Limited impact of Salmonella stress and persisters on antibiotic clearance

Antimicrobial compounds are essential for controlling bacterial infections. Stress-induced bacterial tolerance and persisters can undermine antimicrobial activities under laboratory conditions, but their quantitative effects under physiological conditions remain unclear^1,2. Here we determined constraints on clearance of Salmonella by antimicrobials in infected mice and tissue-mimicking chemostats. The antibiotics enrofloxacin and ceftriaxone exhibited poor anti-Salmonella activity under both conditions, primarily owing to severe nutrient starvation, which restricted Salmonella replication^3-5. Other infection-associated conditions, such as acidic pH, glucose, oxidative stress, nitrosative stress, antimicrobial peptides, osmolarity, oxygen limitation, carbon dioxide and carbonate, as well as drug efflux, toxin-antitoxin modules and cell size had limited effects. A subset of resilient Salmonella appeared as a key obstacle for clearance by enrofloxacin, based on the biphasic decline of Salmonella colony-forming units. However, these data were misleading, because colony formation was confounded by extensive post-exposure killing. More accurate single-cell, real-time assays showed uniformly slow damage, indicating high resilience across the entire Salmonella population. The resulting extensive survival of bulk bacteria minimized the effect of hyper-resilient persisters. Thus, starvation-induced general resilience of Salmonella was the main cause of poor antibiotic clearance. These findings highlight the importance of quantifying antibiotic activity with real-time, single-cell assays under physiological conditions.