Purine and pyrimidine synthesis differently affect the strength of the inoculum effect for aminoglycoside and β-lactam antibiotics.

The inoculum effect has been observed for nearly all antibiotics and bacterial species. However, explanations accounting for its occurrence and strength are lacking. Previous work found that the relationship between [ATP] and growth rate can account for the strength and occurrence of the inoculum effect for bactericidal antibiotics. However, the molecular pathway(s) underlying this relationship, and therefore determining the inoculum effect, remain undiscovered. Using a combination of flux balance analysis and experimentation, we show that nucleotide synthesis can determine the relationship between [ATP] and growth and thus the strength of inoculum effect in an antibiotic class-dependent manner. If the [ATP]/growth rate is sufficiently high as determined by exogenously supplied nitrogenous bases, the inoculum effect does not occur. This is consistent for both IMPORTANCE: If a bacterial population can grow and reach a sufficiently high density, routine doses of antibiotics can be ineffective. This phenomenon, called the inoculum effect, has been observed for nearly all antibiotics and bacterial species. It has also been reported to result in antibiotic failure in the clinic. Understanding how to reduce the inoculum effect can make high-density infections easier to treat. Here, we show that purine and pyrimidine synthesis affect the strength of the inoculum effect; as the transcriptional activity of pyrimidine synthesis increases, the strength of the inoculum effect for aminoglycosides decreases. Conversely, as the transcriptional activity of purine synthesis increases, the strength of the inoculum effect for β-lactam antibiotics decreases. Our work highlights the importance of nucleotide synthesis in determining the strength of the inoculum effect, which may lead to the identification of new ways to treat high-density infections in the clinic.