As an important source of human food, milk can be a carrier of human pathogenic bacteria, including tuberculous and nontuberculous mycobacteria (NTM), in its raw and unpasteurized state. In this research, 175 raw milk samples and 175 traditional cheese samples were collected from traditional dairy stores in 22 regions of Tehran in a 9- month period from August 2019 to May 2020. Samples were prepared and transferred to a specialized laboratory, where they were inoculated in Lowenstein-Jensen (LJ) medium containing glycerol or sodium pyruvate, as well as Herrold's egg-yolk with and without Mycobactin J. to determine the sample's identity of samples. The recommended 16S rRNA (1436 bp) and hsp65 (644 bp) gene fragments from the positive isolates identified in Ziehl-Neelsen (Z-N) staining were amplified and sequenced using PCR and compared with the sequences of the gene fragments of reference strains available in the global GenBank database. No mycobacterial species were isolated from traditional cheese samples in microbial culture. In case of raw milk samples, a total of four bacteria were collected, all of which were found in the genetic differential testing to be NTM, including n = 1 Mycobacterium heraklionense, n = 2 Mycolicibacterium fortuitum, and n = 1 Mycobacterium thermoresistibile. The analysis of the results obtained by isolate sequencing using the 16S rRNA gene showed higher discriminatory power and percentage similarities in the identification of the isolates than the hsp65 gene.

Agricultural Research Education and Extension Organization Tuberculin and Mallein Research and Production Department Razi Vaccine and Serum Research Institute Bovine Tuberculosis Laboratory Karaj Iran

Bovine Tuberculosis Reference Laboratory Agricultural Research Education and Extension Organization Razi Vaccine and Serum Research Institute Karaj Iran

Department of Microbiology Faculty of Basic Science Qom Branch Islamic Azad University Qom Iran

Zobrazit více v PubMed

Abedalthagafi M, Rosenberg O, Miller S (2014) First report of tenosynovitis in an immunocompetent person caused by Mycobacterium heraklionense. JMM Case Rep 1(2):e002071.  https://doi.org/10.1099/jmmcr.0.002071

Ali ZI, Hanafy M, Hansen C, Saudi AM, Talaat AM (2021) Genotypic analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin. J Dairy Sci 104(1):211–220. https://doi.org/10.3168/jds.2020-18214 PubMed DOI

Arend SM, van Soolingen D, Ottenhoff TH (2009) Diagnosis and treatment of lung infection with nontuberculous mycobacteria. Curr Opin Pulm Med 15(3):201–208. https://doi.org/10.1097/MCP.0b013e3283292679 PubMed DOI

Bolaños CA, Franco MM, Souza Filho AF, Ikuta CY, Burbano-Rosero EM, Ferreira Neto JS, Ribeiro MG (2018) Nontuberculous mycobacteria in milk from positive cows in the intradermal comparative cervical tuberculin test: implications for human tuberculosis infections. Rev Inst Med Trop Sp 60. https://doi.org/10.1590/S1678-9946201860006

Cloud JL, Meyer JJ, Pounder JI, Jost KC Jr, Sweeney A, Carroll KC, Woods GL (2006) Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens. Int J Syst Evol Micr 56(6):1413–1418. https://doi.org/10.1099/ijs.0.64194-0 DOI

Comas I, Coscolla M, Luo T, Borrell S, Holt KE, Kato-Maeda M, Gagneux S (2013) Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat Genet 45(10):1176–1182. https://doi.org/10.1038/ng.2744 PubMed DOI PMC

Gupta RS, Lo B, Son J (2018) Phylogenomics and comparative genomic studies robustly support division of the genus Mycobacterium into an emended genus Mycobacterium and four novel genera. Front Microbiol 9:67. https://doi.org/10.3389/fmicb.2018.00067 PubMed DOI PMC

Haghighat M, Shahmoradi AH, Tadayon K, Keshavarz R, Ghaderi R, Sekhavati M, Mosavari N (2017) Molecular identification of Mycobacterium avium “subsp. ” paratuberculosis 316F and III & V strains by a multi-approach strategy. Vet Res Biol Prod 30(2):89–100. https://doi.org/10.22034/VJ.2017.109226 DOI

Hamada S, Ito Y, Hirai T, Murase K, Tsuji T, Fujita K, Mishima M (2016) Impact of industrial structure and soil exposure on the regional variations in pulmonary nontuberculous mycobacterial disease prevalence. Int J Mycobacteriol 5(2):170–176. https://doi.org/10.1016/j.ijmyco.2016.02.006 PubMed DOI

