Disturbing the Spatial Organization of Biofilm Communities Affects Expression of agr-Regulated Virulence Factors in Staphylococcus aureus.

Staphylococcus aureus uses quorum sensing and nutrient availability to control the expression of agr-regulated virulence factors. Quorum sensing is mediated by autoinducing peptide (AIP), which at a high concentration reduces expression of surface attachment proteins (coa, fnbpA) and increases expression of exotoxins (lukS) and proteases (splA). Nutrient availability can be sensed through the saeS/saeR system. Low nutrients increase expression of saeR, which augments expression of coa and fnbpA, distinct from the activity of AIP. The formation of spatial structure, such as biofilms, can alter quorum sensing and nutrient acquisition. In natural environments, biofilms encounter forces that may alter their spatial structure. These forces may impact quorum sensing and/or nutrient acquisition and thus affect the expression of agr-regulated virulence factors. However, this has not been studied. We show that periodically disturbing biofilms composed of S. aureus using a physical force affected the expression of agr-regulated virulence factors. In nutrient-poor environments, disturbance increased the expression of coa, fnbpA, lukS, and splA. Disturbance in a nutrient-rich environment at low or high disturbance amplitudes moderately reduced expression of coa and fnbpA but increased expression of lukS and splA. Interestingly, at an intermediate amplitude, the overall expression of agr-regulated virulence factors was the lowest; expression of lukS and splA remained unchanged relative to an undisturbed biofilm, while expression of coa and fnbpA significantly decreased. We hypothesize that these changes are a result of disturbance-driven changes in access to AIP and nutrients. Our results may allow the identification of environments where virulence is enhanced, or reduced, owing to a disturbance. IMPORTANCE Bacteria, such as Staphylococcus aureus, integrate signals from the environment to regulate genes encoding virulence factors. These signals include those produced by quorum-sensing systems and nutrient availability. We show that disturbing the spatial organization of S. aureus populations can lead to changes in the expression of virulence factors, likely by altering the ways in which S. aureus detects these signals. Our work may allow us to identify environments that increase or reduce the expression of virulence factors in S. aureus.

Interactions between metabolism and growth can determine the co-existence of Staphylococcus aureus and Pseudomonas aeruginosa.

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.

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.

Growth productivity as a determinant of the inoculum effect for bactericidal antibiotics.

Understanding the mechanisms by which populations of bacteria resist antibiotics has implications in evolution, microbial ecology, and public health. The inoculum effect (IE), where antibiotic efficacy declines as the density of a bacterial population increases, has been observed for multiple bacterial species and antibiotics. Several mechanisms to account for IE have been proposed, but most lack experimental evidence or cannot explain IE for multiple antibiotics. We show that growth productivity, the combined effect of growth and metabolism, can account for IE for multiple bactericidal antibiotics and bacterial species. Guided by flux balance analysis and whole-genome modeling, we show that the carbon source supplied in the growth medium determines growth productivity. If growth productivity is sufficiently high, IE is eliminated. Our results may lead to approaches to reduce IE in the clinic, help standardize the analysis of antibiotics, and further our understanding of how bacteria evolve resistance.

Periodically Disturbing the Spatial Structure of Biofilms Can Affect the Production of an Essential Virulence Factor in Pseudomonas aeruginosa.

Understanding the environmental factors that affect the production of virulence factors has major implications in evolution and medicine. While spatial structure is important in virulence factor production, observations of this relationship have occurred in undisturbed or continuously disturbed environments. However, natural environments are subject to periodic fluctuations, including changes in physical forces, which could alter the spatial structure of bacterial populations and impact virulence factor production. Using Pseudomonas aeruginosa PA14, we periodically applied a physical force to biofilms and examined production of pyoverdine. Intermediate frequencies of disturbance reduced the amount of pyoverdine produced compared to undisturbed or frequently disturbed conditions. To explore the generality of this finding, we examined how an intermediate disturbance frequency affected pyoverdine production in 21 different strains of P. aeruginosa. Periodic disturbance increased, decreased, or did not change the amount of pyoverdine produced relative to undisturbed populations. Mathematical modeling predicts that interactions between pyoverdine synthesis rate and biofilm density determine the amount of pyoverdine synthesized. When the pyoverdine synthesis rates are high, depletion of the biofilm due to disturbance reduces the accumulation of pyoverdine. At intermediate synthesis rates, production of pyoverdine increases during disturbance as bacteria dispersed into the planktonic state enjoy increased growth and pyoverdine production rates. At low synthesis rates, disturbance does not alter the amount of pyoverdine produced since disturbance-driven access to nutrients does not augment pyoverdine synthesis. Our results suggest that environmental conditions shape robustness in the production of virulence factors and may lead to novel approaches to treat infections. IMPORTANCE Virulence factors are required to cause infections. Previous work has shown that the spatial organization of a population, such as a biofilm, can increase the production of some virulence factors, including pyoverdine, which is produced by Pseudomonas aeruginosa. Pyoverdine is essential for the infection process, and reducing its production can limit infections. We have discovered that periodically changing the spatial structure of a biofilm of P. aeruginosa strain PA14 using a physical force can reduce the production of pyoverdine. A mathematical model suggests that this is due to the disruption of spatial organization. Using additional strains of P. aeruginosa isolated from patients and the environment, we use experiments and modeling to show that this reduction in pyoverdine is due to interactions between biofilm density and the synthesis rate of pyoverdine. Our results identify conditions where pyoverdine production is reduced and may lead to novel ways to treat infections.