Identification of Different Subtypes of Rapid Growing Atypical

International Journal of Mycobacteriology xxx (2016) xxx– xxx

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Identiﬁcation of different subtypes of rapid growing Atypical Mycobacterium from water and soil sources: Using PCR-RFLP using hsp65 and rRNA 16s–23s genes

Mohammad Varahram a, Parissa Farnia a,*, Shima Saif a, Mehran Marashian a, Poopak Farnia a,b, Jaladein Ghanavi a, Ali Akbar Velayati a a Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran b Department of Biotechnology Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

ARTICLE INFO ABSTRACT

Article history: Objective/Background: Nontuberculosis mycobacteria (NTM) are a diverse group of microor- Received 13 September 2016 ganisms that cause a variety of diseases in humans including skin, respiratory, and gas- Accepted 14 September 2016 trointestinal tract infection. Generally, NTM are classified into two categories: rapid Available online xxxx (<7 days) and slow growing (>7 days). In this study, we aimed to investigate NTM frequency and prevalence in environmental samples. Additionally, we tried to identify various sub- Keywords: types of isolated rapid growing mycobacteria (RGM). Iran Methods: Through a prospective descriptive cross-sectional study, water and soil samples Mycobacterium fortuitum were gathered from four neighboring towns around Tehran, the capital of Iran, at different 2 RGM geographic directions. Every 100 m of the studied areas gave one sample containing 6 g of Soil soil in 3–5 cm depth deposited in 50 mL sterile water as sampling media. After digestion Water and decontamination, DNA from culture-positive specimens (RGM) were extracted using phenol–chloroform methods. Then the molecular identification of species and subspecies were performed using 16s–23s rRNA and hsp65 gene. Results: In total, 341 RGM were found, out of which 322 (94.4%) were identified and 20 (5.8%) could not be identified. The most frequent RGM was, Mycobacterium fortuitum (72; 22%), Mycobacterium senegalense (58; 17.7%), Mycobacterium parafortuitum (44; 13.4%) and Mycobac- terium conceptionense type 1 (24; 7.2%), and Mycobacterium cheloni type 1 (20; 6.0%). As shown in Table 1, M. fortuitum had more subtypes (8), and the frequency of subtypes 1 (27.7%), 4 (16.6%), and 5 (13.8%) were higher. Among subtypes of M. senegalense, subtype 1 had a higher frequency (70.4%) in comparison to subtype 2 (29.5%). M. cheloni had just one subtype.

* Corresponding author. Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran. E-mail addresses: [email protected], [email protected] (P. Farnia). Peer review under responsibility of Asian African Society for Mycobacteriology. http://dx.doi.org/10.1016/j.ijmyco.2016.09.057

Please cite this article in press as: M Varahram et al. Identiﬁcation of diﬀerent subtypes of rapid growing Atypical Mycobacterium from water and soil sources: Using PCR-RFLP using hsp65 and rRNA 16s–23s genes. Int. J. Mycobacteriol. (2016), http://dx.doi.org/10.1016/j.ijmyco.2016.09.057 2 International Journal of Mycobacteriology xxx (2016) xxx– xxx

Conclusion: Our results showed M. fortuitum as the most prominent strain isolated from environmental samples. The frequency was similar in different places, irrespective of cli- matic variations. Availability of various subtypes of M. fortuitum might indicate a large cir- culation of this RGM in soil and water of Iranian territory. This high prevalence of M. fortuitum might raise the risk infection, especially in children, immunocompromised patients, diabetics, and cancer cases.

Table 1 – The frequency of rapid growing NTMs found by the current study. Rapid growing mycobacteria (RGM) 341 Identified RGM 322 (94.4%) Unidentified RGM 20 (5.8%) Mycobacterium fortuitum (Types 1, 2, 4, 5, 6, 8) 72 (22.0%) Mycobacterium senegalense (Types 1 & 2) 58 (17.7%) Mycobacterium parafortuitum (Types 1 & 2) 44 (13.4%) Mycobacterium conceptionense Type 1 24 (7.3%) Mycobacterium cheloni (Type 1) 20 (6.1%) Mycobacterium peregrinum (Types 1, 2, 3) 13 (4.0%) Mycobacterium abscessus Type1 12 (3.6%) Mycobacterium aurum Type 1 10 (3.0%) Mycobacterium mucogenicum Type 1 10 (3.0%) Mycobacterium brumae Type 1 10 (3.0%) Mycobacterium phlei Type1 8 (2.4%) Mycobacterium obuense Type1 7 (2.1%) Mycobacterium poriferae Type 1 7 (2.1%) Mycobacterium neoaurum Type1 6 (1.8%) Mycobacterium novocastrense Type 1 5 (1.5%) Mycobacterium rhodesia Type1 3 (0.9%) M. aurum Type 2 3 (0.9%) Mycobacterium confluentis Type 1 3 (0.9%) Mycobacterium fluorantherivorans Type1 3 (0.9%) Mycobacterium smegmatise Type 1 2 (0.6%) Mycobacterium gadium Type 1 2 (0.6%) NTM = nontuberculosis mycobacteria; RGM = rapid growing mycobacteria.

Conﬂicts of interest

The authors have no conﬂicts of interest to declare.