ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1990, p. 2133-2136 0066-4804/90/112133-040$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 34, No. 11
Comparative In Vitro and In Vivo Susceptibilities of the Lyme Disease Spirochete Borrelia burgdorferi to Cefuroxime and Other Antimicrobial Agents RUSSELL C.
JOHNSON,'*
CARRIE B. KODNER,1 PATTI J.
JURKOVICH,1
AND JEFFREY J. COLLINS2
Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455,1 and Clinical Development, Glaxo Inc., Research Triangle Park, North Carolina 277092 Received 6 November 1989/Accepted 31 August 1990
The in vitro and in vivo susceptibilities of the Lyme disease pathogen Borrelia burgdorferi to cefuroxime were compared with those of several other antibiotics commonly used to treat this disease. Cefuroxime demonstrated a higher MBC in vitro (1.0 ,ug/ml) than ceftriaxone (0.08 ,Ig/ml) or erythromycin (0.32 ,ug/ml), but the MBC was similar to that of amoxicillin (0.8 ,ug/ml) and doxycycline (1.6 ,ig/ml). B. burgdorferi was considerably less susceptible to tetracycline (3.2 ,ug/mI) and penicillin G (6.4 ,ug/ml). Of the three other Borrelia species tested, two (Borrelia turicatae and Borrelia anserina) also demonstrated susceptibility to cefuroxime, while the third (Borrelia hernsii) was less susceptible. Results obtained with four antimicrobial agents in the in vivo hamster model parallel the antibiotic susceptibilities in the in vitro study. The three antibiotics with similar MBCs in vitro, i.e., cefuroxime, doxycycline, and amoxicillin, demonstrated comparable activities in preventing borreliosis in B. burgdorferi-chalHenged hamsters (50% curative doses = 28.6, 36.5 and 45.0 mg/kg, respectively). Penicillin G, which demonstrated the highest MBC in vitro, had very weak protective activity in the hamster model system. These results indicate that the in vitro and in vivo activities of cefuroxime against B. burgdorfefi are comparable to those of several oral antibiotics currently being used in the treatment of early Lyme disease and suggest that the oral form of this cephalosporin may be an effective alternative therapy for this disease.
Lyme disease, which is caused by the spirochete Borrelia burgdorferi and transmitted to humans via the deer tick Ixodes dammini, is a multisystem disorder that mimics the signs and symptoms of various other conditions (5, 9, 10, 29). The progression of the disease is currently divided into three stages. During localized stage 1, there is usually a rapidly expanding skin lesion known as erythema chronicum migrans or simply erythema migrans. Disseminated stage 2 is characterized by neurological, musculoskeletal, and/or cardiac abnormalities which appear within weeks or months of stage 1 onset. Stages 1 and 2 have come to be commonly referred to as early Lyme disease (2, 27). Persistent stage 3 (also called late Lyme disease), the chronic phase of the disease, is manifested by prolonged arthritic episodes in the large joints (Lyme arthritis). This is frequently accompanied by chronic neurological abnormalities and can occur months to years after the clinical onset of the spirochetal infection. While the best clinical marker of stage 1 Lyme disease is erythema migrans (28), some patients with B. burgdorferi infection never develop this lesion, complicating the diagnosis of early Lyme disease. As the pathogenesis and the spectrum of clinical manifestations of Lyme disease have become better known, the diagnosis of the disease, the recognition of the progression of the disease, and the treatment of symptomatic patients have been updated and improved. Nevertheless, because of variable symptomatology, inconsistent results in the serological assays *
used to detect anti-Borrelia antibodies, and the difficulty of culturing or directly visualizing B. burgdorferi in patient specimens, diagnosis of Lyme disease remains problematic. Although recommendations for treatment are likely to be modified as further clinical studies are conducted, at the present time the most commonly used oral antibiotics for treatment of early Lyme disease are tetracycline, amoxicillin, doxycycline, erythromycin, and penicillin V. Oral antibiotics appear to be relatively ineffective in treating late Lyme disease, and such patients usually receive parenteral therapy with penicillin or ceftriaxone. The limited clinical trials (12, 13, 15; N. Y. Liu, H. Dinerman, R. E. Levin, E. Messarotti, P. J. Molloy, and R. J. Schoen, Arthritis Rheum. 32:546, 1989) which have been conducted thus far have provided data which support the need to investigate additional antibiotics for their efficacy in treating early or late Lyme disease or both. Previous in vitro and in vivo antimicrobial susceptibility studies have demonstrated the susceptibility of B. burgdorferi to a number of antibiotics (17, 18, 20, 23, 24; R. C. Johnson and C. B. Kodner, submitted for publication). By using the Syrian hamster model for assessing the in vivo susceptibility of B. burgdorferi, we have reported that the parenteral broad-spectrum cephalosporin ceftriaxone is effective in treating experimentally induced borreliosis (17). In the present report, the results of in vitro and in vivo animal model antimicrobial susceptibility studies are presented which indicate that cefuroxime, a 3-lactamase-stable cephalosporin available in both parenteral and oral forms, has anti-B. burgdorferi activity comparable or superior to that of the other oral antibiotics tested.
