and Coxiella bumetii to Clarithromycin

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1993, p /93/ $02.00/0 Copyright 1993, American Society for Microbiology Vol. 37, No. 12 In Vitro Susceptibilities of Spotted Fever Group Rickettsiae and Coxiella bumetii to Clarithromycin M. MAURIN' AND D. RAOULT* Unite des Rickettsies, Faculte de Medecine, 27 Boulevard Jean Moulin, Marseille Cedex 05, France Received 25 June 1993/Returned for modification 2 August 1993/Accepted 4 October 1993 The in vitro bacteriostatic activity of clarithromycin, a new macrolide derivative, against Rickettsia rickettsii, Rickettsia conorii, and "Rickettsia israeli" was determined by the plaque assay and the dye uptake assay. Both bacteriostatic and bactericidal activities of clarithromycin against the Nine Mile, Q212, Priscilla, and ME9 strains of Coxiella burnetii were evaluated by using three cell culture systems. Clarithromycin showed improved antibacterial activity compared with that of erythromycin. A bacteriostatic activity was obtained at concentrations below the reported maximum concentration of clarithromycin in human serum (about 4,ug/ml) for all tested rickettsiae. MICs ranged from 1 to 2,ug/ml for the three Rickettsia species and from 1 to 4,ug/ml for the C. burnetii strains. No bactericidal activity against C. burnetii was obtained when clarithromycin was used at 4,ug/ml. Rickettsia rickettsii is the etiologic agent of Rocky Mountain spotted fever, Rickettsia conorii is the etiologic agent of Mediterranean spotted fever (MSF), and "Rickettsia israeli" is responsible for a spotted fever without "tache noire" in Israel (20, 21). Coxiella burnetii is the etiologic agent of Q fever, a widely distributed zoonosis. C. burnetii infections may present as an acute disease, such as hepatitis, pneumonitis, and self-limited febrile illness, or a chronic disease, mainly endocarditis. Bacteria of the genera Rickettsia and Coxiella are obligate intracellular pathogens, the former being located in the cytoplasm (16, 42, 45) and the latter being located in the phagolysosomes (1, 22) of infected host cells. Tetracyclines remain the antibiotics of choice for the treatment of rickettsial diseases, with chloramphenicol and the fluoroquinolones used as alternative drugs (27, 32, 33, 37, 46). However, severe forms of Rocky Mountain spotted fever and MSF have been described (38, 44), especially when appropriate antibiotic treatment was delayed. Whereas acute C. burnetii infections respond to antibiotic therapy with tetracyclines, chronic C. burnetii infections are hard to cure, and we have previously demonstrated that the antibiotic compounds used against C. burnetii have no in vitro bactericidal activity (25). On the other hand, because of potential adverse effects, tetracyclines and fluoroquinolones are not recommended for use during pregnancy and childhood, and chloramphenicol may be responsible for aplastic anemia. Erythromycin is considered not to be a reliable treatment for diseases caused by R conorii (33). Authors have emphasized the susceptibility heterogeneity among different strains of C. burnetii to macrolides, which may explain why erythromycin may be considered effective or not effective in treating Q fever (31a). More recently, in vitro experiments have shown that the macrolides josamycin and roxithromycin are effective against R. conorii and R. rickettshi (13, 35). Children (5) and pregnant women (31b) suffering from MSF were successfully treated with josamycin. Clarithromycin, a new macrolide derivative, may be of interest because of its * Corresponding author improved pharmacological and pharmacokinetics properties compared with those of erythromycin and because it is well tolerated during childhood and pregnancy. Previous studies have shown that clarithromycin may reach concentrations in serum of up to 4,ug/ml (9, 19) and that it is highly concentrated within eukaryotic cells (2). MATERIALS AND METHODS Antibiotic preparation. Clarithromycin (Abbott Laboratories, Rungis, France) and erythromycin (Roussel, Paris, France) were dissolved in methanol and were then diluted in aqueous solutions. A stock solution of 1 mg/ml was prepared, divided into aliquots, and frozen at -20 C. Aliquots were quickly thawed and the drug solution was incorporated into minimal essential medium (MEM) for the antibiotic