In vitro susceptibilities of Bartonella henselae, B. quintana, B. elizabethae, Rickettsia rickettsii, R. conorii, R. akari, and R. prowazekii to macrolide antibiotics as determined by immunofluorescent-antibody analysis of infected Vero cell monolayers

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Abstract
The in vitro susceptibilities of Bartonella (Rochalimaea) henselae, B. quintana, B. elizabethae, Rickettsia akari, R. conorii, R. prowazekii, and R. rickettsii to different concentrations of azithromycin, clarithromycin, dirithromycin, erythromycin, and roxithromycin in Vero cell cultures were evaluated. Bartonella and Rickettsia spp. were allowed to initiate infection of the antibiotic-free Vero cell monolayers, which were maintained in 16-chamber microscope slides in the absence of antibiotics at 32 degrees C in a CO2-enriched atmosphere. The monolayers were then incubated for 3 h to allow for initial host cell intracellular penetration by infecting species. After inoculation, inocula were replaced and tested with media containing 12 different concentrations of each antibiotic in replicate (10 wells of each antibiotic dilution) for each species, and the monolayers were reincubated. Tetracycline served as the control. Growth status of Bartonella spp. and Rickettsia spp. was determined by evaluation of immunofluorescent staining bacilli. Five days later, when antibiotic-free, control-infected cell monolayers demonstrated significant fluorescence, media were removed for all cell monolayers, the monolayers were fixed, and all specimens were stained with standard indirect immunofluorescent antibody reagents. Fluorescent foci were enumerated by counting such foci on random fields visualized with an epifluorescence microscope. The extent of antibiotic-induced focus inhibition was recorded for each dilution of antibiotic and compared with that of an antibiotic-negative control. Effective antibiotic dilution endpoints for inhibition of Bartonella and Rickettsia proliferation, as judged by absence of increase of significant fluorescence (as compared with no-growth controls), were enumerated by determining the number of cell culture chambers at various antibiotic dilutions that were negative or positive for significant Bartonella- or Rickettsia-specific fluorescence. All of the macrolide agents tested were readily active against all three Bartonella organisms, and azithromycin, clarithromycin, and roxithromycin may have potential in the treatment of Rickettsia infections. Animal model-based clinical trials are warranted to define the specific treatment role of the newer macrolide antibiotics.