title: Identification of an Antimicrobial Agent Effective against Methicillin-Resistant Staphylococcus aureus Persisters Using a Fluorescence-Based Screening Strategy.
abstract: Persisters are a subpopulation of normal bacterial cells that show tolerance to conventional antibiotics. Persister cells are responsible for recalcitrant chronic infections and new antibiotics effective against persisters would be a major development in the treatment of these infections. Using the reporter dye SYTOX Green that only stains cells with permeabilized membranes, we developed a fluorescence-based screening assay in a 384-well format for identifying compounds that can kill methicillin-resistant Staphylococcus aureus (MRSA) persisters. The assay proved robust and suitable for high throughput screening (Z`-factor: >0.7). In screening a library of hits from a previous screen, which identified compounds that had the ability to block killing of the nematode Caenorhabditis by MRSA, we discovered that the low molecular weight compound NH125, a bacterial histidine kinase inhibitor, kills MRSA persisters by causing cell membrane permeabilization, and that 5 μg/mL of the compound can kill all cells to the limit of detection in a 108 CFU/mL culture of MRSA persisters within 3h. Furthermore, NH125 disrupts 50% of established MRSA biofilms at 20 μg/mL and completely eradicates biofilms at 160 μg/mL. Our results suggest that the SYTOX Green screening assay is suitable for large-scale projects to identify small molecules effective against MRSA persisters and should be easily adaptable to a broad range of pathogens that form persisters. Since NH125 has strong bactericidal properties against MRSA persisters and high selectivity to bacteria, we believe NH125 is a good anti-MRSA candidate drug that should be further evaluated.
authors.affiliation: Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, United States of America.
authors.forename: Wooseong
authors.initials: W
authors.lastname: Kim
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Annie L
authors.initials: AL
authors.lastname: Conery
authors.affiliation: Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, United States of America; Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Rajmohan
authors.initials: R
authors.lastname: Rajamuthiah
authors.affiliation: Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, United States of America; Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Beth Burgwyn
authors.initials: BB
authors.lastname: Fuchs
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Frederick M
authors.initials: FM
authors.lastname: Ausubel
authors.affiliation: Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, United States of America.
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Mylonakis
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000493:pharmacokinetics*; D018441:Biofilms / Q000187:drug effects*; D017173:Caenorhabditis elegans / Q000382:microbiology*; D004353:Drug Evaluation, Preclinical; D005453:Fluorescence; D055624:Methicillin-Resistant Staphylococcus aureus / Q000502:physiology*; D009930:Organic Chemicals / Q000737:chemistry; D013203:Staphylococcal Infections / Q000188:drug therapy*
chemical_list: D000900:Anti-Bacterial Agents; D009930:Organic Chemicals; C402795:SYTOX Green