challenges. Bacterial strains. R. rickettsii Sheila Smith, R. conorii Moroccan, and "R. israeli" were cultivated in Vero cells. The Nine Mile, Q212, Priscilla, and ME9 strains of C. burnetii (36) were used in the present study. They were grown in mouse P388D1 macrophage-like cells. Infected cells were cultured in MEM supplemented with 10% fetal calf serum and 2 mm glutamine. Susceptibility testing. The bacteriostatic activity of clarithromycin against three Rickettsia species was evaluated by the plaque assay and the dye uptake assay. The bacteriostatic activity of clarithromycin against C. burnetii was assessed by using an acute-infection cell model, whereas two chronic-infection cell models served to evaluate the bactericidal effect of clarithromycin. During antibiotic challenges, the lack of a bacteriostatic effect and the toxicity of methanol to cells were verified. (i) Plaque assay. The plaque assay was used to determine the MICs of clarithromycin for R rickettsii, R. conorii, and "R. israeli" (32, 44). A suspension of 104 Vero cells per ml was prepared in Eagle MEM with 5% fetal calf serum and 2 mm glutamine. Tissue culture petri dishes (60-mm diameter; Corning Glass Works, Corning, N.Y.) were filled with 5 ml of the cell suspension, and the mixture was incubated for 24 h at 37 C in a 5% CO2 incubator. The incubation medium was then discarded and the confluent cell monolayers were

2 2634 MAURIN AND RAOULT inoculated with 104 PFU of rickettsiae. After rocking the petri dishes for 1 h at 22 C, infected cells were overlaid with 5 ml of Eagle MEM containing 2% fetal calf serum and 0.5% agar. Clarithromycin and erythromycin were added to final concentrations of 0 (negative control), 0.125, 0.25, 0.5, 1, 2, 4, 8, or 16,ug/ml. Petri dishes were incubated for 4 days at 37 C in a 5% CO2 atmosphere, and monolayers were stained by adding Eagle MEM containing 0.5% agar and 0.01% neutral red dye. The MIC was the lowest concentration of antibiotic allowing complete inhibition of plaque formation. Experiments were repeated twice for confirmation of the results. (ii) Dye uptake assay. The MICs of clarithromycin were also determined by the dye uptake assay (32). A suspension of 104 Vero cells per ml was dispensed in 96-well flat-bottom microtiter plates (CEB, Nancy, France) at 100,ul per well. The inoculum ofr. nickettsii, R. conorii, or "R israeli" was added at a final volume of 50,ul per well so that 2,000 PFU of rickettsiae was added to each well of the first row, 200 PFU was added to each well of the second row, and 20 PFU was added to each well of the third row. No rickettsiae were added to the fourth row, which served as an uninfected control. Two thousand PFU of rickettsiae was added to each well of the eight remaining rows for the antibiotic assay. Clarithromycin or erythromycin was added to a volume of 50 p,l per well at twofold serial final concentrations ranging from to 16,ug/ml. The plates were incubated for 4 days at 37 C in a 5% CO2 incubator. The incubation medium was then discarded, 50 p,l of neutral red dye (0.15% in saline [ph 5.5]) was added to each well, and the plates were incubated for 1 h at 37 C. The wells were washed three times with phosphate-buffered saline, and the incorporated red dye was extracted by adding 100,ul of phosphate-ethanol buffer (10% ethanol in phosphate-buffered saline [ph 4.2]) to each well. The optical density at 492 nm of the solution was determined with a spectrophotometer (Flow Laboratories, McLean, Va.). The MIC was the first dilution for which the mean optical density at 492 nm of the row was between those of the cell control row and the row containing 20 PFU per well. Experiments were repeated twice for confirmation of the results. (iii) Model of acute infection with C. burnetii. The bacteriostatic activity of clarithromycin against the Nine Mile, Q212, Priscilla, and ME9 strains of C. bumetii was evaluated by the shell vial technique described previously (36). Briefly, HEL cell monolayers cultured in shell vials were infected with 10-fold serial dilutions of a C. burnetii inoculum for each strain tested. Shell vials were centrifuged at 700 x g for 1 h and were incubated at 37 C in a 5% CO2 atmosphere for 6 days. Infected cells were then revealed by indirect immunofluorescence by using a locally prepared rabbit hyperimmune serum to C