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.

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.

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.

Most bacteria exist and interact within polymicrobial communities. These interactions produce unique compounds, increased virulence and augmented 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 towards extinction. However, when found in vivo, both species can co-exist. Previous work has noted that this may due to altered gene expression or mutations. However, little is known about how the growth environment could influence 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 final population composition. We found that changing the carbon source in growth medium 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 absolute growth for one species, that species will 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 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, and may offer a novel mechanism to manipulate polymicrobial populations.

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