In vitro activity of trimethoprim againstBorrelia burgdorferi

458

6.

7.

8.

9.

10.

11.

12.

Notes

therapy for urinary tract infections in adults. Infection 1992, 20, Supplement 3: 164-169. Wahlig H, Langenberg G, von Kobyletzki D: Erg~inzende Untersuchungen zur Pharmakokinetik von Gentamicin. Infection 1975, 3:217-222. Nordstr6m L, Lerner SA: Single daily dose therapy with aminoglycosides. Journal of Hospital Infection 1991,18, Supplement A: 117-129. Isaksson B, Nilsson L, Mailer R, S6ren L: Postantibiotic effect of aminoglycosides on gram-negative bacteria evaluated by a new method. Journal of Antimicrobial Chemotherapy 1988, 22: 23-33. Kampmann J, Siersbaek-Nietsen K, Kristensen M, M61holm-Hansen J: Rapid evaluation of creatinine clearance. Acta Medica Scandinavica 1974, 196: 517-520. The Swedish Reference Group for Antibiotics: A revised system for antibiotic sensitivity testing. Scandinavian Journal of Infectious Diseases 1981, 13: 148-152. Norrby SR: Design of clinical trials in patients with urinary tract infections: Infection 1992, 20, Supplement 3: 181-188. Norrby SR: Evaluation of antibiotics for treatment of urinary tract infections. Journal of Antimicrobial Chemotherapy 1994, 33, Supplement A: 43-50.

Eur. J. Clin. Microbiol. Infect. Dis.

Effective oral antibiotic treatment for late Lyme disease is not yet established. As reported earlier (1), oral therapy with a combination of trimethoprim, sulfamethoxazole, and roxithromycin has been successful in treating late Lyme borreliosis unresponsive to conventional treatment. This observation has been confirmed by others (2). The benefit derived from treatment with cotrimoxazole has been questioned on grounds of this compound's inactivity against spirochetes in vitro (3). In recent studies we demonstrated that both trimethoprim and sulfamethoxazole are antagonized by several ingredients of Barbour-StoennerKelly (BSK) II medium (4), which is commonly used for susceptibility testing of Borrelia burgdorferi. Therefore, previously published data (5, 6) showing that trimethoprim and sulfamethoxazole are inactive against borreliae in BSK II medium may be inaccurate. In order to determine whether trimethoprim and sulfamethoxazole are active against Borrelia burgdorferi in vitro, we developed a specially modified BSK II medium.

Materials and Methods. The components of BSK

In Vitro Activity of Trimethoprim against Borrelia

burgdorferi E.C. Reisinger*, I. Wendelin, R. Gasser

A new culture m e d i u m has been d e v e l o p e d to evaluate the activity of trimethoprim and sulfametho x a z o l e against Borrelia burgdorferi in vitro. In this specially modified Barbour-Stoenner-Kelly medium, in which antagonizing substances were reduced to a minimum, trimethoprim was more active against Borrelia burgdorferi than against a sensitive strain of Escherichia coil, but s u l f a m e t h o x a z o l e was not active against Borrelia burgdorferi.

Division of Infectious Diseases, Department of Medicine, Karl-FranzensUniversity,Auenbruggerplatz15,A-8036Graz, Austria.

II medium known to inactivate trimethoprim and sulfamethoxazole (4) were either replaced with an acceptable substitute or reduced to the minimal concentrations required for unaffected growth of Borrelia burgdorferi. The components of this minimal trimethoprim-inactivating (MTI) medium are listed in Table 1. The ingredients (except Dulbecco's modified Eagle medium and human serum) were dissolved in double-distilled water. The solution was then filter-sterilized and stored at -20~ Before use, Dulbecco's modified Eagle medium and human serum were added. The pH of the medium was adjusted to 7.4 with sterile 0.1 N NaOH. The growth rate of Borrelia burgdorferi strain B31 in MTI medium was tested as described elsewhere (7). Cultures with nominal inoculum densities of 1, 2, 5,10,102,103, and 104 spirochetes per culture, already adapted to MTI medium, as well as medium alone were incubated at 35~ The presence of spirochetes was observed by dark-field microscopy (400x) for three weeks. In order to validate the MTI medium for broth macrodilution susceptibility testing of Borrelia burgdorferi, the MICs of penicillin, ceftriaxone (Sigma, Germany), roxithromycin (A. Roussel, Austria), and ciprofioxacin (Miles, USA) obtained in MTI medium were compared with those obtained in BSK II medium. Medium (MTI

