- Email: [email protected]
Therapeutic effects of rifampin and erythromycin in experimental murine brucellosis
Concise Communications
an epidemiologic survey is necessary to evaluate and prevent this illness from affecting persons at risk. 5.
ACKNOWLEDGEMENTS
6.
We thank Joceline Rocourt for serotyping and phage-typing our strains and Edouard Bingen for the molecular typing and helpful discussions.
7.
REFERENCES
8.
1. Schuchat A, Swaminathan B, Broome CV. Epidemiology of human listeriosis. Clin Microbiol Rev 1991; 4: 169–83. 2. Benallegue A, Benhassine M, Grangaud JP, Mazouni M. Me´ningite a` Listeria monocytogenes. Alge´rie me´dicale 1968; V: 29–32. 3. Boukadida J, Sboui H, Monastiri K et al. La liste´riose humaine en Tunisie: deux nouveaux cas chez le nouveau ne´. Med Mal Infect 1994; 24: 117–8. 4. Bingen E, Boissinot C, Desjardins P et al. Arbitrary primed poly-
9.
10.
111
merase chain reaction provides rapid differentiation of Proteus mirabilis isolates from a pediatric hospital. J Clin Microbiol 1993; 31: 1055–9. Campbell AN, Sili PR, Wardle JK. Listeria meningitis acquired by cross infection in a delivery suite. Lancet 1981; 3: 752–3. Nelson KE, Warren D, Tomasi AM et al. Transmission of neonatal listeriosis in a delivery room. Am J Dis Child 1985; 139: 903– 5. Mazurier SI, Audurier A, Marquet-Van Der Meen Notermans S, Wernars K. A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates. Res Microbiol 1992; 143: 507–12. Louie M, Jayaratne P, Luchsinger.I, Devenish J et al. Comparison of ribotyping, arbitrary primed PCR, and pulsed field gel electrophoresis for molecular typing of Listeria monocytogenes. J Clin Microbiol 1996; 34: 15–9. Jaquet C, Catimel B, Brosch R et al. Investigations related to the epidemic strain involved in the French listeriosis outbreak in 1992. Appl Environ Microbiol 1995; 61: 2242–6. Williams JGK, Kubelik AR, Livak J et al. DNA polymorphysms amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res 1990; 18: 6531–5.
Therapeutic effects of rifampin and erythromycin in experimental murine brucellosis S. Felek*, K. Demirdagˇ, A. Kalkan and A. Akbulut ¨ niversitesi, Firat Tip Merkezi, 23200 Elazig`, Turkey Department of Clinical Microbiology and Infectious Diseases, Firat U *Tel: +90 4242333555 Fax: +90 4242379138 E-mail: [email protected] Accepted 31 May 1999
Clin Microbiol Infect 2000: 6: 111–114
Brucellosis is a serious health problem because of its severe complications and its tendency to be a chronic disease [1]. The most effective and least toxic therapy for human brucellosis is still questionable. Streptomycin, gentamicin, tetracycline, doxycycline, rifampin and trimethoprim–sulfamethoxazole are among the widely used antibiotics in the treatment of human brucellosis. The use of a single antibiotic is not recommended because of reduced efficacy. The combination most frequently recommended is rifampin plus doxycycline for 45 days. Other recommended regimens are streptomycin plus doxycycline and rifampin plus trimethoprim–sulfamethoxazole [2–4]. In spite of these suggested treatments, the relapse rate is still about 10% [1,3]. The treatment of brucellosis in children (under 6–8 years of age) is a problem because of tetracycline accumulation in bones and tooth structures [5]. Brucellosis is also a problem in pregnant women, since for them the usual drugs are contraindicated [2].
