In Vitro and In Vivo Antibiotic Susceptibilities of ELB Rickettsiae

1 downloads 0 Views 317KB Size Report
The activities of doxycycline, rifampin, chloramphenicol, and erythromycin against ELB .... tion of the bacteria by the host immune response, we collected.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1995, p. 2564–2566 0066-4804/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 39, No. 11

In Vitro and In Vivo Antibiotic Susceptibilities of ELB Rickettsiae S. RADULOVIC, J. A. HIGGINS, D. C. JAWORSKI,

AND

A. F. AZAD*

Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore, Baltimore, Maryland 21201 Received 14 June 1995/Returned for modification 1 August 1995/Accepted 24 August 1995

The activities of doxycycline, rifampin, chloramphenicol, and erythromycin against ELB rickettsiae (Rickettsia azadi) were determined by dye uptake and plaque assays. Plaque formation in Vero cells was inhibited by 0.12 mg of doxycycline per ml. The data presented demonstrate the susceptibility of ELB rickettsiae to commonly used antibiotics for the treatment of rickettsial diseases. washed three times with phosphate-buffered saline (PBS) to remove unincorporated dye. Incorporated dye was then extracted from the well by using 100 ml of phosphate ethanol buffer (10% ethanol in PBS [pH 4.2]). The optical density of the solution was determined at 492 nm by using an enzyme immunoassay microplate reader (model 2550; Bio-Rad, Melville, N.Y.). The mean optical densities of the control wells and the wells infected with 2,000 PFU of ELB were assigned values of 1 and 0, respectively. A given antibiotic concentration was judged to be active if the mean optical density was between those of the inoculum containing 20 PFU and those of uninfected cells. For the plaque assay, Vero cells were seeded in 6-well tissue culture plates (Costar, Cambridge, Mass.) and infected with 0.2 ml of an ELB suspension containing 3 3 104 PFU. After a 2-h incubation at 378C, the cultures were overlaid with 5 ml of Dulbecco’s modified Eagle’s medium (DMEM)–1% fetal bovine serum–0.5% agarose (Amresco, Solon, Ohio). The antibiotic solutions were added to obtain final concentrations of 0, 0.06, 0.125, 0.250, 0.500, 1, 2, and 4 mg/ml. After incubation for 9 days at 348C, the monolayers were stained by the addition of a second overlay containing 1% fetal bovine serum, 0.5% agarose, and 0.01% neutral red in DMEM. For each of the antibiotic concentrations, the plaques from three parallel plates were counted. In vitro, the ELB agent was susceptible to all four antibiotics tested (Table 1). The MIC of rifampin (0.25 to 0.50 mg/ml) was slightly lower than those of erythromycin and chloramphenicol. A reduction of ELB plaque formation was observed for antibiotic concentrations of 1.0 to 4.0 mg/ml for erythromycin, 0.25 mg/ml for rifampin, and 2.0 mg/ml for chloramphenicol. Compared with these antibiotic concentrations, a lower concentration (0.12 mg/ml) of doxycycline was capable of reducing the number of ELB plaques in vitro (Fig. 1). On the basis of the slightly lower MIC in vitro, we used doxycycline and rifampin for in vivo experiments. SpragueDawley male rats (weights of 200 to 250 g) (Charles River, Wilmington, Mass.) were inoculated intraperitoneally with 3 3 102 PFU of ELB. The rats were bled 4 days postinfection prior to antibiotic treatment. The infection was confirmed by PCR and isolation by tissue culture plaque assay. At 4 days postinoculation, the rats were divided into three groups (three animals per group). While groups 1 and 2 received intraperitoneal injections of 0.2 ml of doxycycline and rifampin (25 mg/kg of body weight), respectively, the third group received a sham inoculation. An attempt to grow ELB isolated from rat blood collected on days 4 and 11 postinoculation was made in Vero cells. In addition, the intracellular growth of ELB was moni-