bumetii. The percentage of infected cells was assessed at x 100 magnification by counting at least 200 cells. The inoculum dilution that resulted in 30 to 50% infected cells was determined (36). For each C bumetii strain, the antibiotic challenge was performed by inoculating a series of shell vials with the inoculum which resulted in 30 to 50% infected cells after 6 days of incubation. Clarithromycin or erythromycin was incorporated into the incubation media at concentrations of 0 (control), 0.5, 1, 2, 4, and 8 jig/ml. Bacterial growth was evaluated after 6 days of incubation as described previously (36). The percentages of infected cells in the presence or absence of the antibiotic was determined as described above. The results were scored as follows: R, resistant, growth comparable to that of the control; I, intermediately ANTimICROB. AGENTS CHEMOTHER. susceptibility, fewer than 10% infected cells; and S, susceptible, the absence of infected cells or the presence of isolated bacteria. The experiments were performed in duplicate and were repeated twice for the confirmation of the results. (iv) Models of chronic infection with C. burnetii. The bactericidal effect of clarithromycin against C. burnetii was assessed by using two different chronic infection models. The Nine Mile and Q212 strains were tested in these experiments. First, we used the model of L929 cells persistently infected with C. bumetii and blocked with cycloheximide (34). Infected cells were grown on 30-mm-diameter coverslips in six-well plates. On day 0 of the experiments, clarithromycin (4,ug/ml) or erythromycin (1,ug/ml) was added to the incubation medium (four wells per drug), whereas no drug was added to the control wells. The percentage of infected cells, as determined by direct immunofluorescence experiments, was determined on day 0 of the experiments and after 3, 5, 7, and 10 days of antibiotic exposure and was compared with the percentage of infected cells for untreated controls. Cell monolayers were fixed with methanol for 10 min, and an immunofluorescence assay was performed by using a locally produced rabbit anti-c. bumetii serum and a mouse anti-rabbit immunoglobulin (reference 0233, Immunotech). Coverslips were examined under a fluorescence microscope at a x400 magnification, and the percentage of infected cells was determined by counting at least 200 cells. The tests were repeated three times. The bactericidal activity of clarithromycin was also assessed by using the quantitative method described previously (25). L929 cells chronically infected with C. bumetii and grown in a 150-cm2 culture flask were harvested and dispensed into 25-cm2 flasks so that each flask received the same primary inoculum. After allowing 30 min for cell attachment, clarithromycin was added to the culture medium at 4,ug/ml. Flasks without antibiotic served as negative controls. Flasks were incubated at 37 C in a 5% CO2 atmosphere for 24 h. Cell monolayers were then harvested. Infected cells were lysed by three freeze-thaw cycles (-170 and 37 C). Tenfold serial dilutions of cell lysates were distributed into shell vials containing uninfected HEL cells. After 6 days of incubation of the shell vials, the C. burnetii isolates were stained by indirect immunofluorescence* as described above for the acute-infection cell model. Viable organisms were recorded as infecting units per milliliter of culture medium by counting the number of infected cells at the highest dilution showing fluorescent vacuoles. The primary inoculum dose was determined by the same procedure. Bactericidal activity corresponded to a significant reduction in bacterial titters (using the Student t test at the 95% confidence limit) after antibiotic exposure compared with that in the primary inoculum dose. The bactericidal assay mixtures were made in duplicate and were repeated three times. RESULTS Clarithromycin was not toxic for Vero cells or L929 cells at concentrations of up to 4,ug/ml. MIC determinations were reproducible by all methods used. As determined by the plaque assay, the MICs of clarithromycin were 2,ug/ml for R. rickettsii, R. conorii, and "R. israeli." As determined by the dye uptake assay, the MICs were 2, 2, and 1,ug/ml for R. rickettsii, R conorii, and "R. israeli," respectively. The MICs of erythromycin were 4,ug/ml for R. conorii and 8,ug/ml for R rickettsii and "R. israeli." Clarithromycin showed bacteriostatic activity against C. bumetii. When

