Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

Development of liquid culture media mimicking the conditions of sinuses and lungs in cystic fibrosis and health.

The respiratory tract is a compartmentalised and heterogenous environment. The nasopharynx and sinuses of the upper airways have distinct properties from the lungs and these differences may shape bacterial adaptation and evolution. Upper airway niches act as early colonisation sites for respiratory bacterial pathogens, including those, such as Pseudomonas aeruginosa, that can go on to establish chronic infection of the lungs in people with cystic fibrosis (CF). Despite the importance of upper airway environments in facilitating early adaptation to host environments, currently available in vitro models for study of respiratory infection in CF focus exclusively on the lungs. Furthermore, animal models, widely used to bridge the gap between in vitro systems and the clinical scenario, do not allow the upper and lower airways to be studied in isolation. We have developed a suite of culture media reproducing key features of the upper and lower airways, for the study of bacterial adaptation and evolution in different respiratory environments. For both upper and lower airway-mimicking media, we have developed formulations that reflect airway conditions in health and those that reflect the altered environment of the CF respiratory tract. Here, we describe the development and validation of these media and their use for study of genetic and phenotypic adaptations in P. aeruginosa during growth under upper or lower airway conditions in health and in CF.