Evaluating the Impact of Coculture and Cell Proximity of Different Listeria monocytogenes Strains on Time of First Division of Single Cells

This study examined how cellular proximity and interstrain coculture influence the time to first division in L. monocytogenes C5 (4b serotype) and 6179 (1/2a serotype) strains, previously shown to exhibit strong competitive interactions, using time-lapse microscopy. The strains were inoculated on 4.84 cm² Tryptic Soy Agar with 0.6% Yeast Extract as single or mixed 1:1 cocultures, under two population densities: dense proximity (DP) and sparse proximity (SP). Phase-contrast images were acquired every 5 min for 2 h at 37 °C. An in-house-developed program detected and tracked the coordinates of single cells across the image sequence. A total of 105-143 cells from two independent time-lapse experiments were analyzed to determine the times of the first and second divisions. Across both proximity conditions, singly cultured 6179 cells showed a higher percentage of first-division events within 2 h, along with fewer nondividing cells and fewer cells reaching a second division. For both strains, the median time to first division was unaffected by cell proximity. In single cultures, 50% of C5 cells completed their first division within 35-40 min, whereas 6179 cells required approximately 60-65 min. In coculture in SP, 50% of the cells divided for the first time within the first 30 min, almost faster than the singly cultured strains. In contrast, under DP, the median division time shifted to 50-55 min, indicating that close interpopulation proximity can delay first division. These findings indicate that cellular proximity may influence the behavior of the different coexisting strains at a single-cell level.