Trophic cooperation promotes bacterial survival of Staphylococcus aureus and Pseudomonas aeruginosa

Laura Camus, Paul Briaud, Sylvère Bastien, Sylvie Elsen, Anne Doléans-Jordheim, François Vandenesch, Karen Moreau

Posted on: 26 June 2020 , updated on: 25 March 2021

Preprint posted on 18 June 2020

Article now published in The ISME Journal at

Co-existence of S. aureus and P. aeruginosa in cystic fibrosis infections is promoted by shared metabolic pathways

Selected by Josie Gibson

Categories: microbiology


The lung is populated by many microorganisms, including pathogenic species. Cystic fibrosis (CF) is a disease which causes production of sticky mucus, which can promote microbial infections. Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent pathogens found in CF patients and interactions between P. aeruginosa and S. aureus have been established. In some cases, defined as competitive, P. aeruginosa can target S. aureus with virulence factors. However, there are some non-competitive interactions which are associated with chronic infection in CF, defined as co-existing. P. aeruginosa adaption to the infection environment and antimicrobial treatments is thought to be responsible for the establishment of co-existing infections [1]. The aim of this study is to compare gene expression of P. aeruginosa in co-culture with S. aureus between competitive and co-existing strains using transcriptomics.

Key findings:

The primary finding of this study is that co-existence of P. aeruginosa and S. aureus may be caused by trophic cooperation in chronic cystic fibrosis cases. Changes in P. aeruginosa gene expression in the presence of S. aureus were compared between competitive or co-existing pairs of each pathogen, which had been isolated together from patients. In competitive scenarios, the presence of S. aureus significantly altered the expression of 68 P. aeruginosa genes. In co-existence settings, 105 P. aeruginosa genes where significantly altered by S. aureus in co-culture, where the majority of genes identified were involved in metabolism, specifically an up-regulation of genes required for the use of alternative carbon sources, including amino acids and acetoin. These observations were confirmed with RT-qPCR using further patient samples.

The role of altered P. aeruginosa metabolism gene expression to cause the ability to co-exist with S. aureus was next examined. The authors make the important observations that S. aureus produces acetoin, and that acetoin is present in CF patients. Next, it is demonstrated that acetoin produced by S. aureus induces changes in P. aeruginosa gene expression, which subsequently promotes P. aeruginosa acetoin catabolism, useful as an alternative carbon source. Interestingly, the production of acetoin by S. aureus was higher in the co-existing strains in comparison to competitive strains. Equally, co-existing P. aeruginosa was better able to catabolise acetoin than competitive strains. The authors suggest these attributes of co-existing strains are due to adaptation in chronic CF infections. Although acetoin represents a useful alternative carbon source for P. aeruginosa, high concentrations of acetoin were shown to reduce S. aureus growth, so a positive role of acetoin for S. aureus was next examined. S. aureus had reduced survival in co-culture with P. aeruginosa strains deficient in acetoin catabolism in comparison to wild-type P. aeruginosa. This indicates that P. aeruginosa acetoin catabolism is beneficial for S. aureus too, by protecting against toxic levels of acetoin. Therefore, both S. aureus and P. aeruginosa benefit from acetoin through a shared metabolic pathway.


Why I chose this Preprint:

Understanding complex polymicrobial interactions in human infection may be important for future therapeutic developments. P. aeruginosa and S. aureus are often found together in CF cases, and although the ability of P. aeruginosa to kill S. aureus is established, in chronic CF infections P. aeruginosa and S. aureus can co-exist. This study reveals that two pathogens benefit from shared metabolic pathways, based on acetoin production and catabolism, which likely promote their ability to cause chronic infection.


Questions to the authors:

  1. Could acetoin be a carbon source produced by other microbiota species which may indirectly promote S. aureus co-existence with P. aeruginosa?


  1. The changes to P. aeruginosa gene expression of liuA (involved in leucine metabolism) requires an interaction with S. aureus, do you have a suggestion of what role liuA may play in co-existing infections?


