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• W1510576068 abstract "Brucellosis is a serious health problem because of its severe complications and its tendency to be a chronic disease [1Young EJ Brucella species.in: Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases. 4th edn. Churchill Livingstone, New York1995: 2053-2060Google Scholar]. 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 [2Gotuzzo E Cellillo C Brucella.in: Gorbach SL Bartlett JG Blacklow NR Infectious diseases. W. B. Saunders Company, Philadelphia1992: 1513-1521Google Scholar, 3Hall WH Modern chemotherapy for brucellosis in humans.Rev Infect Dis. 1990; 12: 1060-1099Crossref PubMed Scopus (143) Google Scholar, 4Bertrand A Antibiotic treatment of brucellosis.Presse Med. 1994; 23: 1128-1131PubMed Google Scholar]. In spite of these suggested treatments, the relapse rate is still about 10% [1Young EJ Brucella species.in: Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases. 4th edn. Churchill Livingstone, New York1995: 2053-2060Google Scholar, 3Hall WH Modern chemotherapy for brucellosis in humans.Rev Infect Dis. 1990; 12: 1060-1099Crossref PubMed Scopus (143) Google Scholar]. The treatment of brucellosis in children (under 6-8 years of age) is a problem because of tetracycline accumulation in bones and tooth structures [5Moellering RC Principles of anti-infective therapy.in: Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases. 4th edn. Churchill Livingstone, New York1995: 199-212Google Scholar]. Brucellosis is also a problem in pregnant women, since for them the usual drugs are contraindicated [2Gotuzzo E Cellillo C Brucella.in: Gorbach SL Bartlett JG Blacklow NR Infectious diseases. W. B. Saunders Company, Philadelphia1992: 1513-1521Google Scholar]. Erythromycin is not teratogenic and is safe to be used during pregnancy and for children [5Moellering RC Principles of anti-infective therapy.in: Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases. 4th edn. Churchill Livingstone, New York1995: 199-212Google Scholar, 6Steigbigel NH Macrolides and clindamycin.in: Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases. 4th edn. Churchill Livingstone, New York1995: 334-346Google Scholar]. 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 [7Shasha B Lang R Rubinstein E Efficacy of complications of doxycycline and rifampicin in the therapy of experimental mouse brucellosis.J Antimicrob Chemother. 1994; 33: 545-551Crossref PubMed Scopus (16) Google Scholar, 8Shasha 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: 973-976Crossref PubMed Scopus (28) Google Scholar, 9Lang R Shasha B Ifrach N Tinman S Rubinstein E Therapeutic effects of roxithromycin and azithromycin in experimental murine brucellosis.Chemotherapy. 1994; 40: 252-255Crossref PubMed Scopus (12) Google Scholar]. In total, 104 male Balb/c mice (25-30g) 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 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 [10Isenberg HD Clinical microbiology procedures handbook. American Society for Microbiology, Washington1992: 5.16-5.17Google Scholar]. 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 intra-peritoneally. 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 50mg/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 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 [8Shasha 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: 973-976Crossref PubMed Scopus (28) Google Scholar, 11Vogelman 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: 831-847Crossref PubMed Scopus (627) Google Scholar]. 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 [8Shasha 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: 973-976Crossref PubMed Scopus (28) Google Scholar, 11Vogelman 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: 831-847Crossref PubMed Scopus (627) Google Scholar]. 