Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa

Abstract

Significance Obtaining cultures of microbes is essential for developing knowledge of bacterial genetics and physiology, but many microbes with potential biomedical significance identified from metagenomic studies have not yet been cultured due to the difficulty of identifying growth conditions, isolation, and characterization. We developed a microfluidics-based, genetically targeted approach to address these challenges. This approach corrects sampling bias from differential bacterial growth kinetics, enables the use of growth stimulants available only in small quantities, and allows targeted isolation and cultivation of a previously uncultured microbe from the human cecum that belongs to the high-priority group of the Human Microbiome Project’s “Most Wanted” list. This workflow could be leveraged to isolate novel microbes and focus cultivation efforts on biomedically important targets.

High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

“…In recent years, droplet microfluidics has attracted substantial interest because of the capacity for high-throughput screening and single-cell sorting (23). Great efforts have been dedicated to microbial cultivation by developing large-scale storage of droplets in microchannels (24,25) or microfabricated chambers (26)(27)(28). Compared with diffusive compartments, isolation in droplets offers advantages, including prevention of interspecific competition and elimination of biases due to differences in growth rates (29), and is more likely to recover species for scale-up with well-defined cultivation conditions.…”

“…Compared with diffusive compartments, isolation in droplets offers advantages, including prevention of interspecific competition and elimination of biases due to differences in growth rates (29), and is more likely to recover species for scale-up with well-defined cultivation conditions. Recently, splitting of individual microcultures with a SlipChip device has enabled PCR identification of target species even under conditions of high background noise (i.e., interfering species) and resulted in the isolation of a previously uncultivated gut bacterium (26). However, further development is needed to effectively cultivate encapsulated cells over pro-longed time periods and to be able to access specific droplets containing targeted species at multiple time points.…”

Most of the Dominant Members of Amphibian Skin Bacterial Communities Can Be Readily Cultured

“…It is estimated that only 0.001% to 15% of microbes can be cultured by use of commonly used techniques and media (2,4), although some recent attempts at culturing the uncultured have been successful. For example, a prevalent and abundant human gut bacterium with potential biomedical importance was recently cultured by a gene-targeted approach (5). Still, only a third of known bacterial phyla have cultured representatives (6).…”

The pumping lid: investigating multi-material 3D printing for equipment-free, programmable generation of positive and negative pressures for microfluidic applications

“…Experiments taking place in a research lab can benefit from this compact and equipment-free approach, reducing the need for external connectors and simplifying the workflow, especially when experiments are conducted in the controlled environments of a cell culture incubator [42][43][44] or an anaerobic chamber. 45 Additionally, contamination from the external environment and evaporation are minimized because the sample is contained in the pumping cup during the entire experiment. The pumping lid also allows flow rates to be tuned in real time while the experiment is in progress.…”