Erythromycin is not teratogenic and is safe to be used during pregnancy and for children [5,6]. Therefore, in the treatment of brucellosis, new antibiotics with better activity and a higher degree of safety in pregnant women and children are needed. This study was undertaken to investigate the possibility of erythromycin treatment for brucellosis. The murine brucellosis model, which is a well-known and commonly used animal model with reproducibility described previously, was used in this study [7–9]. In total, 104 male Balb/c mice (25–30 g) were used. Mice were fed ad libitum with standard mouse diet. In order to estimate the average water consumption per day per mouse, water consumption was measured for a week and daily consumption was calculated for each mouse. Brucella melitensis M16 was obtained from The Department of Microbiology, School of Veterinary Medicine, Firat University. Minimum inhibitory concentrations (MICs) of antibiotics were
© 2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 6, 111–114
112
Clinical Microbiology and Infection, Volume 6 Number 2, February 2000
determined by routine broth microdilution procedures in microtiter trays. Minimum bactericidal concentrations (MBCs) were determined by subculture on brucella agar (Becton Dickinson and Company, Cockeysville, MD, USA) from tubes showing no turbidity [10]. Bacteria were cultured on brucella agar at 37 °C for 72 h and then suspended in physiologic saline which contained 4– 8 × 104 colony-forming units (CFUs) per mL. From this suspension, 0.5 mL was inoculated into each mouse intraperitoneally. Seven days after inoculation, four mice were sacrificed under ether anaesthesia, and spleen cultures were carried out. All four mice were found to be infected. Eighty mice were randomly divided into five groups, each group consisting of 16 mice. Tap water was given to the first (control) group. Rifampin (Rifadin oral suspension, Hoechst, Istanbul, Turkey) was given to the second group, and erythromycin (Erythrocin oral suspension, Abfar, Fako Drug, Levent, Istanbul, Turkey) to the other three groups, for 15 days. Rifampin and erythromycin were suspended in tap water, and added to the drinking water. Accordingly, mice were given rifampin 50 mg/kg per day and erythromycin 50, 100 and 200 mg/kg per day. Fresh drinking water with antibiotics was prepared daily. Mice were sacrificed under anaesthesia after 3 days without treatment; spleens were removed aseptically and homogenized in 1 mL, of sterile physiologic saline. Volumes of 1 mL, of undiluted and of three dilutions (l/10, 1/100 and 1/1000) of the homogenates in physiologic saline were placed onto brucella agar plates. After 72 h of incubation at 37 °C, Brucella melitensis colonies were counted. Colonies were evaluated according to growth characteristics, colony morphology and Gram-staining. Each procedure was performed in triplicate. The number of CFUs per plate was calculated, and the results of all four dilutions were averaged and expressed as mean log10 of Brucella per mL of homogenate. Plates with no bacterial growth were incubated for an additional 4 days before being considered sterile. The presence of growth was considered as a failure. Blood for determination of antibiotic level was collected by orbital puncture from five mice from each of the four groups. Blood samples were obtained from each mouse on days 3 and
Table 1 Failure rates and mean log CFU of the control, rifampin- and erythromycintreated groups
7. The antibiotic levels were determined in duplicate. Blood was soaked onto a paper disk, and the disk was placed on agar seeded with appropriate microorganisms for bioassays of antibiotic concentrations [8,11]. Indicator organisms and media were Bacillus subtilis ATCC 6633 and antibiotic medium no. 2 (Difco Laboratories, Detroit, MI, USA) for rifampin, and Sarcinia lutea ATCC 9341 and antibiotic medium no. 11 (Difco) for erythromycin [8,11]. Blood levels were calculated by comparing the zone of inhibition with a curve of known concentrations of the antibiotics. Statistical evaluation of failure rates was performed using chi-square and Fisher’s exact tests. Since the number of positive cultures was low in some groups, no statistical evaluation was performed for CFUs. The MIC and MBC values for rifampin were 1 and 2 mg/L, respectively, and for erythromycin they were 4 and 8 mg/L, respectively. In the control group, all spleen cultures were positive on day 18. In the rifampin-treated group, all spleen cultures were negative; this result was significantly different from that of the control group (P ³ 0.000001). Failures were found in nine (56.2%), three (18.7%) and two mice (12.5%) in groups which were given erythromycin at doses of 50, 100 and 200 mg/kg, respectively. These results were statistically different compared to the control group (P ³ 0.01, P ³ 0.000005, P ³ 0.000001, respectively). The lowest dose of the erythromycin-treated group (50 mg/kg per day) was found to be different from that of the rifampin-treated group (P ³ 0.001), but the other erythromycin-treated groups (100 and 200 mg/kg per day) were not found to be statistically different from the rifampintreated group (P × 0.05) (Table 1). Mean log CFU was 4.72 in the control group. In positive cultures of the erythromycin 50, 100 and 200 mg/kg per day groups, means log CFU were 4.45, 4.56 and 3.52, respectively. Mean blood levels of antibiotics are shown in Table 2. The mean blood level of rifampin was 41.6 mg/L in the rifampintreated group. The mean blood levels of erythromycin were 0.41, 0.74 and 1.13 mg/L in the 50, 100 and 200 mg/kg per day groups, respectively. Over a long period, macrolide antibiotics were investigated
Failure Therapy Control Rifampin Erythromycin Erythromycin Erythromycin a
mg/kg/day
n
%
Mean log CFU
0 50 50 100 200
16/16 0/16a 9/16a 3/16a 2/16a
100 0 56.2 18.7 12.5
4.72 – 4.45 4.56 3.52
P ³ 0.01 (according to the control group).