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 2mg/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 < 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 < 0.01, P < 0.000005, P < 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 < 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).Table 1Failure rates and mean log CFU of the control, rifampin- and erythromycintreated groupsFailureTherapymg/kg/dayn%Mean log CFUControl016/161004.72Rifampin500/16aP < 0.01 (according to the control group).0−Erythromycin509/16aP < 0.01 (according to the control group).56.24.45Erythromycin1003/16aP < 0.01 (according to the control group).18.74.56Erythromycin2002/16aP < 0.01 (according to the control group).12.53.52a P < 0.01 (according to the control group). Open table in a new tab 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 inTable 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.Table 2Mean blood levels of antibiotics in the antibiotictreated groupsTherapymg/kg/dayMean blood antibiotic level (mg/L)Rifampin5041.6Erythromycin500.41Erythromycin1000.74Erythromycin2001.13 Open table in a new tab Over a long period, macrolide antibiotics were investigated in the treatment of brucellosis. Lang et al. [9Lang R Shasha B Ifrach N Tinman S Rubinstein E Therapeutic effects of roxithromycin and azithromycin in experimental murine brucellosis.Chemotherapy. 1994; 40: 252-255Crossref PubMed Scopus (12) Google Scholar] 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. [12Domingo S Gastearena I Vitas AI et al.Comparative activity of azithromycin and doxycycline against Brucella spp. infection in mice.J Antimicrob Chemother. 1995; 36: 647-656Crossref PubMed Scopus (20) Google Scholar] 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 [2Gotuzzo E Cellillo C Brucella.in: Gorbach SL Bartlett JG Blacklow NR Infectious diseases. W. B. Saunders Company, Philadelphia1992: 1513-1521Google Scholar]. Also, the antibiotic must be active in vitro [4Bertrand A Antibiotic treatment of brucellosis.Presse Med. 1994; 23: 1128-1131PubMed Google Scholar]. The macrolide erythromycin is widely used in the treatment of intracellular pathogens such as Legionella, Chlamydia, Mycoplasma and Ricketsia [6Steigbigel NH Macrolides and clindamycin.in: Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases. 4th edn. Churchill Livingstone, New York1995: 334-346Google Scholar, 13Calia FM Erythromycin.in: Gorbach SL Bardett JG Blacklow NR Infectious diseases. W. B. Saunders Company, Philadelphia1992: 223-231Google Scholar]. Erythromycin diffuses readily into the intracellular fluids and actively accumulates intracellularly in polymorphonuclear leukocytes and in alveolar macrophages [6Steigbigel NH Macrolides and clindamycin.in: Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases. 4th edn. Churchill Livingstone, New York1995: 334-346Google Scholar, 14Washington JA Wilson WR Erythromycin: a microbial and clinical perspective after 30 years of clinical use.Mayo Clin Proc. 1985; 60: 189-203Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar]. 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 < 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 [13Calia FM Erythromycin.in: Gorbach SL Bardett JG Blacklow NR Infectious diseases. W. B. Saunders Company, Philadelphia1992: 223-231Google Scholar]. 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 [7Shasha B Lang R Rubinstein E Efficacy of complications of doxycycline and rifampicin in the therapy of experimental mouse brucellosis.J Antimicrob Chemother. 1994; 33: 545-551Crossref PubMed Scopus (16) Google Scholar, 8Shasha 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: 973-976Crossref PubMed Scopus (28) Google Scholar]. 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 [7Shasha B Lang R Rubinstein E Efficacy of complications of doxycycline and rifampicin in the therapy of experimental mouse brucellosis.J Antimicrob Chemother. 1994; 33: 545-551Crossref PubMed Scopus (16) Google Scholar]. In the present study, we used rifampin at a 50 mg/kg per day dose and found a failure rate of 0%. We used 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 4mg/L for the strain used in this study. Since the reported erythromycin MIC range is 0.2-16 mg/L [15Garcia-Rodriguez JA Muñoz Bellido JL Fresnadillo MJ Trujillano I In vitro activities of new macrolides and rifapentine against Brucella spp.Antimicrob Agents Chemother. 1993; 37: 911-913Crossref PubMed Scopus (34) Google Scholar, 16Mortensen JE Moore DG Clarradge JE Young EJ Antimicrobial susceptibility of clinical isolates of Brucella.Diagn Microbiol Infect Dis. 1986; 5: 163-169Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 17Loza 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: 856-866Crossref PubMed Scopus (21) Google Scholar], 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." @default.
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• W1510576068 title "Therapeutic effects of rifampin and erythromycin in experimental murine brucellosis" @default.
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