BackgroundThe empirical antibiotic therapies for bacterial infections cause the emergence and propagation of multi-drug resistant bacteria, which not only impair the effectiveness of existing antibiotics but also raise healthcare costs. To reduce the empirical treatments, rapid antimicrobial susceptibility testing (AST) of causative microorganisms in clinical samples should be conducted for prescribing evidence-based antibiotics. However, most of culture-based ASTs suffer from inoculum effect and lack differentiation of target pathogen and commensals, hampering their adoption for evidence-based antibiotic prescription. Therefore, rapid ASTs which can specifically determine pathogens’ susceptibilities, regardless of the bacterial load in clinical samples, are in urgent need. ResultsWe present a pathogen-specific and inoculum size-insensitive AST to achieve the reliable susceptibility determination on Escherichia coli (E. coli) in urine samples. The developed AST is featured with an 1 h sample-to-result workflow in a filter, termed on-filter AST. The AST results can be obtained by using an inducible enzymatic assay to in-situ measure the cell response of E. coli collected from urine after 20 min of antibiotic exposure. The calculated detection limit of our AST (1.95 × 104 CFU/mL) is much lower than the diagnosis threshold of urinary tract infections. The specific expression of the inducible enzyme enables on-filter AST to correctly profile the susceptibilities of target pathogen to multi-type antibiotics without the interference from commensals. We performed the on-filter AST on 1 mL urine samples with bacterial loads varying from 105 CFU/mL to 107 CFU/mL and compared the results to that of standard method, demonstrating its insensitivity to inoculum size. SignificanceThe developed AST is demonstrated to be of high sensitivity, specificity, and insensitive to inoculum size. With further developments for additional bacteria and clinical validation, on-filter AST is promising as a rapid and reliable surrogate of culture-based AST to promote the evidence-based prescription at the first visit and minimize the emergency of new multi-drug resistant microorganisms.