Pseudomonas aeruginosa (Pa) is an important bacterial pathogen in people with cystic fibrosis and other chronic lung conditions. During early infection, Pa adapts to the environment of the respiratory tract, leading to long-lasting, difficult to treat infections. The ways in which Pa adapt to the lung environment, and the host factors driving this adaptation are poorly understood. Better understanding of Pa in-host adaptation would help identity new diagnosis and treatment approaches and help in the fight against accelerating antimicrobial resistance (AMR).
This project aims to pioneer new models for studying bacterial adapatations to the respiratory tract, by developing culture media that capture key features of the lung and upper airway (nasopharynx) environments. Animal models don't allow nasopharynx and lungs to be studied in isolation, but the two environments may shape bacterial evolution in different ways. Furthermore, as a cheap, rapid means for screening bacterial virulence or adaptation to the host, of testing efficacy of novel therapeutics or of providing information on the AMR profile of bacterial isolates, the media will enable reduction, and in some cases replacement of animal usage.
Our previous work identified the nasopharynx as a protective environment for Pa, enabling gradual adaptation to the host. Host immune molecules, including antimicrobial enzymes such as lysozyme, drive the bacterial adaptation process and influence the emergence of AMR, even in the absence of antibiotic treatment. This work revealed that bacterial mutations leading to susceptibility to antibiotics in liquid culture or on agar may not confer susceptibility in the more relevant in vivo environments of nasopharynx or lung. For example, polyamines, which are abundant in the respiratory tract, can profoundly influence AMR, in some cases counteracting the deleterious effects of mutations that appear to confer antimicrobial susceptibility in MIC assays conducted in broth. There is a pressing need to develop more relevant culture methods for testing AMR and determining the effects of bacterial mutations on pathogen survival and virulence.
Microbiologists grow bacteria for long-periods (months-years) in liquid culture to study their evolution or adaptation to antibiotic stresses. This technique (experimental evolution) has been useful in developing our understanding of bacterial adaptation processes. However, we hypothesise that the conditions of the host environment are key drivers of bacterial evolution and that acquisition of AMR may occur by alternative mechanisms in vivo to those identified in liquid culture experiments. We therefore propose the following:
1. Design of culture conditions replicating key features of nasopharynx and lung environments. We will reproduce factors including temperature, pH, oxygen and carbon dioxide availability, sugar and protein abundance, host-derived antimicrobial levels, and polyamine availability. All of these factors differ between lungs and nasopharynx and are not captured in current bacterial culture media.
2. Experimental evolution of Pa in nasopharynx- and lung-like media over a period of 3-6 months in the presence or absence of antibiotics.
3. Characterisation of experimentally-evolved Pa to identify mutations conferring advantages in the respiratory environment or resistance to antibiotics. This work will also determine the extent to which nasopharynx- and lung-like media accurately reflect the respiratory tract environment.
These studies will determine how different host environments shape bacterial evolution and pathways to emergence of AMR. Infection-relevant culture media will provide a platform for future studies on bacterial adaptive evolution, host-pathogen interactions, virulence and AMR. They will be of use to the wider research community and could be adapted to reflect other host environments such as the brain, intestines or stomach, reducing animal usage across disciplines.