This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sicklinger, M.
Right arrow Articles by Neubert, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sicklinger, M.
Right arrow Articles by Neubert, U.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, April 2003, p. 1791-1793, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1791-1793.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Vitro Susceptibility Testing of Four Antibiotics against Borrelia burgdorferi: a Comparison of Results for the Three Genospecies Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto

Martin Sicklinger, Ralf Wienecke, and Uwe Neubert*

Department of Dermatology, Ludwig-Maximilians University of Munich, D-80337 Munich, Germany

Received 1 October 2002/ Returned for modification 6 November 2002/ Accepted 20 December 2002


arrow
ABSTRACT
 
MICs and minimal bactericidal concentrations (MBCs) were evaluated for the four antibiotics azithromycin, amoxicillin, ceftriaxone, and doxycycline against the three main genospecies of Borrelia burgdorferi sensu lato. In MBC testing, statistically significant differences between the genospecies could be found in 7 out of 12 comparative evaluations (P < 0.05).


arrow
TEXT
 
Lyme borreliosis is a multisystemic disease caused by the spirochete Borrelia burgdorferi, which was discovered in 1981 (5). It is transmitted by tick vectors of the genus Ixodes (13, 15).

Today, several different genospecies of the spirochete Borrelia are known. Three of them are of a special clinical relevance: B. burgdorferi sensu stricto is the only one endemic in North America, whereas B. afzelii and B. garinii are predominant in Europe (1, 4, 14, 16, 17).

Many in vitro (3, 6-8, 10, 12) and in vivo (11) studies have been performed to find an optimal treatment for Lyme disease; what form such a treatment would take is still not quite clear (12). On the other hand, only few data (3, 6-8, 10, 12) are available about possible differences in the antibiotic sensitivities of the three main genospecies, and the results obtained thus far are quite controversial.

Considering the geographic pattern of distribution mentioned above, we found it necessary to look for differences in the antibiotic responses of B. garinii, B. afzelii, and B. burgdorferi sensu stricto. The results could contribute to answering the question of whether Lyme disease should be treated differently in America and Europe.

The four antibiotics used for this study were azithromycin (Pfizer, New York, N.Y.), amoxicillin, ceftriaxone, and doxycycline (Sigma, Munich, Germany).

All strains of B. burgdorferi except strain B31 (imported from the United States) were low-passage isolates (not more than 10 passages). The isolates were obtained from either ticks or skin lesions at the Department of Dermatology, Ludwig-Maximilians University, Munich, Germany, or kindly provided by A. Vogt, University Clinic of Freiburg, Freiburg, Germany. Identification of the strains was performed by PCR typing as previously described (1, 16, 17). We chose a total of 24 strains (8 strains of each genospecies) (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. MICs and MBCs of four antimicrobial agents against Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto after 72 h of incubation at 33°C

The antibiotics were dissolved in BSK-H medium (Sigma-Aldrich, Deisenhofen, Germany) supplemented with 6% rabbit serum and were stored in 2.5-ml test tubes. The final concentration ranges were 0.125 to 16 µg/ml for doxycycline and 0.002 to 0.5 µg/ml for the other tested antimicrobial substances. They were chosen according to the expected MICs and minimal bactericidal concentrations (MBCs) described in the literature (3, 6-8, 10, 12).

The MIC was defined as the lowest concentration of antibiotics at which the number of countable cells after 72 h of incubation at 33°C did not exceed the basis concentration. To determine the MIC, 50 µl of BSK-H medium containing B. burgdorferi was added to the test tubes described above. Using the method of dark-field microscopy, the basis concentration was adjusted to 105 cells/ml. From each tube, 5 µl of BSK-H medium was pipetted on a cover glass. The vital spirochetes in 10 fields of view were counted under the microscope. The concentration of bacteria in the test tubes was determined by using the following formula:

In our study, the technical data were as follows: cover glass size, 400 mm2; field of view size, 0.159 mm2.

This testing was done in triplicate for each strain. A test tube containing BSK medium without antibiotics was used as a control. The test tubes were then incubated for 72 h at 33°C. After that period, the cells were counted again.

The MBC was defined as the lowest concentration of antibiotics at which no viable spirochetes could be detected after 2 weeks of subcultivation in BSK-H medium free of antibiotics at 33°C. To determine the MBC, 50 µl of the contents of each of the test tubes which had been used earlier for MIC determination and which contained antibiotics at the MIC or a higher concentration was added to 2.5 ml of BSK-H without antibiotics. The mixtures were incubated for 14 days at 33°C, and then the cells were counted again.

Statistical analysis was done by comparisons of the confidence intervals and by the Kruskal-Wallis H test and the Wilcoxon ranking test. The level of significance was P < 0.05.

