Objectives: Sublethal bactericidal antibiotics promote the formation of multidrug-tolerant persisters. Clinically, serum drug concentration increases gradually and reaches the peak level with high killing efficiency some time after administration. This study aimed to investigate if the initial low antibiotic concentration would promote persister formation in Klebsiella pneumoniae, an increasingly important nosocomial pathogen. Methods: Time-dependent killings of K. pneumoniae by different types

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... of bactericidal antibiotics were conducted to determine the existence of multidrug-tolerant K. pneumoniae persisters. Killing experiments with antibiotic gradient feeding were then conducted for a K. pneumoniae laboratory strain (ATCC 10031) and a clinical isolate (YWSCU-03) by adding antibiotics step by step until the drug peak serum concentration was attained. Results: Multidrug-tolerant persisters indeed existed in K. pneumoniae and the persistence decreased with increasing drug concentrations or prolonged treatments. Antibiotic gradient feeding, to simulate a gradual increase in serum drug concentration, not only significantly elevated the persistence of ATCC 10031 and YWSCU-03, but also increased the frequency of drug-resistant mutant formation in YWSCU-03. Conclusions: After administration, the initial low serum drug concentration could promote the formation of multidrug-tolerant bacterial persisters, which could survive the lethal drug concentrations attained later and potentially render the antibiotic treatment fruitless. Therefore, antibiotic treatments should be based on the comprehensive analysis of, not only drug pharmacokinetics, but also the synergistic effect between pharmacokinetics and persister formation. KAN 2.0 KAN gradient KAN 20.0 CIP 0.03 CIP gradient CIP 5.0 CRO 0.1 CRO gradient CRO 80.0 CRO 0.1 CRO gradient CRO 80.0 KAN 4.0 KAN gradient KAN 35.0