Interactions between metabolism and growth can determine the co-existence of Staphylococcus aureus and Pseudomonas aeruginosa
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eLife assessment
How the pathogens Pseudomonas aeruginosa and Staphylococcus aureus compete and co-occur within opportunistic infections is a topic of broad significance, but the major drivers of these interactions remain unclear. Here the authors defined parameters that predict the coexistence of these microbes using their absolute growth in certain nutritional conditions, leading to questions about how other nutrients lead to the dominance of one or the other during infections. Within a confined context, this valuable study provides solid support for a novel framework in which to evaluate this clinically important species interaction.
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Abstract
Most bacteria exist and interact within polymicrobial communities. These interactions produce unique compounds, increase virulence and augment antibiotic resistance. One community associated with negative healthcare outcomes consists of Pseudomonas aeruginosa and Staphylococcus aureus . When co-cultured, virulence factors secreted by P. aeruginosa reduce metabolism and growth in S. aureus . When grown in vitro, this allows P. aeruginosa to drive S. aureus toward extinction. However, when found in vivo, both species can co-exist. Previous work has noted that this may be due to altered gene expression or mutations. However, little is known about how the growth environment could influence the co-existence of both species. Using a combination of mathematical modeling and experimentation, we show that changes to bacterial growth and metabolism caused by differences in the growth environment can determine the final population composition. We found that changing the carbon source in growth media affects the ratio of ATP to growth rate for both species, a metric we call absolute growth. We found that as a growth environment increases the absolute growth for one species, that species will increasingly dominate the co-culture. This is due to interactions between growth, metabolism, and metabolism-altering virulence factors produced by P. aeruginosa . Finally, we show that the relationship between absolute growth and the final population composition can be perturbed by altering the spatial structure in the community. Our results demonstrate that differences in growth environment can account for conflicting observations regarding the co-existence of these bacterial species in the literature, provides support for the intermediate disturbance hypothesis, and may offer a novel mechanism to manipulate polymicrobial populations.
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Author Response
Reviewer #1 (Public Review):
This is thorough, quantitative microbial ecology research on one of the most important problems of species coexistence in infection biology. The intermediate disturbance hypothesis is supported once again, and they show unsurprisingly that nutrition matters for their ratio of coexistence, but more specifically as a novel function of the ratio of metabolic fueling to reproductive rate, which the authors term absolute growth. I like this study for its care and completeness even though the results are fairly intuitive to those in the field of cystic fibrosis microbial ecology.
We would like to thank the reviewer for acknowledging the importance, care, and completeness of our original manuscript. We have continued to employ our standards of rigor for this revision.
Reviewer #2 (Public Review):
Author Response
Reviewer #1 (Public Review):
This is thorough, quantitative microbial ecology research on one of the most important problems of species coexistence in infection biology. The intermediate disturbance hypothesis is supported once again, and they show unsurprisingly that nutrition matters for their ratio of coexistence, but more specifically as a novel function of the ratio of metabolic fueling to reproductive rate, which the authors term absolute growth. I like this study for its care and completeness even though the results are fairly intuitive to those in the field of cystic fibrosis microbial ecology.
We would like to thank the reviewer for acknowledging the importance, care, and completeness of our original manuscript. We have continued to employ our standards of rigor for this revision.
Reviewer #2 (Public Review):
The authors present a manuscript that addresses an important topic of bacterial co-existence. Specifically modeling infection-relevant scenarios to determine how two highly antibiotic-resistant pathogens will develop over time. Understanding how such organisms can persist and tolerate therapeutic interventions has important consequences for the design of future treatment strategies.
We would like to thank the reviewer for acknowledging the importance of our work.
A major strength of this paper is the methodical approach taken to assess the dynamics between the two bacterial species. Using carbon sources to regulate growth to test different community structures provides a level of control to be able to directly assess the impact of one dominant pathogen over another.
The modeling aspect of this manuscript provides a basis for testing other disturbances and/or the impact of additional incoming pathogens. This could easily be applied to other infection settings where multiple microbes are observed ( for example viral/bacterial interactions in the lung).
Thank you for acknowledging the rigor in our experimental and modeling approaches.
The authors clearly show that by altering the growth rate and metabolism of various carbon sources, population structure can be modified, with one out-competing the other. Both modeling and experimental approaches support this.
The exploration of the role of virulence factors is less clear, for example how strains unable to produce virulence factors are impacted in regard to their overall growth and whether S. aureus is able to sense virulence factors without transcriptional assays here. Although the hypothesis is strong, the experimental data does not fully support this conclusion.
