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

Abstract

<jats:p>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.</jats:p>

Microfluidic devices for studying bacterial taxis, drug testing and biofilm formation

“…Watterson et al isolated bacteria from faecal microbiota transplants, and by optical density, they selected those that showed slower growth. Genome identification showed the presence of bacteria previously uncultured by traditional methods (Watterson et al, 2020).…”

Towards culturing the microbe of your choice

“…More tech‐heavy cultivation methods include microdroplet encapsulation, where individual cells or small groups of cells are separated into droplet emulsions which can be sorted by a variety of means into wells of a 96‐well plate, separate tubes, or even solid media (Zengler et al ., 2002; Watterson et al ., 2020). These microdroplets ensure effective cell separation and can keep them among closely associated organisms on which they may depend.…”

“…These microdroplets ensure effective cell separation and can keep them among closely associated organisms on which they may depend. Microdroplet encapsulation has been successful in a variety of environments from aquatic to human gut (Zengler et al ., 2002; Boedicker et al ., 2009; Jiang et al ., 2016; Villa et al ., 2020; Watterson et al ., 2020). Another less conventional approach is the application of electrochemical methods whereby microbes grow on or near electrode surfaces.…”

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

“…A complementary trend is the miniaturization of culture vessels to save material and time while increasing throughput [11,13,[25][26][27][28][29][17][18][19][20][21][22][23][24]. Thereby, the chance of finding non-dormant, culturable variants of naturally occurring species, as stochastic events [30], rises.…”

“…At the same time, the miniaturized vessels 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 [17][18][19][20][21][22][23][24][25]27] and techniques that keep the compartments not fixed to positions but rather mobile within the bulk population [11,13,26,28,29]. The clear advantage of arrays is the possibility to address the same culture several times since the compartments are identified by their coordinates.…”