A fluorescence-based protocol to monitor bacterial antagonism

Bacteria are often found in polymicrobial communities where competition for limited space and resources drives antagonistic interactions. Therefore, bacteria have evolved various antibacterial weapons to outcompete their neighbors. These antagonistic interactions can have profound effects on the structure, dynamics, and composition of bacterial communities. To study interactions between two bacterial species, co-culture assays are often employed. The most common approach utilizes selection plates for each bacterial species to quantify recovery after co-incubation. Although this method is relatively accurate and inexpensive, there are some limitations. These assays can be time-consuming, low-throughput, and may present difficulties if the bacteria of interest show similar resistance patterns to one another or if the tolerance to selective agents is unknown. Here, we have developed and validated a method that uses fluorescence as a proxy to screen antibacterial interactions between two species. We utilized two fluorescently tagged bacteria, Staphylococcus aureus JE2 and Escherichia coli DH5⍺, both expressing a red fluorescent protein (RFP), and competed them against non-fluorescent bacteria: two strains of Pseudomonas aeruginosa or one strain of Stenotrophomonas maltophilia. We observed that RFP production correlates with growth in the reporter strains and that a reduction in relative fluorescent units from the reporter strains corresponds with a reduction in colony-forming units. This method is fast, semi-quantitative, semi-high-throughput, and can be used to rapidly screen for antagonistic activity during bacterial co-cultures. We propose that our protocol can be a useful tool to detect antibacterial activity using fluorescently labeled target bacteria.IMPORTANCEIn nature, bacteria often reside in communities where limited space and resources drive competition. Bacterial antagonistic interactions can profoundly affect microbial communities. A common approach to study these interactions is to measure the recovery of each bacterium after competition by using selective media. While relatively accurate and inexpensive, this approach has a few limitations: the assay can be labor-intensive and time-consuming, is low throughput, and can present issues when the bacterial strains of interest have similar antimicrobial resistance or if their resistance profile is unknown. We developed and validated a fast and semi-high-throughput protocol that gauges antagonistic bacterial interactions using fluorescence as a proxy. As proof of principle, this screening protocol was tested with known antagonistic bacteria, using a fluorescently labeled target bacterium.