Evaluating the impact of co-culture and cell proximity of different Listeria monocytogenes strains on time of first division of single-cells

• Density and cell proximity affected the time to first division at single-cell level. • C5 strain exhibited faster time to first division and greater adaptability. • Competition and signalling potentially slowed down division in dense cultures. • Phenotypic diversity in L. monocytogenes C5 strain represents a survival strategy. This study examined how cellular proximity and inter-strain co-culture influence the time to first division in Listeria 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 cm 2 Tryptic Soy Agar with 0.6% Yeast Extract as single or mixed 1:1 co-cultures, under two population densities: dense proximity (DP) and sparse proximity (SP). Phase-contrast images were acquired every 5 min for 2h 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 2h, along with fewer non-dividing 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 co-culture 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 inter-population proximity can delay first division. These findings indicate that cellular proximity may influence the behavior of the different co-existing strains at a single-cell level.