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Year : 2014  |  Volume : 3  |  Issue : 4  |  Page : 286-289

Detection of Mycobacterium gilvum first time from the bathing water of leprosy patient from Purulia, West Bengal

Stanley Browne Laboratory, TLM Community Hospital, Nand Nagari, Delhi 110093, India

Date of Web Publication27-Feb-2017

Correspondence Address:
Mallika Lavania
Stanley Browne Laboratory, TLM Community Hospital, Nand Nagari, Delhi 110093
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.ijmyco.2014.10.005

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In this present study for the first time the authors are reporting the isolation of Mycobacterium gilvum from the accumulated water in the drain connected to the bathing place of leprosy patients residing in an endemic region. The identification and characterization of this isolate was carried out by various conventional and molecular tests, including 16S rDNA sequencing. These findings might shed further light and association with amoeba in the leprosy endemic area of this rare Mycobacterium species.

Keywords: M. gilvum, Acanthamoeba, 16S rRNA sequencing, Leprosy, NTM, Purulia

How to cite this article:
Lavania M, Turankar R, Singh I, Nigam A, Sengupta U. Detection of Mycobacterium gilvum first time from the bathing water of leprosy patient from Purulia, West Bengal. Int J Mycobacteriol 2014;3:286-9

How to cite this URL:
Lavania M, Turankar R, Singh I, Nigam A, Sengupta U. Detection of Mycobacterium gilvum first time from the bathing water of leprosy patient from Purulia, West Bengal. Int J Mycobacteriol [serial online] 2014 [cited 2021 Apr 20];3:286-9. Available from: https://www.ijmyco.org/text.asp?2014/3/4/286/200980

  Introduction Top

Amongst the reported list of bacteria, there are 169 species and 13 subspecies known to be present in the genus Mycobacterium (2014). Genus Mycobacterium has been further categorized into strict pathogens (Mycobacterium tuberculosis complex) and potential pathogens, non-tuberculous mycobacteria (NTM) (http://www.bacterio.net/mycobacterium.html accessed on 23 September 2014) [1]. Mycobacteria are aerobic, non-motile and acid fast bacteria. Members of the genus Mycobacterium are widespread in nature. NTM have been designated as “atypical” or anonymous mycobacterial species or mycobacteria other than tuberculosis (MOTT). Environmental mycobacteria consist of both rapid and slow-growing bacteria. These microbes share many common properties, such as acid fastness and the ability to cause pulmonary and extrapulmonary granulomatous disorders. As a group, they are comprised of diverse organisms with dissimilarities in their cultural characteristics and pathogenicity to humans compared with Mycobacterium tuberculosis (MTB).

NTM are widely distributed in the environment and are passed on by ingestion, inhalation, and inoculation from such sources. Studies estimate that 30% of mycobacterial species isolated from water [2],[3], soil [4], air and patients do not belong to any identified species [5]. The large number of isolated environment Mycobacterium remains non-identified by phenotypic and molecular techniques, which are usually employed for identification of clinical samples. However, all the previous studies suggest that there is the existence of diversity in this group of bacteria and such techniques are not discriminating enough for identification of mycobacterial species in environment samples.

  Methods Top

Water samples

Water samples were collected from the drain connected to the bathing place of patients of leprosy in an endemic region and has been investigated for this study. Decontamination was done by the protocol published by Parashar et al. [6] by treating water samples with 3% SDS, 4% NaOH and 2% cetrimide. Water samples were centrifuged for 8000 × g for 15 min at 4 °C. Pellets were re-suspended in 20 ml of treatment solution (3% SDS+4% NaOH) and divided into two parts: A and B. Part A was incubated for 15 min and B was incubated for 30 min at room temperature to obtain the growth of rapid and slow growers, respectively. Both the suspensions were centrifuged for 15 min after incubation at 8000 × g. Pellets were treated with 2% cetrimide and incubated for 5 and 15 min for rapid and slow growers, respectively. After incubation sediments were washed twice with sterile water and 100 μl of decontaminated suspension was inoculated on Lowenstein Jenson (LJ medium), slants were incubated at 37 °C and primary colonies were subcultured on LJ medium again. Each sample was processed two times. Identification of growth was done by growth rate and morphology of colonies. The colonies were pale yellow in color and were smooth and pleomorphic with rapid growth in 7days.