Harris KA, Hartley JC (2003) Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol 52(8):685–691. https://doi.org/10.1099/jmm.0.05213-0 PubMed DOI

Ho HT, Chang PL, Hung CC, Chang HT (2004) Capillary electrophoretic restriction fragment length polymorphism patterns for the mycobacterial hsp65 gene. J Clin Microbiol 42(8):3525–3531. https://doi.org/10.1128/JCM.42.8.3525-3531.2004 PubMed DOI PMC

Huard RC, de Oliveira Lazzarini LC, Butler WR, van Soolingen D, Ho JL (2003) PCR-based method to differentiate the subsp. of the Mycobacterium tuberculosis complex on the basis of genomic deletions. J Clin Microbiol 41(4):1637–1650. https://doi.org/10.1128/JCM.41.4.1637-1650.2003 PubMed DOI PMC

Johnson MM, Odell JA (2014) Nontuberculous mycobacterial pulmonary infections. J Thorac Dic 6(3):210. https://doi.org/10.3978/j.issn.2072-1439.2013.12.24 DOI

Keikha M (2017) The importance of molecular techniques in identification and phylogenetic studies of nontuberculosis mycobacteria. Iran J Med Microbiol 10(6):78–81

Khosravi AD, Hashemzadeh M, Hashemi Shahraki A, Teimoori A (2017) Differential identification of mycobacterial species using high-resolution melting analysis. Front Microbiol 8:2045. https://doi.org/10.3389/fmicb.2017.02045 PubMed DOI PMC

Khosravi AD, Hashemzadeh M, Rokhfirooz P (2022) Molecular identification of nontuberculous mycobacteria using the rpoB, argH and cya genes analysis. AMB Express 12(1):1–10. https://doi.org/10.1186/s13568-022-01463-1 DOI

Kim H, Kim SH, Shim TS, Kim MN, Bai GH, Park YG, Kim BJ (2005) Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int J Syst Evol Micr 55(4):1649–1656. https://doi.org/10.1099/ijs.0.63553-0 DOI

Ledesma Y, Echeverría G, Claro-Almea FE, Silva D, Guerrero- Freire S, Rojas Y, Bastidas-Caldes C, Navarro JC, de Waard JH (2022) The re-identification of previously unidentifiable clinical non-tuberculous mycobacterial isolates shows great species diversity and the presence of other acid-fast genera. Pathogens 11:1159. https://doi.org/10.3390/pathogens11101159 PubMed DOI PMC

Lee CK, Gi HM, Cho Y, Kim YK, Lee KN, Song KJ, Bai GH (2004) The genomic heterogeneity among Mycobacterium terrae complex displayed by sequencing of 16S rRNA and hsp65 genes. Microbiol Immunol 48(2):83–90. https://doi.org/10.1111/j.1348-0421.2004.tb03492.x PubMed DOI

Leite CQ, Anno IS, Leite SR, Roxo E, Morlock GP, Cooksey RC (2003) Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil. Mem Inst Oswaldo Cruz 98:319–323. https://doi.org/10.1590/S0074-02762003000300005 PubMed DOI

Makovcova J, Babak V, Slany M, Slana I (2015) Comparison of methods for the isolation of mycobacteria from water treatment plant sludge. A Van Leeuw J Microb 107(5):1165–1179. https://doi.org/10.1007/s10482-015-0408-4 DOI

Mun HS, Park JH, Kim H, Yu HK, Park YG, Cha CY, Kim BJ (2008) Mycobacterium senuense sp. nov., a slowly growing, non-chromogenic species closely related to the Mycobacterium terrae complex. Int J Syst Evol Micr 58(3):641–646. https://doi.org/10.1099/ijs.0.65374-0 DOI

Namkoong H, Kurashima A, Morimoto K, Hoshino Y, Hasegawa N, Ato M, Mitarai S (2016) Epidemiology of pulmonary nontuberculous mycobacterial disease. Emerg Infect Dis. https://doi.org/10.3201/eid2206.151086 PubMed DOI PMC

Nasiri MJ, Dabiri H, Fooladi AA, Amini S, Hamzehloo G, Feizabadi MM (2018) High rates of nontuberculous mycobacteria isolation from patients with presumptive tuberculosis in Iran. New Microbe and New Infect 21:12–17. https://doi.org/10.1016/j.nmni.2017.08.008 DOI