There were no statistically significant differences between the MICs of the tested antibiotics against B. afzelii, B. garinii, and B. burgdorferi sensu stricto (Table 1).

In 7 out of 12 comparative evaluations (P < 0.05), MBCs were significantly different among the three genospecies (Table 1). B. garinii seemed to be especially susceptible to azithromycin, while amoxicillin had a significantly greater effect on B. burgdorferi sensu stricto compared to the other genospecies. Ceftriaxone had the lowest MBC with B. afzelii and increasingly higher MBCs with B. garinii and B. burgdorferi sensu stricto. Doxycycline did not show any remarkable differences in its effects on the three genospecies.

The results of our study contradict those of Baradaran-Dilmaghani et al., who found no statistically significant differences between the genospecies (3), but partially confirm those of Preac-Mursic et al. (12), who described susceptibilities of B. garinii to amoxicillin, doxycycline, cefotaxime, ceftriaxone, azithromycin, and penicillin G that were higher than those of B. afzelii. Hunfeld et al., too, found B. garinii to be the genospecies most susceptible to macrolides and cephalosporins (9). As in our study, Henneberg and Neubert describe B. burgdorferi sensu stricto as being comparatively less susceptible to cephalosporins, but they also found B. garinii to be more sensitive than the other genospecies to penicillin (6).

With the exception of doxycycline, the antibotics tested by us showed excellent efficacy against all 24 borrelial strains. Indeed, we found some statistically significant differences between antibiotic MBCs for the three genospecies.

B. burgdorferi sensu stricto, the etiologic agent of North American Lyme disease, showed the highest susceptibility of the three species to amoxicillin. B. afzelii, the predominant borrelial species in Central Europe and the causative organism of acrodermatitis chronica atrophicans (2, 17), proved to be especially susceptible to ceftriaxone. B. garinii, the most frequently isolated species from cerebrospinal fluid in European neuroborreliosis (14), demonstrated excellent sensitivity to azithromycin. However, these differences in antibiotic sensitivity seem not sufficiently pronounced to be of fundamental clinical relevance.

Beyond these findings, one has to consider that the different pharmacokinetics of the particular antibiotics do not allow a simple transfer of our in vitro results to practical treatment. For instance, the concentration of azithromycin in the cerebrospinal fluid is too low to represent a promising alternative to standard therapeutic regimens of neuroborreliosis such as intravenous infusion of ceftriaxone (9).

All together, the results of our study do not justify specific recommendations concerning antibiotic therapy of Lyme borreliosis for the suspected causative borrelial species.


arrow
ACKNOWLEDGMENTS
 
We thank J. Laude and G. Weishaar (Ludwig-Maximilians University, Munich, Germany) for excellent technical assistance.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Irisstr. 5, D-82194 Gröbenzell, Germany. Phone: 04981427102. Fax: 0498142592749. E-mail: UES.Neubert{at}gmx.de. Back