Identification of mycobacterial isolates at species level was done by growth rate, colony morphology and pigment production.

Identification by 16S rRNA sequencing

DNA from the growth from LJ slants were extracted by a procedure established earlier using lysozyme and proteinase K.

Sequencing of the 16S rRNA gene

Sequencing was performed by 16S rRNA gene region [7]. Each reaction mixture contained approximately 10ng of DNA, with 1× PCR master mix from Qiagen, USA; 0.3 μg/reaction of each forward and reverse primer was added in a final volume of 25 μl. The PCR was performed using the Corbett PCR system (USA) 95 °C for 5 min for initial denaturation followed by 37 cycles, each cycle consisting of denaturation at 95 °C for 30 s, annealing at 60 °C for 2 min and extension at 72 °C for 4 min with a final extension at 72 °C for 10 min. 10 μl of PCR product was subjected to electrophoresis to ensure that successful amplification had occurred and that the correct fragment was obtained which was purified by Qiagen PCR purification kit (Qiagen, USA). PCR product was sent for commercial sequencing (an Xplorigen Pvt., Ltd., Delhi, India). Sequences were blasted on the NCBI Blast http://blast.ncbi.nlm.nih.gov/Blast.cgi to compare the sequences of the study strains with the ones in the databases.

  Results and discussion Top

In this study the presence of M. gilvum was found for the first time from a water sample collected from an endemic area wherein a study on the transmission of Mycobacterium leprae in the population was being conducted. Other species which were found in the environment were Mycobacterium asiaticum and Mycobacterium parascrofulaceum.

Sequencing of the nearly complete 16S rRNA gene revealed a unique organism M. glivum distantly related to the Mycobacterium vaccae group ([Figure 1]). Blast results showed the similarity of these sequences with M. gilvum. This mycobacterial species was first reported by Stanford and Gunthorpe [8] from sputum samples. The species M. gilvum is derived from the Latin word gilvus, which means pale yellow. This Mycobacterium species is a rapid grower (growth within 7days) and shows resistance to isoniazid, rifampicin, and sodium aminosalicylate. M. gilvum is not a pathogenic mycobacterial species and is able to degrade most polycyclic aromatic hydrocarbons (PAH). It has also been isolated from river sediments [9],[10]. This mycobacteria recovered from water samples also colonized in free living amoebae (FLA) [11],[12]. It was shown that M. gilvum after phagocytosis was entering the trophozoites of Acanthamoeba polyphaga and survived at this location over a period of 5days like other NTMs. Many of these NTMs were found to be resistant to the bactericidal activity of amoeba. They can survive easily within the cysts or trophozoites of FLA [13],[14]. However, Lamrabet and Drancourt [15] observed that M. gilvum, Mycobacterium rhodesiae and Mycobacterium thermoresistibile mycobacteria did not multiply within amoebae and did not kill the amoebae ([Figure 2]).
Figure 1: Nucleotide sequence of the 16S rRNA gene region of isolate identified as M. gilvum by rRNA sequencing.

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Figure 2: Pairwise alignment of blast results among known Mycobacterial species.

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In this study this environmental mycobacterial species M. gilvum was isolated from a leprosy endemic village (Simonpur of Purulia district of West Bengal). Water samples from the bathing places of the leprosy patients were collected for the study. These environmental samples were investigated for the presence of mycobacterial species. This area is highly endemic with a small leprosy colony of old treated leprosy patients situated nearby The Leprosy Mission Community Hospital, Purulia, West Bengal. The population of the village is 1607 (2011 Census). A total of 66 new leprosy cases were reported from Simonpur during the period 2011–2014.

