Vol. 11, 1992
-..-_..
All Clostridium difficile strains were susceptible to tosufloxacin, piperacillin, imipenem, metronidazole and chloramphenicol (Table 1). The MIC of ciprofloxacin for 50 % of the strains tested Was 8.0 mg/l and for 90 % of the strains 16 mg/1. Ninety percent of the Clostridium perfringens isolates were inhibited by 128 mg/1 of cefoxitin and 8 rag/1 of clindamycin. Tosufloxacin was more active than ciprofloxacin against the Bacteroides fragilis strains tested (Table 1). Imipenem was the most active beta-lactam agent tested against the isolates. Most strains Were susceptible to piperacillin, cefoxitin and clindamycin, while all of them were susceptible to raetronidazole and chloramphenicol. The other Bactero/des species were also more susceptible to tosufloxacin than ciprofloxacin (Table 1). Imipenem and clindamycin had the highest antirnicrobial activities. Piperacillin, cefoxitin and Chloramphenicol were active against all strains tested. Four Bacteroides strains were resistant to metronidazole. All fusobacteria were susceptible to tosufloxacin, piperacillin, cefoxitin, imipenem, clindamycin, metronidazole and chloramphenicol, while a few Strains were resistant to ciprofloxacin (Table 1). The in vitro results presented here indicate that tosufloxacin is a promising new fluoroquinolone Which may be useful in the treatment and prophylaxis of anaerobic infections. Clinical Studies are thus warranted.
References 1. FernandesPB, Chu DTW, Swanson RN, Ramer NR, Hanson CW, Bower RR, Stature JM, Hardy DJ: A61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 1988,32: 27-32. 2. Holdeman LV, Cato EP, Moore WEC: Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, VA, 1977, p. 1-156. 3. Dornbusch K, Nord CE, Oisson-Liljequisl B: Antibiotic susceptibility of anaerobic bacteria with special reference to Bacteroides fragitis. Scandinavian Journal of Infectious Diseases 1979, 19: 17-25. 4. National Committee for Clinical Laboratory Standards: Methods for antimierobial susceptibilitytesting of anaerobic bacteria. 2nd ed. Document Mll-A2. NCCLS, Villanova, PA, 1990.
265
In Vitro Activity of Azithromycin and Tetracycline against 358 Clinical Isolates of Brucella melitensis R. Landfnez 1., J. Lifiares 2, E. Loza 3, J. Mart[nez-Beltr~in 3, R. Martfn 2, E B a q u e r o 3
The in vitro activity of azithromycin against human pathogenic strains of Brucella melitensis was tested at three centres and compared to that of tetracycline, the standard antibiotic currently used for the treatment of human brucellosis. MIC determination was carried out. Tested concentration ranges for both azithromycin and tetracycline were between 0.03 and 16/ag/mL Brucella melitensis biotype 1, strain M16 was employed as a control microorganism. A total of 358 Brucella rnelitensis strains from human blood cultures were tested. MIC90 (~g/ml) values ranged from 0.5 to 1.00 for azithromycin and were 0.25 for tetracycline. It was concluded that there was little difference in the sensitivity of pathogenic Brucella melitensis to azithromycin and tetracycline isolated from three different regions in Spain. These results encourage further investigations on the possible therapeutic role of azithromycin in brucellosis.
Current treatment of acute brucellosis combining tetracycline and streptomycin or rifampicin usually achieves excellent clinical and microbiological results (1-3). Nevertheless, a variable percentage of failures and recurrence are reported in most published series. In addition, these therapeutic regimes pose difficulties in patients' compliance due to their duration; moreover, tetracyclines are contraindicated in children under eight years of age and in pregnant women. Thus, it is felt that new antibiotics overcoming these problems would represent a significant therapeutic advancement in the treatment of brucellosis. 1Departamento de Microbiologfa, Facultad de Medicina, Universidad de Valladolid, Ramon y Cajal, 7, 47005 Valladolid, Spain. 2Departamento de Mierobiologfa, Hospital "Pr/neipes de Espafia', Hospitalet de Llobregat, Barcelona, Spain. 3Departamento de Microbiologfa, Hospital 'Ram6n y Cajal", Madrid, Spain.
266
Eur. J. Clin. Microbiol. Infect. Dis.
