Phenazines and toxoflavin act as interspecies modulators of resilience to diverse antibiotics

Abstract

Bacterial opportunistic pathogens make diverse secondary metabolites both in the natural environment and when causing infections, yet how these molecules mediate microbial interactions and their consequences for antibiotic treatment are still poorly understood. Here, we explore the role of three redox‐active secondary metabolites, pyocyanin, phenazine‐1‐carboxylic acid, and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin and phenazine‐1‐carboxylic acid are made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. All molecules alter the susceptibility profile of pathogenic species within the “Burkholderia cepacia complex” to different antibiotics, either antagonizing or potentiating their effects, depending on the drug’s class. Defense responses regulated by the redox‐sensitive transcription factor SoxR potentiate the antagonistic effects these metabolites have against fluoroquinolones, and the presence of genes encoding SoxR and the efflux systems it regulates can be used to predict how these metabolites will affect antibiotic susceptibility of different bacteria. Finally, we demonstrate that inclusion of secondary metabolites in standard protocols used to assess antibiotic resistance can dramatically alter the results, motivating the development of new tests for more accurate clinical assessment.

Optical O2 sensors also respond to redox active molecules commonly secreted by bacteria

“…All strains were grown in a glucose minimal medium (GMM) comprising 10 mM glucose 50 mM KH2PO4/K2HPO4 (pH 7.2), 42.8 mM NaCl, 9.35 mM NH4Cl, 1 mM MgSO4 and a trace elements solution. The medium was prepared by autoclaving all the components together, except for the glucose and the 1000× trace elements stock solution, which were sterilized through filtration and added after autoclaving (26). For the experiments reported in Figure 1E-F, 1× MEM Amino Acids solution (MilliporeSigma, Cat.…”

“…As PA14 makes substantial PYO in our minimal medium (26), we used lifetime spectroscopy to verify that PYO quenches three distinct phosphorescent O 2 sensors in vitro: RTDP, O 2 NS, and a commercial sensor called OXNANO (Figure 1E-G). We next tested a variety of small molecules with diverse biological functions in a serial dilution assay.…”

“…Platinum (II) meso-tetra(pentafluorophenyl)porphine, or PtTFPP, is the O 2 -sensing component of O 2 NS nanosensors, which encapsulate the Pt-porphyrin with a reference dye in a spherical ≈200 nm PVC matrix (13). (B) Strains were grown in a minimal medium where P. aeruginosa (PA14) reliably produces pyocyanin (26). Serially-diluted PA14 supernatants quenched RTDP in a concentration-dependent manner.…”

Optical O 2 Sensors Also Respond to Redox Active Molecules Commonly Secreted by Bacteria

“…As PA14 makes substantial PYO in our minimal medium (26), we used lifetime spectroscopy to verify that PYO quenches three distinct phosphorescent O2 sensors in vitro: RTDP, O2NS, and a commercial sensor called OXNANO (Figure 1E-G). We next tested a variety of small molecules with diverse biological functions in a serial dilution assay.…”

“…The medium was prepared by autoclaving all the components together, except for the glucose and the 1000x trace elements stock solution, which were sterilized through filtration and added after autoclaving (26). For the experiments reported in Figure 1E-F, 1x MEM Amino Acids solution (MilliporeSigma, Cat.…”