  1. The production of acetoin by S. aureus was increased in the presence of the co-existing P. aeruginosa strain, perhaps suggesting that S. aureus senses and responds to P. aeruginosa, do you know how this effect is caused?



  1. Baldan R, Cigana C, Testa F, Bianconi I, De Simone M, Pellin D, et al. Adaptation of Pseudomonas aeruginosa in Cystic Fibrosis airways influences virulence of Staphylococcus aureus in vitro and murine models of co-infection. PLoS One. 2014;9(3):e89614.

Tags: acetoin, cystic fibrosis, metabolism, p. aeruginosa, s. aureus


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Author's response

Laura Camus shared

  1. Could acetoin be a carbon source produced by other microbiota species which may indirectly promote aureus co-existence with P. aeruginosa?

Acetoin is indeed known to be produced by other microbial species; Bacillus subtilis is for instance a great acetoin producer (1). Regarding microorganisms colonizing CF environment, clinical isolates of Stenotrophomonas maltophilia were sometimes shown to produce the molecule, although it is not a generality (2, 3). Acetoin dosed in the CF sputa can thus arise from production by S. aureus but also by other microbial species present in the sample, as well as other microorganisms than P. aeruginosa might catabolize acetoin in these conditions (1). This question could be addressed by evaluating the abilities of acetoin production and catabolism of all microorganisms isolated from the CF sputa, which can represent a lot of work due to the number of samples.

We observed that acetoin production and catabolism were both more efficient for coexisting strains of P. aeruginosa and S. aureus. However, if acetoin can promote the coexisting interaction between P. aeruginosa and S. aureus remains unclear. Coexistence seems to be related to P. aeruginosa adaptation to CF environment, that induces a decreased production of virulence and thus anti-staphylococcal factors. This low-virulent state was shown to be selected in response to selective pressures mainly induced by the host immune system (4, 5).

In one hand, acetoin could also constitute a selective force and directly favor a non-virulent state of P. aeruginosa. In this case, acetoin presence, produced by S. aureus or other microorganisms, would play a role in the coexistence establishment. In the other hand, evolution in the CF lungs also goes along with trophic adaptation according to the available resources in this environment (6). The acetoin metabolism between our two pathogens might arise from this trophic adaptation, independently of any virulence modifications. If so, acetoin presence would probably not affect the anti-staphylococcal behavior of P. aeruginosa, and thus coexistence.

  1. The changes to aeruginosa gene expression of liuA (involved in leucine metabolism) requires an interaction with S. aureus, do you have a suggestion of what role liuA may play in co-existing infections?

P. aeruginosa trophic adaptation to CF lung goes along with auxotrophy and could explain why leucine degradation seems to be an important feature of our clinical strains (4). We suppose that P. aeruginosa overexpresses genes of the leucine catabolism pathway in co-culture with S. aureus due to elevated leucine levels in these conditions, in comparison to P. aeruginosa monoculture. Although this amino acid is already part of the rich medium we use (BHI), elevated leucine levels could come from a production by S. aureus. Interestingly, leucine and acetoin biosynthesis pathways are connected and they share a direct and common precursor (1, 7). This suggests that leucine, besides acetoin, may be overproduced by S. aureus during the coexisting interaction. We hypothesize that leucine, and thus P. aeruginosa’s liuA gene, might be the factors of another trophic cooperation between the two bacteria.

Why liuA overexpression relies on S. aureus presence requires further investigations. Contrary to acetoin, leucine may not be secreted by S. aureus in the medium in absence of P. aeruginosa. This could explain why S. aureus supernatant (grown in monoculture) does not induce the liuA overexpression in P. aeruginosa. It is also possible that other signals than leucine play a role in liuA induction. In all cases, leucine dosages in mono- and co-culture would certainly answer many questions about this mechanism.