New antibiotics exhibiting therapeutical potential in the treatment of this disease are routinely assayed in vitro against Brucella metitensis as a preliminary step to decide further clinical development. The objective of this study was to determine the in vitro susceptibility of a large number of human pathogenic strains of Brucella melitensis to azithromycin, a new azalide antibiotic related to erythromycin, in comparison to tetracycline, the standard treatment of brucellosis. Materials and Methods. A total of 358 Brucelta melitensis strains isolated from blood cultures in patients with acute brucellosis between 1987 and 1989 were tested. Each strain was recovered from a different patient; the possibility of a source of infection common to various patients was considered likely in only very few cases. The strains were stored in skimmed milk at -70 °C. These strains constitute one of the largest samples of this microorganism ever studied from three different areas in Spain: Vanadolid (223 strains), Barcelona (73 strains) and Madrid (62 strains).
Azithromycin (Pfizer Central Research, UK) and tetracycline hydrochloride (Antibi6ticos, Spain) supplied as assayed active substance powder were employed. Minimum inhibitory concentrations (MICs) were determined using the agar-dilution method. The culture medium employed was Iso-sentitest Agar CM471 (Oxoid, UK) to which serial twofold dilutions (from 0.03 to 16 ~tg/ml) of one of the two antibiotics were added. The inoculum was prepared from a smooth-phase Brucella melitensis culture in trypticase soy agar (BBL Microbiology Systems, USA). Three to five colonies were subcultured in brain-heart infusion broth (Difco Laboratories, USA) for 48 hours at 37 °C. A post-incubation dilution (1/20) in brain-heart infusion
broth was carried out. This resulted in an inoculum of approximately 105 cfu applied to the agar surface. Plates were inoculated with a Steers replicator and incubated at 37 °C in air. A first reading was performed at 48 hours and a confirmatory second one at 72 hours. MIC was defined as the lowest concentration of antibiotic at which there was no visible growth (>__ 3 isolated colonies). A control microorganism, Brucella melitensis biotype 1 strain M16 (Central Veterinary Laboratory, Weybridge, UK) was tested at each centre to assess inter-group variability. Results andDiscussion. Table I shows the in vitro MIC50 and MIC90 values found in each of the three participating centres for azithromycin and tetracycline, respectively.
MICs of azithromycin ranged between 0.03 and 2.00 ~g/ml. MIC90 was 0.50 ~tg/ml in two of the centres, while it was 1.00/ag/ml at the centre in Barcelona (73 strains). Comparative results for tetracycline showed MICs ranging from 0.06 to 0.50 /ag/ml. MIC90 was 0.25 ~g/ml in all three centres. No difference in results was found between the readings at 48 hours and 72 hours. MIC90 values for the control microorganism ranged between 0.25 and 0.50/ag/ml for azithromycin and between 0.10 and 0.25 ~ag/ml for tetracycline. A remarkable uniformity in the results (except for the small discrepancy mentioned above) was found in this study. Azithromycin is a novel 15-membered ring azalide antibiotic, chemically related to macrolides (4). This agent exhibits certain pharmacokinetic and pharmacodynamic properties which can be useful in the treatment of infections caused by intracellular microorganisms. Peak
Table 1: Activityof azithromycinand tetracyclineagainst 358 human pathogenicstrains of Brucella melitensis. Source
No. of strains
Antibiotics
MIC (/ag/ml) Range
MIC50
MIC90
Valladolid
223
azithromycin tetracycline
0.03 - 2.00 0.12 - 0.50
0.50 0.25
0.50 0.25
Barcelona
73
azithromycin tetracycline
0.12 -2.00 0.06 - 0.50
0.50 0.25
1.00 0.25
Madrid
62
azithromycin tetracycline
0.12 - 1.00 0.06 - 0.50
0.50 0.25
0.50 0.25
Vol. 11, 1992
Serum concentration after a single oral dose of 500 trig of azithromycin reaches 0.4 lag/ml, but tissue Concentrations of azithromycin are much greater (10 to 100 times) and persist for longer periods (5). In addition to high tissue concentration, the in vivo performance of azithromycin may be related to its intracellular concentration and release at sites of infection. Azithromycin concentrates Within human and mouse polymorphonuclear cells, peritoneal macrophages from the mouse and alveolar cells from the rat and mouse. In vitro intracellular concentrations of this antibiotic are up to 226-fold higher than those measured outside the cell (6). The results obtained in this study from a very large selection of Brucella melitensis strains from three different regions of Spain are very consistent. MIC results indicate in vitro susceptibility of Brucella melitensis to azithromycin. The MIC Values are usually one dilution.higher than those obtained for tetracycline. As shown in further Studies, the in vitro activity of azithromycin against Brucella meIitensis is similar to that of Streptomycin and rifampicin (R. Martin, 7th Mediterranean Congress of Chemotherapy, 1990. Barcelona, Abstract 68). These results encourage further investigations on the potential usefulness of azithromycin in the treatment of human brucellosis.
