Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India

Accepted Manuscript Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India

Ravindra P. Turankar, Vikram Singh, Hariom Gupta, Vinay Kumar Pathak, Madhvi Ahuja, Itu Singh, Mallika Lavania, Amit Dinda, Utpal Sengupta PII: DOI: Reference:

S1567-1348(18)30883-9 https://doi.org/10.1016/j.meegid.2018.11.010 MEEGID 3709

To appear in:

Infection, Genetics and Evolution

Received date: Revised date: Accepted date:

29 May 2018 31 August 2018 11 November 2018

Please cite this article as: Ravindra P. Turankar, Vikram Singh, Hariom Gupta, Vinay Kumar Pathak, Madhvi Ahuja, Itu Singh, Mallika Lavania, Amit Dinda, Utpal Sengupta , Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India. Meegid (2018), https://doi.org/10.1016/ j.meegid.2018.11.010

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ACCEPTED MANUSCRIPT Association of Non-Tuberculous Mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India Ravindra

P.

Turankara,*

[email protected],

[email protected],

Vikram

Singha, Hariom Guptaa, Vinay Kumar Pathaka, Madhvi Ahujaa, Itu Singha, Mallika Lavaniaa,

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The leprosy Mission Trust India, Stanley Browne Laboratory, TLM community Hospital

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a

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Amit Dindab and Utpal Senguptaa

Nand Nagari, New Delhi-110093, India b

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Department of Pathology. Institute/University: All India Institute of Medical Sciences.

*

M

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Address: Ansari Nagar, New Delhi, 110 092, India

Corresponding author at: Stanley Browne Laboratory, TLM community Hospital Nand Nagari,

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Delhi-110093, India.

Abstract: Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The present study was conducted to find out the presence of various species of NTM in leprosy endemic region along with Mycobacterium (M) leprae. Water and wet soil samples from

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ACCEPTED MANUSCRIPT the periphery of ponds used by the community were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India.

Samples were processed and decontaminated

followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by

M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by

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using

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sequencing method. Indirect immune-fluorescent staining of M. leprae from soil was performed

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using MAFFT software. From 380 soil samples 86 NTM were isolated, out of which 34(40%) isolates were rapid growing mycobacteria (RGM) and 52(60%) isolates were slow growing

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mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out

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of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabinose and M. asiaticum from Indian

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environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out

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of all SGM, M. holsaticum, M. yongonense, M. seouense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was

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the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect PCR method from the same environmental samples.

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immunofluorescent microcopy and

Phylogenetic tree was showing a close association between these NTMs and M. leprae in these

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samples. Several species of pathogenic and nonpathogenic NTM species along with M. leprae were isolated from soil and pond water samples from leprosy endemic regions and these might be playing a role in causing disease and maintaining leprosy endemicity in India.

Keywords 2

ACCEPTED MANUSCRIPT Mycobacterial species, 16S rRNA gene target, sequencing, FITC, PGL-1, M. leprae Abbreviations NTM non-tuberculous

RGM rapid

growing

SGM slow

growing

MAFFT

multiple

alignment

using

Lowenstein

ZN

Ziehl-Neelsen

fast

PBS

phosphate

staining

sodium

trimethyl

acid saline

ammonium

bromide

dodecyl

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SDS

sulfate

NaOH sodium

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Tris-EDTA

EDTA ethylene

transform

Jensen

buffer

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CTAB cetyl

mycobacteria

bacilli

DNAdeoxyribonucleic

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FITC fluorescein isothiocyanate

mycobacteria

antigen

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LJ

Fourier

M

acid

fast

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PGL-1 phenol-glycolipid-1 AFB

reaction

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chain

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polymerase

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PCR

mycobacteria

hydroxide (T.E.) diamine

tetra

NCBI the National Center for Biotechnology Information.

1. Introduction:

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buffer acetic

acid

ACCEPTED MANUSCRIPT Nontuberculous mycobacteria (NTM), are ubiquitous environmental opportunistic pathogens found in soil and water (Lavania et al., 2014; Parashar et al., 2004; Brook et al., 1984; Wang et al., 2004). Mycobacterium (M.) species are acid fast bacilli (AFB) and aerobic in nature and include

more

than

196

different

mycobacterial

species

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(http://www.bacterio.net/mycobacterium.html, accessed on 21th April 2018; Euzéby, 1997).

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Several species of environmental mycobacterial species are known to be important human

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pathogens and from the recent past reports suggest that there is an increasing trend in the incidence of NTM infections in human (Moore et al., 2010; Shojaei et al., 2011; Donohue et al.,

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2015). NTMs have been classified according to their growth rate and pigment formation

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(Runyon 1959). Recently, major four group of human pathogens were recognized in mycobacterium genus such as 1. M. tuberculosis complex, 2. M. leprae, 3. slow growing

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mycobacteria (SGM) and rapid growing mycobacteria (RGM) (Porvaznik et al., 2017). SGM

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included M. avium complex and are known to cause disseminated disease in immunodeficient subjects (Reichenbach et al., 2001; Newport et al., 1996). RGM included species from M.

