Tissue compartmentalization enables persistence during chemotherapy.

Antimicrobial chemotherapy can fail to eradicate the pathogen, even in the absence of antimicrobial resistance. Persisting pathogens can subsequently cause relapsing diseases. In vitro studies suggest various mechanisms of antibiotic persistence, but their in vivo relevance remains unclear because of the difficulty of studying scarce pathogen survivors in complex host tissues. Here, we localized and characterized rare surviving in mouse spleen using high-resolution whole-organ tomography. Chemotherapy cleared >99.5% of the but was inefficient against a small subset in the white pulp. Previous models could not explain these findings: drug exposure was adequate, continued to replicate, and host stresses induced only limited drug tolerance. Instead, antimicrobial clearance required support of -killing neutrophils and monocytes, and the density of such cells was lower in the white pulp than in other spleen compartments containing higher loads. Neutrophil densities declined further during treatment in response to receding loads, resulting in insufficient support for clearance from the white pulp and eradication failure. However, adjunctive therapies sustaining inflammatory support enabled effective clearance. These results identify uneven tissue colonization and spatiotemporal inflammation dynamics as main causes of persistence and establish a powerful approach to investigate scarce but impactful pathogen subsets in complex host environments.