The explanation for the relation between leprosy and M. gilvum is beyond the scope of the present study. Other environmental Mycobacterium species like M. vaccae have been shown to be immunomodulator which suppress the immune system of the host towards susceptibility to leprosy. M. gilvum belongs to the group of M. vaccae and might be responsible for such an immunomodulation of the host. Further studies will be needed to explore the interactions between free-living amoebae and the majority of waterborne Mycobacterium species and whether these species may influence the susceptibility to infectious diseases like leprosy.

  Conflict of interest Top

The authors have no conflict of interest.

  Acknowledgements Top

This work was supported by a grant from the host organization The Leprosy Mission India. We thank Mr. Atul Roy for his support in sample collection.

  References Top

J.P. Euzbey, List of bacterial names with standing in nomenclature genus Mycobacterium, Available: from http:// www.bacterio.net/mycobacterium.html (accessed 23.09.14).  Back to cited text no. 1
M.R. Ettinger, S.R. Webb, S.A. Harris, S.P. McIninch, C.G. Garman, B.L. Brown, Distribution of free living amoebae in James River, Virginia, USA, Parasitol. Res. 89 (2003) 6–15.  Back to cited text no. 2
J.O. Falkinham III, C.D. Norton, M.W. LeChevallier, Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare and other mycobacteria in drinking water distribution systems, Appl. Environ. Microbiol. 67 (2001) 1225– 1231.  Back to cited text no. 3
R. Narang, P. Narang, D.K. Mendiratta, Isolation and identification of nontuberculous mycobacteria from water and soil in central India, Indian J. Med. Microbiol. 27 (2009) 247–250.  Back to cited text no. 4
E. Tortoli, A. Bartoloni, E.C. Bottger, S. Emler, C. Garzelli, E. Magliano, Burden of unidentifiable mycobacteria in a reference laboratory, J. Clin. Microbiol. 39 (2001) 4058–4065.  Back to cited text no. 5
D. Parashar, D.S. Chauhan, V.D. Sharma, A. Chauhan, S.V.S. Chauhan, V.M. Katoch, Optimization of procedures for isolation of environmental mycobacteria from soil and water samples of North India, Appl. Environ. Microbiol. 70 (2004) 3751–3753.  Back to cited text no. 6
R.S. Jadhav, R.R. Kamble, V.S. Shinde, S. Edward, V.K. Edward, Use of reverse transcription polymerase chain reaction for the detection of Mycobacterium leprae in the slit-skin smears of leprosy patients, Indian J. Lepr. 77 (2005) 116–127.  Back to cited text no. 7
J.L. Stanford, W.J. Gunthorpe, A study of some fast growing scotochromogenic mycobacteria including species descriptions of Mycobacterium gilvum (new species) and Mycobacterium duvalii (new species), Br. J. Exp. Pathol. 52 (1971) 627–637.  Back to cited text no. 8
B. Brezna, A.A. Khan, C.E. Cerniglia, Molecular characterization of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp., FEMS Microbiol. Lett. 223 (2003) 177–183.  Back to cited text no. 9
D. Dean-Ross, C.E. Cerniglia, Degradation of pyrene by Mycobacterium flavescens, Appl. Microbiol. Biotechnol. 46 (1996) 307–312.  Back to cited text no. 10
T. Adekambi, S. Ben Salah, M. Khlif, D. Raoult, M. Drancourt, Survival of environmental mycobacteria in Acanthamoeba polyphaga, Appl. Environ. Microbiol. 72 (2006) 5974–5981.  Back to cited text no. 11
I.B. Salah, E. Ghigo, M. Drancourt, Free-living amoeba, a training field for macrophage resistance of mycobacteria, Clin. Microbiol. Infect. 15 (2009) 894–905.  Back to cited text no. 12
G. Greub, D. Raoult, Microorganisms resistant to free-living amoebae, Clin. Microbiol. Rev. 17 (2004) 413–433.  Back to cited text no. 13
O. Lamrabet, F. Medie, M. Drancourt, Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis, PLoS One. 7 (2012) e29833.  Back to cited text no. 14
O. Lamrabet, M. Drancourt, Mycobacterium glivum illustrates size correlated relationships between Mycobacteria and Acanthamoeba polyphaga, Appl. Environ. Microbiol. 79 (2013) 1606–1611.  Back to cited text no. 15


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