The ELB agent was initially recognized by electron microscopy as a rickettsia-like microorganism that was present in a colonized population of the cat flea, Ctenocephalides felis, and successfully propagated in vitro (7). This typhus-like rickettsia was initially designated ELB after the EL laboratory (Soquel, Calif.) from which the infected flea colony was obtained. After further molecular and biological characterizations of the isolate, the name Rickettsia azadi has been proposed (4). Both Rickettsia typhi and the ELB agent were found in fleas and in opossum tissues from the areas of murine typhus endemicity in southern California and Texas (11, 14). A retrospective investigation of five murine typhus patients from Texas subsequently demonstrated that four patients were infected with R. typhi and that the fifth had been infected with the ELB agent (10). This documentation of human infection by ELB rickettsiae and their presence in opossums and their fleas and possibly in other wildlife associated with human habitations has raised concerns for ELB spread into human populations. The successful cultivation of the ELB agent in Vero cells allowed us to evaluate its susceptibility to antibiotics that are commonly used to treat human rickettsioses. We used two in vitro methods, dye uptake assay and the plaque assay, to evaluate the antibiotic susceptibilities of ELB rickettsiae. An ELB isolate (CF1) from cat flea homogenates was recovered following infection of laboratory rats and passage of liver and spleen homogenates through the yolk sacs of embryonated chicken eggs, with further propagation in Vero cells (7). The dye uptake assay was done as described previously (8). Briefly, the monolayers of Vero cells seeded in 96-well flat-bottom culture plates were infected with 2,000, 200, and 20 PFU of ELB per well. The wells which were used for antibiotic assay contained 200 PFU of ELB per well. Uninfected Vero cells were used as controls. Stock solutions (100 mg/ml) of rifampin, doxycycline, chloramphenicol, and erythromycin (Sigma Chemical Co., St. Louis, Mo.) were diluted to give the following concentrations: 0.06, 0.125, 0.250, 0.500, 1, 2, and 4 mg/ml; 50 ml of each was added to 0.25 ml of medium. The plates were incubated for 9 days at 348C. Then, the medium was removed, 50 ml of neutral red dye (Gibco, Grand Island, N.Y.) per ml in 0.15% saline (pH 5.5) (Travenol Lab. Inc., Deerfield, Ill.) was dispensed, and the plates were incubated for 1 h at 348C. The wells were * Corresponding author. Mailing address: Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore, 655 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 706-3335. Fax: (410) 706-0282. Electronic mail address: aazad@ umabnet.ab.umd.edu. 2564

VOL. 39, 1995

NOTES

2565

TABLE 1. Susceptibility of ELB agent to antibiotics MIC (mg/ml) Drug

Chloramphenicol Doxycycline Rifampin Erythromycin

Dye uptake assay

Plaque assaya

1.0 0.06 0.5 1.0

2.0 0.12 0.25 0.50

a The MICs reported are the averages of three plaque assays (two experiments).

