Establishing culture conditions for uncultured microbes

PROJECT SUMMARY Microbes play crucial roles in human health. They contribute as pathogens, as sources of enzymes and bioactive compounds, and through their roles in the communities that make up the microbiome which impacts numerous diseases and physiological processes. Cultivating microbes in isolation is a key step for understanding their biology and utilizing their products. Culture allows controlled manipulations, genetic perturbations, analysis of metabolites, and testing interactions with the host and other microbes. However, it is currently difficult to establish culture conditions for uncultured microbes, and many important microbes have not yet been cultured in isolation. Recent advances in the field have made this challenge more tractable, and here we propose to unlock the study and utilization of uncultured microbes by establishing their growth conditions. This work builds on the PIs background in microbiology and microfluidics, especially utilizing two recent contributions: the “SPOTs” liquid handling platform, which allows high-throughput, high-performance liquid handling with unprecedented ease; and a scalable pipeline for 16S genotyping which allows cost effective parallel processing of samples for high- throughput sequencing. This work will be focused on two related projects. In the first project we seek to establish the first axenic culture of the intracellular bacteria Wolbachia, from order Rickettsiales. Wolbachia is prevalent in insects and nematodes playing a key role in and filarial diseases and the spread of viral diseases through insect vectors. To culture Wolbachia, we will generate many thousands of diverse media compositions using the SPOTs platform then monitor cell metabolic activity with redox active dyes and cell division with microscopy to determine which conditions are most favorable. Iteratively we incorporate data from each experimental cycle to refine the media compositions until they can support growth. Axenic Wolbachia growth would be a major advance that may also unlock culture of additional organisms from Rickettsiales, of which there are dozens of important uncultured pathogens. In the second project we will utilize a “many-by-many” strategy, generating many diverse medias, using SPOTs, to simultaneously grow many different microbes from different starting communities including fecal and soil samples. Growth will be monitored by microscopy and which organisms grow in which media compositions will be determined using our high throughput 16S genotyping method. We expect that this approach will culture a wide range of yet-uncultured organisms, enabling their characterization and use and providing a rich dataset linking different microbes to the conditions in which they grow. The impacts of this work include catalyzing the study and use of new microbes by determining their culture conditions and contributing a powerful new approach for addressing combinatorial questions in biomedicine.