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Pathogenic rapidly growing--Mycobacterium manitobense in the environment of Agra, north India.

Sir,

Environmental mycobacteria, also called atypical mycobacteria or nontuberculous mycobacteria (NTM), or mycobacteria other than tuberculosis (MOTT) are common saprophytes in all natural ecosystems, including water, soil and aerosols (1-3).

For many years, the identification of mycobacteria to the species level has relied on investigation of colony morphology, growth kinetics and biochemical characteristics (4,5). From this approach, phenotypic patterns with features that do not exactly match any established species may frequently emerge. Among strains with overlapping phenotypic patterns the conventional procedures are unable to distinguish organisms belonging to a certain taxon from other different species (6). In recent years, increasing numbers of clinically significant mycobacteria have been isolated especially due to availability of automated DNA sequencing protocol at the diagnostic level. In 2003, 95 mycobacterial species were reported and the number has increased to 127 by 2007 due to availability of the sequencing facilities (7).

Environmental mycobacteria include both slow growing and rapid growing species. The rapidly growing pathogenic mycobacteria capable of producing disease in humans consist primarily of the Mycobacterium fortuitum, M. chelonae, M. abscessus species group and the M. smegmatis group. M. fortuitum group has been expanding and recently a third biovar was identified. M. boenickei, M. houstonense, M. neworleansense and M. brisbanense were new species identified within this biovar (8). Clinical features of infections due to rapidly growing mycobacteria include post-traumatic wound infections, disseminated cutaneous disease, bone and joint infections, catheter-related infections and chronic pulmonary disease (3).

In this communication we report M. manitobense for first time from a soil sample from Shaganj area of Agra city. This sample was collected as a part of our study on environmental mycobacteria (9). One hundred and fifteen soil samples were processed for this study and only one was found positive for M. manitobense. Decontamination was done by the procedure optimized earlier (9) by 3 per cent sodium dodecyl sulfate SDS, 4 per cent NaOH and 2 per cent cetrimide and the processed suspension was inoculated on Lowenstein-Jensen (L-J) slants in duplicate which were incubated at 30 and 37[degrees]C respectively. Colonies on primary culture were picked up, streaked on LJ media and then other standard methods for identification of mycobacteria (conventional and molecular) were applied.

Identification of mycobacterial isolate at species level was done by growth rate, morphology of colonies and standard biochemical tests like catalase, nitrate reduction, Tween 80 hydrolysis, aryl sulphatase, growth on L-J medium containing 5 per cent NaCl, MacConkey agar and pigment production tests as per the manual of Centers for Disease Control and Prevention, Atlanta (4). The pigmented colony of mycobacteria grew both at 30 and 37[degrees]C temperature within 7 days and did not show any distinct features. Biochemical results were positive for semi quantitative catalase and Tween 80 hydrolysis.

Negative results were observed for nitrate reductase, tolerance to 5 per cent NaCl, aryl sulphatase at 3 days and no growth appeared at MacConkey agar medium. Overall, biochemical results did not match with any known species of rapid growing mycobacteria.

Gene amplification restriction analysis was performed by an in-house developed system (10) targeting 16S-23S rRNA spacer and flanking gene region which amplified 1.8 kb fragment followed by restriction analysis by Hha I, Hinf I and Rsa I from MBI Fermantas. The resulting fragments were 450, 230, 200, 150, 100 bp in size with Hha I, 520, 480, 460, 230, 190 bp with Hinf I and 770, 700, 550, 420, 220 bp with Rsa I enzyme (Fig. 1). The restriction patterns were found different from other rapid growers such as M. fortuitum, M. chelonae and M. smegmatis.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Sequencing of 16S rRNA gene region was performed by the method of Edward et al (11) (Fig. 2). For sequencing, amplicons of this gene region were recovered from agarose gel after electrophoresis using a Qiagen gel Extraction Kit (Qiagen, USA). Determination of sequences was performed using a Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin Elmer, USA) in a 310 Analyzer (Perkin Elmer, USA). The BLAST program (version 2.0), European Molecular Biology Laboratory (http://dove. Embl-Heidelberg.De/Blast2/) was used to compare the sequence of the study strains with the ones in the databases. Sequencing of the nearly complete 16S rRNA gene revealed a unique organism distantly related to the Mycobacterium smegmatis group. Results showed the similarity of these sequences with M.manitobense (12).

