MicroMPN: methods and software for high-throughput screening of microbe suppression in mixed populations

Abstract

ABSTRACT Screening assays are used to test if one or more microbes suppress a pathogen of interest. In the presence of more than one microbe, the screening method must be able to accurately distinguish viable pathogen cells from non-viable and non-target microbes in a sample. Current screening methods are time-consuming and require special reagents to detect viability in mixed microbial communities. Screening assays performed using soil or other complex matrices present additional challenges for screening. Here, we develop an experimental workflow based on the most probable number (MPN) assay for testing the ability of synthetic microbial communities to suppress a soil-borne pathogen. Our approach, fluorMPN, uses a fluorescently labeled pathogen and microplate format to enable high-throughput comparative screening. In parallel, we developed a command-line tool, MicroMPN, which significantly reduces the complexity of calculating MPN values from microplates. We compared the performance of the fluorMPN assay with spotting on agar and found that both methods produced strongly correlated counts of equal precision. The suppressive effect of synthetic communities on the pathogen was equally recoverable by both methods. The application of this workflow for discriminating which communities lead to pathogen reduction helps narrow down candidates for additional characterization. Together, the resources offered here are meant to facilitate and simplify the application of MPN-based assays for comparative screening projects.IMPORTANCEWe created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.

Keywords

• most probable number
• MicroMPN software
• fluorMPN protocol
• synthetic bacterial communities
• soil microcosms
• pathogen suppression