Corresponding author. 2133
2134
ANTIMICROB. AGENTS CHEMOTHER.
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TABLE 1. In vitro antimicrobial susceptibility of B. burgdorferi 297
MATERIALS AND METHODS
Origin and cultivation of Borrelia isolates. Eight human Lyme disease B. burgdorferi isolates and three other Borrelia species were used in this study. One isolate, B. burgdorferi 297, was obtained from human cerebrospinal fluid (29). This hamster-virulent isolate was not subcultured more than three times before use in the in vitro and in vivo studies. The seven human skin isolates used were obtained from Bernard Berger, Southampton, N.Y. (8). Borrelia anserina was obtained from Richard Kelly, Baptist Memorial Hospital, Memphis, Tenn. Borrelia turicatae was obtained from the Rocky Mountain Laboratories, Hamilton, Mont., and Borrelia hermsii M1001 (ATCC 35209) was from the American Type Culture Collection, Rockville, Md. All cultures were grown at 30°C in Barbour-Stoenner-Kelly (BSK) medium (3). Animal isolation medium was modified by the addition of 0.15% SeaKem LE agarose (FMC Corp., Marine Colloids Div., Rockland, Maine). Antibiotics. The antibiotics tested were cefuroxime sodium (Glaxo Inc., Research Triangle Park, N.C.), tetracycline hydrochloride, doxycycline, erythromycin, and amoxicillin (Sigma Chemical Co., St. Louis, Mo.), ceftriaxone (Hoffmann-La Roche Inc., Nutley, N.J.), and penicillin G potassium (Pfizer Inc., New York, N.Y.). Recommendations by Anhalt and Washington were used for preparation and storage of antibiotic solutions (1). In vitro susceptibility procedures. The MBCs of amoxicillin, ceftriaxone, cefuroxime, doxycycline, erythromycin, penicillin G, and tetracycline were determined by using the broth dilution method of Johnson et al. (17). Cultures were in the log phase of growth and were counted with a PetroffHausser counting chamber. Concentrations of the test antibiotics ranged from 0.02 to 32 p.g/ml. Duplicate tubes containing BSK medium with the appropriately diluted antimicrobial agents and control (no antibiotic) tubes were inoculated to a final density of 105 cells per ml. After incubation at 30°C for 3 weeks, the tubes were checked under dark-field microscopy for the presence of spirochetes. All negative tubes (no spirochetes observed) were blind transferred (10% vol/vol) to BSK medium without antibiotics, incubated at 30°C for anther 3 weeks, and examined for spirochetes. The MBC was taken as the lowest antibiotic concentration at which spirochetes could not be subcultured. In vivo susceptibility procedures. Golden Syrian hamsters, 4 to 6 weeks old and weighing 80 to 100 g, were injected intraperitoneally with 1 ml of a culture (108 organisms per ml) of B. burgdorferi 297. Two weeks later, the test animals received daily subcutaneous injections with one-fifth of their total test antibiotic dosage for a period of 5 days. The antibiotics tested were cefuroxime, amoxicillin, penicillin G, and doxycycline, and the total dosages are given in Table 2. Control hamsters received only saline injections. Two weeks after the final antibiotic treatment, the hamsters were sacrificed, and the blood, spleen, kidneys, and bladder were collected and cultured. Each organ was aseptically placed into 6 ml of sterile BSK medium in a Stomacher bag and homogenized with a Tekmar Stomacher Lab-blender (Tekmar Co., Cincinnati, Ohio). A 1:10 dilution of the supernatant was placed into duplicate tubes of BSK medium. After 3 weeks of incubation at 30°C, the cultures were examined by dark-field microscopy for the presence of spirochetes. An animal was considered infected if at least one tissue yielded positive cultures. In most instances, five animals were tested
MBC (,ug/ml)
Antimicrobial agent
Ceftriaxone .........................................
0.08 0.32
Erythromycin ......................................... Amoxicillin ......................................... Cefuroxime .........................................
Doxycycline ......................................... Tetracycline ......................................... Penicillin G ................
.........................
0.8 1.0 1.6 3.2 6.4
per antimicrobial dose. The 50% curative dose (CD50) was calculated by using a log-logistic model (11).
