Infections caused by multiple types of bacteria are tough to treat. For example, co-infections with Staphylococcus aureus and Pseudomonas aeruginosa are so difficult to cure they may persist for years in humans and cause serious illness. But when these two types of bacteria are grown together in the laboratory, P. aeruginosa kills off all the S. aureus.

Learning why these two types of bacteria can coexist in people but not in the laboratory may lead to new treatments to clear infections. It may also help scientists grow beneficial bacteria mixes that break down pollution or produce biofuels.

Pajon and Fortoul et al. show that interactions between bacterial metabolism and growth rate determine whether S. aureus and P. aeruginosa coexist. In the experiments, they grew both types of bacteria in different environments with different food sources. They measured their growth and metabolism and how many bacteria of each species survived over time. Then, they used their data to develop a mathematical model and tested its predictions in the laboratory again. The type of bacteria that had more energy also grew faster and outcompeted the other species. Measuring the growth rate of the two species allowed the scientists to predict which one would win out and what the tipping point would be. Physically disrupting the mix of bacteria disrupted this relationship.

These results may help explain what allows these bacteria to coexist in some settings but not others. It may enable scientists to develop new ways to treat infections with P. aeruginosa and S. aureus that work by manipulating growth in the two species.

Bacterial growth and metabolism are known to drive antibacterial resistance. Studies in mice using drugs or other therapies to manipulate growth and metabolism may help scientists thwart these resistance mechanisms. The results may also help scientists design and grow beneficial multispecies bacteria communities.

Microbes rarely exist in isolation. It is more common to find them as part of diverse polymicrobial communities comprised of multiple microbial species (e.g. Peters et al., 2012). Interactions within these communities can be synergistic or antagonistic. While cooperation can augment the stability of the community (Cavaliere et al., 2017), competition amongst community members can stimulate the expression of gene products in an effort to drive competitors toward extinction (Burgess et al., 1999). In both cases, these interactions can produce novel behaviors that are not otherwise observed when community members are grown in isolation. On the one hand, they can have beneficial functions; they can produce useful products, such as biofuels (Mittermeier et al., 2023), and degrade harmful environmental pollutants (Brune and Bayer, 2012). On the other hand, they can produce behaviors detrimental to human welfare, including augmented virulence (Stacy et al., 2014) and increased antibiotic resistance (Perez et al., 2014). Accordingly, understanding the general principles that shape the interactions and co-existence of members within a polymicrobial community has wide-ranging implications, such as enhancing the production of beneficial products, and strategies to attenuate antibiotic resistance.

One polymicrobial community that is commonly observed in the clinic and is of increasing concern owing to the emergence of antibiotic resistant strains is composed of Pseudomonas aeruginosa and Staphylococcus aureus. These bacteria co-colonize burns (Norbury et al., 2016), individuals with cystic fibrosis (Sagel et al., 2009), and chronic infection sites (Serra et al., 2015; Pastar et al., 2013). Their co-occurrence leads to significantly worse healthcare outcomes (Hotterbeekx et al., 2017; Maliniak et al., 2016), including increased inflammation during pneumonia (Sagel et al., 2009) and increased wound healing time (Pastar et al., 2013; Dalton et al., 2011; Seth et al., 2012). The interactions between S. aureus and P. aeruginosa are well understood and, at a fundamental level, influence the growth and metabolism of both species (Figure 1A). Their co-culture induces interspecies competition, which enhances antibiotic resistance and virulence. In the presence of P. aeruginosa, S. aureus upregulates the expression of virulence factors, including staphylococcal protein A, α-hemolysis and Panton-Valentine leucocidin (Pastar et al., 2013). While S. aureus can initially aid in the establishment of the P. aeruginosa population (Alves et al., 2018), production of N-acetylglucosamine from S. aureus augments the virulence of P. aeruginosa by upregulating expression of multiple secreted virulence factors. Many of these are regulated by quorum sensing and include pyocyanin (Korgaonkar and Whiteley, 2011), pyoverdine (Orazi et al., 2019), 2-Heptyl-4-hydroxyquinoline N-oxide (Machan et al., 1992), and 4-hydroxy-2-heptylquinoline N-oxide (HQNO) (Li et al., 2015; Déziel et al., 2004; Nadal Jimenez et al., 2012; Korgaonkar et al., 2013). Some of these virulence factors reduce metabolism in S. aureus through multiple mechanisms. Pyocyanin induces the production of reactive oxygen species in S. aureus by reacting with menadione, which results in oxidative damage (Noto et al., 2017). Pyocyanin has been found to interfere with electron transport, reducing respiration (Biswas et al., 2009). The siderophores pyoverdine and pyochelin drive S. aureus into fermentation (Filkins et al., 2015). While exposure to HQNO can initially promote biofilm formation in S. aureus (Fugère et al., 2014), it eventually interferes with the activity of cytochrome b, which reduces ATP production (Nguyen and Oglesby-Sherrouse, 2016). N-3-oxo-dodecanoyl-L-homoserine lactone (3OC12HSL) produced by P. aeruginosa for the purposes of quorum sensing has been shown to reduce the growth of S. aureus (Qazi et al., 2006). Overall, multiple virulence factors interfere with growth and metabolism in S. aureus, ultimately leading to facultative respiration and increased antibiotic tolerance (DeLeon et al., 2014). Without a fast-growing competitor, and with additional nutrients released by S. aureus (Mashburn et al., 2005), P. aeruginosa can increase its growth and density (Figure 1A). Overall, co-occurrence of P. aeruginosa and S. aureus alters growth and metabolism, which ultimately results in a highly virulent and antibiotic-resistant infection.