Nasr-Esfahani B, Sarikhani E, Moghim S, Faghri J, Fazeli H, Hoseini N, Rezaei-Yazdi H (2012) Molecular characterization of environmental nontuberculous mycobacteria using PCR-RFLP analysis of 441 Bp heat shock protein 65 fragments. Iran J Public Health 41(4):108 PubMed PMC

Nørgaard Eskesen A, Skråmm I, Steinbakk M (2007) Infectious tenosynovitis and osteomyelitis caused by Mycobacterium nonchromogenicum. Scand J Infect Dis 39(2):179–180. https://doi.org/10.1080/00365540600798817 DOI

Patiño EAA (2019) Species identification of nontuberculous mycobacteria. Invest Clin 60(1):79–103. https://doi.org/10.22209/IC.v60n1a07 DOI

Pennington KM, Vu A, Challener D, Rivera CG, Shweta FNU, Zeuli JD, Temesgen Z (2021) Approach to the diagnosis and treatment of nontuberculous mycobacterial disease. J Clin Tuberc Other Mycobact Dis 24:100244. https://doi.org/10.1016/j.jctube.2021.100244

Rezaeyan MH, Havaei SA, Moghim S, Riyahi F, Rahdar H, Rouzbahani M, Nasr-Esfahani B (2016) Determination of nontuberculosis mycobacteria species genotypes present in cattle milk samples using 16S rRNA gene direct sequencing. J Isfahan Med School 34(373):175–181

Shin SJ, Lee BS, Koh WJ, Manning EJ, Anklam K, Sreevatsan S, Collins MT (2010) Efficient differentiation of Mycobacterium avium complex species and subsp. by use of five-target multiplex PCR. J Clin Microbiol 48(11):4057–4062. https://doi.org/10.1128/JCM.00904-10 PubMed DOI PMC

Slana I, Liapi M, Moravkova M, Kralova A, Pavlik I (2009) Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR. Prev Vet Med 89(3–4):223–226. https://doi.org/10.1016/j.prevetmed.2009.02.020 PubMed DOI

Tortoli E (2014) Microbiological features and clinical relevance of new species of the genus Mycobacterium. Clin Microbiol Rev 27(4):727–752. https://doi.org/10.1128/CMR.00035-14 PubMed DOI PMC

Tortoli E (2019) The taxonomy of the genus Mycobacterium. In nontuberculous mycobacteria (NTM). pp 1–10 Academic Press. https://doi.org/10.1016/B978-0-12-814692-7.00001-2

Tortoli E, Gitti Z, Klenk HP, Lauria S, Mannino R, Mantegani P, Neonakis I (2013) Survey of 150 strains belonging to the Mycobacterium terrae complex and description of Mycobacterium engbaekii sp. nov., Mycobacterium heraklionense sp. nov. and Mycobacterium longobardum sp. nov. Int J Syst Evol Micr 63(2):401–411. https://doi.org/10.1099/ijs.0.038737-0 DOI

Tsukamura M (1971) Differentiation between Mycobacterium phlei and Mycobacterium thermoresistibile. Am Rev Respir Dis 103(2):280–282 PubMed

Tsukamura M, Mizuno S, Tsukamura S (1981) Numerical analysis of rapidly growing, Scotochromogenic mycobacteria, including Mycobacterium obuense sp. nov., nom. rev., Mycobacterium rhodesiae sp. nov., nom. rev., Mycobacterium aichiense sp. nov., nom. rev., Mycobacterium chubuense sp. nov., nom. rev., and Mycobacterium tokaiense sp. nov., nom. rev. Int J Syst Evol Micr 31(3):263–275. https://doi.org/10.1099/00207713-31-3-263 DOI

Umrao J, Singh D, Zia A, Saxena S, Sarsaiya S, Singh S, Dhole TN (2016) Prevalence and species spectrum of both pulmonary and extrapulmonary nontuberculous mycobacteria isolates at a tertiary care center. Int J Mycobacteriol 5(3):288–293. https://doi.org/10.1016/j.ijmyco.2016.06.008 PubMed DOI

Van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, Shinnick TM (1993) Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 31(2):406–409. https://doi.org/10.1128/jcm.31.2.406-409.1993 PubMed DOI PMC

Van Soolingen D, Hoogenboezem T, De Haas PE, Hermans PW, Koedam MA, Teppema KS, Van Embden JD (1997) A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. Int J Syst Evol Micr 47(4):1236–1245. https://doi.org/10.1099/00207713-47-4-1236 DOI