RESULTS In vitro susceptibility of the Borrelia isolates to antibiotics. The MBCs of the tested antibiotics at the end of the 6-week observation period ranged from 0.08 to 6.4 ,ug/ml (Table 1). The lowest MBC was obtained with ceftriaxone, followed by erythromycin. Similar MBCs were demonstrated with amoxicillin, cefuroxime, and doxycycline (0.8 to 1.6 p.g/ml). Penicillin G had the highest MBC among the antibiotics tested. Six of the seven human skin B. burgdorferi isolates tested were as susceptible to cefuroxime as the human cerebrospinal fluid isolate (MBC = 1.0 ,ug/ml; data not shown). Cefuroxime MBCs for B. turicatae and B. anserina were 1.0 and 2.0 p.g/ml, respectively. Only B. hermsii demonstrated an MBC for cefuroxime, i.e., >2.0 ,ug/ml (data not shown). Antibiotic treatment of B. burgdorferi-infected Syrian hamsters. The four antibiotics examined were tested at doses ranging between 0.4 to 149 mg/kg (Table 2). Although all four TABLE 2. Therapeutic evaluation of antimicrobial agents in Syrian hamsters experimentally infected with B. burgdorferi 297 Drug
Cefuroxime
Doxycycline
Amoxicillin
Penicillin G
Control
Total dose Total (mg/kg)
89.3 44.6 22.3 4.5 0.5
No. culture-positive hamsters/total no. treated
CD50' (mg/kg)
1/5
28.6 (15.34-53.14)
2/5 3/5 4/5 4/4
96.2 48.1 24.0 4.8 0.5
0/5
47.1 36.5 18.3 3.7 0.4
2/5 2/5 4/5
149.0 74.5 37.3 18.6
0/3
36.5 (4.07-327.54)
1/5 5/5 5/5 5/5
45.0 (13.58-149.31)
5/5 5/5
93.9 (73.80-119.49)
5/5 5/5
5/5 4/5
a Numbers in parentheses are 95% confidence limits.
ANTIMICROBIAL SUSCEPTIBILITY OF B. BURGDORFERI
VOL. 34, 1990
antibiotics provided some protection against borreliosis in this animal model system, the poorest results were obtained with penicillin G, in that no protection was obtained at doses as high as 74.5 mg/kg. In contrast, at least 60% protection (three of five animals) was provided by cefuroxime, amoxicillin, and doxycycline at doses ranging from 36.5 to 48.1 mg/kg. These three antibiotics, which demonstrated similar in vitro MBCs against B. burgdorferi (Table 1), also had comparable CD50 values in this in vivo protection system (Table 2), although the CD50 for cefuroxime indicated that it was somewhat more effective than amoxicillin or doxycy-
cline. DISCUSSION The two-stage scheme for categorizing Lyme disease (i.e., early and late) has the advantage of simplifying its diagnosis and, in addition, has an impact on the choice of drug therapy. The diagnosis of Lyme disease in the acute phase of the spirochetal infection should be based on a combination of clinical signs and symptoms, serological testing, and epidemiological evidence of tick exposure (4, 5). The serological response to B. burgdorferi is the basis of an important approach in recognizing the chronic stage of the disease (14), but its value as a diagnostic tool in early Lyme disease is diminished by the slow and variable antibody response in some individuals as well as by the assay-to-assay and laboratory-to-laboratory variability of the commonly used serological procedures. A monoclonal antibody-based detection method for B. burgdorferi antigens in the urine of mice experimentally infected with B. burgdorferi and in humans with Lyme disease, which could provide an alternative diagnostic method to serological testing (16), has been reported recently. Antibiotic therapy appears to be of value for patients with either early or late Lyme disease. However, certain antibiotics may vary in their efficacy depending on the stage of the disease. For patients with early Lyme disease, oral tetracycline, doxycycline, and amoxicillin are currently recommended in adults, whereas oral amoxicillin, penicillin V, and erythromycin are recommended for children s8 years of age (21, 22, 27). For patients presenting with mild neurological symptoms, ceftriaxone or oral doxycycline can be used. Patients with more severe neurological abnormalities and with arthritic symptoms may be treated with intravenous penicillin or ceftriaxone (12, 13, 21, 22, 27, 30; Liu et al., Arthritis Rheum., 1989). Despite these recommended therapies, the optimal treatment of patients at different stages of Lyme disease is still not clear. Although oral antibiotic therapy is generally thought to shorten the duration of the skin rash and to prevent the development of later illness in most patients, Dattwyler et al. (14) reported that patients treated with oral antibiotics at the early stages of the disease may occasionally become seronegative and yet still develop symptoms characteristic of late Lyme disease. Findings such as those reported by Dattwyler et al. (14) underscore the value of the in vitro susceptibility system as an efficient means for identifying antibiotics with activity against B. burgdorferi as well as the usefulness of the Syrian hamster (17, 18) and gerbil (23, 24) in vivo systems, in which eradication of the spirochetes is an important treatment endpoint. Both susceptibility systems should prove useful in evaluating drug therapy for early Lyme disease. The present data demonstrate that the MBC of cefuroxime for B. burgdorferi is similar to those of amoxicillin and doxycycline,