3 VOL. 37, 1993 SUSCEPTIBILITIES OF RICKETTSIAE TO CLARITHROMYCIN 2635 TABLE 1. Bacteriostatic activity of clarithromycin against C. burnetii determined by the shell vial technique Compound Concn Susceptibility' (~±gm1 ) Nine Mile Q212 Priscilla ME9 Clarithromycin 0.5 I R R I 1.0 S I R I 2.0 S S I S 4.0 S S S S Erythromycin 0.5 R R R R 1.0 R R R R 2.0 R R R R 4.0 I R R I 8.0 I R R S a S, susceptible; I, intermediately susceptible; R, resistant. using the shell vial technique, complete bacterial growth inhibition was obtained with 1,ug of clarithromycin per ml for the Nine Mile strain, 2,ug/ml for the Q212 and ME9 strains, and 4,ug/ml for the Priscilla strain (Table 1). Thus, all strains were susceptible to clarithromycin at a maximum concentration of 4.g/ml. Only the strains Nine Mile and Q212 were tested during bactericidal experiments. No bactericidal activity was demonstrated with 4,ug of clarithromycin per ml. By using the cycloheximide-blocked cell model, cell cultures remained nearly 100% infected when clarithromycin (4,ug/ml) was added for 10 days. With the Nine Mile strain, the percentages of infected cells (mean + standard deviation) in untreated controls were , , and on days 3, 5, 7, and 10 of incubation, respectively. The percentages were (not significant [NS] compared with the control), 94 ± 5.3 (NS), 99 ± 1 (NS), and 92.6 ± 2.1 (NS) at 3, 5, 7, and 10 days of incubation, respectively, in cultures receiving 4,ug of clarithromycin per ml. With the Q212 strain, the percentages of infected cells were , 91.3 ± 1.52, 93 ± 2, and 95.6 ± 0.57 on days 3, 5, 7, and 10 of incubation, respectively. The percentages were 98 ± 3.4 (NS compared with the control), 94.3 ± 1.15 (NS), 94.3 ± 1.52 (NS), and 96.6 ± 3.05 (NS) on days 3, 5, 7, and 10 of incubation, respectively, in cultures receiving 4,ug/ml of clarithromycin per ml. By the quantitative technique, no significant differences, as determined by the Student t test at the 95% confidence limit, in bacterial titers were noted between untreated controls and cultures receiving 4,ug of clarithromycin per ml for 24 h. When using the Nine Mile strain, the mean + standard deviation bacterial titers were 121.9% % (NS) and 139.2% % (NS) of the primary inoculum in cultures receiving 4,tg of clarithromycin per ml and untreated controls, respectively. They were, respectively, 132.9% % (NS) and 120.5% % (NS) of the primary inoculum, with and without clarithromycin, when using strain Q212. However, infected L929 cells showed dramatic morphological changes when clarithromycin was added in the incubation medium. Within 24 h, the percentage of infected cells showing large vacuoles full of bacteria was markedly reduced (from nearly 30% to less than 10% with strain Q212). C. bumetii-infected cells recovered the morphology of uninfected cells, although the number of intracellular organisms did not appear to be reduced when examined after Gimenez staining (Fig. 1). DISCUSSION Clarithromycin, an erythromycin derivative, has previously been reported to be effective in vitro against several intracellular pathogens, including Mycobacterium avium-m. intracellulare (18, 23, 29) and other nontuberculous mycobacteria (6, 39, 40), Legionella pneumophila (24), Chlamydia spp. (8, 10, 17, 41), and Toxoplasma gondii (3, 7, 12). In comparison with erythromycin, clarithromycin showed improved pharmacokinetic properties, including the maximum concentration of drug in the serum of patients treated with up to 4 p,g/ml (9, 19), and a high intracellular concentration of the drug, with intracellular to extracellular concentration ratios of 30 to 40 (2). In the present study, the efficacy of clarithromycin against R rickettsii Sheila Smith, R conorii Morrocan, "R. israeli," and four strains of C. burnetii was determined. We used the plaque assay and the dye uptake assay, which are the methods currently used for determination of the bacteriosta- FIG. 1. Intracellular multiplication of C. burnetii Q212 in untreated L929 cell cultures (A) and clarithromycin-containing cultures (B) determined by immunofluorescence assay. Large vacuoles (arrows) full of bacteria were seen in untreated controls, but the large vacuoles disappeared after 24 h of incubation with clarithromycin.