Vol. 16, 1997

Notes

Table 1: Components of the minimal trimethoprim-inactivating medium. Ingredient

Borrelia burgdorferi

Escherichia coil

>512

Quantity per liter

Neopeptone (Difco, USA, no. 0119-01) 2.5 g Bovine serum albumin Fraction V powder (Sigma, Germany, no. A-7409) 2.5 g HEPES (Sigma, Germany, no. H-3375) 2.5 g Glucose (Merck, Germany, no. 8342) 3.5 g Sodium citrate (Sigma, Germany, no. C-7254) 0.07 g Sodium pyruvate (Sigma, Germany, no. P-2256) 0.07 g N-acetyl glucosamine (Sigma, Germany, no. A-8625) 0.2 g Sodium bicarbonate (Merck, Germany, no. 6329) 0.8 g Sodium chloride (Merck, Germany, no. 6405) 2.0 g Dulbecco's MEM (Gibco, UK, no. 12501-029) 50 ml Human serum* 60 ml Gelatine (Merck, Germany, no. 4070), 2% aqueous solution 10 ml Water (double distilled) 880 ml * Free of antibodiesagainstBorrelia burgdorferi, heat-inactivatedat 56~ for 30 rain, and filter-sterilized.

or BSK II medium), serial twofold antibiotic dilutions, and a culture of actively growing spirochetes (precultured in the respective medium) were dispensed into the wells of a 24-well tissue culture plate (Costar, USA) to a total volume of 2 ml (8). The final inoculum was 5 x 105 cells/ml. The growth control wells contained medium and spirochetes without antibiotics, while the negative control wells contained medium only. The 24-well tissue culture plates were hermetically sealed and incubated for four days at 35~ The MIC was defined as the lowest concentration causing visible inhibition of growth (a lack of color change of medium from pink to yellow) and a cell density _< 105 motile cells/ml as determined with a PetroffHausser counting chamber and dark-field microscopy. To evaluate the activity of trimethoprim and sulfamethoxazole in MTI medium, the MICs of both trimethoprim and sulfamethoxazole were determined for a strain of Escherichia coli (a clinical isolate sensitive in Mueller-Hinton broth) and compared with those obtained in Mueller-Hinton broth (Merck, Germany) and BSK II medium (9). Medium, serial twofold dilutions of trimethoprim or sulfamethoxazole (Hofmann La Roche, Switzerland), and a culture of actively growing Escherichia coli (precultured in the respective medium) were dispensed into the wells of a 24-well tissue culture plate as described above. The tissue culture plates were incubated at 37~ for 20 h. The MIC was defined as the lowest concentration resulting in no visible turbidity (10).

459

IIBSK II medium E3MTI medium DMH medium 8

2 1 0.25 TMP

SMZ

TMP

SMZ

Figure 1= MICs of trimethoprim (TMP) and sulfamethoxazole (SMZ) for Borrelia burgdorferi strain B31 and Escherichia coil in Barbour-Stoenner-Kelly (BSK II) medium, MTI medium, and Mueller-Hinton (MH) medium.

Minimal trimethoprim-inactivating medium was used for broth macrodilution susceptibility testing of Borrelia burgdorferi strains ATCC 35210 (B31, tick isolate from Ixodes dammini), ATCC 35211 (tick isolate from Ixodes ricinus), and strain ATCC 53899 (isolate from cerebrospinal fluid) (German Collection of Microorganisms, Braunschweig, Germany) as described above. Results and Discussion. Growth of Borrelia burgdorferi strain B31 in MTI medium was detected in one of six cultures originally inoculated with one spirochete, in two of six cultures originally inoculated with two spirochetes, in five of six cultures originally inoculated with five spirochetes, and in all cultures originally inoculated with 10, 102, 103, and 104 spirochetes. The final density of organisms was > 108 cells/ml as determined with a Petroff-Hausser counting chamber and dark-field microscopy. The calculated generation time was 11 to 12 h. No spirochetes were detected in the negative controls. These data demonstrate that growth of Borrelia burgdorferi in MTI medium is comparable with that in BSK II and BSK-H medium (7, 9). The MICs of penicillin, ceftriaxone, and ciprofloxacin for Borrelia burgdorferi B31 obtained in BSK II medium were equal to those obtained in MTI medium: 0.06 txg/ml, 0.03 txg/ml, and 1 ixg/ml, respectively.The MICs of roxithromycin for B31 were 0.06 txg/ml in MTI medium and 0.03 txg/ml in BSK II medium. These data show that MTI medium is appropriate for in vitro susceptibility testing of Borrelia burgdorferi. The MICs of trimethoprim for Escherichia coli were > 512 txg/ml in BSK II medium, 8 v~g/mlin MTI medium, and 0.25 ~g/ml in Mueller-Hinton broth. The MICs of sulfamethoxazole for Esche-