  1. The production of acetoin by aureus was increased in the presence of the co-existing P. aeruginosa strain, perhaps suggesting that S. aureus senses and responds to P. aeruginosa, do you know how this effect is caused?

We indeed observed an increased acetoin production by coexisting S. aureus strains in comparison to competitive ones, but only when the strains were cultivated in P. aeruginosa supernatant.

This phenomenon could rely only on nutritional and metabolism clues. Indeed, it is likely that P. aeruginosa supernatant presents a highly different composition than unaltered rich medium, with different proportions of each resource usable by S. aureus. This specific nutrient composition may dysregulate metabolic pathways of S. aureus, including acetoin biosynthesis. Coexisting S. aureus thus appears to regulate their metabolism differentially than competitive ones according to the medium composition. Such metabolic adaptations were already described for P. aeruginosa in the CF context (6).

However, it is also possible that this effect relies on P. aeruginosa quorum-sensing (QS) molecules, that are diffused in the medium and thus in the culture supernatant. Besides their role in intra-species interactions, it is well-known that these signals can be sensed by other microorganisms and affect their activities (8). It would be possible that some QS-molecules of P. aeruginosa are able to directly modify the regulation of acetoin production in S. aureus, especially in the coexistence context. Interestingly, we observed that a lasR defective mutant of PA14 was able to catabolize acetoin more efficiently that the wild-type strain, supporting the role of QS in this activity (not published).

In all cases, the exact signals and mechanisms of this increased production of acetoin in presence of P. aeruginosa supernatant remain to be studied. By performing experimental evolution, we aim to confirm that this phenomenon arises from co-evolution of P. aeruginosa and S. aureus.


  1. Xiao Z, Xu P. 2007. Acetoin Metabolism in Bacteria. 2. Crit Rev Microbiol 33:127–140.
  2. Dryahina K, Sovová K, Nemec A, Španěl P. 2016. Differentiation of pulmonary bacterial pathogens in cystic fibrosis by volatile metabolites emitted by their in vitro cultures: Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia and the Burkholderia cepacia complex. 3. J Breath Res 10:037102.
  3. Amoli RI, Nowroozi J, Sabokbar A, Rajabniya R. 2017. Isolation of Stenotrophomonas maltophilia from clinical samples: An investigation of patterns motility and production of melanin pigment. 9. Asian Pac J Trop Biomed 7:826–830.
  4. Cullen L, Weiser R, Olszak T, Maldonado RF, Moreira AS, Slachmuylders L, Brackman G, Paunova-Krasteva TS, Zarnowiec P, Czerwonka G, Reilly J, Drevinek P, Kaca W, Melter O, De Soyza A, Perry A, Winstanley C, Stoitsova SR, Lavigne R, Mahenthiralingam E, Sá-Correia I, Coenye T, Drulis-Kawa Z, Augustyniak D, Valvano MA, McClean S. 2015. Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains. Microbiol Read Engl 161:1961–1977.
  5. Hotterbeekx A, Kumar-Singh S, Goossens H, Malhotra-Kumar S. 2017. In vivo and In vitro Interactions between Pseudomonas aeruginosa and Staphylococcus spp. Front Cell Infect Microbiol 7.
  6. La Rosa R, Johansen HK, Molin S. 2019. Adapting to the Airways: Metabolic Requirements of Pseudomonas aeruginosa during the Infection of Cystic Fibrosis Patients. 10. Metabolites 9.
  7. Chaudhari SS, Thomas VC, Sadykov MR, Bose JL, Ahn DJ, Zimmerman MC, Bayles KW. 2016. The LysR-type transcriptional regulator, CidR, regulates stationary phase cell death in Staphylococcus aureus: Metabolic control of cell death in S. aureus. 6. Mol Microbiol 101:942–953.
  8. Tashiro Y, Yawata Y, Toyofuku M, Uchiyama H, Nomura N. 2013. Interspecies interaction between Pseudomonas aeruginosa and other microorganisms. 1. Microbes Environ 28:13–24.

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