4 2636 MAURIN AND RAOULT tic activity of antibiotics against rickettsiae (14). By these techniques, previous in vitro experiments have shown that josamycin and roxithromycin are effective against R. rickettsii and R conorii (MICs, 1 j,g/ml) in vitro, whereas other macrolides such as erythromycin (MICs, 4 to 8,ug/ml) and spiramycin (MICs, 16 to 32 jig/ml) are not (13, 35). These results correlate well with clinical data since successful treatment has been reported with josamycin in children (5) and pregnant women (31b) suffering from MSF. Thus, the methods used may be reliable for determination of the activity of clarithromycin against rickettsiae and for comparison of the activity of clarithromycin with those of other macrolides. R. rickettsii, R. conorii, and R israeli were susceptible to clarithromycin (MIC range, 1 to 2 p,g/ml). Thus, regardless of the method used, MICs for the three Rickettsia species were comparable and were lower than the maximum concentration in serum (4 ;g/ml) reported for clarithromycin. These results were better than those for erythromycin, which could inhibit bacterial growth at concentrations ranging from 4 to 8 jig/ml, as reported previously (13, 35). To our knowledge, this is the first report on the in vitro antibiotic susceptibility of "R. israeli." The in vitro results presented here should be interpreted with caution because they do not necessarily predict clinical efficacy. In fact, for rifampin, despite its outstanding in vitro efficacy, treatment failures were reported when rifampin was used in children with MSF (4). Determination of the bactericidal activity of clarithromycin against rickettsiae was not performed during our antibiotic challenges but should be of interest. Clarithromycin was also active against four strains of C. burnetii, including the Nine Mile strain, which is the reference strain for acute disease; the Q212 and Priscilla strains, which are reference strains for chronic disease; and the ME9 strain, which we isolated from the blood of a patient suffering from endocarditis and which was shown to be resistant to most antibiotics tested, including erythromycin, ofloxacin, pefloxacin, ciprofloxacin, and chloramphenicol (36). Complete bacterial growth inhibition was obtained for all strains at a concentration of 4,ug/ml or less. This is the first report of a macrolide that is effective against C. bumetii. The usefulness of erythromycin and, more recently, clarithromycin (30) for the treatment of community-acquired pneumonia has been discussed, since these drugs are highly effective against L. pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci. However, in vitro experiments have shown that erythromycin is not effective against C. burnetii, which may also be involved in acute pneumonia, as determined by using either the chick embryo model (27, 43) or the culture cell model (47). Using the shell vial bacteriostatic method, Raoult et al. (36) reported a susceptibility heterogeneity among 13 C burnetii isolates, with some being moderately susceptible to 1,ug of erythromycin per ml and others being resistant to the drug at that concentration. On the other hand, although the macrolides are considered not to be a reliable treatment of Q fever diseases (26), successful treatments of Q fever pneumonia with erythromycin have been reported (11, 15, 28, 31). The antibiotic susceptibility heterogeneity