2135
and that this cephalosporin has greater in vitro activity against B. burgdorferi than tetracycline and penicillin, two of the recommended antibiotics for early Lyme disease. The in vivo activities of cefuroxime, amoxicillin, and doxycycline were similar, and all were more effective than penicillin G in preventing experimentally induced borreliosis in Syrian hamsters. The in vitro and in vivo anti-B. burgdorferi results obtained in this study with cefuroxime, which appear favorable when compared with results with other antibiotics currently recommended for the treatment of Lyme disease, suggest that future follow-up studies are warranted. Recent reports have shown that Lyme arthritis can be experimentally induced in LEWIN rats (7), mice (6, 25), and irradiated LAK hamsters (26). Investigation of parenteral and/or oral cefuroxime or other parenteral cephalosporins that are less protein bound than ceftriaxone in these animal models for the treatment of late Lyme disease would be of interest. In summary, the present results suggest that oral cefuroxime may be an effective alternative treatment for early Lyme disease. This possibility is currently under investigation in a clinical trial designed to compare the safety and efficacy of cefuroxime axetil with one of the currently recommended oral antibiotics in treating early Lyme disease and preventing the development of late Lyme disease in these patients. ACKNOWLEDGMENTS
This work was supported in part by a grant from Glaxo Inc. and Public Health Service grant AR 34744 from the National Institutes of Health to R.C.J. The assistance of Joseph Quinn in the calculation of the CD50 values and of Thomas Wong and Rose Mills in the preparation of the manuscript is gratefully acknowledged. LITERATURE CITED 1. Anhalt, J. P., and J. A. Washington II. 1985. Preparation and storage of antimicrobial solutions, p. 1019-1020. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 2. Asbrink, D., and A. Hovmark. 1988. Early and late cutaneous 3.
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manifestations in Ixodes-borne borreliosis (erythema migrans borreliosis, Lyme borreliosis). Ann. N.Y. Acad. Sci. 539:4-15. Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521-525. Barbour, A. G. 1988. Laboratory aspects of Lyme borreliosis. Clin. Microbiol. Rev. 1:399-414. Barbour, A. G. 1989. The diagnosis of Lyme disease: rewards and perils. Ann. Intern. Med. 110:501-502. Barthold, S. W., D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody. 1990. Lyme borreliosis in selected strains and ages of laboratory mice. J. Infect. Dis. 162:133-138. Barthold, S. W., K. D. Moody, G. A. Terwilliger, R. 0. Jacoby, and A. C. Steere. 1988. An animal model for Lyme arthritis. Ann. N.Y. Acad. Sci. 539:264-273. Berger, B. W., and R. C. Johnson. 1989. Clinical and microbiologic findings in six patients with erythema migrans of Lyme disease. J. Am. Acad. Dermatol. 21:1188-1191. Berger, B. W., M. H. Kaplan, I. R. Rothenberg, and A. G. Barbour. 1985. Isolation and characterization of the Lyme disease spirochete from the skin of patients with erythema chronicum migrans. J. Am. Acad. Dermatol. 13:444 449. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis. 1982. Lyme disease-a tick-borne spirochetosis? Science 216:1317-1319. Cox, D. R. 1970. Analysis of binary data. Chapman & Hall, Ltd., London. Dattwyler, R. J., J. J. Halperin, H. Pass, and B. J. Luft. 1987. Ceftriaxone as effective therapy in refractory Lyme disease. J.
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Infect. Dis. 155:1322-1325. 13. Dattwyler, R. J., J. J. Halperin, D. J. Volkman, and B. J. Luft. 1988. Treatment of late Lyme borreliosis-randomized comparison of ceftriaxone and penicillin. Lancet i:1191-1194. 14. Dattwyler, R. J., D. J. Volkman, B. J. Luft, J. J. Halperin, J. Thomas, and M. C. Golightly. 1988. Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte response to Borrelia burgdorferi. N. Engl. J. Med. 319:1441-1446. 15. Hansen, K., P. Hindersson, and N. S. Pedersen. 1988. Measurement of antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease. J. Clin. Microbiol. 26:338-346. 16. Hyde, F. W., R. C. Johnson, T. J. White, and C. E. Shelburne. 1989. Detection of antigens in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease. J. Clin. Microbiol. 27:58-61. 17. Johnson, R. C., C. Kodner, and M. Russell. 1987. In vitro and in vivo susceptibility of the Lyme disease spirochete, Borrelia burgdorferi, to four antimicrobial agents. Antimicrob. Agents Chemother. 31:164-167. 18. Johnson, R. C., C. Kodner, M. Russell, and D. Girard. 1990. In vitro and in vivo susceptibility of Borrelia burgdorferi to azithromycin. Antimicrob. Chemother. 25(Suppl. A):33-38. 19. Johnson, R. C., N. Marek, and C. Kodner. 1984. Infection of Syrian hamsters with Lyme disease spirochetes. J. Clin. Microbiol. 20:1099-1101. 20. Johnson, S. E., G. C. Klein, G. P. Schmid, and J. C. Feeley. 1984. Susceptibility of the Lyme disease spirochete to seven antimicrobial agents. Yale J. Biol. Med. 57:549-553. 21. Medical Letter. 1988. Treatment of Lyme disease. Med. Lett. Drugs Ther. 30:65-66.