This mycobacterial species was first time reported from Canada by Turenne et al (12) whereas ours is perhaps the first report from India. Since this is a clinically significant mycobacteria which has been reported to cause soft tissue infection (12), it would be important to watch for infections by this mycobacteria in India and not discard it as a contaminant. Correct identification of clinically relevant mycobacteria including such rare species could be important for patient management, and support of reference laboratory with sequencing facility will be thus required.

This environmental mycobacteria was isolated from densily populated area of Agra city in north India and for that reason merits special attention as various environmental mycobacteria have been implicated in human infections particularly in immunocompromised patients, who may be exposed through inhalation, ingestion or broken skin. Understanding the sources, frequency and consequences of human exposure to various NTM is also important because immune responses following exposure to environmental mycobacteria may influence susceptibility to tuberculosis and leprosy as well as may also affect the protective efficacy of BCG vaccination against tuberculosis (13). In depth studies are required to isolate and study dynamics of presence of such rapidly growing potentially pathogenic mycobacteria in India.

References

(1.) Kazda JF. The principles of the ecology of mycobacteria. In: Ratledge C, Stanford J, editors. The biology of mycobacteria, vol.2. London, United Kingdom: Academic Press; 1983 p. 323-41.

(2.) Falkinham JO III. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 1996; 9 : 177-215.

(3.) Katoch VM. Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res 2004; 120 : 290-304.

(4.) Vestal AL. In : Procedure for the isolation and identification of mycobacteria. US Department of Health, Education and Welfare Pub no. (CDC) 77-8230. Atlanta, Georgia: Centers for Disease Control and Prevention; 1977 p.15-90.

(5.) Wayne LG, Kubica GP. The Mycobacteria. In: Sheath PHA, Mair NS, Sharpe ME, Holt JG, editors. Bergey's manual of systematic bacteriology, vol II. Baltimore: Williams and Wilkins; 1986 p. 1435-57.

(6.) Springer B, Stockman L, Teschner K, Roberts GD, Bottger EC. Two laboratory collaborative study on identification of mycobacteria: Molecular versus phenotypic methods. J Clin Microbiol 1996; 34 : 296-303.

(7.) Euzeby JP. (2007) List of bacterial names with standing in nomenclature genus Mycobacterium. Available: from http:// www.bacterio.cict.fr/m/mycobacterium, accesed on March 2, 2007.

(8.) Schinsky MF, Morey RE, Steigerwatt AG, Douglas MP, Wilson RW, Floyd MM, et al. Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates. Int J Syst Evol Microbiol 2004; 54 : 1653-67.

(9.) Parashar D, Chauhan DS, Sharma VD, Chauhan A, Chauhan SVS, Katoch VM. Optimization of procedures for isolation of environmental mycobacteria from soil and water samples of north India. Appl Environ Microbiol 2004; 70 : 3751-3.

(10.) Katoch VM, Parashar D, Chauhan DS, Singh D, Sharma VD, Ghosh S. Rapid identification of mycobacteria by gene amplification restriction analysis technique targeting 16S-23S ribosomal DNA spacer and flanking region Indian J Med Res 2007; 125 : 155-62.

(11.) 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 : 7843-53.

(12.) Turenne CY, Suchak AA, Wolfe JN, Kabani A, Nicolle LE. Soft tissue infection caused by a novel pigmented, rapidly growing Mycobacterium species. J Clin Microbiol 2003; 41 : 2779-82.

(13.) Stanford JL, Shield MJ, Rook GAW. Hypothesis: How environmental mycobacteria may predetermine the protective efficacy of BCG. Tubercle 1981; 62 : 55-62.

Deepti Parashar, Ram Das, V.D. Sharma D.S. Chauhan & V.M. Katoch *

Department of Microbiology & Molecular Biology National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR) Tajganj, Agra 282001, India * For correspondence: e-mail: vishwamohan_katoch@yahoo.co.in
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Title Annotation:Correspondence
Author:Parashar, Deepti; Das, Ram; Sharma, V.D.; Chauhan, D.S.; Katoch, V.M.
Publication:Indian Journal of Medical Research
Article Type:Letter to the editor
Date:Sep 1, 2007
Words:1373
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