among various C. burnetii strains may explain such contradictory reports on the effectiveness of antibiotic treatment with macrolides during acute Q fever. As for clarithromycin, bacterial growth inhibition was obtained with 1,g of clarithromycin per ml for the Nine Mile strain and with 2,g/ml for the Q212 and ME9 strains, whereas the Priscilla strain was more resistant ANTimICROB. AGENTS CHEMOTHER. (MIC, 4,g/ml). However, it must be noted that the MICs for all strains tested were within the range of concentrations attainable in the serum of patients treated with clarithromycin. The usefulness of clarithromycin during communityacquired pneumonia must be evaluated further. We have previously demonstrated that doxycycline, rifampin, and the fluoroquinolones are not bactericidal against C. burnetii (25, 34). Clarithromycin at 4,ug/ml was not bactericidal against the Nine Mile or the Q212 strain, although surprising cell morphology modifications were observed. In conclusion, our results show that clarithromycin possesses promising in vitro MICs for bacteria of the genera Rickettsia and Coxiella. The molecule may be of particular interest for the treatment of children and pregnant women, since the use of tetracyclines and quinolones is contraindicated in such groups. ACKNOWLEDGMENTS This study received partial support from a grant from Abbott Laboratories, Rungis, France. We thank G. Vestris for technical assistance. REFERENCES 1. Akporiaye, E. T., J. D. Rowatt, A. A. Aragon, and 0. G. Baca Lysosomal response of a murine macrophage-like cell line persistently infected with Coaiella bumetii. Infect. Immun. 40: Anderson, R., G. Joone, and C. E. J. von Rensburg An in-vitro evaluation of the cellular uptake and intraphagocytic bioactivity of clarithromycin (A-56268, TE-031), a new macrolide antimicrobial agent. J. Antimicrob. Chemother. 22: Araujo, F. G., P. Prokocimer, T. Lin, and J. S. Remington Activity of clarithromycin alone or in combination with other drugs for treatment of murine toxoplasmosis. Antimicrob. Agents Chemother. 36: Bella, F., E. Espejo, S. Uriz, J. A. Serrano, M. D. Alegre, and J. Tort Randomized trial of five-day rifampin versus oneday doxycycline therapy for Mediterranean spotted fever. J. Infect. Dis. 164: Bella, F., B. Font, S. Uriz, T. Munoz, E. Espejo, J. Traveria, J. A. Serrano, and F. Segura Randomized trial of doxycycline versus josamycin for Mediterranean spotted fever. Antimicrob. Agents Chemother. 34: Brown, B. A., R. J. Wallace, and G. 0. Onyi Activities of clarithromycin against eight slowly growing species of nontuberculous mycobacteria, determined by using a broth microdilution MIC system. Antimicrob. Agents Chemother. 36: Chang, H. R., F. C. Rudareanu, and J. C. Pechere Activity of A (TE-031), a new macrolide, against Toxoplasma gondii in mice. J. Antimicrob. Chemother. 22: Chirgwin, K., P. M. Roblin, and M. R. Hammerschlag In vitro susceptibilities of Chlamydia pneumoniae (Chlamydia sp. strain TWAR). J. Antimicrob. Chemother. 33: Chu, S. Y., L. T. Sennello, S. T. Bunnell, L. L. Varga, D. S. Wilson, and R. C. Sonders Pharmacokinetics of clarithromycin, a new macrolide, after single ascending oral doses. Antimicrob. Agents Chemother. 36: Cooper, M. A., D. Baldwin, R. S. Matthews, J. M. Andrews, and R. Wise In-vitro susceptibility of Chlamydia pneumoniae (TWAR) to seven antibiotics. J. Antimicrob. Chemother. 28: D'Angelo, L. J., and R. Hetherington Q fever treated with erythromycin. Br. Med. J. 2: Derouin, F., and C. Chastang Activity in vitro against Toxoplasma gondii of azithromycin and clarithromycin alone and with pyrimethamine. J. Antimicrob. Chemother. 25: Drancourt, M., and D. Raoult In vitro susceptibilities of