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Previous studies have reported the co-existence dynamics of S. aureus and P. aeruginosa in human hosts. S. aureus is the predominant bacterial species isolated from pediatric patients with cystic fibrosis. However, during adolescence and into adulthood, the frequency of S. aureus decreases, while the frequency of P. aeruginosa increases (Filkins et al., 2015; Rajan and Saiman, 2002). One leading hypothesis to explain this trend is that P. aeruginosa outcompetes S. aureus through the production of HQNO and siderophores. Indeed, when co-cultured in vitro, P. aeruginosa has been shown to competitively exclude S. aureus over 24 hr (Filkins et al., 2015). However, long-term co-existence of these species has been observed in infected hosts, which has prompted investigations into mechanisms that can account for these conflicting observations. For example, overproduction of alginate by P. aeruginosa (Limoli et al., 2017; Price et al., 2020), upregulation of super-oxide dismutase (Treffon et al., 2018), decreased production of HQNO (Frydenlund Michelsen et al., 2016), and spatial segregation (DeLeon et al., 2014) are expected to facilitate co-existence. Differences in growth environments have also been suggested to play a role in facilitating co-existence. These include differences in environmental albumin concentration (Smith et al., 2017) and the production of proteins by the immune system (Wakeman et al., 2016). Interestingly, previous work has demonstrated that differences in nutrient availability in the growth environment can affect growth and metabolism of bacteria (Pereira and Berry, 2017). Indeed, the presence of different nutrients and metabolites in host microenvironment has been previously shown to impact metabolism and growth of bacteria (Maslowski, 2019; Bjarnsholt et al., 2022). Thus, it is possible that differences in nutrients that define the growth environment can affect the co-existence of P. aeruginosa and S. aureus. A growth environment that provides for a high metabolic rate in S. aureus could potentially buffer it against virulence factors produced by P. aeruginosa that reduce metabolism and growth. In turn, this could facilitate co-existence and perhaps allow S. aureus to outcompete P. aeruginosa. Alternatively, growth environments that reduce growth and metabolism of S. aureus could make that population more sensitive to these virulence factors, thus allowing dominance of P. aeruginosa and reducing co-existence. While these hypotheses are plausible, they have yet to be investigated. Thus, we ask whether growth environment-driven changes in growth rate and metabolism affect the co-existence of P. aeruginosa and S. aureus in co-culture. Addressing the role of growth environment on species interactions has implications in the bottom-up design of polymicrobial communities and may lead to novel treatment strategies in the clinic.

Through a series of empirical and analytical investigations, we have shown that differences in absolute growth, a combined metric that encompasses the relationship between growth rate and metabolism, can determine the population composition of S. aureus and P. aeruginosa. In a stationary environment, as the absolute growth ratio increases, the final density ratio of P. aeruginosa and S. aureus increases. The absolute growth ratio also determines the tipping point of the co-cultured population. Importantly, many virulence factors produced by P. aeruginosa (e.g., HQNO, pyocyanin) that impact growth and metabolism in S. aureus are regulated by quorum sensing. Quorum sensing involves the production and exchange of small diffusible molecules, called autoinducers (Waters and Bassler, 2005). Since each P. aeruginosa bacterium produces autoinducers, the concentration of autoinducer increases as a function of cell density. At a sufficiently high concentration, the autoinducer will activate expression of multiple virulence factors in P. aeruginosa, which begin to impact growth and metabolism in S. aureus. Given this density-dependent activation of virulence factors, it is likely that the tipping point in our systems is being determined by quorum sensing, although we did not explicitly investigate this hypothesis in this manuscript.

