Quantifying Chemotaxis in Escherichia coli and Bdellovibrio bacteriovorus Using A Flow-Based Microfluidic Device

Abstract

Bdellovibrio bacteriovorus is a small, highly motile, predatory bacterium. It preys on a wide variety of gram-negative bacteria, including some human pathogens. As a result, Bdellovibrio has been proposed as a non-pharmaceutical method of biofilm eradication. Biofilms are communities of microorganisms that grow at interfaces. They are notoriously difficult to destroy and do not respond to conventional treatments such as antibiotics. Biofilms can easily contaminate medical devices thus posing a serious threat to humans. Though Bdellovibrio has been shown to consume bacteria in biofilms, its methods of prey location and detection are not yet understood. Genomic insight indicates that chemotaxis is a likely detection method. Bdellovibrio contains 20 chemotaxis receptor genes, four times more than the number in E. coli, a known chemotatic species. Previous studies have revealed that Bdellovibrio containing a mutation in one of these receptor genes is a much less efficient predator, further supporting the chemotaxis theory.

The focus of our research was to determine whether Bdellovibrio is a chemotactic species and, if so, to identify its chemoattractants and chemorepellants. In order to detect whether Bdellovibrio moves toward stimuli, we used a flow based microfluidic assay. We chose these assays due to their transparency, facile fabrication and straightforward gradient formation. At the sub-millimeter scale, fluid flow is laminar, allowing for consistent automatic linear gradient formation over long periods of time. The device used for this project, based upon the design proposed by Mao et al., consists of a main channel, where the chemical gradient forms, which is then partitioned into smaller outlet channels. Each of these outlets contains a different chemical concentration. By measuring the cell population in each outlet, the chemotactic effects of a chemical, as a function of concentration, can be calculated. Cells were visualized by staining with a LIVE/DEAD fluorescent dye to ensure only live cells were included in calculations and observed under an inverted fluorescence microscope. We assessed the devices’ functionality by running control trials with E. coli as its chemotactic ability has been well documented. These control and experimental tests verified that the design and construction of the devices could successfully be used to study bacterial motility in chemical gradients. Trial runs using Bdellovibrio were then performed yielding promising control results with both cell distributions fitting a Gaussian curve robustly. Bdellovibrio was then tested using casamino acids as a chemoeffector yielding inconclusive results. While more experimental runs using Bdellovibrio are necessary, one major finding of this work is the importance of a large cell population.