title: Rickettsia tsutsugamushi infection in cell culture: antibiotic susceptibility determined by flow cytometry.
abstract: Recent unpublished reports from northern Thailand of severe and sometimes fatal cases of scrub typhus, despite appropriate antibiotic therapy, suggest that resistance may occur. Current antibiotic susceptibility methods that use direct microscopic counts of Giemsa-stained cells or mouse protection assays are slow, labor-intensive, and expensive. We explored the use of flow cytometry to measure rickettsial infection in vitro in L-929 cells treated with and without doxycycline, ciprofloxacin, erythromycin, and chloramphenicol. It was possible to detect the rickettsiae down to a level of 83% of the cells infected, mean of 37 rickettsiae per cell, and 40% of cells with too many rickettsiae to count. This level of sensitivity was sufficient to determine the inhibitory effect of all four drugs at standard screening concentrations. At lower concentrations of doxycycline, flow cytometry detected inhibition of rickettsial growth at a concentration of 6.25 x 10(-2) micrograms/ml but not at 6.25 x 10(-3) micrograms/ml, suggesting that the minimum inhibitory concentration is somewhere between these two values. The data from this study show that flow cytometry permits the rapid screening of numerous rickettsial isolates for their susceptibility to a variety of antibiotics, but that visual counts of infected cells provide a more precise indication of rickettsial growth.
authors.affiliation: Department of Clinical Investigation and Allergy/Immunology Service, Walter Reed Army Medical Center, Washington, District of Columbia, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Kelly
authors.forename: K F
authors.initials: KF
authors.lastname: Salata
authors.forename: D
authors.initials: D
authors.lastname: Strickman
authors.forename: J N
authors.initials: JN
authors.lastname: Hershey
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000907:Antibodies, Bacterial / Q000276:immunology; D002452:Cell Count; D002460:Cell Line; D002478:Cells, Cultured; D002701:Chloramphenicol / Q000494:pharmacology; D002939:Ciprofloxacin / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D004917:Erythromycin / Q000494:pharmacology; D005347:Fibroblasts / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D051379:Mice; D008826:Microbial Sensitivity Tests; D012285:Orientia tsutsugamushi / Q000187:drug effects / Q000254:growth & development* / Q000276:immunology
chemical_list: D000900:Anti-Bacterial Agents; D000907:Antibodies, Bacterial; D002939:Ciprofloxacin; D004917:Erythromycin; D002701:Chloramphenicol; D016650:Fluorescein-5-isothiocyanate; D004318:Doxycycline