While the ability of the absolute growth ratio to predict final population composition is also true in a continually disturbed environment, this relationship falls apart in a periodically disturbed environment. Our model and experiments suggest that this is owing to changes in access to metabolism altering virulence factors produced by P. aeruginosa; in stationary or continuously disturbed environments, the spatial overlap of both bacteria is predicted to be high, which facilitates interactions between these virulence factors and S. aureus. However, in periodically disturbed environments, the overlap between bacteria is reduced; this limits interactions between S. aureus and virulence factors. S. aureus then dominates the population. Absolute growth appears to be the most accurate metric within the scope of our study to quantify the changes in the final ratio between P. aeruginosa and S. aureus. While we did find a significant relationship between the final ratio of both species and the ratio of [ATP] of both species when grown in the stationary condition and in TSB medium, the relationship was not as strong as the absolute growth ratio (R²=0.97 (absolute growth ratio) vs. 0.79 ([ATP] ratio)). Moreover, we did not find a significant relationship between the final ratio of both species and the ratio of [ATP] when grown in TSB medium in the continuously disturbed condition, and in either AMM medium or SCFM in the stationary condition (Figure 3—figure supplement 2).

Given the clinical importance of co-infection with both P. aeruginosa and S. aureus, multiple previous studies have identified mechanisms of co-existence. Indeed, long-term co-existence of both species can result in physiological changes that reduce their competitive interactions. Strains of P. aeruginosa isolated from patients that enter into a mucoid state show reduced production of siderophores, pyocyanin, rhamnolipids, and HQNO, which facilitates the survival of S. aureus (Limoli et al., 2017; Frydenlund Michelsen et al., 2016). These strains can also overproduce the polysaccharide alginate, which in itself is sufficient to decrease the production of these virulence factors. Moreover, exogenously supplied alginate can reduce the production of pyoverdine and expression from the PQS quorum sensing system, which is responsible for the production of HQNO (Price et al., 2020). Changes in the physiology of S. aureus can also facilitate co-existence. Strains of S. aureus isolated from patients with cystic fibrosis show multiple changes in the abundance of proteins including super oxide dismutase, the GroEL chaperone protein, and multiple surface-associated proteins (Treffon et al., 2018). Interestingly, the majority of proteins that show changes in abundance in S. aureus are related to central metabolism, which is consistent with our findings demonstrating that metabolism can influence the co-existence of both species. While it is unclear as to how long-term co-culture would affect the ratio of absolute growth, our findings provide an additional mechanism that can determine the co-existence of these bacterial species.

Previous work has shown that niches in the human body, which can vary in environmental conditions, can promote bacterial growth and persistence (Fung et al., 2019). Spatiotemporal differences in nutrient availability within these niches can promote the growth of different bacteria (Pereira and Berry, 2017; Hoffman et al., 2010). Even within the same niche, such as sputum isolated from individuals with cystic fibrosis, the concentration of critical nutrients, such as amino acids and carbon sources, can differ from host to host (Palmer et al., 2007). Variations in nutrient availability within or between niches may alter growth and metabolism of the resident bacteria, and may serve to perturb interactions within polymicrobial communities. In the case of P. aeruginosa and S. aureus, differences in nutrient availability in sputum (Palmer et al., 2007) or the epithelium (Chen et al., 2018) may alter growth, metabolism and the interactions between these species. This could alter their co-existence dynamics leading to host and/or niche specific colonization dynamics. We note that our findings may be relevant to infections occurring in both high and low O₂ environments. While P. aeruginosa is limited in its ability to perform fermentation (Glasser et al., 2014), we have provided evidence that the absolute growth ratio can affect community composition in both aerobic (Figures 2—5) and more anaerobic environments (Figure 2—figure supplement 1). The limited ability of P. aeruginosa to grow in anaerobic environments was apparent in SCFM as we could not obtain reliable or robustly quantifiable growth of this bacteria when succinate or α-ketoglutarate was provided as a carbon source. One drawback of our approach in using different carbon sources to manipulate absolute growth is that some carbon sources are osmotically active, whereas others are not, which could have additional physiological effects on the bacteria outside of changing growth and metabolism. Moreover, both species of bacteria have different carbon source preferences; as above S. aureus tends to prefer carbon sources such as glucose (Halsey et al., 2017), whereas P. aeruginosa prefers organic and amino acids (Rojo, 2010). Given the carbon source preferences of each species, in complex medium such as TSB, there is the potential that P. aeruginosa consumes amino acids prior to consuming the supplied carbon source. This is perhaps less of a concern in AMM medium or SCFM where the concentration of amino acids and additional nutrient components is reduced as compared to TSB medium. Along this line, it is certainly worth investigating how each nutrient component and its ordered utilization by both species contributes to changes in absolute growth. Minor or transient changes in absolute growth owing to preferential nutrient consumption may provide windows of opportunity for one species to increase its relative density to the other. Nevertheless, differences between in vitro and in vivo growth and metabolism owing to the nutritional composition of the growth environment may also help account for differences in the co-existence dynamics reported previously (Filkins et al., 2015; Rajan and Saiman, 2002).

