Competitivity Identification

At each sampling point of the competition experiments above, the colonies were gently scraped out from the plates by using autoclaved bamboo sticks with flat-shaped shovel at one end and dispersed in 1.0 ml of sterile saline solution, and then the bacterial cells were washed for three times. A fraction of appropriately diluted bacterial suspension was spread on M9-milk plate and cultured overnight (17 h) for species discrimination. WT PAO1 would form large colony size with clear proteolytic halo around the colony, P. aeruginosa lasR mutant would form relatively small colony without proteolytic halo, and K. pneumonia would form large colony size with no or faint proteolytic halo. The strains were further confirmed by IVIS XRII imaging to check the fluorescent colors or by colony PCR using specific primer pair 5′-CTTCATCGTCGGCAACTAC-3′ and 5′-GTCTGGTAGATGGACGGTTC-3′ to amplify the partial lasR gene of P. aeruginosa. Positive PCR-amplicon could only be obtained from WT PAO1. After calculating the proportion of each species in the mixed colonies at each sampling points, the relative fitness (v) of one species was then calculated by comparing the initial (x₀) and final frequencies (x₁) using the modified equation v = log10[x₁ (1-x₀)/x₀ (1-x₁)] based on previous description (Diggle et al., 2007a).