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Pseudomonas aeruginosa  and  Staphylococcus aureus  frequently occur together in polymicrobial infections, and their interactions can complicate disease progression and treatment options. While interactions between  P. aeruginosa  and  S. aureus  have been extensively described using planktonic batch cultures, little is known about whether and how individual cells interact with each other on solid substrates. This is important because both species frequently colonize surfaces to form aggregates and biofilms in infections. Here, we performed single-cell time-lapse fluorescence microscopy, combined with automated image analysis, to describe interactions between  P. aeruginosa  PAO1 with three different  S. aureus  strains (Cowan I, 6850, JE2) during microcolony growth on agarose surfaces. While  P. aeruginosa  is usually considered the dominant species, we found that the competitive balance tips in favor of  S. aureus  on surfaces. We observed that all  S. aureus  strains accelerated the onset of microcolony growth in competition with  P. aeruginosa  and significantly compromised  P. aeruginosa  growth prior to physical contact. Upon direct contact, JE2 was the most competitive  S. aureus  strain, simply usurping  P. aeruginosa  microcolonies, while 6850 was the weakest competitor itself suppressed by  P. aeruginosa . Moreover,  P. aeruginosa  reacted to the assault of  S. aureus  by showing increased directional growth and expedited expression of quorum sensing regulators controlling the synthesis of competitive traits. Altogether, our results reveal that quantitative single-cell live imaging has the potential to uncover microbial behaviors that cannot be predicted from batch culture studies, and thereby contribute to our understanding of interactions between pathogens that co-colonize host-associated surfaces during polymicrobial infections.

Single-Cell Imaging Reveals That Staphylococcus aureus Is Highly Competitive Against Pseudomonas aeruginosa on Surfaces