ANTIMICROB. AGENTS CHEMOTHER. 22. Medical Letter. 1989. Treatment of Lyme disease. Med. Lett. Drugs Ther. 31:57-59. 23. Mursic, V., P. Wilske, G. Schierz, M. Holmburger, and E. Suss. 1987. In vitro and in vivo susceptibility of Borrelia burgdorferi. Eur. J. Clin. Microbiol. 4:424-426. 24. Preac-Mursic, V., B. Wilske, G. Schierz, E. Suss, and B. Gross. 1989. Comparative antimicrobial activity of the new macrolides against Borrelia burgdorferi. Eur. J. Clin. Microbiol. Infect. Dis. 8:651-653. 25. Schaible, V. E., M. D. Kramer, C. Museteanu, G. Zimmer, H. Mossman, and M. M. Simon. 1989. The severe combined immunodeficiency (scid) mouse: a laboratory model for the analysis of Lyme arthritis and carditis. J. Exp. Med. 170:14271432. 26. Schmitz, J. L., R. F. Schell, A. Jejka, D. M. England, and L. Konick. 1988. Induction of Lyme arthritis in LSH hamsters. Infect. Immun. 56:2336-2342. 27. Steere, A. C. 1989. Lyme disease. N. Engl. J. Med. 321:586-596. 28. Steere, A. C., N. H. Bartenhagen, J. E. Craft, G. J. Hutchinson, J. H. Newman, D. W. Rahn, L. H. Sigal, P. N. Spieler, K. S. Stenn, and S. E. Malawista. 1983. The early clinical manifestations of Lyme disease. Ann. Intern. Med. 99:22-26. 29. Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista. 1983. The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308:733-740. 30. Steere, A. C., G. J. Hutchinson, D. W. Rahn, L. H. Sigal, J. E. Craft, E. T. DeSanna, and S. E. Malawista. 1983. Treatment of the early manifestation of Lyme disease. Ann. Intern. Med. 99:22-26.
Vol. 34, No. 11
Comparative In Vitro and In Vivo Susceptibilities of the Lyme Disease Spirochete Borrelia burgdorferi to Cefuroxime and Other Antimicrobial Agents RUSSELL C.
JOHNSON,'*
CARRIE B. KODNER,1 PATTI J.
JURKOVICH,1
AND JEFFREY J. COLLINS2
Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455,1 and Clinical Development, Glaxo Inc., Research Triangle Park, North Carolina 277092 Received 6 November 1989/Accepted 31 August 1990
The in vitro and in vivo susceptibilities of the Lyme disease pathogen Borrelia burgdorferi to cefuroxime were compared with those of several other antibiotics commonly used to treat this disease. Cefuroxime demonstrated a higher MBC in vitro (1.0 ,ug/ml) than ceftriaxone (0.08 ,Ig/ml) or erythromycin (0.32 ,ug/ml), but the MBC was similar to that of amoxicillin (0.8 ,ug/ml) and doxycycline (1.6 ,ig/ml). B. burgdorferi was considerably less susceptible to tetracycline (3.2 ,ug/mI) and penicillin G (6.4 ,ug/ml). Of the three other Borrelia species tested, two (Borrelia turicatae and Borrelia anserina) also demonstrated susceptibility to cefuroxime, while the third (Borrelia hernsii) was less susceptible. Results obtained with four antimicrobial agents in the in vivo hamster model parallel the antibiotic susceptibilities in the in vitro study. The three antibiotics with similar MBCs in vitro, i.e., cefuroxime, doxycycline, and amoxicillin, demonstrated comparable activities in preventing borreliosis in B. burgdorferi-chalHenged hamsters (50% curative doses = 28.6, 36.5 and 45.0 mg/kg, respectively). Penicillin G, which demonstrated the highest MBC in vitro, had very weak protective activity in the hamster model system. These results indicate that the in vitro and in vivo activities of cefuroxime against B. burgdorfefi are comparable to those of several oral antibiotics currently being used in the treatment of early Lyme disease and suggest that the oral form of this cephalosporin may be an effective alternative therapy for this disease.