5 VOL. 37, 1993 SUSCEPTIBILITIES OF RICKETTSIAE TO CLARITHROMYCIN 2637 Rickettsia rickettsii and Rickettsia conorii to roxithromycin and pristinamycin. Antimicrob. Agents Chemother. 33: Drancourt, M., and D. Raoult Antimicrobial therapy of rickettsial spotted fever, p In D. Raoult (ed.), Antimicrobial agents and intracellular pathogens. CRC Press, Inc., Boca Raton, Fla. 15. Ellis, M. E., and E. M. Dunbar In vivo response of acute Q fever to erythromycin. Thorax 37: Ewing, E. P., Jr., A. Takeuchi, A. Shirai, and J. V. Osterman Experimental infection of mouse peritoneal mesothelium with scrub typhus rickettsiae: an ultrastructural study. Infect. Immun. 19: Fenelon, L. E., G. Mumtaz, and G. L. Ridgway The in-vitro antibiotic susceptibility of Chlamydia pneumoniae. J. Antimicrob. Chemother. 26: Fernandes, P. B., D. J. Hardy, D. McDaniel, C. W. Hanson, and R. N. Swanson In vitro and in vivo activities of clarithromycin against Mycobacterium avium. Antimicrob. Agents Chemother. 33: Gan, V. N., S. Y. Chu, and H. T. Kusmiesz Pharmacokinetics of a clarithromycin suspension in infants and children. Antimicrob. Agents Chemother. 36: Goldwasser, R. A., M. A. Klingberg, W. Klingberg, Y. Steiman, and T. A. Swartz Laboratory and epidemiologic studies of rickettsial spotted fever in Israel, p In Frontiers of internal medicine. Proceedings of the 12th International Congress of Internal Medicine, Tel Aviv. 21. Goldwasser, R. A., Y. Steiman, W. Klingberg, T. A. Swartz, and M. A. Klingberg The isolation of strains of rickettsiae of the spotted fever group in Israel and their differentiation from other members of the group by immunofluorescence methods. Scand. J. Infect. Dis. 6: Hackstadt, T., and J. C. Williams Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc. Natl. Acad. Sci. USA 78: Heifets, L. B., P. J. Lindholm-Levy, and R. 0. Comstock Clarithromycin minimal inhibitory and bactericidal concentrations against Mycobacterium avium. Am. Rev. Respir. Dis. 145: Johnson, D. M., M. E. Erwin, M. S. Barrett, B. Briggs Gooding, and R. N. Jones Antimicrobial activity of ten macrolide, lincosamine and streptogramin drugs tested against Legionella species. Eur. J. Clin. Microbiol. Infect. Dis. 11: Maurin, M., A. M. Benoliel, P. Bongrand, and D. Raoult Phagolysosomal alkalinization and the bactericidal effect of antibiotics: the Coxiella burnetii paradigm. J. Infect. Dis. 166: Murray, H. W., and C. Tuazon Atypical pneumonias. Med. Clin. North Am. 64: Ormsbee, R. A., H. Parker, and E. G. Pickens The comparative effectiveness of aureomycin, terramycin, chloramphenicol, erythromycin, and thiomycetin in suppressing experimental rickettsial infections in chick embryos. J. Infect. Dis. 96: Perez-del-Molino, A., J. M. Aguado, J. A. Riancho, I. Sampredo, P. Matorras, and J. Gonzales-Macias Erythromycin and the treatment of Coxiella burnetii pneumonia. J. Antimicrob. Chemother. 28: Perronne, C., A. Gikas, C. Truffot-Pernot, J. Grosset, J. J. Pocidalo, and J. L. Vilde Activities of clarithromycin, sulfisoxazole, and rifabutin against Mycobacterium avium complex multiplication within human macrophages. Antimicrob. Agents Chemother. 