It has been previously recognized that spatial organization facilitated by an undisturbed growth environment can facilitate the co-existence of bacteria, including P. aeruginosa and S. aureus (Barraza and Whiteley, 2021). Importantly, the production of HQNO from P. aeruginosa can impact the pattern of spatial organization of this co-cultured population (Barraza and Whiteley, 2021). While we did not explicitly quantify spatial organization experimentally owing to technical limitations of our microplate reader and microscope setups, in theory, co-culture in an undisturbed condition should facilitate the creation of spatial organization. We note that the use of microscopy would be a superior approach as compared to our ABM. We are currently working toward developing a system that would allow us to monitor the effect of disturbance on the spatial positioning of bacteria in real time. Nevertheless, we found that, in an undisturbed growth environment, the absolute growth ratio could predict the final population composition; increasing the absolute growth ratio increased the final density of P. aeruginosa relative to S. aureus. When disturbed periodically using a physical force, S. aureus dominated the final population composition. However, a clear trend between the absolute growth ratio and the final population composition was not observed when using a linear regression or WLS regression analysis. Our model suggests that these disturbances decrease the spatial overlap of both populations, and limit the amount of virulence factors that are sensed by S. aureus. Importantly, physical forces that are encountered where bacteria can infect hosts, including luminal areas (Thomas et al., 2004; Lipowsky et al., 1978), can fluctuate, which could serve to periodically alter the spatial positions, or overlap, between both populations. Thus, the degree to which a population of S. aureus and P. aeruginosa is disturbed may influence their ability to interact; this would impact the population composition. Importantly, we cannot rule out that increasing the amount of disturbances might have also impacted oxygenation of the medium, or caused unknown transcriptional responses in the bacteria. However, our model, and experiments using knockout strains, suggest that co-existence of S. aureus and P. aeruginosa in periodically disturbed environments is largely due to interactions governed by HQNO, and to a lesser extent, pyoverdine and pyochelin. While these disturbances have been previously shown to impact bacteria cooperation (Wilson et al., 2017) and the production of virulence factors (Quinn et al., 2021), to our knowledge, this is the study reporting that such disturbances can alter the population composition in a polymicrobial community. Thus, these disturbances represent a new method by which the composition, and potentially the functionality, of a polymicrobial community might be perturbed.

From an ecological perspective, our findings provide support for the intermediate disturbance hypothesis. This hypothesis suggests that the diversity of competing species is maximized at intermediate disturbance frequencies or intensities (Connell, 1978). While the intermediate disturbance hypothesis has faced criticism (Fox, 2013), evidence of its influence on biodiversity has been observed multiple times (Roxburgh et al., 2004), has helped to guide conservation programs (Olff and Ritchie, 1998), and can help explain the establishment of invasive species (Catford et al., 2012). Herein, our results provide evidence that further supports the intermediate disturbance hypothesis; disturbance at intermediate frequencies facilitates the co-existence of both P. aeruginosa and S. aureus, whereas in continually and undisturbed environments P. aeruginosa dominated the co-culture (Figure 5). This observation was found in both TSB medium and SCFM suggesting that it is robust in multiple environments. While previous work has also found that intermediate disturbance can influence the biodiversity of bacterial populations (Brockhurst et al., 2007), our work provides additional support for the intermediate disturbance hypothesis using two clinically important bacterial pathogens.