Lyme disease, which is caused by the spirochete Borrelia burgdorferi and transmitted to humans via the deer tick Ixodes dammini, is a multisystem disorder that mimics the signs and symptoms of various other conditions (5, 9, 10, 29). The progression of the disease is currently divided into three stages. During localized stage 1, there is usually a rapidly expanding skin lesion known as erythema chronicum migrans or simply erythema migrans. Disseminated stage 2 is characterized by neurological, musculoskeletal, and/or cardiac abnormalities which appear within weeks or months of stage 1 onset. Stages 1 and 2 have come to be commonly referred to as early Lyme disease (2, 27). Persistent stage 3 (also called late Lyme disease), the chronic phase of the disease, is manifested by prolonged arthritic episodes in the large joints (Lyme arthritis). This is frequently accompanied by chronic neurological abnormalities and can occur months to years after the clinical onset of the spirochetal infection. While the best clinical marker of stage 1 Lyme disease is erythema migrans (28), some patients with B. burgdorferi infection never develop this lesion, complicating the diagnosis of early Lyme disease. As the pathogenesis and the spectrum of clinical manifestations of Lyme disease have become better known, the diagnosis of the disease, the recognition of the progression of the disease, and the treatment of symptomatic patients have been updated and improved. Nevertheless, because of variable symptomatology, inconsistent results in the serological assays *
used to detect anti-Borrelia antibodies, and the difficulty of culturing or directly visualizing B. burgdorferi in patient specimens, diagnosis of Lyme disease remains problematic. Although recommendations for treatment are likely to be modified as further clinical studies are conducted, at the present time the most commonly used oral antibiotics for treatment of early Lyme disease are tetracycline, amoxicillin, doxycycline, erythromycin, and penicillin V. Oral antibiotics appear to be relatively ineffective in treating late Lyme disease, and such patients usually receive parenteral therapy with penicillin or ceftriaxone. The limited clinical trials (12, 13, 15; N. Y. Liu, H. Dinerman, R. E. Levin, E. Messarotti, P. J. Molloy, and R. J. Schoen, Arthritis Rheum. 32:546, 1989) which have been conducted thus far have provided data which support the need to investigate additional antibiotics for their efficacy in treating early or late Lyme disease or both. Previous in vitro and in vivo antimicrobial susceptibility studies have demonstrated the susceptibility of B. burgdorferi to a number of antibiotics (17, 18, 20, 23, 24; R. C. Johnson and C. B. Kodner, submitted for publication). By using the Syrian hamster model for assessing the in vivo susceptibility of B. burgdorferi, we have reported that the parenteral broad-spectrum cephalosporin ceftriaxone is effective in treating experimentally induced borreliosis (17). In the present report, the results of in vitro and in vivo animal model antimicrobial susceptibility studies are presented which indicate that cefuroxime, a 3-lactamase-stable cephalosporin available in both parenteral and oral forms, has anti-B. burgdorferi activity comparable or superior to that of the other oral antibiotics tested.
Corresponding author. 2133
2134
ANTIMICROB. AGENTS CHEMOTHER.
JOHNSON ET AL.
TABLE 1. In vitro antimicrobial susceptibility of B. burgdorferi 297
MATERIALS AND METHODS
Origin and cultivation of Borrelia isolates. Eight human Lyme disease B. burgdorferi isolates and three other Borrelia species were used in this study. One isolate, B. burgdorferi 297, was obtained from human cerebrospinal fluid (29). This hamster-virulent isolate was not subcultured more than three times before use in the in vitro and in vivo studies. The seven human skin isolates used were obtained from Bernard Berger, Southampton, N.Y. (8). Borrelia anserina was obtained from Richard Kelly, Baptist Memorial Hospital, Memphis, Tenn. Borrelia turicatae was obtained from the Rocky Mountain Laboratories, Hamilton, Mont., and Borrelia hermsii M1001 (ATCC 35209) was from the American Type Culture Collection, Rockville, Md. All cultures were grown at 30°C in Barbour-Stoenner-Kelly (BSK) medium (3). Animal isolation medium was modified by the addition of 0.15% SeaKem LE agarose (FMC Corp., Marine Colloids Div., Rockland, Maine). Antibiotics. The antibiotics tested were cefuroxime sodium (Glaxo Inc., Research Triangle Park, N.C.), tetracycline