460

Notes

richia coil were > 512 ixg/ml in BSK II medium, 2 txg/ml in MTI medium, and 2 txg/ml in MuellerHinton broth (Figure 1). These data show that trimethoprim and sulfamethoxazole were active in MTI medium and Mueller-Hinton medium but not in BSK II medium. In MTI medium, trimethoprim was active against Borrelia burgdorferi strains B31, ATCC 35211, and ATCC 53899, with MICs of 1 txg/ml, 0.5 txg/ml, and 1 Ixg/ml, respectively (Figure 1). When exposed to trimethoprim, the spirochetes lost their motility and produced filaments with a morphology similar to that of the filaments produced with [3-lactam antibiotics (11). In MTI medium, sulfamethoxazole was inactive against Borrelia burgdorferi, with MICs of > 512 p.g/ml (Figure 1). The higher MIC of trimethoprim for Escherichia coli in MTI medium compared to that in Mueller-Hinton medium indicates that the activity of trimethoprim is still inhibited in MTI medium. Therefore, the MICs of trimethoprim for Borrelia burgdorferi in a medium causing no inhibition may be even lower than the ones described. Our results are interesting in view of the observations regarding effective oral treatment of late Lyme disease with the combination of trimethoprim, sulfamethoxazole, and roxithromycin (1, 2, 12). Since sulfamethoxazole is not active in MTI medium and roxithromycin has been ineffective in vivo (13, 14), trimethoprim may be a better candidate for oral treatment of Lyme disease.

Acknowledgement

Eur. J. Clin. Microbiol. Infect. Dis.

6. Morshed MG, Konishi H, Nishimura T, Nakazawa T: Evaluation of agents for use in medium for selective isolation of Lyme disease and relapsing fever Borrelia species. European Journal of Clinical Microbiology & Infectious Diseases 1993, 12: 512-518. 7. Pollack RJ, Telford III SR, Spielman A: Standardisation of medium for culturing Lyme disease spirochetes. Journal of Clinical Microbiology 1993, 31 : 1251-1255. 8. Reisinger EC, Wendelin I, Gasser R, Halwachs G, Wilders-Truschnig M, Krejs G: Antibiotics and increased temperature against Borrelia burgdorferi in vitro. Scandinavian Journal of Infectious Diseases 1996, 28: 155-157. 9. Barbour AG: Isolation and cultivation of Lyme disease spirochetes. Yale Journal of Biology and Medicine 1984, 57: 521-525. 10. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7A2. NCCLS, Villanova, PA, 1990. 1I. Kersten A, Poitschek C, Rauch S, Aberer E: Effects of penicillin, ceftriaxone, and doxycycline on morphology of Borrelia burgdorferi. Antimicrobial Agents and Chemotherapy 1995, 39:1127-1133. 12. Gasser R, Wendelin I, Reisinger E, Bergloff J, Feigl B, Schafhalter I, Eber B, Grisold M, Klein W: Roxithromycin in the treatment of Lyme disease - update and perspectives. Infection 1995, 23, Supplement 1: 39-43. 13. Preac-Mursic V, Wilske B, Schierz G, S08 E, Gro8 B: Comparative antimicrobial activity of the new macrolides against Borrelia burgdorferi. European Journal of Clinical Microbiology & Infectious Diseases 1989, 8: 651-653. 14. Hansen K, Hovmark A, Lebech AM, Lebech K, Olsson I, Sorensen L: Roxithromycin in Lyme borreliosis: discrepant results of an in-vitro and in-vivo animal susceptibility study and a clinical trial in patients with erythema migrans. Acta Dermatologica Venerologica 1992, 72: 297-300.

We thank Mrs. S. Pailer,M. Siwetz,and M. Resel for their excellent technical assistance.

References 1. Gasser R, Dusleag J: Oral treatment of late borreliosis with roxithromycin plus co-trimoxazole. Lancet 1990, 336: 1189-1190. 2. Pedersen LM, Friis-Moller A: Late treatment of chronic Lyme arthritis. Lancet 1991, 337: 241. 3. Bowman CA: Oral treatment of late borreliosis with roxithromycin plus co-trimoxazole. Lancet 1990, 336:1514. 4. Reisinger EC, Wendelin I, Gasser R: Inactivation of diaminopyrimidines and suifonamides in Barbour-StoennerKelly medium for isolation of Borrelia burgdorferi. European Journal of Clinical Microbiology & Infectious Diseases 1995, 14: 732-733. 5. Preac-Mursic V, Wilske B, Schierz G: European Borrelia burgdorferi isolated from humans and ticks. Culture conditions and antibiotic susceptibility. Zentralblatt for Bakteriologie und Hygiene (A) 1986, 268:112-118.

Fatal Scedosporium prolificans Infection in a Leukemic Patient M . C . W . F e l t k a m p 1, M.J. K e r s t e n 2, J. v a n d e r L e l i e 2, J.D. B u r g g r a a f 3, G.S. d e H o o g 4, E.J. K u i j p e r 1.

1 Departmentof MedicalMicrobiology,2Departmentof Internal Medicine,Divisionof Haematology,and 3Departmentof Pathology,AcademicMedicalCentre,Universityof Amsterdam,Meibergdreef9, NL-1105AZ Amsterdam,The Netherlands. 4 Centraalbureau voor Schimmelcultures,PO Box 273, NL3740 AG Baarn,The Netherlands.