Co-culture of S. aureus and P. aeruginosa leads to changes in antibiotic susceptibility. For example, HQNO produced by P. aeruginosa interferes with ATP production in S. aureus. This causes a reduction in the metabolism of S. aureus and increases its resistance to aminoglycoside antibiotics. Importantly, a reduction in metabolism has been previously noted to decrease antibiotic efficacy across a wide range of bactericidal antibiotics (Lopatkin et al., 2019). Interestingly, HQNO can also increase membrane permeability of S. aureus, which increases the efficacy of antimicrobials such as chloroxylenol and fluoroquinolones (Orazi et al., 2019). Manipulation of absolute growth using different carbon sources may serve to rationally increase the efficacy of antibiotics. For example, carbon sources that cause S. aureus to have a significantly greater absolute growth relative to P. aeruginosa would limit the growth of P. aeruginosa, and thus reduce the total concentration of HQNO produced. This would allow the metabolism of S. aureus to remain relatively high, thus promoting the efficacy of antibiotics, such as aminoglycosides. Conversely, promoting the production of P. aeruginosa by using carbon sources that increase its absolute growth relative to S. aureus would augment the production of HQNO. This could increase the membrane permeability of S. aureus found in biofilms, thus augmenting sensitivity to certain antibiotics, such as fluoroquinolones. Thus, rational manipulation of absolute growth in polymicrobial interactions may serve to differentially alter susceptibility to existing antibiotics.

All raw experimental data is currently deposited in Dryad at https://doi.org/10.5061/dryad.fn2z34tz3.

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Decision letter after peer review:

Thank you for submitting your article "Interactions between metabolism and growth can determine the co-existence of Staphylococcus aureus and Pseudomonas aeruginosa" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Aleksandra Walczak as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The correlation between ATP levels and relative growth rates for Staph is interesting and informative, but the relatively flat growth responses and ATP concentration responses of Pa are because they are primarily eating the amino acids in the TSB. This becomes a major limiter for understanding carbon source impacts on Pa, while still potentially valid for Staph in an environment where Pa is mostly growing on amino acids.

The predictive premise of the model derived from the TSB-based experiments should be tested in two different (non-TSB, non-LB, non-BHI) media formulations and the relative relationship between absolute growth observed. Currently, the study only retests competitive outcomes in the same media from which the prediction was derived.

2) Virulence mutants of Pa should be grown in each media condition to determine if there is a growth deficit of the mutant compared to wt. If this is already in the text it was not clear and should be highlighted in the text as a control.

3) More explicit mention of the intermediate disturbance hypothesis and possible underlying effects of disturbance would be worthwhile, to connect readers to this foundational concept in ecology. In addition, a large literature exploring interactions between these species is cited but under-discussed.

Reviewer #1 (Recommendations for the authors):

The analysis of virulence factors is somewhat less thorough given that their induction is often tied to many factors beyond nutrition, but it is remarkable how well fueling explains who wins and loses. Additional discussion of the wide range of inducers of virulence factors is worthwhile.

More explicit mention of the intermediate disturbance hypothesis and possible underlying effects of disturbance are worthwhile, to connect readers to this foundational concept in ecology.

Reviewer #2 (Recommendations for the authors):

I have some recommendations for additional required data/controls and comments:

- line 282 – authors discuss 'tipping point' in their model, without the mention of quorum sensing which is likely what this is modeling. E.g a threshold cell density. This should be discussed in the context of the relevant data here and in the discussion. many of the virulence factors assessed here are also QS dependent. A way to test this experimentally may be to use a dual carbon source of casamino acids and those selected previously to control an initial density or tipping point (for future studies, Darch 2012 PNAS).

- Virulence mutants of Pa should be grown in each media condition to determine if there is a growth deficit to mutation compared to wt. If this is already in the text it was not clear and should be highlighted in the text as a control.

- Each of the virulence mutants should be complemented and the experiments performed again to show the phenotype is lost in co-culture with SA.

- Overall the paper is very well written and the figures are presented nicely. There are some spots where italics are needed for organism names etc.

Reviewer #3 (Recommendations for the authors):

I have no additional major comments other than those described in the Public Review. If this study had been conducted within the SCFM2 media background via constituent substitution it could have been groundbreaking and immediately relevant to the field. Occurring, as it does, in TSB, it is subject to too many caveats to enhance or supplant existing work demonstrating situational contributions to co-culture.

Essential revisions:

1) The correlation between ATP levels and relative growth rates for Staph is interesting and informative, but the relatively flat growth responses and ATP concentration responses of Pa are because they are primarily eating the amino acids in the TSB. This becomes a major limiter for understanding carbon source impacts on Pa, while still potentially valid for Staph in an environment where Pa is mostly growing on amino acids.

The predictive premise of the model derived from the TSB-based experiments should be tested in two different (non-TSB, non-LB, non-BHI) media formulations and the relative relationship between absolute growth observed. Currently, the study only retests competitive outcomes in the same media from which the prediction was derived.

We have tested the relationship between the final density ratio of P. aeruginosa to S. aureus and the ratio of absolute growth in synthetic minimal medium (AMM), which allows for the growth of S. aureus (see more below in response to Reviewer 3) and synthesis cystic fibrosis medium (SCFM). In both cases, we continue to find strong and significant linear relationships between the final population composition and the ratio of absolute growth. This is consistent when we applied either a simple linear regression or a weighted least squares (WLS) regression, which considers error on the y-axis (in this case error in the final density ratio). This new data is presented in Figure 3.

In addition, we also confirmed that periodically disturbing co-cultured populations grown in SCFM allows S. aureus to gain a competitive advantage at intermediately, but not infrequent or frequently, disturbed environments. This data is noted in the Results section and is presented in Figure 5 —figure supplement 3.

2) Virulence mutants of Pa should be grown in each media condition to determine if there is a growth deficit of the mutant compared to wt. If this is already in the text it was not clear and should be highlighted in the text as a control.

We measured the growth rate of all three mutants and compared it to the wildtype growth rate in all growth conditions presented in our original manuscript. Using a Dunnett’s test with the wildtype growth rate as the control, we did not a find a significant decrease in the growth rate of the mutant strains across all conditions tested. Thus, a reduction in growth rate cannot account for the differences in final density ratio when using the knockout strains. This data is highlighted in the Results section and shown in Figure 2 —figure supplement 1, panels K-L. All P values and biological replicate numbers are found in Supplementary File 2.

3) More explicit mention of the intermediate disturbance hypothesis and possible underlying effects of disturbance would be worthwhile, to connect readers to this foundational concept in ecology. In addition, a large literature exploring interactions between these species is cited but under-discussed.

1)We have made mention of the intermediate disturbance hypothesis in the revised abstract. We have also discussed the implications of our finding for this hypothesis in the discussion where we write:

“From an ecological perspective, our findings provide support for the intermediate disturbance hypothesis. This hypothesis suggests that the diversity of competing species is maximized at intermediate disturbance frequencies or intensities [1]. While the intermediate disturbance hypothesis has faced criticism [2], evidence of its influence on biodiversity has been observed multiple times [3], has helped to guide conservation programs [4], and can help explain the establishment of invasive species [5]. Herein, our results provide evidence that further supports the intermediate disturbance hypothesis; disturbance at intermediate frequencies facilitates the co-existence of both P. aeruginosa and S. aureus whereas in continually and undisturbed environments P. aeruginosa dominated the co-culture (Figure 5). This observation was found in both TSB medium and SCFM suggesting that it is robust in multiple environments. While previous work has also found that intermediate disturbance can influence the biodiversity of bacterial populations [6], our work provides additional support for the intermediate disturbance hypothesis using two clinically important bacterial pathogens.”

2) We have expanded the information provided on the interactions between P. aeruginosa and S. aureus. In the introduction, we have expanded on the mechanisms by which both species interact and its impact on growth and metabolism. This section now reads:

“…Their co-culture induces interspecies competition, which enhances antibiotic resistance and virulence. In the presence of P. aeruginosa, S. aureus upregulates the expression of virulence factors including staphylococcal protein A, α-hemolysis and Panton-Valentine leucocidin [7]. While S. aureus can initially aid in the establishment of the P. aeruginosa population [8], production of N-acetylglucosamine from S. aureus augments the virulence of P. aeruginosa by upregulating expression of multiple secreted virulence factors. Many of these are regulated by quorum sensing and include pyocyanin [9], pyoverdine [10], 2-Heptyl-4-hydroxyquinoline N-oxide [11], and 4-hydroxy-2-heptylquinoline N-oxide (HQNO)[12-15]. Some of these virulence factors reduce metabolism in S. aureus through multiple mechanisms. Pyocyanin induces the production of reactive oxygen species in S. aureus by reacting with menadione, which results in oxidative damage [16]. Pyocyanin has been found to interfere with electron transport, reducing respiration [17]. The siderophores pyoverdine and pyochelin drive S. aureus into fermentation [18]. While exposure to HQNO can initially promote biofilm formation in S. aureus [19], it eventually interferes with the activity of cytochrome b, which reduces ATP production [20]. N-3-oxo-dodecanoyl-L-homoserine lactone (3OC12HSL) produced by P. aeruginosa for the purposes of quorum sensing has been shown to reduce growth of S. aureus [21]. Overall, multiple virulence factors interfere with growth and metabolism in S. aureus, ultimately leading to facultative respiration and increased antibiotic tolerance [22]...”

In the Discussion section, and in response to a comment by reviewer 3 (below), we have also expanded on the interactions that facilitate co-existence of both species. Specifically, we write:

“Given the clinical importance of co-infection with both P. aeruginosa and S. aureus, multiple previous studies have identified mechanisms of co-existence. Indeed, long term co-existence of both species can result in physiological changes that reduce their competitive interactions. Strains of P. aeruginosa isolated from patients that enter into a mucoid state show reduced production of siderophores, pyocyanin, rhamnolipids and HQNO, which facilitates the survival of S. aureus [23, 24]. These strains can also overproduce the polysaccharide alginate, which in itself is sufficient to decrease the production of these virulence factors. Moreover, exogenously supplied alginate can reduce the production of pyoverdine and expression from the PQS quorum sensing system, which is responsible for the production of HQNO [25]. Changes in the physiology of S. aureus can also facilitate co-existence. Strains of S. aureus isolated from patients with cystic fibrosis show multiple changes in the abundance of proteins including super oxide dismutase, the GroEL chaperone protein, and multiple surface associated proteins [26]. Interestingly, the majority of proteins that show changes in abundance in S. aureus are related to central metabolism, which is consistent with our findings demonstrating that metabolism can influence the co-existence of both species. While it is unclear as to how long-term co-culture would affect the ratio of absolute growth, our findings provide an additional mechanism that can determine the co-existence of these bacterial species.”

Overall, we hope that all three Reviewers agree that we have satisfactorily addressed the essential revisions.

Reviewer #1 (Recommendations for the authors):

The analysis of virulence factors is somewhat less thorough given that their induction is often tied to many factors beyond nutrition, but it is remarkable how well fueling explains who wins and loses. Additional discussion of the wide range of inducers of virulence factors is worthwhile.

To address this comment, we have included information of how quorum sensing also leads to the induction of virulence factor expression (which also helped to address a comment made by Reviewer 2). We have made this modification in both the introduction and the Discussion sections.

Introduction: “While S. aureus can initially aid in the establishment of the P. aeruginosa population [8], production of N-acetylglucosamine from S. aureus augments the virulence of P. aeruginosa by upregulating expression of multiple secreted virulence factors. Many of these are regulated by quorum sensing and include pyocyanin [9], pyoverdine [10], 2-Heptyl-4-hydroxyquinoline N-oxide [11], and 4-hydroxy-2-heptylquinoline N-oxide (HQNO)[12-15].

Discussion: “The absolute growth ratio also determines the tipping point of the co-cultured population. Importantly, many virulence factors produced by P. aeruginosa (e.g., HQNO, pyocyanin) that impact growth and metabolism in S. aureus are regulated by quorum sensing. Quorum sensing involves the production and exchange of small diffusible molecules, called autoinducers [27]. Since each P. aeruginosa bacterium produces autoinducers, the concentration of autoinducer increases as a function of cell density. At a sufficiently high concentration, the autoinducer will activate expression of multiple virulence factors in P. aeruginosa, which begin to impact growth and metabolism in S. aureus. Given this density-dependent activation of virulence factors, it is likely that the tipping point in our systems is being determined by quorum sensing, although we did not explicitly investigate this hypothesis in this manuscript.”

More explicit mention of the intermediate disturbance hypothesis and possible underlying effects of disturbance are worthwhile, to connect readers to this foundational concept in ecology.

As noted in our response in the ‘essential revisions’ section, we have included mention of the intermediate disturbance hypothesis in the abstract and have discussed our findings in the scope of this hypothesis in the Discussion section.

Reviewer #2 (Recommendations for the authors):

I have some recommendations for additional required data/controls and comments:

- line 282 – authors discuss 'tipping point' in their model, without the mention of quorum sensing which is likely what this is modeling. E.g a threshold cell density. This should be discussed in the context of the relevant data here and in the discussion. many of the virulence factors assessed here are also QS dependent. A way to test this experimentally may be to use a dual carbon source of casamino acids and those selected previously to control an initial density or tipping point (for future studies, Darch 2012 PNAS).

We would like to thank the reviewer for the suggestion for future work.