267
References 1. Land[nez R, Abad R, Rodriguez Torres A: Estudio
sobre el tratamiento de la brucelosis. Infcctologika 1984, 5: 7-14. 2. Ariza J, Gudi01 F, Pallares R, Ruff G, FernandezViladrich P: Comparative trial of rifampin-doxycycline
versus tetracycline-streptomycin in the therapy of human brucellosis. Antimicrobial Agents and Chemotherapy 1985, 28: 548-551. 3. Acocella G, Bertrand A, Beylout J., Duriande JB, Garcia Rodriguez JA, Kosmidis J, Micoud M, Rey M, Rodriguez Zapala M, Roux J, Slahl JP- Com-
parison of three different regimens in the treatment of acute brucellosis: a multicenter multinational study. Journal of Antimicrobia/Chemotherapy 1989, 23: 433439. 4. Anonymous: Azithromycin. Drugs of the Future 1988, 13: 9-12. 5. Foulds G, Shepard RM, Johnson RB: The pharmacokinetics of azithromycin in human serum and tissues. Journal of Antimicrobial Chemotherapy 1990,25, Supplement A: 73-82. 6. Gladue RP, Bright GM, Isaacson RE, Newborg MF:
In vitro and in vivo uptake of azithromycin by phagocytic cells: possible mechanisms of delivery and release at sites of infection. Antimicrobial Agents and Chemotherapy 1989, 33: 277-282.
-..-_..
All Clostridium difficile strains were susceptible to tosufloxacin, piperacillin, imipenem, metronidazole and chloramphenicol (Table 1). The MIC of ciprofloxacin for 50 % of the strains tested Was 8.0 mg/l and for 90 % of the strains 16 mg/1. Ninety percent of the Clostridium perfringens isolates were inhibited by 128 mg/1 of cefoxitin and 8 rag/1 of clindamycin. Tosufloxacin was more active than ciprofloxacin against the Bacteroides fragilis strains tested (Table 1). Imipenem was the most active beta-lactam agent tested against the isolates. Most strains Were susceptible to piperacillin, cefoxitin and clindamycin, while all of them were susceptible to raetronidazole and chloramphenicol. The other Bactero/des species were also more susceptible to tosufloxacin than ciprofloxacin (Table 1). Imipenem and clindamycin had the highest antirnicrobial activities. Piperacillin, cefoxitin and Chloramphenicol were active against all strains tested. Four Bacteroides strains were resistant to metronidazole. All fusobacteria were susceptible to tosufloxacin, piperacillin, cefoxitin, imipenem, clindamycin, metronidazole and chloramphenicol, while a few Strains were resistant to ciprofloxacin (Table 1). The in vitro results presented here indicate that tosufloxacin is a promising new fluoroquinolone Which may be useful in the treatment and prophylaxis of anaerobic infections. Clinical Studies are thus warranted.
References 1. FernandesPB, Chu DTW, Swanson RN, Ramer NR, Hanson CW, Bower RR, Stature JM, Hardy DJ: A61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 1988,32: 27-32. 2. Holdeman LV, Cato EP, Moore WEC: Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, VA, 1977, p. 1-156. 3. Dornbusch K, Nord CE, Oisson-Liljequisl B: Antibiotic susceptibility of anaerobic bacteria with special reference to Bacteroides fragitis. Scandinavian Journal of Infectious Diseases 1979, 19: 17-25. 4. National Committee for Clinical Laboratory Standards: Methods for antimierobial susceptibilitytesting of anaerobic bacteria. 2nd ed. Document Mll-A2. NCCLS, Villanova, PA, 1990.