In addressing your comments below, we hope that we have increased your confidence in our hypotheses presented in our manuscript as it pertains to the involvement of virulence factors.
Spatial disturbance has a significant impact on community structure. Although using one approach to assess this, it is not clear if the spatial structure is impacted without the comparable microscopy evaluation.
We have indeed acknowledged this short coming in our revised manuscript. In the discussion, we write:
“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.”
In fact, we would really like to be able to track the position of each bacterium during shaking events. However, the plate reader cannot accommodate a microscope setup. While we could remove the plate from the plate reader and transport it to the microscope (two floors down), we cannot be certain that the position of the bacterium would not be altered during transport. We have thought about fixing the bacterium in place prior to transport. However, the injection of liquid for the purposes of fixation would likely alter the positioning of bacteria. Thus, we chose a modeling approach using an agent based model that is parametrized based on our experimental approach. Accordingly, we agree that this is a limitation of our current study. We hope that acknowledging this limitation in the discussion sits well with the reviewer.
Overall this paper highlights the use of modeling approaches in combination with wet lab experiments to predict microbial interactions in changing environments.
Reviewer #3 (Public Review):
This is an intriguing manuscript with a rigorous experimental and computational methodology looking at the interaction of Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa). These two pathogens frequently co-habit infections but in standard liquid media often show a winner-take-all outcome. This study seeks to be mechanistically predictive as to the outcome of the co-culture based on the addition of specific carbon sources as filtered through the lens of metabolic efficiency or, as the authors term - absolute growth. Overall, the study is sound, but there are some specific caveats that I would like to present:
We would like to thank the reviewer for acknowledging the rigor of our work.
- The study undersells the knowledge in the literature of what allows or prohibits the stability of the Pa and Sa co-cultures. While most of the correct papers are cited, the outcomes of those studies are downplayed in favor of the current predictive study. While the current study is indeed more "predictive", it strays exceedingly far from an infection-relevant media, whereas other studies show reasonable co-existence in host-relevant media.
We have addressed this comment two different ways. First, we have included an entire paragraph in the discussion that acknowledges previous work and how our results fit into previous findings. 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.”
Second, as noted in our response in the ‘essential revisions’ section, we have tested the relationship between the final density ratio and the absolute growth ratio in SCFM medium, which we believe is host relevant. Our findings were fully consistent with the trends that we saw in our original submission. This data is presented in Fig. 3 and Figure 5 – figure supplement 3.
- The major weakness in the ability of this study to be extrapolatable to infection conditions is the basal media selected for this analysis. The authors choose TSB, which is an incredibly rich media from the start, and proceed to alter only 11% of the available carbon (per mass) with their carbon source manipulations. This suggests an underappreciation for the amino acid metabolism routes of these two pathogens that are taking advantage of the roughly 89% of carbon as amino acid content in the TSB components of tryptone and soytone (17g and 3g, respectively vs the 2.5g carbon source). There are a few major issues with this basal formulation:
a) Comparison to all extant literature on Pa - The media historically used to assess Pa include (rich) LB, BHI, MH; (minimal) MOPS, M63, M9; (host-associated) ASM, SCFM, SCFM2, Serum, and DMEM. TSB is not a historically evaluated formulation for Pa (though it is often for non-mammalian pathogenic Pseudomonads and environmental species). Thus, this study is not inherently integrated into the Pa literature and presents an offshoot study for which a direct connection to extant literature is difficult. Explicitly testing these predictions in the most minimal media possible and then in a host-relevant model would be optimal.
We would truly like to thank the reviewer for their rigor in reviewing our manuscript. We, admittedly, overlooked how amino acids might be influencing the growth of P. aeruginosa in TSB medium. We originally chose TSB medium as previous studies that have examined the co-culture of S. aureus and P. aeruginosa, or their mechanisms of interaction, have used this medium (e.g., [29-34]).
To address this comment directly, we grew co-cultures in AMM minimal medium. This medium, to our knowledge, is the only minimal medium that allows growth of S. aureus. We, and others, have not reported growth of S. aureus in M9 or MOPS minimal medium despite the addition of components such as casamino acids and increases in the concentration of thiamine.
While AMM as reported is quite complex relative to media such as MOPS and M9, we removed several vitamins (nicotinic acid, thiamine, calcium pantothenate, biotin), decreased the concentration of some salts, used a low concentration of casamino acids (0.01%), and used a higher concentration of carbon source (0.04%). In doing so, we hoped to reduce any ‘background effect’ of media components and thus absolute growth could be driven more by carbon source.