hydrochloride, doxycycline, erythromycin, and amoxicillin (Sigma Chemical Co., St. Louis, Mo.), ceftriaxone (Hoffmann-La Roche Inc., Nutley, N.J.), and penicillin G potassium (Pfizer Inc., New York, N.Y.). Recommendations by Anhalt and Washington were used for preparation and storage of antibiotic solutions (1). In vitro susceptibility procedures. The MBCs of amoxicillin, ceftriaxone, cefuroxime, doxycycline, erythromycin, penicillin G, and tetracycline were determined by using the broth dilution method of Johnson et al. (17). Cultures were in the log phase of growth and were counted with a PetroffHausser counting chamber. Concentrations of the test antibiotics ranged from 0.02 to 32 p.g/ml. Duplicate tubes containing BSK medium with the appropriately diluted antimicrobial agents and control (no antibiotic) tubes were inoculated to a final density of 105 cells per ml. After incubation at 30°C for 3 weeks, the tubes were checked under dark-field microscopy for the presence of spirochetes. All negative tubes (no spirochetes observed) were blind transferred (10% vol/vol) to BSK medium without antibiotics, incubated at 30°C for anther 3 weeks, and examined for spirochetes. The MBC was taken as the lowest antibiotic concentration at which spirochetes could not be subcultured. In vivo susceptibility procedures. Golden Syrian hamsters, 4 to 6 weeks old and weighing 80 to 100 g, were injected intraperitoneally with 1 ml of a culture (108 organisms per ml) of B. burgdorferi 297. Two weeks later, the test animals received daily subcutaneous injections with one-fifth of their total test antibiotic dosage for a period of 5 days. The antibiotics tested were cefuroxime, amoxicillin, penicillin G, and doxycycline, and the total dosages are given in Table 2. Control hamsters received only saline injections. Two weeks after the final antibiotic treatment, the hamsters were sacrificed, and the blood, spleen, kidneys, and bladder were collected and cultured. Each organ was aseptically placed into 6 ml of sterile BSK medium in a Stomacher bag and homogenized with a Tekmar Stomacher Lab-blender (Tekmar Co., Cincinnati, Ohio). A 1:10 dilution of the supernatant was placed into duplicate tubes of BSK medium. After 3 weeks of incubation at 30°C, the cultures were examined by dark-field microscopy for the presence of spirochetes. An animal was considered infected if at least one tissue yielded positive cultures. In most instances, five animals were tested
MBC (,ug/ml)
Antimicrobial agent
Ceftriaxone .........................................
0.08 0.32
Erythromycin ......................................... Amoxicillin ......................................... Cefuroxime .........................................
Doxycycline ......................................... Tetracycline ......................................... Penicillin G ................
.........................
0.8 1.0 1.6 3.2 6.4
per antimicrobial dose. The 50% curative dose (CD50) was calculated by using a log-logistic model (11).
RESULTS In vitro susceptibility of the Borrelia isolates to antibiotics. The MBCs of the tested antibiotics at the end of the 6-week observation period ranged from 0.08 to 6.4 ,ug/ml (Table 1). The lowest MBC was obtained with ceftriaxone, followed by erythromycin. Similar MBCs were demonstrated with amoxicillin, cefuroxime, and doxycycline (0.8 to 1.6 p.g/ml). Penicillin G had the highest MBC among the antibiotics tested. Six of the seven human skin B. burgdorferi isolates tested were as susceptible to cefuroxime as the human cerebrospinal fluid isolate (MBC = 1.0 ,ug/ml; data not shown). Cefuroxime MBCs for B. turicatae and B. anserina were 1.0 and 2.0 p.g/ml, respectively. Only B. hermsii demonstrated an MBC for cefuroxime, i.e., >2.0 ,ug/ml (data not shown). Antibiotic treatment of B. burgdorferi-infected Syrian hamsters. The four antibiotics examined were tested at doses ranging between 0.4 to 149 mg/kg (Table 2). Although all four TABLE 2. Therapeutic evaluation of antimicrobial agents in Syrian hamsters experimentally infected with B. burgdorferi 297 Drug
Cefuroxime
Doxycycline
Amoxicillin
Penicillin G
Control
Total dose Total (mg/kg)
89.3 44.6 22.3 4.5 0.5
No. culture-positive hamsters/total no. treated
CD50' (mg/kg)
1/5
28.6 (15.34-53.14)
2/5 3/5 4/5 4/4
96.2 48.1 24.0 4.8 0.5
0/5
47.1 36.5 18.3 3.7 0.4
2/5 2/5 4/5
149.0 74.5 37.3 18.6
0/3
36.5 (4.07-327.54)
1/5 5/5 5/5 5/5
45.0 (13.58-149.31)
5/5 5/5
93.9 (73.80-119.49)
5/5 5/5
5/5 4/5
a Numbers in parentheses are 95% confidence limits.
