Multi-tiered high through-put screen for compounds effective against bacterial biofilm and compounds effective for inhibiting and eradicating bacterial biofilm
Inventors
Hassett, Daniel J. • Lamkin, Thomas J. • Panmanee, Warunya • Taylor, Deborah E. • Shea, Chloe J. A.
Assignees
University of Cincinnati • United States Department of the Air Force
Publication Number
US-10704076-B2
Publication Date
2020-07-07
Expiration Date
2034-03-14
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Abstract
A high through-put screening method for identifying agents effective for inhibiting biofilm formation and/or killing established biofilm are disclosed. The method includes three tiers, and each tier includes three specific biological process assays. The tier levels are a primary screen, a confirmation screen, and a dose-response screen, and the biological process assays include assays for total bacterial growth, bacterial metabolic activity, and biofilm formation. The series of assays may be run once or more than once at each tier. A library of compounds is subject to tier A and only compounds meeting a primary parameter advance to tier B, and only tier B compounds meeting a confirmation parameter advance to tier C, and only tier C compounds meeting a dose-response parameter are identified as putative agents effective for inhibiting and/or eradicating a biofilm, further wherein the assays are conducted for each compound subject to the respective tier. The method is effective and validated for identifying agents which inhibit and/or kill Staphylococcus epidermidis, Pseudomonas aeruginosa, and Acinetobacter baumannii biofilms. Agents identified according to the high through-put screen and validated in follow-up experiments as effective for inhibiting and/or killing bacterial biofilms are also disclosed.
Core Innovation
The invention provides a multi-tiered high through-put screening (HTS) method for identifying agents effective in inhibiting bacterial biofilm formation and killing established biofilms. The method includes three tiers: a primary screen, a confirmation screen, and a dose-response screen. Each tier includes three specific biological process assays focused on total bacterial growth, bacterial metabolic activity, and biofilm formation. The screening applies a filtration approach where compounds must meet specific parameters at each tier to advance to the next, culminating in the identification of putative agents effective against biofilms formed by bacteria such as Staphylococcus epidermidis, Pseudomonas aeruginosa, and Acinetobacter baumannii.
The problem being solved addresses the challenges posed by bacterial biofilms that form on various surfaces in industrial, medical, and residential settings. Biofilms protect embedded bacteria from environmental stresses and increase resistance to biocides and antibiotics, resulting in persistent infections and contamination. Present biofilm control approaches are limited, often relying on toxic antimicrobial agents that present environmental risks and may not effectively kill established biofilms. Furthermore, compounds effective against planktonic bacteria may not work against biofilm-associated bacteria. There is thus a need for safe, non-toxic agents capable of inhibiting biofilm formation and eradicating established biofilms across multiple bacterial species, and methods for efficiently screening large compound libraries for such agents.
The HTS method developed addresses these needs by combining multiple assays reflecting different mechanisms of action across several screening tiers to reliably identify compounds that inhibit biofilm formation or kill established biofilms. Confirmatory and validation processes, including confocal laser scanning microscopy (CLSM), ensure robust identification of hits, with further testing such as toxicity and minimum inhibitory concentration (MIC) determination performed on confirmed compounds. The invention also discloses specific novel compounds effective for inhibiting and/or killing biofilms formed by the targeted bacteria, along with compositions comprising these compounds.
Claims Coverage
The patent contains 13 claims focusing on compounds effective against bacterial biofilm formation and established biofilms, highlighting various bacterial species and compounds identified from the screening method. The main inventive features cover effective compounds selective for specific bacteria, combinations effective for multiple species, and particular compounds characterized by their structures.
Compounds effective against one or more bacterial biofilms from Table 1
A compound effective for inhibiting bacterial biofilm formation and/or killing established bacterial biofilm wherein the bacteria is one or more of S. epidermidis, P. aeruginosa, and A. baumannii, selected from compounds set forth in Table 1.
Compounds effective specifically for killing established A. baumannii biofilm
Compounds selected from compounds 1, 2, 12, 17, 18, and 31, characterized by their specified chemical structures, effective for killing established A. baumannii biofilm.
Compounds effective for inhibiting A. baumannii biofilm formation
Compounds selected from compounds 1, 2, 12, 17, and 18, characterized by their specified chemical structures, providing inhibition of A. baumannii biofilm formation.
Compounds effective for inhibiting and killing P. aeruginosa biofilms
Compounds selected from compounds 1, 2, 12, and 14 are effective for inhibiting P. aeruginosa biofilm formation, with compounds 1, 2, and 14 further effective for killing established P. aeruginosa biofilms.
Compounds effective for inhibiting both P. aeruginosa and A. baumannii biofilms
Compounds 1, 2, and 12 are effective for inhibiting biofilm formation of both P. aeruginosa and A. baumannii, with compounds 1 and 2 also functioning as bactericides against both species.
Compounds effective against biofilms of S. epidermidis, P. aeruginosa, and A. baumannii
Compounds 3, 7, and 8 are effective for inhibiting biofilm formation of any one of the three bacteria, with compounds 3 and 7 further effective for killing any established biofilm of the three, and compound 8 effective for killing established S. epidermidis and A. baumannii biofilms.
Compounds effective specifically against S. epidermidis biofilms
Compounds Se-3, Se-4, Se-9, Se-14, Se-15, and Se-16 are effective for inhibiting S. epidermidis biofilm formation, with compounds Se-3, Se-4, and Se-15 further effective for killing S. epidermidis biofilms.
Specific compound effective for inhibiting and/or killing biofilms
Compound 34 (3,4,5',6'-tetrahydro-6',6'-dimethyl-4-phenyl-2'-(2-propen-1-ylthio)-spiro[2H-1-benzopyran-2,4'(3'H)-pyrimidine]-7,8-diol) is identified as effective for inhibiting bacterial biofilm formation and/or killing established biofilms of S. epidermidis, P. aeruginosa, and A. baumannii.
The claims collectively cover identification and use of specific compounds effective in inhibiting and killing biofilms of major problematic bacteria, spanning single species, dual species, and broad spectrum activities. The coverage focuses on compounds identified through the HTS screening method and includes detailed chemical structures of key active agents.
Stated Advantages
The high through-put screening method provides an effective and validated platform for identifying agents capable of inhibiting biofilm formation and killing established biofilms across multiple bacterial species.
The multi-tiered screening approach with confirmation by confocal microscopy ensures reliability and robustness of identified compounds, reducing false positives.
Identified compounds include novel single, dual, and broad spectrum agents that show efficacy in inhibiting and killing biofilms of clinically relevant bacteria.
Broad spectrum agents demonstrate lower cytotoxicity in human cell lines and show low minimum inhibitory concentrations against planktonic bacteria, highlighting potential therapeutic safety and effectiveness.
Documented Applications
Identification of antimicrobial agents for inhibiting and eradicating bacterial biofilms formed by Staphylococcus epidermidis, Pseudomonas aeruginosa, and Acinetobacter baumannii in clinical, industrial, and environmental settings.
Application in medical device coatings and compositions to prevent or reduce bacterial biofilm formation on surfaces such as catheters, prostheses, tubing, and hospital equipment.
Use in disinfectant formulations for controlling bacterial contamination in food processing equipment, water and sewage pipes, cooling systems, and other infrastructure vulnerable to biofilm-related corrosion and infection.
Screening large chemical libraries to discover new anti-biofilm compounds effective against biofilms with multiple bacterial species, contributing to drug development for biofilm-associated infections.
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