title: Real-time monitoring of intracellular Staphylococcus aureus replication.
abstract: A high-throughput system to rapidly assess the intracellular replication of Staphylococcus aureus has been developed utilizing S. aureus transformed with a dual gfp-luxABCDE reporter operon under the control of a growth-dependent promoter. Replication of tagged bacteria internalized into bovine mammary epithelial cells (MAC-T) could be measured by monitoring fluorescence and bioluminescence from the reporter operon following removal of extracellular bacteria from the plates. Bacterial replication inside cells was confirmed by a novel ex vivo time-lapse confocal microscopic method. This assay of bacterial replication was used to evaluate the efficacy of antibiotics which are commonly used to treat staphylococcal infections. Not all antibiotics tested were able to prevent intracellular replication of S. aureus and some were ineffective at preventing replication of intracellular bacteria at concentrations above the MIC determined for bacteria in broth culture. Comparison of the fluorescence and bioluminescence signals from the bacteria enabled effects on protein synthesis and metabolism to be discriminated and gave information on the entry of compounds into the eukaryotic cell, even if bacterial replication was not prevented. Elevated resistance of S. aureus to antibiotics inside host cells increases the likelihood of selecting S. aureus strains which are resistant to commonly used antimicrobial agents within the intracellular niche. The approach presented directly assesses intracellular efficacy of antibiotics and provides an evidence-based approach to antibiotic selection for prescribing physicians and medical microbiologists.
authors.affiliation: Institute of Infection, Immunity, and Inflammation. Institute of Cell Signalling, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom.
authors.forename: S N A
authors.initials: SN
authors.lastname: Qazi
authors.forename: S E
authors.initials: SE
authors.lastname: Harrison
authors.forename: T
authors.initials: T
authors.lastname: Self
authors.forename: P
authors.initials: P
authors.lastname: Williams
authors.forename: P J
authors.initials: PJ
authors.lastname: Hill
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D008163:Luminescent Measurements; D008321:Mammary Glands, Animal / Q000166:cytology / Q000382:microbiology; D018613:Microscopy, Confocal; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development*
chemical_list: D000900:Anti-Bacterial Agents