Bacterial iron acquisition systems are potential targets for new antibiotics, because iron plays a central role in energy production, DNA synthesis, intermediate metabolism, nitrogen fixation, and oxygen detoxification. Bacteria actively acquire iron by secreting siderophores that chelate Fe3+, and then transporting the ferric siderophore complexes. Pathogenic K. pneumoniae produces at least four siderophores: enterobactin, glycosylated enterobactin, yersiniabactin, and aerobactin. Like all Gram‐negative species, K. pneumoniae obtains iron with TonB dependent transporters (TBDT) in its outer membrane (OM). For example, aided by the inner membrane protein TonB, that provides energy, the OM protein FepA actively transports ferric enterobactin (FeEnt). K. pneumoniae strain Kp52.145 contains three chromosomal and one plasmid‐mediated fepA homologs, that we individually cloned in pHSG575 and genetically engineered to encode Cys residues at strategic sites for attachment of maleimide fluorophores. Any of these FepA homologs efficiently functioned in the spectroscopic assay: FeEnt binding quenched light emissions from fluoresceinated FepA, but intensity recovered as the bacteria transported and depleted the ferric siderophore from solution. Furthermore, inhibitors that blocked the uptake of FeEnt prevented fluorescence recovery. This individualized, “species specific” method was sensitive and accurate, but genetic and biochemical manipulations of ESKAPE pathogens are potentially hazardous and technically difficult. So, we modified the approach to permit measurements of iron transport in clinical isolates without genetically engineering them. A TonB‐deficient E. coli strain with fluorescently labeled FepA in its OM binds, but cannot transport FeEnt, and thereby constitutes a “fluorescent decoy” (FD) sensor of [FeEnt]. When cohabiting an environment with ESKAPE bacteria (A. baumannii, P. aeruginosa, or E. cloacae), the E. coli sensor monitored their TonB‐dependent uptake of FeEnt. Such sensor strains are broadly applicable, in that they allow direct, in vivo measurements of the high‐affinity transport of virtually any ligand, in any organism. FD sensors also allowed high‐throughput screening of chemical libraries to find antagonists of TonB‐dependent iron uptake by K. pneumoniae or other bacteria. The increasing antibiotic resistance of Gram‐negative pathogens calls for novel agents against new microbial targets, and chemicals that block TonB action may thwart pathogenesis by preventing bacterial iron acquisition in humans and animals.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.