Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes.

Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and evolutionary factors. New strategies are needed for ample representation of rare taxa and slow-growers that are often outcompeted by fast-growers in cultivation experiments. Here we describe a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts them based on colony density to enhance slow-growing organisms. We applied our strategy to a fecal microbiota transplant (FMT) donor stool using multiple growth media, and found significant increase in taxonomic richness and larger representation of rare and clinically relevant taxa among droplet-grown cells compared to conventional plates. Furthermore, screening the FMT donor stool for antibiotic resistance revealed 21 populations that evaded detection in plate-based assessment of antibiotic resistance. Our method improves cultivation-based surveys of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.

Watterson WJ ，Tanyeri M ，Watson AR ，Cham CM ，Shan Y ，Chang EB ，Eren AM ，Tay S ... -  《eLife》

Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities.

Antibiotics from few culturable microorganisms have saved millions of lives since the 20th century. But with resistance formation, these compounds become increasingly ineffective, while the majority of microbial and with that chemical compound diversity remains inaccessible for cultivation and exploration. Culturing recalcitrant bacteria is a stochastic process. But conventional methods are limited to low throughput. By increasing (i) throughput and (ii) sensitivity by miniaturization, we innovate microbiological cultivation to comply with biological stochasticity. Here, we introduce a droplet-based microscale cultivation system, which is directly coupled to a high-throughput screening for antimicrobial activity prior to strain isolation. We demonstrate that highly parallelized in-droplet cultivation starting from single cells results in the cultivation of yet uncultured species and a significantly higher bacterial diversity than standard agar plate cultivation. Strains able to inhibit intact reporter strains were isolated from the system. A variety of antimicrobial compounds were detected for a selected potent antibiotic producer.

Mahler L ，Niehs SP ，Martin K ，Weber T ，Scherlach K ，Hertweck C ，Roth M ，Rosenbaum MA ... -  《eLife》

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

Culture and screening of gut bacteria enable testing of microbial function and therapeutic potential. However, the diversity of human gut microbial communities (microbiota) impedes comprehensive experimental studies of individual bacterial taxa. Here, we combine advances in droplet microfluidics and high-throughput DNA sequencing to develop a platform for separating and assaying growth of microbiota members in picoliter droplets (MicDrop). MicDrop enabled us to cultivate 2.8 times more bacterial taxa than typical batch culture methods. We then used MicDrop to test whether individuals possess similar abundances of carbohydrate-degrading gut bacteria, using an approach which had previously not been possible due to throughput limitations of traditional bacterial culture techniques. Single MicDrop experiments allowed us to characterize carbohydrate utilization among dozens of gut bacterial taxa from distinct human stool samples. Our aggregate data across nine healthy stool donors revealed that all of the individuals harbored gut bacterial species capable of degrading common dietary polysaccharides. However, the levels of richness and abundance of polysaccharide-degrading species relative to monosaccharide-consuming taxa differed by up to 2.6-fold and 24.7-fold, respectively. Additionally, our unique dataset suggested that gut bacterial taxa may be broadly categorized by whether they can grow on single or multiple polysaccharides, and we found that this lifestyle trait is correlated with how broadly bacterial taxa can be found across individuals. This demonstration shows that it is feasible to measure the function of hundreds of bacterial taxa across multiple fecal samples from different people, which should in turn enable future efforts to design microbiota-directed therapies and yield new insights into microbiota ecology and evolution.IMPORTANCE Bacterial culture and assay are components of basic microbiological research, drug development, and diagnostic screening. However, community diversity can make it challenging to comprehensively perform experiments involving individual microbiota members. Here, we present a new microfluidic culture platform that makes it feasible to measure the growth and function of microbiota constituents in a single set of experiments. As a proof of concept, we demonstrate how the platform can be used to measure how hundreds of gut bacterial taxa drawn from different people metabolize dietary carbohydrates. Going forward, we expect this microfluidic technique to be adaptable to a range of other microbial assay needs.

Villa MM ，Bloom RJ ，Silverman JD ，Durand HK ，Jiang S ，Wu A ，Dallow EP ，Huang S ，You L ，David LA ... -  《mSystems》

High throughput cultivation-based screening on porous aluminum oxide chips allows targeted isolation of antibiotic resistant human gut bacteria.

Versluis D ，de J Bello González T ，Zoetendal EG ，Passel MWJV ，Smidt H ... -  《PLoS One》

Droplet Microfluidics for High-Throughput Analysis of Antibiotic Susceptibility in Bacterial Cells and Populations.

Postek W ，Garstecki P 《-》