© 2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 6, 111–114
Concise Communications
Table 2 Mean blood levels of antibiotics in the antibiotictreated groups
Therapy Rifampin Erythromycin Erythromycin Erythromycin
mg/kg/day
Mean blood antibiotic level (mg/L)
50 50 100 200
41.6 0.41 0.74 1.13
in the treatment of brucellosis. Lang et al. [9] reported that, in an experimental murine brucellosis, azithromycin cured 10 of 10 mice, whereas roxithromycin cured one of 10 mice. These authors recommended azithromycin in the treatment of brucellosis in young children and pregnant females. Domingo et al. [12] reported that a short oral treatment course with azithromycin was able to reduce the infection significantly, but it was not able to cure the animals as effectively as the classic regimen with doxycycline administered for a longer period of time. Since Brucella spp. are intracellular pathogens, antibiotics for treatment of brucellosis must have adequate intracellular concentrations [2]. Also, the antibiotic must be active in vitro [4]. The macrolide erythromycin is widely used in the treatment of intracellular pathogens such as Legionella, Chlamydia, Mycoplasma and Ricketsia [6,13]. Erythromycin diffuses readily into the intracellular fluids and actively accumulates intracellularly in polymorphonuclear leukocytes and in alveolar macrophages [6,14]. To the best of our knowledge, there is no report concerning the in vivo use of erythromycin for the treatment of brucellosis. Since erythromycin is effective in vivo with good activity in macrophages, it may be effective in the treatment of brucellosis. In this study, all spleen-culture results were found to be positive in the control group, whereas we found that erythromycin was indeed effective in the treatment of murine brucellosis at doses of 50, 100 and 200 mg/kg (P ³ 0.01). Erythromycin blood levels were found to be less than the MIC value of the strain used in this study, but the concentrations of erythromycin detected in alveolar macrophages and neutrophils were reported to be 9–23 times and 10–13 times greater than the extracellular fluid concentrations, respectively [13]. We found mean blood levels of erythromycin of 0.41, 0.74 and 1.13 mg/L and obtained failure rates of 56.2%, 18.7% and 12.5%, respectively, in groups of mice treated with erythromycin. Drug efficacy is primarily related to the dose [7,8]. In an experimental mouse brucellosis, when 1.4, 3, 6, 12.5, 25 and 50 mg/kg per day doses of rifampin were used, the failure rate was found to be 100%, 82%, 40%, 0%, 12.5% and 0%, respectively [7]. In the present study, we used rifampin at a 50 mg/kg per day dose and found a failure rate of 0%. We used
113
erythromycin at doses of 50, 100 and 200 mg/kg per day and obtained failure rates of 56.2%, 18.7% and 12.5%, respectively. The MIC of erythromycin was 4 mg/L for the strain used in this study. Since the reported erythromycin MIC range is 0.2– 16 mg/L [15–17], if the dose of erythromycin is greater than 200 mg/kg per day or if it is used against brucellosis caused by Brucella strains with low MIC values, the failure rate could be even lower. Our results indicate that erythromycin could be an alternative choice in the treatment of human brucellosis. It may offer particular advantages, especially in young children and pregnant women with brucellosis.
