How Bacterial Adaptation to Cystic Fibrosis Environment Shapes Interactions Between Pseudomonas aeruginosa and Staphylococcus aureus

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus are the two most prevalent bacteria species in the lungs of cystic fibrosis (CF) patients and are associated with poor clinical outcomes. Co-infection by the two species is a frequent situation that promotes their interaction. The ability of P. aeruginosa to outperform S. aureus has been widely described, and this competitive interaction was, for a long time, the only one considered. More recently, several studies have described that the two species are able to coexist. This change in relationship is linked to the evolution of bacterial strains in the lungs. This review attempts to decipher how bacterial adaptation to the CF environment can induce a change in the type of interaction and promote coexisting interaction between P. aeruginosa and S. aureus. The impact of coexistence on the establishment and maintenance of a chronic infection will also be presented, by considering the latest research on the subject.

Keywords: P. aeruginosa; S. aureus; cystic fibrosis; evolution; interaction.

FIGURE 1

Evolution of bacterial interactions with Staphylococcus aureus related to Pseudomonas aeruginosa adaptation to CF environment. (A) Reference, environmental and acute infection P. aeruginosa isolates produce high amounts of virulence factors (LasA, HQNO, pyocyanin) or QS signals (3OC12-HSL). These factors allow P. aeruginosa to outcompete S. aureus by lysis mechanisms, growth, metabolism and virulence inhibition. Mixed-species biofilms can be formed through SpA-Psl binding, but can be disrupted by rhamnolipids and cis-2-decenoic acid. (B) P. aeruginosa isolates evolved in CF context coexist with S. aureus due to genetic and transcriptomic adaptations reducing the production of virulence and anti-staphylococcal factors. P. aeruginosa QS networks are rewired and regulated through the C4-HSL signal, that does not affect S. aureus physiology. CF-adapted isolates present a slowed and adjusted metabolism to the carbon sources of the CF environment and/or produced by S. aureus. Matrix of mixed-species biofilm are dominated by the alginate exopolysaccharide. (C) P. aeruginosa adaptation and its interaction with S. aureus are influenced by environmental factors that characterize in vivo conditions. Among them, antibiotic and oxidative stresses, depletion in oxygen, zinc and manganese, and high availability of nutrients and iron were shown to promote a coexisting interaction between the two pathogens. QS, quorum-sensing; SCV, small-colony variants; HQNO, 2-heptyl-4-hydroxyquinoline N-oxide; 3OC12-HSL, N-3-oxo-dodecanoyl homoserine lactone; C4-HSL, butyryl homoserine lactone.

FIGURE 2

Main genetic and phenotypic adaptations of Staphylococcus aureus during its evolution in the CF lung environment. EPS, exopolysaccharide; TD, thymidine; SCV, small colony variants; NETs, Neutrophil Extracellular Traps.

FIGURE 3

Identification methods of the coexisting interaction between Pseudomonas aeruginosa and Staphylococcus aureus. (A) P. aeruginosa and S. aureus are cultivated in liquid mono- and co-culture in a rich medium during 8-h and S. aureus cells are counted in each condition. Competition is characterized by a rapid elimination of S. aureus in co-culture in comparison to the monoculture, whereas S. aureus growth is not affected during the whole kinetic in the case of a coexisting interaction (Briaud et al., 2019; Camus et al., 2020). (B) P. aeruginosa and S. aureus suspensions are prepared from overnight precultures in a rich medium and S. aureus lawn is uniformly plated on a rich agar medium. A P. aeruginosa spot is deposited in the center of the lawn and the plate is incubated 24 h. Competition is characterized by an inhibition halo of S. aureus growth in contact with the spot of P. aeruginosa, whereas S. aureus growth is not affected by P. aeruginosa during the coexisting interaction. Size of the inhibition halo can be measured (Baldan et al., 2014; Briaud et al., 2019, 2020; Camus et al., 2020). (C) P. aeruginosa streak is performed on a rich agar medium, after what a perpendicular S. aureus streak is added. The plate is incubated during 24 h. Competition is characterized by an inhibition of S. aureus growth in contact with the streak of P. aeruginosa and a low S. aureus proportion in the post-streak. Coexistence is characterized by a S. aureus growth in contact with the streak of P. aeruginosa and a high S. aureus proportion in the post-streak. Both of these parameters are visually determined (Michelsen et al., 2016; Minandri et al., 2016; Hotterbeekx et al., 2017). Arrows indicate when or where the competitive (red arrow) or coexisting (blue arrow) interaction is observed.

FIGURE 4

Cooperative behaviors between Pseudomonas aeruginosa and Staphylococcus aureus during coexistence. (A) Antibiotic resistance of both pathogens can be increased by the formation of mixed-species biofilm, especially through the production of alginate and poly-N-acetylglucosamine (PNAG) by P. aeruginosa and S. aureus, respectively. P. aeruginosa and S. aureus formation of small-colony variants (SCV) is also promoted during their interaction. P. aeruginosa presence was also shown to induce the over-expression of efflux pumps from the Nor family in S. aureus, enhancing its antibiotic resistance. (B) S. aureus produces acetoin from pyruvate thanks to AlsSD. P. aeruginosa catabolizes the acetoin produced by S. aureus and uses it as an alternative carbon source thanks to the aco system to feed the Krebs cycle. It improves its growth during co-culture with S. aureus. This catabolism also increases S. aureus survival potentially through a feed-back on acetoin production: the medium depletion in acetoin would promote AlsSD activity, limiting acetate production from pyruvate and thus cell acidification. (C) Resistance to immune system. P. aeruginosa-induced overexpression of tet38 improves S. aureus internalization in epithelial cells, allowing to hide from the immune system. S. aureus can limit P. aeruginosa-induced immune responses, notably through the binding of S. aureus protein A (SpA) to anti-P. aeruginosa antibodies (Anti-Psl IgG).