tored by Diff Quik (Baxter HealthCorp., Miami, Fla.) staining and immunofluorescence assay on days 7, 9, and 11 (6). ELB infection of rats was confirmed by PCR and immunofluorescence assay prior to antibiotic treatment. The predicted 232-bp product was amplified from all ELB-infected rats 4 days postinoculation (Fig. 2). In addition, we have recovered rickettsiae in Vero cells from all the rat blood samples collected on day 4 (before antibiotic treatment). Treatment with either rifampin or doxycycline 3 days after intraperitoneal inoculation of 103 rickettsiae reduced the duration of rickettsemia in rats compared with that in drug-free controls. Blood samples collected from antibiotic-treated groups on day 11 postinoculation (7 days after antibiotic treatment) yielded no ELB rickettsiae. In contrast, ELB rickettsiae were recovered in Vero cells from all control rats. At the same time point we were able to amplify a 17-kDa antigen gene segment in control and antibiotic-treated rats by PCR. The PCR amplifications were done as previously described, with a pair of oligonucleotide primers (59-CATTAC TTGGTTCTCAATTCGGT-3 and 59-GTTTTATTAGTGGT TACGTAACC-39) based on the DNA sequences for the 17kDa antigen gene of R. typhi (2, 11, 12). The length of the genome targeted for amplification was predicted to be 232 bp. The PCR amplification of the 17-kDa antigen gene segment yielded no product by 21 days postinoculation (18 days postantibiotic treatment) in either controls or antibiotic-treated animals. The rats had mounted an ELB-specific antibody response as determined by immunofluorescence assay 4 days postinoculation (mean, 1/5,518; range, 1/512 to 1/8,192; n 5 9). However, the antibiotic treatments had no significant effect on the ELB-specific antibody titers by day 21 postinoculation (drug-free control mean, 1/3,093; range, 1/1,024 to 1/8,192; n 5 3; doxycycline group mean, 1/4,096; n 5 3; rifampin group mean, 1/1,280; range, 1/512 to 1/2,048; n 5 3). ELB growth in culture resembled the typhus group more strongly than the spotted fever group of rickettsiae, in its light microscopic morphology, growth pattern in Vero cells, and delayed formation of small plaques (7). Among the macrolide compounds, only erythromycin was tested, and it was found to be effective, with MICs of 0.50 mg/ml by plaque assay and 1.0 mg/ml by dye uptake assay. These values compare with a MIC of 1 mg/ml reported for R. typhi (9). In contrast, in cell culture models, R. prowazekii (Madrid E strain), the etiologic agent of epidemic typhus, was found to be resistant to erythromycin. Rifampin has activity against R. conorii, the agent of Mediterranean spotted fever, and was shown to be clinically effective among adults but failed to cure children (8). In addition, rifampin treatment showed slower resolution of symptoms than treatment with doxycycline (3). Although there has been no report indicating naturally acquired antibiotic resistance among rickettsial isolates, the administration of rifampin should be monitored since high mutation rates are attributed to the development of rifampin resistance among other bacteria (Staphylococcus aureus [1027] and Escherichia coli [1028])

FIG. 1. Neutral red staining of plaques showing reduction in ELB-infected Vero cells (cultures 1 and 4) treated with doxycycline (0.12 mg/ml, culture 3) rifampin (0.25 mg/ml, culture 2), and erythromycin (1.0 mg/ml, culture 5) or untreated (culture 6, control). Magnification, 33.2.

(13). On the basis of their slightly lower MICs in vitro, we used doxycycline and rifampin in our in vivo model. We have previously shown that laboratory rats develop rickettsemia following intraperitoneal or subcutaneous inoculation of #1,000 ELB rickettsiae per inoculum, similar to the inoculum sizes reported for R. typhi infection in the same host (1). The results of this study demonstrate that treatment with either rifampin or doxycycline reduced the duration of rickettsemia compared with that in the control, thereby confirming the in vitro results. Although the relevance of animal model data is often questioned because animal pharmacokinetics differ dramatically from the kinetic profile in humans, because of the zoonotic nature of rickettsial diseases, animals could provide potentially useful information for antibiotic efficacy and vaccine testing. We have shown that rickettsial clearance from peripheral blood 7 days posttreatment corresponds to the host immune response, e.g., ELB-specific antibody response in infected rats. In addition to a PCR assay, which verified the presence of rickettsial DNA, cell culture was used to determine the viability of the rickettsiae recovered from rat peripheral blood after antibiotic administration. Since rickettsial DNA may persist in the host’s circulation system for some time after the destruction of the bacteria by the host immune response, we collected blood samples 4, 11, and 27 days after the rickettsial inoculation. We found targeted PCR products on days 4 and 11 postrickettsial inoculation (days 0 and 7 posttreatment) but not on day 21 postinoculation (day 17 posttreatment). In contrast, isolation via cell culture was unsuccessful by day 7 posttreat-

FIG. 2. Ethidium bromide-stained agarose gel of ELB 17-kDa antigen gene PCR product amplified from rat blood prior to antibiotic treatment. Lanes: 1, 123-bp molecular size marker; 2 to 4, Chelex-extracted DNA from blood (12) of three rats infected with 3 3 102 ELB prior to antibiotic treatment; 5, ELB isolate propagated in vitro; 6, PCR contamination control.