34: Poirier, R Comparative study of clarithromycin and roxithromycin in the treatment of community-acquired pneumonia. J. Antimicrob. Chemother. 27(Suppl. A): Raoult, D Treatment of Q fever. Antimicrob. Agents Chemother. 37: a.Raoult, D. Antimicrob. Agents Chemother., in press. 31b.Raoult, D. Unpublished data. 32. Raoult, D., P. Bres, M. Drancourt, and G. Vestris In vitro susceptibilities of Coxiella bumneiii, Rickettsia rickettsii, and Rickettsia cononi to the fluoroquinolone sparfloxacin. Antimicrob. Agents Chemother. 35: Raoult, D., and M. Drancourt Antimicrobial therapy of rickettsial diseases. Antimicrob. Agents Chemother. 35: Raoult, D., M. Drancourt, and G. Vestris Bactericidal effect of doxycycline associated with lysosomotropic agents on Coxiella burnetii in P388D1 cells. Antimicrob. Agents Chemother. 34: Raoult, D., P. Roussellier, and J. Tamalet In vitro evaluation of josamycin, spiramycin, and erythromycin against Rickettsia rickettsii and R. conorii. Antimicrob. Agents Chemother. 32: Raoult, D., H. Torres, and M. Drancourt Shell-vial assay: evaluation of a new technique for determining antibiotic susceptibility tested in 13 isolates of Coxiella bumnetii. Antimicrob. Agents Chemother. 35: Raoult, D., M. R. Yeaman, and 0. G. Baca Susceptibility of Coxiella bumetii to pefloxacin and ofloxacin in ovo and in persistently infected L929 cells. Antimicrob. Agents Chemother. 33: Raoult, D., P. Zuchelli, P. J. Weiler, C. Charrel, J. L. San Marco, H. Gallais, and P. Casanova Incidence, clinical observations and risk factors in the severe form of Mediterranean spotted fever among patients admitted to hospital in Marseilles J. Infect. 12: Rastogi, N., and K S. Goh Effect of ph on radiometric MICs of clarithromycin against 18 species of mycobacteria. Antimicrob. Agents Chemother. 36: Rastogi, N., K. S. Goh, and V. Labrousse Activity of clarithromycin compared with those of other drugs against Mycobacterium paratuberculosis and further enhancement of its extracellular and intracellular activities by ethambutol. Antimicrob. Agents Chemother. 36: Ridgway, G. L., G. Mumtaz, and L. Fenelon The in-vitro activity of clarithromycin and other macrolides against the type strain of Chlamydia pneumoniae (TWAR). J. Antimicrob. Chemother. 27(Suppl. A): Rikihisa, Y., and S. Ito Entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes. Infect. Immun. 38: Spicer, A. J., M. G. Peacock, and J. C. Williams Effectiveness of several antibiotics in suppressing chick embryo lethality during experimental infections by Coxiella burnetii, Rickettsia typhi, and R. rickettsii, p In W. Burgdorfer and R. L. Anacker (ed.), Rickettsiae and rickettsial diseases. Academic Press, Inc., New York. 44. Walker, D. H Rocky Mountain spotted fever: a disease in need of microbiological concern. Clin. Microbiol. Rev. 2: Winkler, H. H., and E. T. Miller Phospholipase A and the interaction of Rickeutsia prowazekii and mouse fibroblasts (L- 929 cells). Infect. Immun. 38: Wisseman, C. L., Jr., A. D. Waddell, and W. T. Walsh In vitro studies of the action of antibiotics on Rickettsia prowazeki by two basic methods of cell culture. J. Infect. Dis. 130: Yeaman, M. R., L. A. Mitscher, and 0. G. Baca In vitro susceptibility of Coxiella burnetii to antibiotics, including several quinolones. Antimicrob. Agents Chemother. 31: