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 Table of Contents  
Year : 2018  |  Volume : 3  |  Issue : 2  |  Page : 52-54

Case report of isolation of Mycobacterium setense from a hospital water supply

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

Date of Submission30-Apr-2018
Date of Acceptance07-Jun-2018
Date of Web Publication12-Jul-2018

Correspondence Address:
Masoud Keikha
Department of Microbiology, School of Medicine, Isfahan Medical University, Isfahan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ed.ed_8_18

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Objective: The current study is a report of isolation and identification of Mycobacterium setense from a tap running water in a general surgery ward in a hospital in Isfahan, Iran.
Materials and Methods: This bacterium was obtained from a hospital water sample according to the cetylpyridinium chloride 0.005% method on Lowenstein–Jensen slant and characterized to the species level as M. setense using results of phenotypic test and 16S rRNA sequencing.
Results: This isolate identified to the species level using the conventional and molecular method as M. setense, this bacterium has highest similarity (100%) with those of M. setense CIP109395 16S rRNA gene sequences.
Conclusion: This study can be useful and provide important information for learning about the natural habitats of pathogenic species of nontuberculosis mycobacteria. Furthermore, the molecular tests such as 16S rRNA gene sequencing can be applied for reliable and appropriate identification of mycobacterial species.

Keywords: Hospital infection, Mycobacterium setense, nontuberculosis mycobacteria, 16S rRNA

How to cite this article:
Keikha M. Case report of isolation of Mycobacterium setense from a hospital water supply. Environ Dis 2018;3:52-4

How to cite this URL:
Keikha M. Case report of isolation of Mycobacterium setense from a hospital water supply. Environ Dis [serial online] 2018 [cited 2023 Jun 5];3:52-4. Available from: http://www.environmentmed.org/text.asp?2018/3/2/52/236535

  Introduction Top

Members of Mycobacterium spp. are classified into two groups including the pathogenic groups of Mycobacterium tuberculosis complex and nontuberculosis mycobacteria (NTM), which are recognized as the opportunistic organisms. NTM can enter into the human body through inhalation of aerosols or traumatic inoculation, causing pulmonary, soft-tissue, bone and joints, disseminated, and catheter-related infection in immunodeficient patients and even in healthy individuals.[1] There are several evidences for the presence of NTM in the hospital environmental resources and medical equipment; this group of bacteria is slow growing and able to adapt with harsh conditions, including resistance to chlorine and various disinfectants.[1],[2] Most of the biological markers cannot detect the presence of pathogen microorganisms such as NTM in water; however, several studies have suggested the role of NTM in water resources associated with nosocomial infection.[1],[2],[3],[4] These studies emphasize on identifying the presence of Mycobacterium species in hospital water resources. Moreover, the purpose of these studies is to find a solution for elimination of microbial contaminations such as NTM species.[3],[4]

Due to the similarities of isolation methods and the acid-fast staining results of NTM species and mycobacterium tuberculosis, NTM infection can be misdiagnosed with mycobacterium tuberculosis infections. According to the American Thoracic Society guidelines, identification and differentiation of NTM species are necessary for the detection of emerging species, true diagnosis, patient supervision, correct identification, and epidemiological studies. Thus, the identification and differentiation of nontuberculosis mycobacterial isolates from clinically specimens are considered as the essential steps in the appropriate treatment of NTM infection.[5]

Mycobacterium setense is one of the members of Mycobacterium fortuitum complex which was isolated from clinical specimens and introduced by the French scientist, Lamy et al. However, there have only been three case study reports in the world regarding M. setense infection [6],[7],[8] and the isolation of human infection with these bacteria as well as a report about environmental isolation of this organism, and there has been no further reports considering this species.[9] The current report is describing the unrelated environmental isolate identified as M. setense from the hospital water resource.

  Materials and Methods Top

According to the cetylpyridinium chloride (CPC) protocol, water samples were collected from a general surgery hospital building between August 2017 and January 2018 and treated with CPC. One liter of water was contaminated using CPC 0.005% and passed through a cellulose nitrate membrane filter. Subsequently, the filter was transferred to a sterile tube containing 15 ml of the distilled water and centrifuged for 20 min at 6000 rpm. Finally, the supernatant fluid was discarded and 100 μl of the sediments was transferred to Lowenstein–Jensen slants and the modified Sauton agar in 25°C, 37°C, and 42°C, and characterized by using acid-fast stain and the required biochemical tests.[3],[4]

  Results Top

The isolate, called K6, was identified using Ziehl–Neelsen staining, with smooth and nonpigmented colonies and a growth rate <7 days. The results were positive for arylsulfatase (3 days), iron uptake, pyrazinamidase (PZA) activity, nitrate reductase, and heat-resistant catalase, while they were negative for growth at 42°C and citrate utilization. Based on the results of the phenotypic test, the isolate K6 is closely resembling the rapidly growing mycobacteria (RGM), and also characterized for the species level using nearly full length of the 16S rRNA gene sequencing.[5]

The 16S rRNA gene sequence of the isolate revealed that the isolate has nucleotide signatures of mycobacteria at positions 70–98 (A–T), 293–304 (G–T), 307 (C), 328 (T), 614–626), 661–744 (G–C), (A–T), 631 (G), 824–876 (T–A), 825–875 (A–T), and 843 (C). Moreover, the 16S rRNA sequences of all the RGM contained the short helix 18 at positions 451–482.[5],[10],[11] This sequence was evaluated and matched with other RGM sequences of the nucleotide database using “blastn” and manually aligned and compared with closely related Mycobacterium species. Based on the signature sequences of the hypervariable Region A (positions 128–270 of  Escherichia More Details coli numbering system), the isolate K6 was detected as strain type of M. setense.[9],[10] The 16S rRNA gene sequences of the isolate present the highest similarity (100%) with those of M. setense CIP109395. The GenBank/ENA/DDBJ accession number for the sequences of the 16S rRNA gene sequences of the isolate K6 is MH 041152. The phylogenetic relationship tree of isolate K6 and other closely related RGM was created using the neighbor-joining method, Kimura 2-parameter model, and the 1000 bootstrap value based on the 16S rRNA gene sequences [Figure 1].
Figure 1: Phylogenic relationship tree of Mycobacterium setense (seq1) and closely related species calculated based on the 16S rRNA sequences using the neighbor-joining method and Kimura 2-parameter distance rooted with Nocardia asteroids

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  Discussion Top

M. setense was first introduced in 2008. This bacterium is aerobic, rod shaped, acid-fast, and Gram positive, which have smooth and nonpigmented colonies. M. setense was grown on Lowenstein–Jensen agar within 2–4 days at 25°C, 30°C, and 37°C and also grown in the presence of 5% NaCl, capable of arylsulfatase, and positive for nitrate reductase, urease, heat-resistant catalase (68°C), and iron uptake. This organism can metabolize carbohydrates including D-mannitol and D-glucose as the sole sources of carbon. It can produce PZA, alkaline phosphatase, and gelatinase.[6] This study shows that M. setense can be isolated from hospital water samples and environmental resources, which is a useful finding that provides information about the natural habitats of these group of bacteria.[3]

Mycobacterium spp. are ubiquitous in the environmental resources, which can be transmitted to the human via traumatic inclusion and inhalation of water drop or dust; therefore, appropriate environmental resources of hospitals play a key role in the prevention and control of hospital-acquired infections. It is important to identify the natural habitats of NTM in order to control and prevent the transmission of these organisms.[1],[4],[12]

Identification of NTM according to the phenotypic tests is time consuming, labor intensive, requires a skillful microbiologist, and in some species, it is unable to discriminate [2],[5]; the phenotypic test including growth rate, pigment production, growth at different temperatures, urease production, iron uptake, growth on MacConkey agar without crystal violet, Tween 80 hydrolysis, tolerance to 5% NaCl, aryl-sulfatase production (3 days), semi-quantitative catalase, heat-stable catalase, PZA production, alkaline phosphatase and gelatinase ability, and also nitrate reduction, niacin production, and utilization of various carbohydrates. However, molecular methods such as DNA hybridization, polymerase chain reaction (PCR)-restriction fragment length polymorphism, and sequencing using housekeeping genes such as 16S rRNA, rpoB, hsp65, 16S–23S rRNA internal transcribed spacer, tuf, gyrB, secA, dnaJ, rpoBC, and recA are inexpensive, rapid, reliable, and accurate ways of identifying this group of bacteria.[5],[13] Among housekeeping genes, 16S rRNA gene sequencing has been applied as the golden standard method of unusual NTM identification, but 16S rRNA gene sequencing is unable for the differentiation of the closely related species of mycobacteria, such as Mycobacterium abscessus and Mycobacterium chelonae from Mycobacterium avium complex, Mycobacterium farcinogenes and Mycobacterium senegalense, Mycobacterium kansasii and Mycobacterium gastri, Mycobacterium marinum and Mycobacterium ulceran, or Mycobacterium houstonense and Mycobacterium senegalense. Moreover, the existence of two copies of the 16S rRNA gene with diverse sequences in one distinct bacterium [2],[5],[13] makes the proper identification based on this gene. Recently, multilocus sequence analysis has been established for accurate and reliable identification of mycobacterial isolates and emerging NTM species, which is used from sequences of the selected housekeeping genes.[5],[14],[15] Despite what is mentioned, 16S rRNA sequencing can differentiate mycobacterial species, known as golden standard for identification and differentiation of unusual mycobacterial isolates.[16] In the current study, 16S rRNA was applied for identification of the isolate K6 to the species level using the study of hypervariable region A at positions 125–270 and region B at positions 408–503. We concluded that M. setense has unique 16S rRNA sequence signature and the isolate K6 belonged to M. setense based on the results of the phenotypic features, having short helix 18 (signature of RGM) and highest similarities between 16S rRNA sequences of the isolate K6 with those of M. setense CIP109395.[5],[10]