REFERENCES 1. Young EJ. Brucella species. In Mandell GL, Bennett JE, Dolin R. eds. Principles and practice of infectious diseases, 4th edn. New York: Churchill Livingstone, 1995: 2053–60. 2. Gotuzzo E, Cellillo C. Brucella. In Gorbach SL, Bartlett JG, Blacklow NR, eds. Infectious diseases. Philadelphia: W.B. Saunders Company, 1992: 1513–21. 3. Hall WH. Modern chemotherapy for brucellosis in humans. Rev Infect Dis 1990; 12: 1060–99. 4. Bertrand A. Antibiotic treatment of brucellosis. Presse Med 1994; 23: 1128–31. 5. Moellering RC. Principles of anti-infective therapy In Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases, 4th edn. New York: Churchill Livingstone, 1995: 199– 212. 6. Steigbigel NH. Macrolides and clindamycin. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases, 4th edn. New York: Churchill Livingstone, 1995: 334–46. 7. Shasha B, Lang R, Rubinstein E. Efficacy of complications of doxycycline and rifampicin in the therapy of experimental mouse brucellosis. J Antimicrob Chemother 1994; 33: 545–51. 8. Shasha B, Lang R, Rubinstein E. Therapy of experimental murine brucellosis with streptomycin, co-trimoxazole, ciprofloxacin, ofloxacin, pefloxacin, doxycycline, and rifampin. Antimicrob Agents Chemother 1992; 36: 973–6. 9. Lang R, Shasha B, Ifrach N, Tinman S, Rubinstein E. Therapeutic effects of roxithromycin and azithromycin in experimental murine brucellosis. Chemotherapy 1994; 40: 252–5. 10. Isenberg HD. Clinical microbiology procedures handbook. Washington: American Society for Microbiology, 1992: 5.16–5.17, 11. Vogelman. B, Godmundsson J, Leggett J, Turnidge J, Ebert S, Craig WA. Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. J Infect Dis 1988; 158: 831–47. 12. Domingo S, Gastearena I, Vitas AI et al. Comparative activity of azithromycin and doxycycline against Brucella spp. infection in mice. J Antimicrob Chemother 1995; 36: 647–56. 13. Calia FM. Erythromycin. In: Gorbach SL, Bardett JG, Blacklow NR, eds. Infectious diseases. Philadelphia: W.B. Saunders Company, 1992: 223–31. 14. Washington JA, Wilson WR. Erythromycin: a microbial and clinical perspective after 30 years of clinical use. Mayo Clin Proc 1985; 60: 189–203. 15. Garcia-Rodriguez JA, Mun˜oz Bellido JL, Fresnadillo MJ, Tru-
© 2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 6, 111–114
114
Clinical Microbiology and Infection, Volume 6 Number 2, February 2000
jillano I. In vitro activities of new macrolides and rifapentine against Brucella spp. Antimicrob Agents Chemother 1993; 37: 911–3. 16. Mortensen JE, Moore DG, Clarradge JE, Young EJ. Antimicrobial susceptibility of clinical isolates of Brucella. Diagn Microbiol Infect Dis 1986; 5: 163–9.
17. Loza E, Martinez Beltran J, Baquero E, Leon A, Canton R, Garijo B. Comparative in vitro activity of clarithromycin. Eur J Clin Microbiol Infect Dis 1992; 11: 856–66.
© 2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 6, 111–114
Copyright of Clinical Microbiology & Infection is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
an epidemiologic survey is necessary to evaluate and prevent this illness from affecting persons at risk. 5.
ACKNOWLEDGEMENTS
6.
We thank Joceline Rocourt for serotyping and phage-typing our strains and Edouard Bingen for the molecular typing and helpful discussions.
7.
REFERENCES
8.
1. Schuchat A, Swaminathan B, Broome CV. Epidemiology of human listeriosis. Clin Microbiol Rev 1991; 4: 169–83. 2. Benallegue A, Benhassine M, Grangaud JP, Mazouni M. Me´ningite a` Listeria monocytogenes. Alge´rie me´dicale 1968; V: 29–32. 3. Boukadida J, Sboui H, Monastiri K et al. La liste´riose humaine en Tunisie: deux nouveaux cas chez le nouveau ne´. Med Mal Infect 1994; 24: 117–8. 4. Bingen E, Boissinot C, Desjardins P et al. Arbitrary primed poly-
9.
10.
111
merase chain reaction provides rapid differentiation of Proteus mirabilis isolates from a pediatric hospital. J Clin Microbiol 1993; 31: 1055–9. Campbell AN, Sili PR, Wardle JK. Listeria meningitis acquired by cross infection in a delivery suite. Lancet 1981; 3: 752–3. Nelson KE, Warren D, Tomasi AM et al. Transmission of neonatal listeriosis in a delivery room. Am J Dis Child 1985; 139: 903– 5. Mazurier SI, Audurier A, Marquet-Van Der Meen Notermans S, Wernars K. A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates. Res Microbiol 1992; 143: 507–12. Louie M, Jayaratne P, Luchsinger.I, Devenish J et al. Comparison of ribotyping, arbitrary primed PCR, and pulsed field gel electrophoresis for molecular typing of Listeria monocytogenes. J Clin Microbiol 1996; 34: 15–9. Jaquet C, Catimel B, Brosch R et al. Investigations related to the epidemic strain involved in the French listeriosis outbreak in 1992. Appl Environ Microbiol 1995; 61: 2242–6. Williams JGK, Kubelik AR, Livak J et al. DNA polymorphysms amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res 1990; 18: 6531–5.