We have also modified the Discussion section to acknowledge that quorum sensing is likely playing a role in the tipping point analysis as follows:

“The absolute growth ratio also determines the tipping point of the co-cultured population. Importantly, many virulence factors produced by P. aeruginosa (e.g., HQNO, pyocyanin) that impact growth and metabolism in S. aureus are regulated by quorum sensing. Quorum sensing involves the production and exchange of small diffusible molecules, called autoinducers [27]. Since each P. aeruginosa bacterium produces autoinducers, the concentration of autoinducer increases as a function of cell density. At a sufficiently high concentration, the autoinducer will activate expression of multiple virulence factors in P. aeruginosa, which begin to impact growth and metabolism in S. aureus. Given this density-dependent activation of virulence factors, it is likely that the tipping point in our systems is being determined by quorum sensing, although we did not explicitly investigate this hypothesis in this manuscript. ”

- Virulence mutants of Pa should be grown in each media condition to determine if there is a growth deficit to mutation compared to wt. If this is already in the text it was not clear and should be highlighted in the text as a control.

This was something that we initially overlooked in our original manuscript. However, as noted in response to the ‘essential revisions’, we have indeed performed this analysis. We found that there was not a significant difference in growth rates between all three knockout strains and the wildtype strain. This is noted in the Results section and this new data is presented in Figure 2 —figure supplement 2.

- Each of the virulence mutants should be complemented and the experiments performed again to show the phenotype is lost in co-culture with SA.

We appreciate this suggestion by the reviewer. We chose not to perform these experiments as there was no difference in growth rate between the knockout strains and the wildtype strains. Moreover, multiple previous studies have demonstrated that the removal of these virulence factors from P. aeruginosa allows S. aureus to grow to higher cell densities when in co-culture. Specifically, O’Toole et al. (2015) demonstrated that S. aureus killing is significantly reduced in co-culture with P. aeruginosa ΔpqsL compared to wildtype P. aeruginosa PA14[18]. This was also consistent when S. aureus was co-cultured with P. aeruginosa knockout strains ΔpvdA/ΔpchE and ΔpqsL /ΔpvdA/ΔpchE. These findings were also echoed by Barraza and Whiteley (2021) who demonstrated that P. aeruginosa strain ΔpqsL mutant lysed S. aureus to a lesser degree than wildtype P. aeruginosa under well-mixed conditions in co-culture [28]. Overall, these studies support the conclusion that P. aeruginosa is dependent upon the HQNO pathway and its major siderophores to suppress S. aureus growth in co-culture.

- Overall the paper is very well written and the figures are presented nicely. There are some spots where italics are needed for organism names etc.

Thank you for your kind words and we apologize for this oversight. We have triple checked that all species names in the text are italicized. However, abbreviated genus names that serve as general identifiers in the figures remain unitalicized.

Reviewer #3 (Recommendations for the authors):

I have no additional major comments other than those described in the Public Review. If this study had been conducted within the SCFM2 media background via constituent substitution it could have been groundbreaking and immediately relevant to the field. Occurring, as it does, in TSB, it is subject to too many caveats to enhance or supplant existing work demonstrating situational contributions to co-culture.

We hope that our use of SCFM, along with the additional modifications to our manuscript described here, has increased the relevance of our work in the field.

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Author details

1. Camryn Pajon

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Marla C Fortoul

   Competing interests

   No competing interests declared

2. Marla C Fortoul

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Camryn Pajon

   Competing interests

   No competing interests declared

3. Gabriela Diaz-Tang

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Estefania Marin Meneses

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Ariane R Kalifa

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

6. Elinor Sevy

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Taniya Mariah

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

8. Brandon Toscan

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Maili Marcelin

   Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

10. Daniella M Hernandez

    Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

    Contribution

    Investigation, Writing – review and editing

    Competing interests

    No competing interests declared

11. Melissa M Marzouk

    Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

    Contribution

    Investigation, Writing – review and editing

    Competing interests

    No competing interests declared

12. Allison J Lopatkin

    1. Department of Biology, Barnard College, Columbia University, New York, United States
    2. Data Science Institute, Columbia University, New York, United States
    3. Department of Ecology, Evolution, and Environmental Biology, Columbia University, New York, United States

    Contribution

    Software, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

13. Omar Tonsi Eldakar

    Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States

    Contribution

    Conceptualization, Software, Methodology, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

14. Robert P Smith

    1. Department of Biological Sciences, Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, United States
    2. Cell Therapy Institute, Kiran Patel College of Allopathic Medicine, Nova Southeastern University, Fort Lauderdale, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    rsmith@nova.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2744-7390

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