Development of stable co‐culture of <i>P. aeruginosa</i> and <i>S. aureus</i> in Artificial Sputum Medium

Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are both opportunistic pathogens that are known to cause severe respiratory infections in humans. While these pathogens don't commonly cause infections in healthy individuals, they are often found in patients with chronic lung diseases, such as Cystic Fibrosis (CF). Data from people with CF disease indicates that CF lungs are predominantly infected with SA at a younger age with PA appearing later (<10 years) when it then outcompetes SA. There is, however, significant overlap where both pathogens co‐exist in the patient's lungs. While clinical data on prevalence is abundant, little data exists on mechanisms of competition of two pathogens in CF lungs. Artificial sputum medium (ASM) is a culture medium that was designed to mimic sputum from CF patients. It contains components of CF sputum, such as amino acids, mucin and DNA. It was shown that PA growth in the ASM is similar to the growth in the lungs of CF patients. We hypothesised that the ASM model could be adapted to study the interaction between SA and PA in a controlled environment that is relevant to the in vivo environment of CF lungs. SA (ATCC29213) and PA (H174) were grown in the artificial sputum media (ASM). Approximately 3x10 5 bacteria were added to 15 mL ASM and grown at 37 o C with constant shaking. Conditioned media was prepared by growing SA in ASM for 24 hours and then removing SA. SA appeared to adapt quicker to the nutrient‐poor environment of ASM than PA and enters logarithmic growth immediately upon incubation, as opposed to PA, which exhibited an 8‐hour lag period. When both pathogens are added simultaneously to the ASM, SA outcompeted PA and dominated the culture restricting the growth of PA. To better mimic in vivo conditions, ASM was conditioned by adding SA for 24 hours. This allowed creation of an environment that is similar to that found in the CF lungs with established SA infections. When both pathogens were added into the conditioned media, PA did not exhibit the lag phase and outcompeted SA, suppressing its growth. This effect remained even if SA outnumbered PA 10 000‐fold. In an attempt to create media that is most similar to that of CF sputum, ASM was mixed 50:50 with conditioned media, which represents the constant renewal of secreted products and nutrients seen in the lungs. When both pathogens were added to the media, rapid growth was observed in both pathogens for 24 hours. After 24 hours the population of SA decreased from 1x10 9 CFU/ml to 3x10 6 CFU/ml, while the PA population remained stable at 1x10 8 CFU/ml. After 48 hours, the population of both pathogens remained constant. By modifying ASM with conditioned ASM, we have created a stable in vitro co‐culture of Staphylococcus aureus and Pseudomonas aeruginosa . Our model can be utilised to examine the competition and/or co‐existence between clinical strains and other bacterial species that reside within the lungs and identify factors which suppress pathogen growth.

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