Pseudomonas aeruginosa is a multidrug-resistant nosocomial pathogen. We showed previously that thiostrepton (TS), a Gram-positive thiopeptide antibiotic, is imported via pyoverdine receptors and synergizes with iron chelator deferasirox (DSX) to inhibit the growth of P. aeruginosa and Acinetobacter baumannii clinical isolates. A small number of P. aeruginosa and A. baumannii isolates were resistant to the combination, prompting us to search for other compounds that could synergize with TS against those strains. From literature surveys, we selected 14 compounds reported to have iron-chelating activity, plus one iron analogue, and tested them for synergy with TS. Doxycycline (DOXY), ciclopirox olamine (CO), tropolone (TRO), clioquinol (CLI), and gallium nitrate (GN) synergized with TS. Individual compounds were bacteriostatic, but the combinations were bactericidal. Our spectrophotometric data and chrome azurol S agar assay confirmed that the chelators potentiate TS activity through iron sequestration rather than through their innate antimicrobial activities. A triple combination of TS plus DSX plus DOXY had the most potent activity against P. aeruginosa and A. baumannii isolates. One P. aeruginosa clinical isolate was resistant to the triple combination but susceptible to a triple combination containing higher concentrations of CLI, CO, or DOXY. All A. baumannii isolates were susceptible to the triple combinations. Our data reveal a diverse set of compounds with dual activity as antibacterial agents and TS adjuvants, allowing combinations to be tailored for resistant clinical isolates.

15Pseudomonas aeruginosa is a multidrug-resistant nosocomial pathogen. We showed previously that 16 thiostrepton (TS), a gram-positive thiopeptide antibiotic, was imported via pyoverdine receptors and 17 synergized with iron chelator deferasirox (DSX) to inhibit the growth of P. aeruginosa and Acinetobacter 18 baumannii clinical isolates. A small number of P. aeruginosa and A. baumannii isolates were resistant to the 19 combination, prompting us to search for other compounds that could synergize with TS against those 20 strains. From literature surveys we selected 14 compounds reported to have iron-chelating activity, plus 21 one iron analogue, and tested them for synergy with TS. Doxycycline (DOXY), ciclopirox olamine (CO), 22 during infection. To overcome iron limitation, P. aeruginosa produces the iron-scavenging siderophores 37 pyochelin and pyoverdine that bind iron with low and high affinity, respectively (3-5). Pyoverdine and its 38 outer membrane receptors, FpvA and FpvB, are highly expressed in low-iron conditions (3, 6, 7). Pyoverdine 39 has such a high binding affinity (10 32 M -1 ) for iron that it can strip it from transferrin, a mammalian protein 40 responsible for sequestering iron to impede bacterial growth (8-11). RNA-seq data showed that pyoverdine 41 biosynthetic enzymes and uptake are highly upregulated in vivo in response to the iron-deprived 42 environment (7). P. aeruginosa deficient in iron-uptake mechanisms are less able to cause infections 43 compared to their wild-type counterparts (12). 44 45 3 Natural products often exploit iron acquisition pathways to cross the Gram-negative outer membrane. 46 Pyocin S2, produced by P. aeruginosa to kill competing strains, and related toxins are taken up via FpvA (13, 47