265
In Vitro Activity of Azithromycin and Tetracycline against 358 Clinical Isolates of Brucella melitensis R. Landfnez 1., J. Lifiares 2, E. Loza 3, J. Mart[nez-Beltr~in 3, R. Martfn 2, E B a q u e r o 3
The in vitro activity of azithromycin against human pathogenic strains of Brucella melitensis was tested at three centres and compared to that of tetracycline, the standard antibiotic currently used for the treatment of human brucellosis. MIC determination was carried out. Tested concentration ranges for both azithromycin and tetracycline were between 0.03 and 16/ag/mL Brucella melitensis biotype 1, strain M16 was employed as a control microorganism. A total of 358 Brucella rnelitensis strains from human blood cultures were tested. MIC90 (~g/ml) values ranged from 0.5 to 1.00 for azithromycin and were 0.25 for tetracycline. It was concluded that there was little difference in the sensitivity of pathogenic Brucella melitensis to azithromycin and tetracycline isolated from three different regions in Spain. These results encourage further investigations on the possible therapeutic role of azithromycin in brucellosis.
Current treatment of acute brucellosis combining tetracycline and streptomycin or rifampicin usually achieves excellent clinical and microbiological results (1-3). Nevertheless, a variable percentage of failures and recurrence are reported in most published series. In addition, these therapeutic regimes pose difficulties in patients' compliance due to their duration; moreover, tetracyclines are contraindicated in children under eight years of age and in pregnant women. Thus, it is felt that new antibiotics overcoming these problems would represent a significant therapeutic advancement in the treatment of brucellosis. 1Departamento de Microbiologfa, Facultad de Medicina, Universidad de Valladolid, Ramon y Cajal, 7, 47005 Valladolid, Spain. 2Departamento de Mierobiologfa, Hospital "Pr/neipes de Espafia', Hospitalet de Llobregat, Barcelona, Spain. 3Departamento de Microbiologfa, Hospital 'Ram6n y Cajal", Madrid, Spain.
266
Eur. J. Clin. Microbiol. Infect. Dis.
New antibiotics exhibiting therapeutical potential in the treatment of this disease are routinely assayed in vitro against Brucella metitensis as a preliminary step to decide further clinical development. The objective of this study was to determine the in vitro susceptibility of a large number of human pathogenic strains of Brucella melitensis to azithromycin, a new azalide antibiotic related to erythromycin, in comparison to tetracycline, the standard treatment of brucellosis. Materials and Methods. A total of 358 Brucelta melitensis strains isolated from blood cultures in patients with acute brucellosis between 1987 and 1989 were tested. Each strain was recovered from a different patient; the possibility of a source of infection common to various patients was considered likely in only very few cases. The strains were stored in skimmed milk at -70 °C. These strains constitute one of the largest samples of this microorganism ever studied from three different areas in Spain: Vanadolid (223 strains), Barcelona (73 strains) and Madrid (62 strains).
Azithromycin (Pfizer Central Research, UK) and tetracycline hydrochloride (Antibi6ticos, Spain) supplied as assayed active substance powder were employed. Minimum inhibitory concentrations (MICs) were determined using the agar-dilution method. The culture medium employed was Iso-sentitest Agar CM471 (Oxoid, UK) to which serial twofold dilutions (from 0.03 to 16 ~tg/ml) of one of the two antibiotics were added. The inoculum was prepared from a smooth-phase Brucella melitensis culture in trypticase soy agar (BBL Microbiology Systems, USA). Three to five colonies were subcultured in brain-heart infusion broth (Difco Laboratories, USA) for 48 hours at 37 °C. A post-incubation dilution (1/20) in brain-heart infusion
broth was carried out. This resulted in an inoculum of approximately 105 cfu applied to the agar surface. Plates were inoculated with a Steers replicator and incubated at 37 °C in air. A first reading was performed at 48 hours and a confirmatory second one at 72 hours. MIC was defined as the lowest concentration of antibiotic at which there was no visible growth (>__ 3 isolated colonies). A control microorganism, Brucella melitensis biotype 1 strain M16 (Central Veterinary Laboratory, Weybridge, UK) was tested at each centre to assess inter-group variability. Results andDiscussion. Table I shows the in vitro MIC50 and MIC90 values found in each of the three participating centres for azithromycin and tetracycline, respectively.
