Pseudomonas aeruginosa and Staphylococcus aureus are two key opportunistic pathogens that cause chronic lung infections in cystic fibrosis (CF) patients. Polymicrobial infections with P. aeruginosa and S. aureus are associated with worsened clinical outcomes in CF patients, and unknown still are the mechanisms that cause an increase in patient morbidity and mortality. Studying the interactions between P. aeruginosa and S. aureus is difficult because when co-cultured in vitro, P. aeruginosa drastically outcompetes and eradicates S. aureus cultures. This study explores methods for growing planktonic co-cultures of P. aeruginosa and S. aureus to stationary phase in equal proportions, and this will allow for the examination of changes in quorum-regulated phenotypes.

We grew liquid co-cultures of P. aeruginosa and S. aureus in LB Lennox media and examined their absolute and relative cell densities by plating the co-cultures on selective media. We evaluated the influence of oxygen concentration and co-inoculation vs. staggered inoculation on the ability to achieve a co-cultures with two P. aeruginosa (PA) and two S. aureus (SA) strains. The method that consistently produced PA:SA ratios in the range of 1:1 to 1:100 was to allow a SA mono-culture to reach stationary phase, and then re-suspend the SA cells in fresh media before inoculating with PA. With this method, it is possible to grow both PA and SA to stationary phase, a necessity for studying how PA and SA alter phenotypes in the presence of one another.

P. aeruginosa was found to produce less pyocyanin in the presence of S. aureus, but reduction in pyocyanin expression was depended on the strain of S. aureus. Elastase production differed between the two P. aeruginosa strains as well as between the two S. aureus strains, one increasing and one decreasing in expression. This data indicates that the responses of P. aeruginosa to S. aureus differ depending on both the P. aeruginosa and S. aureus strain present.