Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics.

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. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC cefixime and 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 cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. 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.

Thiostrepton Hijacks Pyoverdine Receptors To Inhibit Growth of Pseudomonas aeruginosa.

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.

A genetic screen identifies a role for oprF in Pseudomonas aeruginosa biofilm stimulation by subinhibitory antibiotics.

Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, potentially as a defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment of Pseudomonas aeruginosa with multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen for stimulation-deficient mutants, we identified the outer membrane porin OprF and the ECF sigma factor SigX as important. Similarly, loss of OmpA - the Escherichia coli OprF homolog - prevented sub-MIC antibiotic stimulation of E. coli biofilms. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not an oprF mutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signaling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.

How to kill Pseudomonas-emerging therapies for a challenging pathogen.

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.