Determination of Nontuberculosis Mycobacteria Species Genotypes Present in Cattle Milk Samples Using 16S rRNA Gene Direct Sequencing

Document Type : Original Article (s)

Authors

1 MSc Student, Department of Microbiology, School of Medicine AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

2 Associate Professor, Nosocomial Infection Research Center AND Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, Iran

3 PhD Student, Department of Microbiology, School of Medicine AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: Nontuberculosis Mycobacterium (NTM) is the most common bacterium transferred to human from cow milk and products. The presence of these bacteria in the cow's milk stands as a public health concern particularly among those individuals consuming raw milk and dairy products. In this study the determination of 16S rRNA gene sequence method was used in order to evaluate the amount of Non tuberculosis Mycobacterium incidence and determine its types in milk samples gathered from Isfahan province.Methods: The cetyltrimethylammonium bromide (CTAB) and phenol chloroform were used for DNA extraction in isolates recognized as Non tuberculosis Mycobacterium, then using designed primers, 16S rRNA polymerase chain reaction was done and the types were determined after sequencing.Findings: 9 cultures, out of 119 samples gathered from Isfahan farms, were recognized positive in which 8 samples had M. fortuitum and one of them was M. gordonae.Conclusion: NTM incidence was 8.6% in evaluated milk samples. By using polymerase chain reaction (PCR) with 16SrRNA and sequencing, the fortuitum isolate type I incidence was 88.9% and Gordonae type III incidence was 11.1%. PCR method and sequencing of 16S rRNA gene which were used in this study are able to recognize isolates up to 100%.

Keywords

References

  1. Cai L, Chen X, Zhao T, Ding BC, Zhang JZ. Identification of Mycobacterium marinum 65 kD heat shock protein gene by polymerase chain reaction restriction analysis from lesions of swimming pool granuloma. Chin Med J (Engl) 2006; 119(1): 43-8.
  2. Franco MM, Paes AC, Ribeiro MG, de Figueiredo Pantoja JC, Santos AC, Miyata M, et al. Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil. BMC Vet Res 2013; 9: 85.
  3. Chye FY, Abdullah A, Ayob MK. Bacteriological quality and safety of raw milk in Malaysia. Food Microbiology 2004; 21(5): 535-41.
  4. Konig B, Tammer I, Sollich V, Konig W. Intra- and interpatient variability of the hsp65 and 16S-23S intergenic gene region in Mycobacterium abscessus strains from patients with cystic fibrosis. J Clin Microbiol 2005; 43(7): 3500-3.
  5. Isfahani BN, Tavakoli A, Salehi M, Tazhibi M. Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods. Mem Inst Oswaldo Cruz 2006; 101(6): 597-602.
  6. Harmsen D, Dostal S, Roth A, Niemann S, Rothganger J, Sammeth M, et al. RIDOM: comprehensive and public sequence database for identification of Mycobacterium species. BMC Infect Dis 2003; 3: 26.
  7. Covert TC, Rodgers MR, Reyes AL, Stelma GN. Occurrence of nontuberculous mycobacteria in environmental samples. Appl Environ Microbiol 1999; 65(6): 2492-6.
  8. Bottger EC. Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA. FEMS Microbiol Lett 1989; 53(1-2): 171-6.
  9. Edwards U, Rogall T, Blocker H, Emde M, Bottger EC. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 1989; 17(19): 7843-53.
  10. Kirschner P, Springer B, Vogel U, Meier A, Wrede A, Kiekenbeck M, et al. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 1993; 31(11): 2882-9.
  11. Balian SC, Pinheiro SR, Guerra JL, Morais ZM, Ferreira F, Ferreira Neto JS. Comparative study of two methods of decontamination of mycobacteria. Arq Inst Biol, Sao Paulo 2002; 69(2): 11-4. [In Portuguese].
  12. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am Rev Respir Dis 1990; 142(4): 940-53.
  13. Katoch VM. Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res 2004; 120(4): 290-304.
  14. Sriyabhaya N, Wongwatana S. Pulmonary infection caused by atypical mycobacteria: A report of 24 cases in Thailand. Rev Infect Dis 1981; 3(5): 1085-9.
  15. Leite CQ, Anno IS, Leite SR, Roxo E, Morlock GP, Cooksey RC. Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil. Mem Inst Oswaldo Cruz 2003; 98(3): 319-23.
  16. Saviola B, Bishai W. The genus mycobacterium-medical. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E, editors. The prokaryotes. 3rd ed. New York, NY: Springer; 2006. p. 919-33.
  17. Aronson T, Holtzman A, Glover N, Boian M, Froman S, Berlin OG, et al. Comparison of large restriction fragments of Mycobacterium avium isolates recovered from AIDS and non-AIDS patients with those of isolates from potable water. J Clin Microbiol 1999; 37(4): 1008-12.
  18. Konuk M, Korcan E, Dulgerbaki S, Altindis M. Isolation and identification of Mycobacteria from raw milk samples in Afyonkarahisar district of Turkey. Int J Food Microbiol 2007; 115(3): 343-7.
  19. Sgarioni SA, Hirata RD, Hirata MH, Leite CQ, de Prince KA, de Andrade Leite SR, et al. Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Parana, Brazil. Braz J Microbiol 2014; 45(2): 707-11.
  20. Jordao Junior CM, Lopes FC, David S, Farache FA, Leite CQ. Detection of nontuberculous mycobacteria from water buffalo raw milk in Brazil. Food Microbiol 2009; 26(6): 658-61.
  21. Mendum TA, Chilima BZ, Hirsch PR. The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil. FEMS Microbiol Lett 2000; 185(2): 189-92.
  22. Padya L, Chin'ombe N, Magwenzi M, Mbanga J, Ruhanya V, Nziramasanga P. Molecular Identification of Mycobacterium Species of Public Health Importance in Cattle in Zimbabwe by 16S rRNA Gene Sequencing. Open Microbiol J 2015; 9: 38-42.
Journal of Isfahan Medical School
Volume 34, Issue 373 - Serial Number 373
January and February 2016
Pages 175-181

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History

  • Receive Date: 15 September 2015
  • Revise Date: 23 May 2024
  • Accept Date: 06 April 2022

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