Importantly, in using AMM medium, we continue to find a strong and significant relationship between the final density ratio and the absolute growth ratio. This data is presented in the Figure 3 and is described in a standalone paragraph in the results, along with our findings using SCFM.
b) TSB is not remotely host-relevant. The Whiteley lab has done monumental work evaluating in vitro models that mimic human infection (scrupulously matching transcriptomes) and TSB is about as far as you can get. Thus, the ability to extrapolate from the current work to infection without testing in host-relevant media is limited.
As noted above, we repeated our core experimental analysis in SCFM. The results are fully consistent with our original submission. This data is presented Figure 3 and in Figure 5- figure supplement 3.
c) The experimental situation has a component that is both good and bad- O2 tension. By overlaying with mineral oil, the authors immediately bias Staph (a more versatile fermenter) to success, whereas Pa deals with most of these carbon sources better at body level or higher O2 levels. The benefit of this is that many of the infection sites in which these two species co-occur are low in O2.
This was an interesting observation that we have partially addressed experimentally and acknowledged in the discussion.
First, we acknowledged the limitations of our experimental approach as it pertains to O2 levels in the discussion as follows:
“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 [35], 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, panel H). 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 -ketoglutarate was provided as a carbon source.”
Second, we tested the effect of placing mineral oil over top of the co-culture experiments, thus increasing the anaerobic nature of the environment. We found that, in general, as the ratio of absolute growth increased, so did the dominance of P. aeruginosa in the growth medium. This new data is presented in Figure 2 - figure supplement 1, panel H.
Taken together, we hope that these two modifications meet the Reviewer’s expectations.
d) Some of the tested metabolites are osmotically active (sucrose), while others are not (acetate), confounding the interpretation of what absolute metabolism means in the context of this study since the concentrations of all tested metabolites vary from above to below physiologic-dependent on the metabolite. A much better approach would have been to vary a single metabolite or combination to alter 'absolute metabolism' and test whether the stability of the co-culture held.
e) The manuscript never goes into the fact that for some of these "the carbon source" sources, they are catabolite repressed compared to the basal TSB amino acids (or not). Both organisms show exquisite catabolite repression control, yet this is not addressed at all within the text of the manuscript. Since this response in both organisms is sensitive to relative proportions of the various C-sources, failure to vary C-sources or compare utilization compared to the massive excess tryptone and soytone in the media makes the 'absolute metabolism' difficult to interpret.
To address comments d and e, and to acknowledge the potential limitations of our findings, we have included the following in the discussion. In this paragraph, we acknowledge the osmotic activity of the different carbon sources and preferential consumption of amino acids in TSB medium.
“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 [36] whereas P. aeruginosa prefers organic and amino acids [37]. 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.”
f) The authors left out the 'favorite' sources of Pa that are known to be relevant in vivo - the TCA intermediates: citrate, succinate, fumarate (and directly relevant to host-pathogen interactions, itaconate)
We have included the analysis of succinate as a carbon source in both TSB medium (Figs. 1 and 2) and AMM medium (Fig. 3). However, we could not achieve reliable or a quantifiable growth rate of P. aeruginosa in SCFM medium supplemented with succinate in our experimental setup. Accordingly, this carbon source was not used in SCFM.
- Statistics: Most of the experiments presented are comparisons in which there are more than two experimental groups and the t-tests employed therefore need to be corrected for multiple comparisons. The standard way to do this is to employ an ANOVA with the appropriate multiple-comparison-corrected post-test. These appear to be appropriate for Dunnett's post-testing but the comparator group is not directly defined within the figure legends. Multiple comparison testing is critical for this analysis, as the H0 is that all are the same - the more samples potentially pulled from the same distribution will result in a higher likelihood that one or more will appear as from a distinct population (i.e. H0 rejected). Multiple comparisons correct for this and are absolutely critical for the evaluation of the data presented in this manuscript.
We have addressed this comment two different ways.
First, where there was a clear control group, we performed either a Dunnett’s (for normally distributed data) or a Dunn’s (for non-parametric data sets) following either an ANOVA or Kruskal-Wallis, respectively. These tests were applied to the data presented in Figure 2B, 5H (top and bottom panels) and in Figure 2 - figure supplement 1, panels K-L.