2566

NOTES

ANTIMICROB. AGENTS CHEMOTHER.

ment, indicating the lack of viable organisms in the blood samples. By day 21 postinoculation, neither treated nor control rats yielded the targeted PCR products. Recently, Murai et al. (5) reported the presence of R. tsutsugamushi DNA in the peripheral blood mononuclear cells of eight patients with scrub typhus disease, as determined by PCR during antibiotic treatment with minocycline or doxycycline. Rickettsial DNA was seen only in the acute phase of the disease. These results correlate well with our in vivo data. Despite the widespread occurrence of ELB infection in opossums and their fleas collected from areas of murine typhus endemicity, our information on human infection is based on a molecular identification of ELB in a patient who was diagnosed as having murine typhus (10). The susceptibility exhibited by ELB rickettsiae to commonly used antibiotics will prove useful in treating suspected cases of rickettsial origin. This investigation was supported by National Institutes of Health grant AI 17828. REFERENCES 1. Arango-Jaramillo, S., A. F. Azad, and C. L. Wisseman, Jr. 1984. Experimental infection with Rickettsia mooseri and antibody response of adult and newborn laboratory rats. Am. J. Trop. Med. Hyg. 33:1017–1025. 2. Azad, A. F., J. B. Sacci, Jr., W. M. Nelson, G. A. Dasch, E. T. Schmidtmann, and M. Carl. 1992. Genetic characterization and transovarial transmission of a typhus-like rickettsia found in cat fleas. Proc. Natl. Acad. Sci. USA 89:43–46. 3. Dumler, J. S., J. P. Taylor, and D. H. Walker. 1991. Clinical and laboratory features of murine typhus in South Texas: 1980 through 1987. JAMA 266: 1365–1370.

4. Higgins, J. A., S. Radulovic, and M. E. Schriefer. Rickettsia azadi: a new species of pathogenic rickettsia isolated from fleas. Submitted for publication. 5. Murai, K., A. Okayama, H. Horinouchi, T. Oshikawa, N. Tachibana, and H. Tsubouchi. 1995. Eradication of Rickettsia tsutsugamushi from patients’ blood by chemotherapy, as assessed by the polymerase chain reaction. Am. J. Trop. Med. Hyg. 52:325–327. 6. Philip, R. N., E. A. Casper, W. Burgdorfer, R. K. Gerloff, L. E. Hughes, and E. J. Bell. 1975. Serological typing of rickettsiae of the spotted fever group by microimmunofluorescence. J. Immunol. 121:1961–1968. 7. Radulovic, S., J. A. Higgins, D. C. Jaworski, G. A. Dasch, and A. F. Azad. Isolation, cultivation and partial characterization of ELB agent associated with cat fleas. Submitted for publication. 8. Raoult, D., P. Bres, M. Drancourt, and G. Vestris. 1991. In vitro susceptibilities of Coxiella burnetii, Rickettsia rickettsii, and Rickettsia conorii to the fluoroquinolone sparfloxacin. Antimicrob. Agents Chemother. 35:88–91. 9. Raoult, D., P. Roussellier, and J. Tamalet. 1988. In vitro evaluation of josamycin, spiramycin, and erythromycin against Rickettsia rickettsii and Rickettsia conorii. Antimicrob. Agents Chemother. 32:255–256. 10. Schriefer, M. E., J. B. Sacci, Jr., J. S. Dumler, M. G. Bullen, and A. F. Azad. 1994. Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J. Clin. Microbiol. 32:949–954. 11. Schriefer, M. E., J. B. Sacci, Jr., J. P. Taylor, J. A. Higgins, and A. F. Azad. 1994. Murine typhus: updated roles of multiple urban components and a second typhuslike rickettsia. J. Med. Entomol. 3:681–685. 12. Schriefer, M. E., J. B. Sacci, Jr., R. A. Wirtz, and A. F. Azad. 1991. Detection of polymerase chain reaction-amplified malarial DNA in infected blood and individual mosquitoes. Exp. Parasitol. 73:311–316. 13. Standiford, H. C. 1995. Tetracyclines and chloramphenicol, p. 306–317. In L. G. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases, 4th ed. Churchill Livingstone Inc., New York. 14. Williams, S. G., J. B. Sacci, Jr., M. E. Schriefer, E. M. Anderson, K. K. Fujioka, F. J. Sorvillo, A. R. Barr, and A. F. Azad. 1992. Typhus and typhuslike rickettsiae associated with opossums and their fleas in Los Angeles County, California. J. Clin. Microbiol 30:1758–1762.