Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and is intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic thiostrepton (TS), which is considered to be inactive against Gram-negative bacteria, stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at subinhibitory concentrations, suggesting that TS was active against P. aeruginosa. Supporting this observation, TS inhibited the growth of a panel of 96 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low-micromolar concentrations. TS also had activity against Acinetobacter baumannii clinical isolates. The expression of Tsr, a 23S rRNA-modifying methyltransferase from TS producer Streptomyces azureus, in trans conferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. The deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed to confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A. baumannii was potentiated by the FDA-approved iron chelators deferiprone and deferasirox and by heat-inactivated serum. Screening of P. aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. We show that the biofilm stimulation phenotype can reveal cryptic subinhibitory antibiotic activity, and that TS has activity against select multidrug-resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

As the number of effective antibiotics dwindled, antibiotic resistance (AR) became a pressing concern. Some Pseudomonas aeruginosa isolates are resistant to all available antibiotics. In this review, we identify the mechanisms that P. aeruginosa uses to evade antibiotics, including intrinsic, acquired, and adaptive resistance. Our review summarizes many different approaches to overcome resistance. Antimicrobial peptides have potential as therapeutics with low levels of resistance evolution. Rationally designed bacteriophage therapy can circumvent and direct evolution of AR and virulence. Vaccines and monoclonal antibodies are highlighted as immune-based treatments targeting specific P. aeruginosa antigens. This review also identifies promising drug combinations, antivirulence therapies, and considerations for new antipseudomonal discovery. Finally, we provide an update on the clinical pipeline for antipseudomonal therapies and recommend future avenues for research.

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon – controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a 'pre-activated' phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions and failed to increase further with the addition of sub-MIC antibiotics. Supporting a role for respiratory stress, the biofilm stimulation response was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of genes encoding the nitrate respiratory machinery abolished its effects, and nitrate respiration increased during growth with sub-MIC antibiotics. In probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors was minimally affected by nitrate supplementation. Finally, using a metabolism-sensitive dye, we showed spatial co-localization of increased respiration with sub-MIC bactericidal antibiotics. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.

17Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and intrinsically resistant to many 18 antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered 19 that the thiopeptide antibiotic, thiostrepton (TS) -considered inactive against Gram-negative 20 bacteria -stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is 21 characteristic of exposure to antimicrobial compounds at sub-inhibitory concentrations, suggesting that 22 TS was active against P. aeruginosa. Supporting this observation, TS inhibited growth of a panel of 96 23 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low micromolar concentrations. TS also had 24 activity against Acinetobacter baumannii clinical isolates. Expression of Tsr -a 23S rRNA-modifying 25 methyltransferase -in trans conferred TS resistance, confirming that the drug acted via its canonical 26 mode of action, inhibition of ribosome function. Deletion of oligopeptide permease systems used by 27 other peptide antibiotics for uptake failed confer TS resistance. TS susceptibility was inversely 28 proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is 29 increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A.30 baumannii was potentiated by FDA-approved iron chelators deferiprone and deferasirox. Screening of P. 31 aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to 32 cross the outer membrane. Our data show that the biofilm stimulation phenotype can reveal cryptic 33 sub-inhibitory antibiotic activity, and that TS has activity against select multidrug resistant Gram-34 negative pathogens under iron-limited growth conditions, similar to those encountered at sites of 35 infection.