title: Validation of a High-Throughput Screening Assay for Identification of Adjunctive and Directly Acting Antimicrobials Targeting Carbapenem-Resistant Enterobacteriaceae.
abstract: We describe development and validation of a high-throughput screen (HTS) for identifying small molecules that restore the efficacy of carbapenems (adjunctives) and/or directly inhibit growth of carbapenem-resistant Enterobacteriaceae (CRE). Our HTS assay is based on a screen-counterscreen approach using a representative multidrug-resistant CRE strain, Klebsiella pneumoniae BIDMC12A. Specifically, we tested the ability of small molecules to inhibit bacterial growth in the presence (screen) or absence (counterscreen) of meropenem, a representative carbapenem antibiotic. Primary screening of 11,698 known bioactive compounds identified 14 with adjunctive activity and 79 with direct antimicrobial effect. Secondary screening identified triclosan as a strongly synergistic meropenem adjunctive (fractional inhibitory concentration = 0.48) and confirmed azidothymidine (AZT) (minimal inhibitory concentration [MIC] = 4 μg mL(-1)), NH125 (MIC = 4 μg mL(-1)), diphenyleneiodonium chloride (MIC = 8 μg mL(-1)), and spectinomycin (MIC = 32 μg mL(-1)) as potent direct antimicrobials. Spectrum of activity of AZT and spectinomycin was tested against a collection of 103 representative Enterobacteriaceae strains (≈50% CRE). AZT, a nucleoside analog used to treat human immunodeficiency virus, demonstrated an MIC50 of 2 μg mL(-1). Spectinomycin, an antibiotic used to treat gonorrhea, had an MIC50 of 32 μg mL(-1). Therefore, a significant percentage of CRE strains appeared relatively susceptible to these antimicrobials. These data identified AZT and spectinomycin as available agents warranting further study for potential treatment of multidrug-resistant CRE infection. Our results provide proof of principle and impetus for performing a large-scale HTS for discovery of novel, small-molecule adjunctives and antibacterial agents directly targeting CRE.
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center , Boston, Massachusetts.
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Smith
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center , Boston, Massachusetts.
authors.forename: James E
authors.initials: JE
authors.lastname: Kirby
mesh_terms: D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000494:pharmacology*; D015780:Carbapenems / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D004755:Enterobacteriaceae / Q000187:drug effects* / Q000254:growth & development; D057166:High-Throughput Screening Assays; D008826:Microbial Sensitivity Tests; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D054852:Small Molecule Libraries