Identification of Nontuberculous Mycobacteria Species Isolated from Water Samples Using Phenotypic and Molecular Methods and Determination of their Antibiotic Resistance Patterns by E- Test Method, in Isfahan, Iran

Document Type : Original Article


Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


Many studies have shown epidemiological links between strains isolated in tap water, and those isolated from patients. Molecular methods linked to PCR are more reliable and faster for identification of             non- tuberculous mycobacteria(NTM). In this study molecular methods were used for identification and typing of NTM.
Materials and Methods
Five hundred ml of 85 water samples was passed through 0.45 μm filters. The filters were transferred directly onto 7H10 Middle Brook solid media, containing 15% OADC. PCR for 16S rRNA was done and the PCR product (1500 bp) was sequenced. PRA of the hsp65 gene was investigated to identify the species of isolates. For evaluation of susceptibility of NTM to antimycobacterial agents, E-test method was used.
The genus of 26 isolated NTM was confirmed by 16s rRNA sequence based method. Nineteen isolates of Mycobacteria were differentiated using hsp65genes PRA. The dominant isolates were M. fortuitum  (26.7%), M. chelonae like organism(13.3%) and M. mucogenicum (13.3%). Seventy one percent of NTM species were resistant to isoniazid, 64% to rifampin, 57% to ethambutol, 35% to tetracycline, 14 % to azithromycin and 7.1 % to amikacin.
The results showed that E-test method is not a proper technique for antimycobacterial assay because some NTM species are slow in growing and have no growth on Muller Hinton agar. Regarding the 16S rRNA sequence analysis, the identification of isolates was restricted to the genus level, because 99% similarity within 16S rRNA of two isolates may or may not determine the same species.


1. Koning B, Amer Ina, Sollich V, Koning W. Intra and interpatient variability of the hsp65 and 16S-23S intergenic gene region in Mycohaterium abscessus strains from patients with cystic fibrosis. J Clin Microbiol 2005;43:3500-3503.
2. Groote MAD, Huitt G. Infections due to rapidly growing Mycobacteria. Clin Infect Dis   2006; 42: 1756 –1763.
3. Khan IUH, Yadav JS. Development of a single – tube, cell lysis – based, genus – specific PCR method for rapid identification of mycobacteria: optimization of cell lysis, PCR primers and conditions and restriction pattern analysis. J Clin Microbiol 2004; 42:453–457.
4. Kotoch VM. Infections due to non– tuberculous mycobacteria (NTM). Indian J Med Res 2004; 120: 290–304.
5. Covert TC, Rodgers MR, Reyes AL, Stelma GN. Occurrence of nontoberculous mycobacteria in environmental sample. Appl Environ Microbiol 1999; 68:3159–3161.
6. Park H, Jang H, kim C, Byungseon C, Chang CL, park SK, et al. Detection and identification of mycobacteria by amplification of the internal transcribed spacer regions with genus and species specific PCR primers. J Clin Microbiol 2000; 38:4080–4085.
7. Harmsen D, Dostal S, Roth A, Niemann S, Rothgänger J, Sammeth M, et al. Ridom comprehensive and public sequence database for identification of mycobacterium species. BMC Infect Dis 2003; 3:1-10.
8. Hafner B, Haag H, Geiss HK, Noite O. Different molecular methods for the identification of rarely isolated nontuberculous mycobacteria and description of new hsp65 restriction fragment length polymorphism patterns. Mol Cell Probes 2004; 18:59-65.
9. Lenor SC, Arnold EG, Andrew DE. Standard methods for the examination of water and wastewater. 20th ed.American Public Health Association, American Water Works Association, Water Environment Federation; United Book Press Inc; 1998. p.1-37.
10. DPD: National drinking water standards.5th ed. Tehran: Iranian industrial research and standards organization publications; 1997; standard no .1053.
11. Cooper C, Revised by Jovell B. Isolation and quantification of genomic DNA from Mycobacterium tuberculosis. 1999.Available at: WWW. Wadsworth. Org / rflp/ tutorials/ DNA isolation.html.
12. Telenti A, Marcehesi F, Marianne Balz. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 1993; 31:175-178.
13. Chang CT, Wang LY, Liao CY, Hvang SP. Identification of nontuberculous mycobacteria existing in tap water by PCR restriction fragment length polymorphism. Appl Environ Microbiol 2002; 68:3159–3161.
14. Steingrube VA, Gibson JL, Brown BA, Zhang Y, Wilson RW, Rajagopalan M, et al. PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene asquence for taxonomic separation of rapidly growing mycobacteria. J Clin Microbiol 2003; 33: 149–153.
15. Brunello F, Ligozzi M. Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 2001; 39:2799–2806.
16. Turenne C, Tschetter L, Wolfe J, Kabani A. Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous mycobacterium species. J Clin Microbiol 2001; 39:3637–3648.
17. Nasr-Esfahani B, Sarikhani E, Moghim S, Faghri J, Fazeli H, Hoseini N, et al. Molecular characterization of environmental non-tuberculous mycobacteria using PCR- RFLP analysis of 441 Bp heat shock protein 65 fragments. Iran J Publ Health? 2012; 41:108-114.
18. lin C, Xue C, Ting ZC, Huan DB, Zhong ZJ. Identification of Mycobacterium marinum 65 ka heat shock protein gene by polymerase chain reaction restriction analysis from lesion of swimming pool granuloma. Chin Med J  2006; 119:43–48.
19. Derek A, Wong PC,YIP W.Simple and Rational Approach to the Identification of Mycobacterium tuberculosis, Mycobacterium avium complex species, and other commonly isolated mycobacteria. J Clin Microbiol 2001; 39:3768–3771.
20. Argueta C, Yoder S, Holtzman AE, Aronson TW, Glover N, Berlin OG, et al. Isolation and identification of nontuberculous mycobacteria. J Food Prot2000; 63:930-933.
21. Wong DA, Yip PC, Tse DL, Tung VW, Cheung DT, Kam KM. Routine use of a simple low-cost genotypic assay for the identification of mycobacteria in a high throughput laboratory. Diagn Microbiol Infect Dis  2003; 47:421-426.
22. Shin JH, Lee EJ, Lee HR, Ryu SM, Kim RH, Chang CL, et al. Prevalence of non tuberculosis mycobacteria in a hospital environment. J Hosp Infect 2007; 65:143-148.