could support future diagnostic efforts to personalize microbiota-43 directed therapies, as well as to provide comprehensive new insights into the ecology of 44 human gut microbiota. 45

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

“…High-throughput anaerobic droplet cultivation has a significant potential to accelerate the study of gut bacteria and the isolation of presently uncultivated species. For instance, in parallel to our work here, Villa et al (2019) recently cultivated human stool microbes in droplets and screened carbohydrate utilization of bacteria from nine stool donors in order to determine the inter-individual variation in metabolic utilization [11]. Here, we demonstrated that our technology enabled an enrichment of low abundance organisms and reduced the variation of the cultivated taxonomic composition between media.…”

“…However, some bacteria prefer defined media over rich media [12] while others utilize surface features such as hydrophobicity, roughness, and surface chemistry to form biofilms and proliferate [42]. Further enrichment of anaerobic organisms within our droplet platform could be achieved by incorporating droplet generation with defined media [11], combinatorially generated gradients of media [43], or varying the droplet surfactant chemistries to improve biofilm formation [44].…”

“…For instance, Jiang et al (2016) isolated environmental soil-associated bacteria in droplets and found an increase in the diversity of taxa with an increased representation of rare organisms [25]. Villa et al (2019) recently cultivated human gut microbes in thousands of nanoliter droplets to characterize metabolic variation in polysaccharide-degrading gut bacteria and analyzed their growth kinetics [11]. Finally, bacteria confined within droplets reach a critical threshold concentration of quorum sensing molecules faster than would occur in bulk culture (due to the inherently small droplet volume), which can lead to improved growth in certain culture media [27].…”

“…Microbial cultivation is an essential first step to address some of these shortcomings as this strategy enables the recovery of complete reference genomes [6], accurate identification of taxonomy and functional potential of new strains [7,8], and validation of causality by perturbation experiments [5]. Microbial cultivation is currently experiencing a pronounced revival [7][8][9][10][11], yet the majority of cultivation efforts require arduous manual picking of thousands of colonies, impeding the efforts to harmonize discoveries that emerge from 'omics strategies with downstream mechanistic investigations in the rapidly advancing field of microbiome research.…”

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

“…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.…”