Quantifying synthetic bacterial community composition with flow cytometry: efficacy in mock communities and challenges in co-cultures

Determination of bacterial community composition in synthetic communities is critical for understanding microbial systems. The community composition is typically determined through bacterial plating or through PCR-based methods, which can be labor-intensive, expensive, or prone to bias. Simultaneously, flow cytometry has been suggested as a cheap and fast alternative. However, since the technique captures the phenotypic state of bacterial cells, accurate determination of community composition could be affected when bacteria are co-cultured. We investigated the performance of flow cytometry for quantifying oral synthetic communities and compared it to the performance of strain specific qPCR and 16S rRNA gene amplicon sequencing. Therefore, axenic cultures, mock communities and co-cultures of oral bacteria were prepared. Random forest classifiers trained on flow cytometry data of axenic cultures were used to determine the composition of the synthetic communities, as well as strain specific qPCR and 16S rRNA gene amplicon sequencing. Flow cytometry was shown to have a lower average root mean squared error and outperformed the PCR-based methods in even mock communities (flow cytometry: 0.11 ± 0.04; qPCR: 0.26 ± 0.09; amplicon sequencing: 0.15 ± 0.01). When bacteria were co-cultured, neither flow cytometry, strain-specific qPCR, nor 16S rRNA gene amplicon sequencing resulted in similar community composition. Performance of flow cytometry was decreased compared with mock communities due to changing phenotypes. Finally, discrepancies between flow cytometry and strain-specific qPCR were found. These findings highlight the challenges ahead for quantifying community composition in co-cultures by flow cytometry.IMPORTANCEQuantification of bacterial composition in synthetic communities is crucial for understanding and steering microbial interactions. Traditional approaches like plating, strain-specific qPCR, and amplicon sequencing are often labor-intensive and expensive and limit high-throughput experiments. Recently, flow cytometry has been suggested as a swift and cheap alternative for quantifying communities and has been successfully demonstrated on simple bacterial mock communities. However, since flow cytometry measures the phenotypic state of cells, measurements can be affected by differing phenotypes. Especially, changing phenotypes resulting from co-culturing bacteria can have a profound effect on the applicability of the technique in this context. This research illustrates the feasibility and challenges of flow cytometry for the determination of community structure in synthetic mock communities and co-cultures.