Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and there is evidence that their interactions negatively affect disease outcome in patients. At the molecular level, interactions between the two bacterial species are well-described, with P. aeruginosa usually being the dominant species suppressing S. aureus through a variety of inhibitory molecules. However, in chronic infections the two species interact over prolonged periods of time, and S. aureus might be able to evolve resistance against inhibitory molecules deployed by P. aeruginosa . Here, we used experimental evolution to test this hypothesis by exposing three different S. aureus strains (Cowan I, 6850, and JE2) to the growth-inhibitory supernatant of P. aeruginosa PAO1 over 30 days. Prior to evolution, we found that S. aureus strains were inhibited by secreted compounds regulatorily controlled by the Pseudomonas quinolone signal quorum-sensing system. Following evolution, inhibitory effects were significantly attenuated, and we observed that adaptations were S. aureus strain specific and involved the upregulation of virulence traits such as staphyloxanthin production and the formation of small colony variants. At the genetic level, mutations in membrane transporters (known to be involved in antibacterial uptake) were the most frequent evolutionary targets. Our work indicates that adaptations of S. aureus to P. aeruginosa occurs rapidly and affect both virulence trait expression and membrane transporter functionality. Thus, pathogen evolution could promote species co-existence and complicate treatment options in infections. IMPORTANCE Polymicrobial infections are common. In chronic infections, the different pathogens may repeatedly interact, which could spur evolutionary dynamics with pathogens adapting to one another. Here, we explore the potential of Staphylococcus aureus to adapt to its competitor Pseudomonas aeruginosa . These two pathogens frequently co-occur, and P. aeruginosa is seen as the dominant species being able to displace S. aureus . We studied three different S. aureus strains and found that all became quickly resistant to inhibitory compounds secreted by P. aeruginosa . Our experimental evolution revealed strains-specific adaptations with three main factors contributing to resistance evolution: (i) overproduction of staphyloxanthin, a molecule protecting from oxidative stress; (ii) the formation of small colony variants also protecting from oxidative stress; and (iii) alterations of membrane transporters possibly reducing toxin uptake. Our results show that species interactions can change over time potentially favoring species co-existence, which in turn could affect disease progression and treatment options.

Experimentally evolved Staphylococcus aureus shows increased survival in the presence of Pseudomonas aeruginosa by acquiring mutations in the amino acid transporter, GltT

“…newly recognized role for the gltT gene influencing S. aureus and P. aeruginosa interactions Interactions between S. aureus and P. aeruginosa have proven to be complex, and dependent on environment and strain background [12,16,[38][39][40][41]. Studies have implicated factors such as the P. aeruginosa mucoid phenotype, Pseudomonas excreted compounds or toxins, and S. aureus metabolic pathways such as the production of acetoin, as important factors in the interspecific interactions between S. aureus and P. aeruginosa [7,8,16,38,42,43].…”

“…Previous studies have demonstrated that the laboratory strains, S. aureus USA300 JE2 and P. aeruginosa PAO1, interact competitively when cultured together and that those interactions are greatly influenced by factors such as alginate and quinolone production by PAO1 [9][10][11]. Additionally, it has been observed that JE2 can adapt to secreted factors present in PAO1 supernatant in short-term evolution experiments [12]. Other studies have shown that adaptations to specific compounds such as 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) and pyocyanin may impact the antibiotic resistance profiles of either species [9,[13][14][15].…”

Mutations in theStaphylococcus aureusGlobal Regulator CodY Confer Tolerance to an Interspecies Redox-Active Antimicrobial

“…Data File 02), we would not expect similar selective pressures in our assay. In a separate study, S. aureus evolved in the presence of P. aeruginosa supernatant showed strain-dependent acquisition of resistance that converged on staphyloxanthin (carotenoid) production and the formation of small colony variants (SCVs) (110). Interestingly, in two different strains, 1 out of 5 populations each encoded a mutation in codY, one being intergenic and the other a non-synonymous mutation.…”

Interactions betweenPseudomonas aeruginosaand six opportunistic pathogens cover a broad spectrum from mutualism to antagonism

“…Especially, interactions between key pathogens like P. aeruginosa and Staphylococcus aureus have been extensively studied as these species often co-occur in patients (Hotterbeekx et al, 2017; Ibberson and Whiteley, 2020; Limoli and Hoffman, 2019; Nguyen and Oglesby-Sherrouse, 2016). Research focused on various aspects including molecular mechanisms of interactions (Armbruster et al, 2016; Limoli et al, 2019; Yarrington et al, 2024; Zarrella and Khare, 2022), variation in interactions between different strains and across environments (Bernardy et al, 2022; Niggli et al, 2021; Niggli and Kümmerli, 2020), the evolution of interactions (Niggli et al, 2023; Tognon et al, 2017) and the consequences of interactions for the host (Orazi and O’Toole, 2017; Radlinski et al, 2017; Rezzoagli et al, 2020). Interactions among other pathogens (e.g.…”