Second, we did not broadly perform multiple comparisons across all data sets. The reason is that this approach would test the significance of relationships that are not relevant to the central premise of the manuscript. For example, a multiple comparison for figure 1B would test the growth rate of all carbon sources against all carbon sources. However, we are only interested if S. aureus or P. aeruginosa grows faster than one another. However, we do understand the need for a corrected P value to reduce the occurrence of Type 1 errors. To accomplish this, we applied a Benjamini-Hochberg Procedure [38] with a 8.5% discovery rate to all P values in the manuscript, including those that tested the distribution of data. This reduced the P value to indicate significance at < 0.0472. We have updated all claims and indications of significance in the figures based on this adjusted P value.
- The authors missed including Alves et Maddocks 2018 in relation to priority effects and other contributing factors to stable Pa/Sa co-culture.
We have indeed included this manuscript and its findings in the introduction where we write:
“While S. aureus can initially aid in the establishment of the P. aeruginosa population [8], production of N-acetylglucosamine from S. aureus augments…..”
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eLife assessment
How the pathogens Pseudomonas aeruginosa and Staphylococcus aureus compete and co-occur within opportunistic infections is a topic of broad significance, but the major drivers of these interactions remain unclear. Here the authors defined parameters that predict the coexistence of these microbes using their absolute growth in certain nutritional conditions, leading to questions about how other nutrients lead to the dominance of one or the other during infections. Within a confined context, this valuable study provides solid support for a novel framework in which to evaluate this clinically important species interaction.
-
Reviewer #1 (Public Review):
This is thorough, quantitative microbial ecology research on one of the most important problems of species coexistence in infection biology. The intermediate disturbance hypothesis is supported once again, and they show unsurprisingly that nutrition matters for their ratio of coexistence, but more specifically as a novel function of the ratio of metabolic fueling to reproductive rate, which the authors term absolute growth. I like this study for its care and completeness even though the results are fairly intuitive to those in the field of cystic fibrosis microbial ecology.
-
Reviewer #2 (Public Review):
The authors present a manuscript that addresses an important topic of bacterial co-existence. Specifically modeling infection-relevant scenarios to determine how two highly antibiotic-resistant pathogens will develop over time. Understanding how such organisms can persist and tolerate therapeutic interventions has important consequences for the design of future treatment strategies.
A major strength of this paper is the methodical approach taken to assess the dynamics between the two bacterial species. Using carbon sources to regulate growth to test different community structures provides a level of control to be able to directly assess the impact of one dominant pathogen over another.
The modeling aspect of this manuscript provides a basis for testing other disturbances and/or the impact of additional …
Reviewer #2 (Public Review):
The authors present a manuscript that addresses an important topic of bacterial co-existence. Specifically modeling infection-relevant scenarios to determine how two highly antibiotic-resistant pathogens will develop over time. Understanding how such organisms can persist and tolerate therapeutic interventions has important consequences for the design of future treatment strategies.
A major strength of this paper is the methodical approach taken to assess the dynamics between the two bacterial species. Using carbon sources to regulate growth to test different community structures provides a level of control to be able to directly assess the impact of one dominant pathogen over another.
The modeling aspect of this manuscript provides a basis for testing other disturbances and/or the impact of additional incoming pathogens. This could easily be applied to other infection settings where multiple microbes are observed ( for example viral/bacterial interactions in the lung).
The authors clearly show that by altering the growth rate and metabolism of various carbon sources, population structure can be modified, with one out-competing the other. Both modeling and experimental approaches support this.
The exploration of the role of virulence factors is less clear, for example how strains unable to produce virulence factors are impacted in regard to their overall growth and whether S. aureus is able to sense virulence factors without transcriptional assays here. Although the hypothesis is strong, the experimental data does not fully support this conclusion.
Spatial disturbance has a significant impact on community structure. Although using one approach to assess this, it is not clear if the spatial structure is impacted without the comparable microscopy evaluation.
Overall this paper highlights the use of modeling approaches in combination with wet lab experiments to predict microbial interactions in changing environments.