MICs of azithromycin ranged between 0.03 and 2.00 ~g/ml. MIC90 was 0.50 ~tg/ml in two of the centres, while it was 1.00/ag/ml at the centre in Barcelona (73 strains). Comparative results for tetracycline showed MICs ranging from 0.06 to 0.50 /ag/ml. MIC90 was 0.25 ~g/ml in all three centres. No difference in results was found between the readings at 48 hours and 72 hours. MIC90 values for the control microorganism ranged between 0.25 and 0.50/ag/ml for azithromycin and between 0.10 and 0.25 ~ag/ml for tetracycline. A remarkable uniformity in the results (except for the small discrepancy mentioned above) was found in this study. Azithromycin is a novel 15-membered ring azalide antibiotic, chemically related to macrolides (4). This agent exhibits certain pharmacokinetic and pharmacodynamic properties which can be useful in the treatment of infections caused by intracellular microorganisms. Peak
Table 1: Activityof azithromycinand tetracyclineagainst 358 human pathogenicstrains of Brucella melitensis. Source
No. of strains
Antibiotics
MIC (/ag/ml) Range
MIC50
MIC90
Valladolid
223
azithromycin tetracycline
0.03 - 2.00 0.12 - 0.50
0.50 0.25
0.50 0.25
Barcelona
73
azithromycin tetracycline
0.12 -2.00 0.06 - 0.50
0.50 0.25
1.00 0.25
Madrid
62
azithromycin tetracycline
0.12 - 1.00 0.06 - 0.50
0.50 0.25
0.50 0.25
Vol. 11, 1992
Serum concentration after a single oral dose of 500 trig of azithromycin reaches 0.4 lag/ml, but tissue Concentrations of azithromycin are much greater (10 to 100 times) and persist for longer periods (5). In addition to high tissue concentration, the in vivo performance of azithromycin may be related to its intracellular concentration and release at sites of infection. Azithromycin concentrates Within human and mouse polymorphonuclear cells, peritoneal macrophages from the mouse and alveolar cells from the rat and mouse. In vitro intracellular concentrations of this antibiotic are up to 226-fold higher than those measured outside the cell (6). The results obtained in this study from a very large selection of Brucella melitensis strains from three different regions of Spain are very consistent. MIC results indicate in vitro susceptibility of Brucella melitensis to azithromycin. The MIC Values are usually one dilution.higher than those obtained for tetracycline. As shown in further Studies, the in vitro activity of azithromycin against Brucella meIitensis is similar to that of Streptomycin and rifampicin (R. Martin, 7th Mediterranean Congress of Chemotherapy, 1990. Barcelona, Abstract 68). These results encourage further investigations on the potential usefulness of azithromycin in the treatment of human brucellosis.
267
References 1. Land[nez R, Abad R, Rodriguez Torres A: Estudio
sobre el tratamiento de la brucelosis. Infcctologika 1984, 5: 7-14. 2. Ariza J, Gudi01 F, Pallares R, Ruff G, FernandezViladrich P: Comparative trial of rifampin-doxycycline
versus tetracycline-streptomycin in the therapy of human brucellosis. Antimicrobial Agents and Chemotherapy 1985, 28: 548-551. 3. Acocella G, Bertrand A, Beylout J., Duriande JB, Garcia Rodriguez JA, Kosmidis J, Micoud M, Rey M, Rodriguez Zapala M, Roux J, Slahl JP- Com-
parison of three different regimens in the treatment of acute brucellosis: a multicenter multinational study. Journal of Antimicrobia/Chemotherapy 1989, 23: 433439. 4. Anonymous: Azithromycin. Drugs of the Future 1988, 13: 9-12. 5. Foulds G, Shepard RM, Johnson RB: The pharmacokinetics of azithromycin in human serum and tissues. Journal of Antimicrobial Chemotherapy 1990,25, Supplement A: 73-82. 6. Gladue RP, Bright GM, Isaacson RE, Newborg MF:
In vitro and in vivo uptake of azithromycin by phagocytic cells: possible mechanisms of delivery and release at sites of infection. Antimicrobial Agents and Chemotherapy 1989, 33: 277-282.
