title: Image-based 384-well high-throughput screening method for the discovery of skyllamycins A to C as biofilm inhibitors and inducers of biofilm detachment in Pseudomonas aeruginosa.
abstract: To date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify Pseudomonas aeruginosa biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of P. aeruginosa and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC50s) of 30 and 60 μM, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Navarro
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Cheng
authors.forename: Kelly C
authors.initials: KC
authors.lastname: Peach
authors.forename: Walter M
authors.initials: WM
authors.lastname: Bray
authors.forename: Valerie S
authors.initials: VS
authors.lastname: Bernan
authors.forename: Fitnat H
authors.initials: FH
authors.lastname: Yildiz
authors.forename: Roger G
authors.initials: RG
authors.lastname: Linington
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017963:Azithromycin / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000187:drug effects*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D047630:Depsipeptides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004357:Drug Synergism; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D057054:Molecular Imaging; D010363:Pattern Recognition, Automated; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure
chemical_list: D000900:Anti-Bacterial Agents; D047630:Depsipeptides; D004338:Drug Combinations; D005456:Fluorescent Dyes; D010456:Peptides, Cyclic; C433003:skyllamycins; D049452:Green Fluorescent Proteins; D017963:Azithromycin