Species-specific PCR for the Diagnosis and Determination of Antibiotic Susceptibilities of Brucella Strains Isolated from Tehran, Iran

Document Type: Original Research

Authors

1 Dept. of Microbiology, Iran University of Medical Sciences, Tehran. Iran

2 Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

3 Dept. of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

4 Dept. of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran

Abstract

Background: Brucellosis is an endemic zoonotic disease in the Middle East. This study intended to design a uniplex PCR assay for the detection and differentiation of Brucella at the species level and determining the antibiotic susceptibility pattern of Brucella in Iran. Methods: Sixty-eight Brucella specimens (38 animal and 30 human specimens) were analyzed using PCR (using one pair of primers). Antibiotic susceptibility patterns were evaluated and compared using the E-Test and disk diffusion susceptibility test. Tigecycline susceptibility pattern was compared with other antibiotics. Results: Thirty six isolates of B. melitensis, 2 isolates of B. abortus and 1 isolate of B. suis from the 38 animal specimens, 24 isolates of B. melitensis and 6 isolates of B. abortus from the 30 human specimens were differentiated. The MIC50 values of doxycycline for human and animal specimens were 125 and 10 μg/ml, respectively, tigecycline 0.064 μg/ml for human specimens and 0.125μg/ml for animal specimens, and trimethoprim/ sulfamethoxazole and ciprofloxacin 0.065 and 0.125μg/ml, respectively, for both human and animal specimens. The highest MIC50 value of streptomycin in the human specimens was 0.5μg/ml and 1μg/ml for the animal specimens. The greatest resistance shown was to tetracycline and gentamicin, respectively.  Conclusion: Uniplex PCR for the detection and differentiation of Brucella at the strain level is faster and less expensive than multiplex PCR, and the antibiotics doxycycline, rifampin, trimethoprim-sulfamethoxazole, ciprofloxacin, and ofloxacin are the most effective antibiotics for treating brucellosis. Resistance to tigecycline is increasing, and we recommend that it be used in a combination regimen.