Therapeutic effects of rifampin and erythromycin in experimental murine brucellosis S. Felek*, K. Demirdagˇ, A. Kalkan and A. Akbulut ¨ niversitesi, Firat Tip Merkezi, 23200 Elazig`, Turkey Department of Clinical Microbiology and Infectious Diseases, Firat U *Tel: +90 4242333555 Fax: +90 4242379138 E-mail: [email protected] Accepted 31 May 1999
Clin Microbiol Infect 2000: 6: 111–114
Brucellosis is a serious health problem because of its severe complications and its tendency to be a chronic disease [1]. The most effective and least toxic therapy for human brucellosis is still questionable. Streptomycin, gentamicin, tetracycline, doxycycline, rifampin and trimethoprim–sulfamethoxazole are among the widely used antibiotics in the treatment of human brucellosis. The use of a single antibiotic is not recommended because of reduced efficacy. The combination most frequently recommended is rifampin plus doxycycline for 45 days. Other recommended regimens are streptomycin plus doxycycline and rifampin plus trimethoprim–sulfamethoxazole [2–4]. In spite of these suggested treatments, the relapse rate is still about 10% [1,3]. The treatment of brucellosis in children (under 6–8 years of age) is a problem because of tetracycline accumulation in bones and tooth structures [5]. Brucellosis is also a problem in pregnant women, since for them the usual drugs are contraindicated [2].
Erythromycin is not teratogenic and is safe to be used during pregnancy and for children [5,6]. Therefore, in the treatment of brucellosis, new antibiotics with better activity and a higher degree of safety in pregnant women and children are needed. This study was undertaken to investigate the possibility of erythromycin treatment for brucellosis. The murine brucellosis model, which is a well-known and commonly used animal model with reproducibility described previously, was used in this study [7–9]. In total, 104 male Balb/c mice (25–30 g) were used. Mice were fed ad libitum with standard mouse diet. In order to estimate the average water consumption per day per mouse, water consumption was measured for a week and daily consumption was calculated for each mouse. Brucella melitensis M16 was obtained from The Department of Microbiology, School of Veterinary Medicine, Firat University. Minimum inhibitory concentrations (MICs) of antibiotics were
© 2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 6, 111–114
112
Clinical Microbiology and Infection, Volume 6 Number 2, February 2000
determined by routine broth microdilution procedures in microtiter trays. Minimum bactericidal concentrations (MBCs) were determined by subculture on brucella agar (Becton Dickinson and Company, Cockeysville, MD, USA) from tubes showing no turbidity [10]. Bacteria were cultured on brucella agar at 37 °C for 72 h and then suspended in physiologic saline which contained 4– 8 × 104 colony-forming units (CFUs) per mL. From this suspension, 0.5 mL was inoculated into each mouse intraperitoneally. Seven days after inoculation, four mice were sacrificed under ether anaesthesia, and spleen cultures were carried out. All four mice were found to be infected. Eighty mice were randomly divided into five groups, each group consisting of 16 mice. Tap water was given to the first (control) group. Rifampin (Rifadin oral suspension, Hoechst, Istanbul, Turkey) was given to the second group, and erythromycin (Erythrocin oral suspension, Abfar, Fako Drug, Levent, Istanbul, Turkey) to the other three groups, for 15 days. Rifampin and erythromycin were suspended in tap water, and added to the drinking water. Accordingly, mice were given rifampin 50 mg/kg per day and erythromycin 50, 100 and 200 mg/kg per day. Fresh drinking water with antibiotics was prepared daily. Mice were sacrificed under anaesthesia after 3 days without treatment; spleens were removed aseptically and homogenized in 1 mL, of sterile physiologic saline. Volumes of 1 mL, of undiluted and of three dilutions (l/10, 1/100 and 1/1000) of the homogenates in physiologic saline were placed onto brucella agar plates. After 72 h of incubation at 37 °C, Brucella melitensis colonies were counted. Colonies were evaluated according to growth characteristics, colony morphology and Gram-staining. Each procedure was performed in triplicate. The number of CFUs per plate was calculated, and the results of all four dilutions were averaged and expressed as mean log10 of Brucella per mL of homogenate. Plates with no bacterial growth were incubated for an additional 4 days before being considered sterile. The presence of growth was considered as a failure. Blood for determination of antibiotic level was collected by orbital puncture from five mice from each of the four groups. Blood samples were obtained from each mouse on days 3 and