arrow
REFERENCES
 
    1
  1. Adam, T., U. Neubert, and U. B. Göbel. 1992. Detection and classification of Borrelia burgdorferi by direct sequencing of 16S rRNA amplified after reverse transcription. Med. Microbiol. Lett. 1:120-126.
    2. 2
  2. Asbrink, E., A. Hovmark, and K. Weber. 1993. Acrodermatitis chronica atrophicans, p. 193-204. In K. Weber and W. Burgdorfer (ed.), Aspects of Lyme borreliosis—1993. Springer-Verlag, Berlin, Germany.
    3. 3
  3. Baradaran-Dilmaghani, R., and G. Stanek. 1996. In vitro susceptibility of thirty Borrelia strains from various sources against eight antimicrobial chemotherapeutics. Infection 24:60-63.[CrossRef][Medline]
    4. 4
  4. Baranton, G., D. Postic, I. Saint Girons, P. Boerlin, J. C. Piffaretti, M. Assous, and P. A. D. Grimont. 1992. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int. J. Syst. Bacteriol. 42:378-383.[Abstract/Free Full Text]
    5. 5
  5. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davies. 1982. Lyme disease—a tick-borne spirochetosis? Science 216:1317-1319.[Abstract/Free Full Text]
    6. 6
  6. Henneberg, J.-P., and U. Neubert. 2002. Borrelia burgdorferi sensu lato—its antibiotic sensitivity in vitro. Orv. Hetil. 21:1195-1198.
    7. 7
  7. Hunfeld, K.-P., P. Kraiczy, T. A. Wichelhaus, V. Schäfer, and V. Brade. 2000. Colorimetric in vitro susceptibility testing of penicillins, cephalosporins, macrolides, streptogramins, tetracyclines, and aminoglycosides against Borrelia burgdorferi isolates. Int. J. Antimicrob. Agents 15:11-17.[CrossRef][Medline]
    8. 8
  8. Janovská, D., D. Hulínská, and T. Godová. 2001. Sensitivity of Borrelia burgdorferi strains isolated in the Czech Republic. Cent. Eur. J. Publ. Health 9:38-40.
    9. 9
  9. Jaruratanasikul, S., R. Hortiwakul, T. Tantisarasart, N. Phuenpathom, and S. Tussanasunthornwong. 1996. Distribution of azithromycin into brain tissue, cerebrospinal fluid, and aqueous humor of the eye. Antimicrob. Agents Chemother. 40:825-826.[Abstract]
    10. 10
  10. Levin, J. M., J. A. Nelson, J. Segreti, B. Harrison, C. A. Benson, and F. Strle. 1993. In vitro susceptibility of Borrelia burgdorferi to 11 antimicrobial agents. Antimcrob. Agents Chemother. 37:1444-1446.[Abstract/Free Full Text]
    11. 11
  11. Neubert, U. 1995. Lyme disease—focus on antibacterial therapy. Consultant Series in Infectious Disease 3. Gardiner-Caldwell, Macclesfield, United Kingdom.
    12. 12
  12. Preac-Mursic, V., W. Marget, U. Busch, D. Pleterski Rigler, and S. Hagl. 1996. Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme borreliosis. Infection 24:9-16.[CrossRef][Medline]
    13. 13
  13. Steere, A. C. 1989. Lyme disease. N. Engl. J. Med. 321:586-594.[Abstract]
    14. 14
  14. van Dam, A. P., H. Kuiper, K. Vos, A. Widjojokusomo, B. M. de Jongh, L. Spanjaard, A. C. P. Ramselaar, M. D. Kramer, and J. Dankert. 1993. Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clin Infect. Dis. 17:708-717.[Medline]
    15. 15
  15. Weber, K. 2001. Aspects of Lyme borreliosis in Europe. Eur. J. Clin. Microbiol. Infect. Dis. 20:6-13.[CrossRef][Medline]
    16. 16
  16. Wienecke, R., O. M. Koch, U. Neubert, U. B. Göbel, and M. Volkenandt. 1993. Detection of subtype-specific gene segments by analysis of conformational polymorphisms of cRNA molecules. Med. Microbiol. Lett. 2:239-246.
    17. 17
  17. Wienecke, R., N. Zöchling, U. Neubert, E.-M. Schlüpen, M. Meurer, and M. Volkenandt. 1994. Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans. J. Investig. Dermatol. 103:19-22.[CrossRef][Medline]

Journal of Clinical Microbiology, April 2003, p. 1791-1793, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1791-1793.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Morgenstern, K., Baljer, G., Norris, D. E., Kraiczy, P., Hanssen-Hubner, C., Hunfeld, K.-P. (2009). In Vitro Susceptibility of Borrelia spielmanii to Antimicrobial Agents Commonly Used for Treatment of Lyme Disease. Antimicrob. Agents Chemother. 53: 1281-1284 [Abstract] [Full Text]  
  • Hodzic, E., Feng, S., Holden, K., Freet, K. J., Barthold, S. W. (2008). Persistence of Borrelia burgdorferi following Antibiotic Treatment in Mice. Antimicrob. Agents Chemother. 52: 1728-1736 [Abstract] [Full Text]  
  • Halperin, J. J., Shapiro, E. D., Logigian, E., Belman, A. L., Dotevall, L., Wormser, G. P., Krupp, L., Gronseth, G., Bever, C. T. Jr (2007). Practice Parameter: Treatment of nervous system Lyme disease (an evidence-based review): Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 69: 91-102 [Abstract] [Full Text]  
  • Hunfeld, K.-P., Ruzic-Sabljic, E., Norris, D. E., Kraiczy, P., Strle, F. (2005). In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy. Antimicrob. Agents Chemother. 49: 1294-1301 [Abstract] [Full Text]  
  • Zambrano, M. C., Beklemisheva, A. A., Bryksin, A. V., Newman, S. A., Cabello, F. C. (2004). Borrelia burgdorferi Binds to, Invades, and Colonizes Native Type I Collagen Lattices. Infect. Immun. 72: 3138-3146 [Abstract] [Full Text]  
  • Hunfeld, K.-P., Wichelhaus, T. A., Rodel, R., Acker, G., Brade, V., Kraiczy, P. (2004). Comparison of In Vitro Activities of Ketolides, Macrolides, and an Azalide against the Spirochete Borrelia burgdorferi. Antimicrob. Agents Chemother. 48: 344-347 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sicklinger, M.
Right arrow Articles by Neubert, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sicklinger, M.
Right arrow Articles by Neubert, U.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»