Totally, our results showed that the environmental hospital resources are the most important probable reservoir for M. setense. Furthermore, identification of mycobacterial isolates limited to the phenotypic tests is insufficient and unreliable. On the other hand, the molecular methods such as 16S rRNA sequencing can be used for appropriate and reliable identification of mycobacterial species, particularly M. setense, having the unique 16S rRNA sequence signature.

Financial support and sponsorship

This paper was financial supported by Isfahan University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.

  References Top

Brown-Elliott BA, Wallace RJ Jr. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 2002;15:716-46.  Back to cited text no. 1
Adékambi T, Berger P, Raoult D, Drancourt M. RpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov. Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int J Syst Evol Microbiol 2006;56:133-43.  Back to cited text no. 2
Keikha M. Isolation of Mycobacterium frederiksbergense from redundant tap water: A case report. Avicenna J Environ Health Eng 2017;4:3.  Back to cited text no. 3
Le Dantec C, Duguet JP, Montiel A, Dumoutier N, Dubrou S, Vincent V, et al. Occurrence of mycobacteria in water treatment lines and in water distribution systems. Appl Environ Microbiol 2002;68:5318-25.  Back to cited text no. 4
Hashemi-Shahraki A, Bostanabad SZ, Heidarieh P, Titov LP, Khosravi AD, Sheikhi N, et al. Species spectrum of nontuberculous mycobacteria isolated from suspected tuberculosis patients, identification by multi locus sequence analysis. Infect Genet Evol 2013;20:312-24.  Back to cited text no. 5
Lamy B, Marchandin H, Hamitouche K, Laurent F. Mycobacterium setense sp. nov. a Mycobacterium fortuitum-group organism isolated from a patient with soft tissue infection and osteitis. Int J Syst Evol Microbiol 2008;58:486-90.  Back to cited text no. 6
Toro A, Adekambi T, Cheynet F, Fournier PE, Drancourt M. Mycobacterium setense infection in humans. Emerg Infect Dis 2008;14:1330-2.  Back to cited text no. 7
Shojaei H, Hashemi A, Heidarieh P, Feizabadi MM, Ataei B, Daei Naser A, et al. First report on isolation and molecular characterization of clinical Mycobacterium setense isolates in Asia. Jpn J Infect Dis 2011;64:234-6.  Back to cited text no. 8
Azadi D, Naser AD, Shojaei H. First isolation of Mycobacterium setense from hospital water. JCoast Life Med 2016;4:331-3.  Back to cited text no. 9
Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a new hierarchic classification system, actinobacteria classis nov. Int J Syst Evol Microbiol 1997;47:479-91.  Back to cited text no. 10
Sydnor ER, Perl TM. Hospital epidemiology and infection control in acute-care settings. Clin Microbiol Rev 2011;24:141-73.  Back to cited text no. 11
Dai J, Chen Y, Dean S, Morris JG, Salfinger M, Johnson JA, et al. Multiple-genome comparison reveals new loci for Mycobacterium species identification. J Clin Microbiol 2011;49:144-53.  Back to cited text no. 12
Achtman M, Wagner M. Microbial diversity and the genetic nature of microbial species. Nat Rev Microbiol 2008;6:431-40.  Back to cited text no. 13
Kim BJ, Hong SH, Kook YH, Kim BJ. Mycobacterium paragordonae sp. nov. a slowly growing, scotochromogenic species closely related to Mycobacterium gordonae. Int J Syst Evol Microbiol 2014;64:39-45.  Back to cited text no. 14
Adékambi T, Colson P, Drancourt M. RpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol 2003;41:5699-708.  Back to cited text no. 15
Keikha M. Comment on Mycobacterium chelonae infection of the buttocks secondary to lipofilling: A case report and review of the literature. Aesthet Surg J 2018;42:610-11.  Back to cited text no. 16


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