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 Escherichia coli and Pseudomonas aeruginosa. Interestingly, and separate from activity through the tricarboxylic acid cycle, we find that transcriptional activity of genes involved in purine and pyrimidine synthesis can predict the strength of the inoculum effect for β-lactam and aminoglycosides antibiotics, respectively. Our work highlights the antibiotic class-specific effect of purine and pyrimidine synthesis on the severity of the inoculum effect, which may pave the way for intervention strategies to reduce the inoculum effect in the clinic