Keywords

Main Subjects


  1. Rubach MP, Halliday JE, Cleaveland S, Crump JA. Brucellosis in low-income and middle-income countries. Curr Opin Infect Dis 2013;26(5):404-12.
  2. Parlak M, Guducuoglu H, Bayram Y, Cikman A, Aypak C, Kilic S, et al. Identification and determination of antibiotic susceptibilities of Brucella strains isolated from patients in van, Turkey by conventional and molecular methods. Int J Med Sci 2013;10(10):1406-11.
  3. Russo G, Pasquali P, Nenova R, Alexandrov T, Ralchev S, Vullo V, et al. Reemergence of human and animal brucellosis, bulgaria. Emerg Infect Dis 2009;15(2):314-6.
  4. Gwida M, Al Dahouk S, Melzer F, Rosler U, Neubauer H, Tomaso H. Brucellosis - regionally emerging zoonotic disease? Croat Med J 2010;51(4):289-95.
  5. Sanodze L, Bautista CT, Garuchava N, Chubinidze S, Tsertsvadze E, Broladze M, et al. Expansion of brucellosis detection in the country of Georgia by screening household members of cases and neighboring community members. BMC Public Health 2015;15:459.
  6. Alavi SM, Alavi L. Treatment of brucellosis: a systematic review of studies in recent twenty years. Caspian J Intern Med 2013;4(2):636-41.
  7. Solera J. Treatment of human brucellosis. J Med Liban 2000;48(4):255-63.
  8. Pappas G, Papadimitriou P, Christou L, Akritidis N. Future trends in human brucellosis treatment. Expert Opin Investig Drugs 2006;15(10):1141-9.
  9. Joint FAO/WHO expert committee on brucellosis. World Health Organ Tech Rep Ser 1986;740:1-132.
  10. Ersoy Y, Sonmez E, Tevfik MR, But AD. Comparison of three different combination therapies in the treatment of human brucellosis. Trop Doct 2005;35(4):210-2.
  11. Solera J, Rodriguez-Zapata M, Geijo P, Largo J, Paulino J, Saez L, et al. Doxycycline-rifampin versus doxycycline-streptomycin in treatment of human brucellosis due to Brucella melitensis. The GECMEI Group. Grupo de Estudio de Castilla-la Mancha de Enfermedades Infecciosas. Antimicrob Agents Chemother 1995;39(9):2061-7.
  12. Yousefi-Nooraie R, Mortaz-Hejri S, Mehrani M, Sadeghipour P. Antibiotics for treating human brucellosis. Cochrane Database Syst Rev 2012;10:CD007179.
  13. Ariza J, Bosilkovski M, Cascio A, Colmenero JD, Corbel MJ, Falagas ME, et al. Perspectives for the treatment of brucellosis in the 21st century: the Ioannina recommendations. PLoS Med 2007;4(12):e317.
  14. Pappas G, Solera J, Akritidis N, Tsianos E. New approaches to the antibiotic treatment of brucellosis. Int J Antimicrob Agents 2005;26(2):101-5.
  15. Al-Mariri A, Safi M. The Antibacterial Activity of Selected Labiatae (Lamiaceae) Essential Oils against Brucella melitensis. Iran J Med Sci 2013;38(1):44-50.
  16. Aliskan H, Can F, Demirbilek M, Colakoglu S, Kilic S, Arslan H. Determining in vitro synergistic activities of tigecycline with several other antibiotics against Brucella melitensis using checkerboard and time-kill assays. J Chemother 2009;21(1):24-30.
  17. Alton GG, Jones LM, Pietz DE. Laboratory techniques in brucellosis. Monogr Ser World Health Organ 1975(55):1-163.
  18. Mirnejad R, Doust RH, Kachuei R, Mortazavi SM, Khoobdel M, Ahamadi A. Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR. Asian Pac J Trop Med 2012;5(1):24-8.
  19. Mirnejad R, Mohamadi M, Piranfar V, Mortazavi SM, Kachuei R. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples. Asian Pac J Trop Med 2013;6(6):453-6.
  20. Bayram Y, Korkoca H, Aypak C, Parlak M, Cikman A, Kilic S, et al. Antimicrobial susceptibilities of Brucella isolates from various clinical specimens. Int J Med Sci 2011;8(3):198-202.
  21. Dean AS, Crump L, Greter H, Schelling E, Zinsstag J. Global burden of human brucellosis: a systematic review of disease frequency. PLoS Negl Trop Dis 2012;6(10):e1865.
  22. Magwedere K, Hemberger MY, Hoffman LC, Dziva F. Zoonoses: a potential obstacle to the growing wildlife industry of Namibia. Infect Ecol Epidemiol 2012;2.
  23. Yu WL, Nielsen K. Review of detection of Brucella spp. by polymerase chain reaction. Croat Med J 2010;51(4):306-13.
  24. Mirnejad R, Mohammadi M, Majdi A, Taghizoghi N, Piranfar V. Molecular Typing of Brucella melitensisand, B. abortus From Human Blood Samples Using PCR-RFLP Method. Jundishapur J Microbiol 2013;6(6):e7197.
  25. Safi M, Al-Mariri A. Efficacy evaluation of some antibiotics against syrian Brucella spp isolates, in vitro. Braz J Microbiol 2012;43(4):1269-73.
  26. Bertrand A. [Antibiotic treatment of brucellosis]. Presse Med 1994;23(24):1128-31.
  27. Herzberg M, Elberg SS, Meyer KF. Immunization against brucella infection. II. Effectiveness of a streptomycin-dependent strain of Brucella melitensis. J Bacteriol 1953;66(5):600-5.
  28. Simon EM, Berman DT. Pathogenicity and immunogenicity of streptomycin-dependent mutants of Brucella. J Bacteriol 1962;83:1347-55.
  29. Abdel-Maksoud M, House B, Wasfy M, Abdel-Rahman B, Pimentel G, Roushdy G, et al. In vitro antibiotic susceptibility testing of Brucella isolates from Egypt between 1999 and 2007 and evidence of probable rifampin resistance. Ann Clin Microbiol Antimicrob 2012;11:24.
  30. Trujillano-Martin I, Garcia-Sanchez E, Martinez IM, Fresnadillo MJ, Garcia-Sanchez JE, Garcia-Rodriguez JA. In vitro activities of six new fluoroquinolones against Brucella melitensis. Antimicrob Agents Chemother 1999;43(1):194-5.
  31. Roushan MR, Mohraz M, Janmohammadi N, Hajiahmadi M. Efficacy of cotrimoxazole and rifampin for 6 or 8 weeks of therapy in childhood brucellosis. Pediatr Infect Dis J 2006;25(6):544-5.
  32. Hashemi SH, Gachkar L, Keramat F, Mamani M, Hajilooi M, Janbakhsh A, et al. Comparison of doxycycline-streptomycin, doxycycline-rifampin, and ofloxacin-rifampin in the treatment of brucellosis: a randomized clinical trial. Int J Infect Dis 2012;16(4):e247-51.
  33. Dizbay M, Kilic S, Hizel K, Arman D. Tigecycline: its potential for treatment of brucellosis. Scand J Infect Dis 2007;39(5):432-4.
  34. Kilic S, Dizbay M, Cabadak H. In vitro activity of tigecycline, tetracycline and fluoroquinolones against Brucella melitensis. J Chemother 2008;20(1):33-7.