Table 1 Failure rates and mean log CFU of the control, rifampin- and erythromycintreated groups
7. The antibiotic levels were determined in duplicate. Blood was soaked onto a paper disk, and the disk was placed on agar seeded with appropriate microorganisms for bioassays of antibiotic concentrations [8,11]. Indicator organisms and media were Bacillus subtilis ATCC 6633 and antibiotic medium no. 2 (Difco Laboratories, Detroit, MI, USA) for rifampin, and Sarcinia lutea ATCC 9341 and antibiotic medium no. 11 (Difco) for erythromycin [8,11]. Blood levels were calculated by comparing the zone of inhibition with a curve of known concentrations of the antibiotics. Statistical evaluation of failure rates was performed using chi-square and Fisher’s exact tests. Since the number of positive cultures was low in some groups, no statistical evaluation was performed for CFUs. The MIC and MBC values for rifampin were 1 and 2 mg/L, respectively, and for erythromycin they were 4 and 8 mg/L, respectively. In the control group, all spleen cultures were positive on day 18. In the rifampin-treated group, all spleen cultures were negative; this result was significantly different from that of the control group (P ³ 0.000001). Failures were found in nine (56.2%), three (18.7%) and two mice (12.5%) in groups which were given erythromycin at doses of 50, 100 and 200 mg/kg, respectively. These results were statistically different compared to the control group (P ³ 0.01, P ³ 0.000005, P ³ 0.000001, respectively). The lowest dose of the erythromycin-treated group (50 mg/kg per day) was found to be different from that of the rifampin-treated group (P ³ 0.001), but the other erythromycin-treated groups (100 and 200 mg/kg per day) were not found to be statistically different from the rifampintreated group (P × 0.05) (Table 1). Mean log CFU was 4.72 in the control group. In positive cultures of the erythromycin 50, 100 and 200 mg/kg per day groups, means log CFU were 4.45, 4.56 and 3.52, respectively. Mean blood levels of antibiotics are shown in Table 2. The mean blood level of rifampin was 41.6 mg/L in the rifampintreated group. The mean blood levels of erythromycin were 0.41, 0.74 and 1.13 mg/L in the 50, 100 and 200 mg/kg per day groups, respectively. Over a long period, macrolide antibiotics were investigated
Failure Therapy Control Rifampin Erythromycin Erythromycin Erythromycin a
mg/kg/day
n
%
Mean log CFU
0 50 50 100 200
16/16 0/16a 9/16a 3/16a 2/16a
100 0 56.2 18.7 12.5
4.72 – 4.45 4.56 3.52
P ³ 0.01 (according to the control group).
© 2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 6, 111–114
Concise Communications
Table 2 Mean blood levels of antibiotics in the antibiotictreated groups
Therapy Rifampin Erythromycin Erythromycin Erythromycin
mg/kg/day
Mean blood antibiotic level (mg/L)
50 50 100 200
41.6 0.41 0.74 1.13
in the treatment of brucellosis. Lang et al. [9] reported that, in an experimental murine brucellosis, azithromycin cured 10 of 10 mice, whereas roxithromycin cured one of 10 mice. These authors recommended azithromycin in the treatment of brucellosis in young children and pregnant females. Domingo et al. [12] reported that a short oral treatment course with azithromycin was able to reduce the infection significantly, but it was not able to cure the animals as effectively as the classic regimen with doxycycline administered for a longer period of time. Since Brucella spp. are intracellular pathogens, antibiotics for treatment of brucellosis must have adequate intracellular concentrations [2]. Also, the antibiotic must be active in vitro [4]. The macrolide erythromycin is widely used in the treatment of intracellular pathogens such as Legionella, Chlamydia, Mycoplasma and Ricketsia [6,13]. Erythromycin diffuses readily into the intracellular fluids and actively accumulates intracellularly in polymorphonuclear leukocytes and in alveolar macrophages [6,14]. To the