Long term exposure of cystic fibrosis associated pathogens to hypoxia contributes to their adaptation to chronic infection

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which causes chronic lung infections in people with cystic fibrosis (CF) and is a major contributor to morbidity and mortality. Despite this, the mechanism driving its adaptation towards chronic colonisation in the CF lung is not yet fully understood. This work focuses on the adaptations of P. aeruginosa to hypoxia, one of the important environmental pressures present in the lung. Long term hypoxia has yet to be examined as a driver of adaptation towards the persistence of this bacterium in CF patients. We have studied the effect of long term adaptation to 6% oxygen for up to 28 days. Interestingly, distinctive colony morphotypes developed, including small colony variants. One of the observed morphotypes appeared exclusively under low-oxygen pressure. Importantly, the colonies with visibly different morphologies were more common in hypoxia-adapted cultures, comprising up to 98% of the population, while they never exceeded 35% in normoxia-adapted cultures. Proteomic analysis showed changes in the abundance of over 200 proteins within 28 days of adaptation, including those involved in antibiotic resistance, stress response, motility and biofilm formation. Phenotype experiments showed two distinct patterns of adaptation to hypoxic conditions. Two of the hypoxia adapted cultures showed increased biofilm formation and swimming and swarming motility, but decreased twitching motility. All three types of motility and biofilm formation were decreased in the third culture. The hypoxia-adapted cultures developed higher resistance to 4 out of 7 antibiotics tested. These results confirm that hypoxia indeed promotes the appearance of many phenotypes in P. aeruginosa that are associated with persistence in the lung and with poor patient outcomes.