-
Reviewer #3 (Public Review):
This is an intriguing manuscript with a rigorous experimental and computational methodology looking at the interaction of Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa). These two pathogens frequently co-habit infections but in standard liquid media often show a winner-take-all outcome. This study seeks to be mechanistically predictive as to the outcome of the co-culture based on the addition of specific carbon sources as filtered through the lens of metabolic efficiency or, as the authors term - absolute growth. Overall, the study is sound, but there are some specific caveats that I would like to present:
1. The study undersells the knowledge in the literature of what allows or prohibits the stability of the Pa and Sa co-cultures. While most of the correct papers are cited, the outcomes of those …
Reviewer #3 (Public Review):
This is an intriguing manuscript with a rigorous experimental and computational methodology looking at the interaction of Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa). These two pathogens frequently co-habit infections but in standard liquid media often show a winner-take-all outcome. This study seeks to be mechanistically predictive as to the outcome of the co-culture based on the addition of specific carbon sources as filtered through the lens of metabolic efficiency or, as the authors term - absolute growth. Overall, the study is sound, but there are some specific caveats that I would like to present:
1. The study undersells the knowledge in the literature of what allows or prohibits the stability of the Pa and Sa co-cultures. While most of the correct papers are cited, the outcomes of those studies are downplayed in favor of the current predictive study. While the current study is indeed more "predictive", it strays exceedingly far from an infection-relevant media, whereas other studies show reasonable co-existence in host-relevant media.
2. The major weakness in the ability of this study to be extrapolatable to infection conditions is the basal media selected for this analysis. The authors choose TSB, which is an incredibly rich media from the start, and proceed to alter only 11% of the available carbon (per mass) with their carbon source manipulations. This suggests an underappreciation for the amino acid metabolism routes of these two pathogens that are taking advantage of the roughly 89% of carbon as amino acid content in the TSB components of tryptone and soytone (17g and 3g, respectively vs the 2.5g carbon source). There are a few major issues with this basal formulation:
a. Comparison to all extant literature on Pa - The media historically used to assess Pa include (rich) LB, BHI, MH; (minimal) MOPS, M63, M9; (host-associated) ASM, SCFM, SCFM2, Serum, and DMEM. TSB is not a historically evaluated formulation for Pa (though it is often for non-mammalian pathogenic Pseudomonads and environmental species). Thus, this study is not inherently integrated into the Pa literature and presents an offshoot study for which a direct connection to extant literature is difficult. Explicitly testing these predictions in the most minimal media possible and then in a host-relevant model would be optimal.
b. TSB is not remotely host-relevant. The Whiteley lab has done monumental work evaluating in vitro models that mimic human infection (scrupulously matching transcriptomes) and TSB is about as far as you can get. Thus, the ability to extrapolate from the current work to infection without testing in host-relevant media is limited.
c. The experimental situation has a component that is both good and bad- O2 tension. By overlaying with mineral oil, the authors immediately bias Staph (a more versatile fermenter) to success, whereas Pa deals with most of these carbon sources better at body level or higher O2 levels. The benefit of this is that many of the infection sites in which these two species co-occur are low in O2.
d. Some of the tested metabolites are osmotically active (sucrose), while others are not (acetate), confounding the interpretation of what absolute metabolism means in the context of this study since the concentrations of all tested metabolites vary from above to below physiologic-dependent on the metabolite. A much better approach would have been to vary a single metabolite or combination to alter 'absolute metabolism' and test whether the stability of the co-culture held.
e. The manuscript never goes into the fact that for some of these "the carbon source" sources, they are catabolite repressed compared to the basal TSB amino acids (or not). Both organisms show exquisite catabolite repression control, yet this is not addressed at all within the text of the manuscript. Since this response in both organisms is sensitive to relative proportions of the various C-sources, failure to vary C-sources or compare utilization compared to the massive excess tryptone and soytone in the media makes the 'absolute metabolism' difficult to interpret.
f. The authors left out the 'favorite' sources of Pa that are known to be relevant in vivo - the TCA intermediates: citrate, succinate, fumarate (and directly relevant to host-pathogen interactions, itaconate)
3. Statistics: Most of the experiments presented are comparisons in which there are more than two experimental groups and the t-tests employed therefore need to be corrected for multiple comparisons. The standard way to do this is to employ an ANOVA with the appropriate multiple-comparison-corrected post-test. These appear to be appropriate for Dunnett's post-testing but the comparator group is not directly defined within the figure legends. Multiple comparison testing is critical for this analysis, as the H0 is that all are the same - the more samples potentially pulled from the same distribution will result in a higher likelihood that one or more will appear as from a distinct population (i.e. H0 rejected). Multiple comparisons correct for this and are absolutely critical for the evaluation of the data presented in this manuscript.
4. The authors missed including Alves et Maddocks 2018 in relation to priority effects and other contributing factors to stable Pa/Sa co-culture. -