best of our knowledge, there is no report concerning the in vivo use of erythromycin for the treatment of brucellosis. Since erythromycin is effective in vivo with good activity in macrophages, it may be effective in the treatment of brucellosis. In this study, all spleen-culture results were found to be positive in the control group, whereas we found that erythromycin was indeed effective in the treatment of murine brucellosis at doses of 50, 100 and 200 mg/kg (P ³ 0.01). Erythromycin blood levels were found to be less than the MIC value of the strain used in this study, but the concentrations of erythromycin detected in alveolar macrophages and neutrophils were reported to be 9–23 times and 10–13 times greater than the extracellular fluid concentrations, respectively [13]. We found mean blood levels of erythromycin of 0.41, 0.74 and 1.13 mg/L and obtained failure rates of 56.2%, 18.7% and 12.5%, respectively, in groups of mice treated with erythromycin. Drug efficacy is primarily related to the dose [7,8]. In an experimental mouse brucellosis, when 1.4, 3, 6, 12.5, 25 and 50 mg/kg per day doses of rifampin were used, the failure rate was found to be 100%, 82%, 40%, 0%, 12.5% and 0%, respectively [7]. In the present study, we used rifampin at a 50 mg/kg per day dose and found a failure rate of 0%. We used
113
erythromycin at doses of 50, 100 and 200 mg/kg per day and obtained failure rates of 56.2%, 18.7% and 12.5%, respectively. The MIC of erythromycin was 4 mg/L for the strain used in this study. Since the reported erythromycin MIC range is 0.2– 16 mg/L [15–17], if the dose of erythromycin is greater than 200 mg/kg per day or if it is used against brucellosis caused by Brucella strains with low MIC values, the failure rate could be even lower. Our results indicate that erythromycin could be an alternative choice in the treatment of human brucellosis. It may offer particular advantages, especially in young children and pregnant women with brucellosis.
REFERENCES 1. Young EJ. Brucella species. In Mandell GL, Bennett JE, Dolin R. eds. Principles and practice of infectious diseases, 4th edn. New York: Churchill Livingstone, 1995: 2053–60. 2. Gotuzzo E, Cellillo C. Brucella. In Gorbach SL, Bartlett JG, Blacklow NR, eds. Infectious diseases. Philadelphia: W.B. Saunders Company, 1992: 1513–21. 3. Hall WH. Modern chemotherapy for brucellosis in humans. Rev Infect Dis 1990; 12: 1060–99. 4. Bertrand A. Antibiotic treatment of brucellosis. Presse Med 1994; 23: 1128–31. 5. Moellering RC. Principles of anti-infective therapy In Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases, 4th edn. New York: Churchill Livingstone, 1995: 199– 212. 6. Steigbigel NH. Macrolides and clindamycin. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases, 4th edn. New York: Churchill Livingstone, 1995: 334–46. 7. Shasha B, Lang R, Rubinstein E. Efficacy of complications of doxycycline and rifampicin in the therapy of experimental mouse brucellosis. J Antimicrob Chemother 1994; 33: 545–51. 8. Shasha B, Lang R, Rubinstein E. Therapy of experimental murine brucellosis with streptomycin, co-trimoxazole, ciprofloxacin, ofloxacin, pefloxacin, doxycycline, and rifampin. Antimicrob Agents Chemother 1992; 36: 973–6. 9. Lang R, Shasha B, Ifrach N, Tinman S, Rubinstein E. Therapeutic effects of roxithromycin and azithromycin in experimental murine brucellosis. Chemotherapy 1994; 40: 252–5. 10. Isenberg HD. Clinical microbiology procedures handbook. Washington: American Society for Microbiology, 1992: 5.16–5.17, 11. Vogelman. B, Godmundsson J, Leggett J, Turnidge J, Ebert S, Craig WA. Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. J Infect Dis 1988; 158: 831–47. 12. Domingo S, Gastearena I, Vitas AI et al. Comparative activity of azithromycin and doxycycline against Brucella spp. infection in mice. J Antimicrob Chemother 1995; 36: 647–56. 13. Calia FM. Erythromycin. In: Gorbach SL, Bardett JG, Blacklow NR, eds. Infectious diseases. Philadelphia: W.B. Saunders Company, 1992: 223–31. 14. Washington JA, Wilson WR. Erythromycin: a microbial and clinical perspective after 30 years of clinical use. Mayo Clin Proc 1985; 60: 189–203. 15. Garcia-Rodriguez JA, Mun˜oz Bellido JL, Fresnadillo MJ, Tru-
© 2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 6, 111–114
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