ANTIMICROBIAL SUSCEPTIBILITY OF B. BURGDORFERI
VOL. 34, 1990
antibiotics provided some protection against borreliosis in this animal model system, the poorest results were obtained with penicillin G, in that no protection was obtained at doses as high as 74.5 mg/kg. In contrast, at least 60% protection (three of five animals) was provided by cefuroxime, amoxicillin, and doxycycline at doses ranging from 36.5 to 48.1 mg/kg. These three antibiotics, which demonstrated similar in vitro MBCs against B. burgdorferi (Table 1), also had comparable CD50 values in this in vivo protection system (Table 2), although the CD50 for cefuroxime indicated that it was somewhat more effective than amoxicillin or doxycy-
cline. DISCUSSION The two-stage scheme for categorizing Lyme disease (i.e., early and late) has the advantage of simplifying its diagnosis and, in addition, has an impact on the choice of drug therapy. The diagnosis of Lyme disease in the acute phase of the spirochetal infection should be based on a combination of clinical signs and symptoms, serological testing, and epidemiological evidence of tick exposure (4, 5). The serological response to B. burgdorferi is the basis of an important approach in recognizing the chronic stage of the disease (14), but its value as a diagnostic tool in early Lyme disease is diminished by the slow and variable antibody response in some individuals as well as by the assay-to-assay and laboratory-to-laboratory variability of the commonly used serological procedures. A monoclonal antibody-based detection method for B. burgdorferi antigens in the urine of mice experimentally infected with B. burgdorferi and in humans with Lyme disease, which could provide an alternative diagnostic method to serological testing (16), has been reported recently. Antibiotic therapy appears to be of value for patients with either early or late Lyme disease. However, certain antibiotics may vary in their efficacy depending on the stage of the disease. For patients with early Lyme disease, oral tetracycline, doxycycline, and amoxicillin are currently recommended in adults, whereas oral amoxicillin, penicillin V, and erythromycin are recommended for children s8 years of age (21, 22, 27). For patients presenting with mild neurological symptoms, ceftriaxone or oral doxycycline can be used. Patients with more severe neurological abnormalities and with arthritic symptoms may be treated with intravenous penicillin or ceftriaxone (12, 13, 21, 22, 27, 30; Liu et al., Arthritis Rheum., 1989). Despite these recommended therapies, the optimal treatment of patients at different stages of Lyme disease is still not clear. Although oral antibiotic therapy is generally thought to shorten the duration of the skin rash and to prevent the development of later illness in most patients, Dattwyler et al. (14) reported that patients treated with oral antibiotics at the early stages of the disease may occasionally become seronegative and yet still develop symptoms characteristic of late Lyme disease. Findings such as those reported by Dattwyler et al. (14) underscore the value of the in vitro susceptibility system as an efficient means for identifying antibiotics with activity against B. burgdorferi as well as the usefulness of the Syrian hamster (17, 18) and gerbil (23, 24) in vivo systems, in which eradication of the spirochetes is an important treatment endpoint. Both susceptibility systems should prove useful in evaluating drug therapy for early Lyme disease. The present data demonstrate that the MBC of cefuroxime for B. burgdorferi is similar to those of amoxicillin and doxycycline,
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and that this cephalosporin has greater in vitro activity against B. burgdorferi than tetracycline and penicillin, two of the recommended antibiotics for early Lyme disease. The in vivo activities of cefuroxime, amoxicillin, and doxycycline were similar, and all were more effective than penicillin G in preventing experimentally induced borreliosis in Syrian hamsters. The in vitro and in vivo anti-B. burgdorferi results obtained in this study with cefuroxime, which appear favorable when compared with results with other antibiotics currently recommended for the treatment of Lyme disease, suggest that future follow-up studies are warranted. Recent reports have shown that Lyme arthritis can be experimentally induced in LEWIN rats (7), mice (6, 25), and irradiated LAK hamsters (26). Investigation of parenteral and/or oral cefuroxime or other parenteral cephalosporins that are less protein bound than ceftriaxone in these animal models for the treatment of late Lyme disease would be of interest. In summary, the present results suggest that oral cefuroxime may be an effective alternative treatment for early Lyme disease. This possibility is currently under investigation in a clinical trial designed to compare the safety and efficacy of cefuroxime axetil with one of the currently recommended oral antibiotics in treating early Lyme disease and preventing the development of late Lyme disease in these patients. ACKNOWLEDGMENTS
This work was supported in part by a grant from Glaxo Inc. and Public Health Service grant AR 34744 from the National Institutes of Health to R.C.J. The assistance of Joseph Quinn in the calculation of the CD50 values and of Thomas Wong and Rose Mills in the preparation of the manuscript is gratefully acknowledged. LITERATURE CITED 1. Anhalt, J. P., and J. A. Washington II. 1985. Preparation and storage of antimicrobial solutions, p. 1019-1020. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 2. Asbrink, D., and A. Hovmark. 1988. Early and late cutaneous 3.
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