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abscessus complex which are known to cause chronic lung infections and skin and soft tissue

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infections. NTMs were isolated and identified from different parts of India such as Chennai (Paramasivan et al., 1981; Kamala et al., 1994), Maharashtra (Hardas et al., 1984), Gujarat

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(Trivedi et al., 1986), Bangalore (Chauhan, 1993), Amritsar (Aggarwal et al., 1993), Agra (Parashar et al., 2009), Sevagram (Narang et al., 2009), but accurate picture of their geographical distribution is still not known. NTM caused diseases are not considered as serious diseases in most countries because of lacking evidence of human-to-human transmission and therefore, they are not considered as a public health problem. However, the organisms are ubiquitous in the environment, and substantial evidence shows that the most common pulmonary, skin and

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ACCEPTED MANUSCRIPT subcutaneous infections and lymphadenitis can be caused by M. intracllulare (Falkingham, 2001), M. fortuitum (Petrini, 2006) and M. scrofulaceum (Wolinsky, 1995) respectively isolated from lining suburban water pipes. M. leprae is a slow growing obligate intracellular pathogen, the causative agent of leprosy

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disease. It is an uncultivable mycobacterium but it can grow in mouse foot pad and armadillo

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(Walsh et al., 1975, 1981). Recently, several reports have shown isolation of M. leprae16S rRNA

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from soil and water collected from the habitation of endemic regions of leprosy (Turankar et al., 2012; 2016; Lavania et al., 2008; Mohanty et al., 2016) indicating its viability and led us to find

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out the role played by the environmental M. leprae in transmission of the disease.

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The sugar epitopes of phenolic glycolipid -I (PGL-I), located on the outermost surface of M. leprae act as a specific antigen and represent a key molecule in penetration to nerve (Harboe et recently PGL-1 has been shown to induce Schwann cells to liberate nitric oxide

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al., 2005) and

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leading to demyelation and nerve damage (Madigan et al., 2017). In contrast, the lipid core, called phenolphthiocerol dimycocerosates, is conserved like other mycobacterial species and

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linked to different species-specific saccharide groups (Spencer et al., 2011; Daffe et al., 1988).

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Modern molecular biology methods are very useful for the identification of NTMs using 16S rRNA gene target by PCR sequencing, providing a faster and better ability to their accurate

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identification. It continues to serve as an important tool as an alternative to phenotypic identification methods. The direct sequencing of amplified deoxyribonucleic acid (DNA) from the 16S rRNA gene of Mycobacterium species is well described (Turenne et al., 2001). Considering the above our attempts have been made to isolate the NTM species from the environment of leprosy endemic regions. Environmental M. leprae was detected by molecular method and immunofluorescent staining using PGL-1 antibody and PCR specific primer 16S

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ACCEPTED MANUSCRIPT rRNA. Isolation of NTMs from pond water and soil by in vitro culture on Lowenstein Jensen (LJ) medium and further confirmed by PCR containing specific primers of 16S rRNA gene targets followed by final confirmation by sequencing method. The neighbor-joining phylogenetic tree was constructed using DNA sequencing of M. leprae and different mycobacterial species by

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software Multiple Alignment using Fast Fourier Transform (MAFFT).

2. Materials:

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2.1 Site of samples collection.

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Prevalence rate (PR) of leprosy at Purulia was recorded as 3.55/10,000 in the year 2010 and that of Champa was recorded as 1.54/10,000 population. (http://www.districthealthstat.com and

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http://rltrird.cg.gov.in). We selected these sites because leprosy patients and their contacts are

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sharing the same pond and environment for their daily routine activities of washing and bathing.

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2.2 Sample collection: Three hundred and fifty soils and 250 pond water samples were collected from 100 leprosy patients’ area. Ground soil was collected from the banks of bathing area of

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ponds by digging 2-5-inch-deep with the help of “hand trowel”. Water samples (50ml) from the

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periphery of the pond of bathing area were collected in clean plastic containers and all samples were labeled with site code and the village name. The collected samples were transported to the laboratory at room temperature (within 2 days) and stored at 4-8 0 C till further use.

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ACCEPTED MANUSCRIPT 3. Methods 3.1 Ziehl-Neelsen staining of environmental samples: Five grams of soil was dissolved in 50ml distilled water and 50 ml water samples were

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centrifuged at 400Xg for 5 min. The supernatants were collected in 50 ml sterile tubes and

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centrifuged again at 8000Xg for 15 min. Pellets were suspended in 0.1ml of distilled water.

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Smears were prepared and subjected to Ziehl-Neelsen (ZN) staining for AFB from all samples

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3.2 Staining method for fluorescence microscopy

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and were observed under binocular microscope (Olympus, Japan).

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Smears from environmental mycobacteria were subjected to fluorescent staining and were observed under fluorescent microscope (Olympus DP72). Smears were made on glass slides,

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kept at 370 C for 30 minutes for drying and fixing. The slides containing the fixed mycobacteria

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were washed twice with phosphate buffer saline (PBS). Primary rabbit polyclonal antibody against PGL-I (1:100) was added on smears and incubated at 370 C for 1 hour in dark humidified

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chamber followed by washing smears two times with PBS. FITC conjugated goat anti-rabbit IgG

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antibody (Catalog No. F0382, Sigma-Aldrich, USA) (1:10000) was added on smears and incubated at 370 C for 1 hour in dark humidified chamber followed by rinsing smears two times with PBS. The stained slides were observed immediately under oil immersion (X400) in a fluorescent microscope (Olympus DP72 Japan).

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ACCEPTED MANUSCRIPT 3.3 Growth of mycobacteria from environmental samples Environmental samples were processed and decontaminated as described by the earlier published protocol (Parashar et al., 2004). Briefly, environmental samples were treated with 3% sodium dodecyl sulfate (SDS), 4% sodium hydroxide (NaOH) and 2% cetrimide. Soil and water samples

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were centrifuged for 8000Xg for 15 min at 4 0 C. Pellets were resuspended in 20 ml of treatment solution (3% SDS + 4% NaOH) and divided into two parts: A and B. Part A was incubated for

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15 min and B was incubated for 30 min at room temperature to obtain the growth of rapid and

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slow growers, respectively. Both the suspensions were centrifuged for 15 min after incubation at 8000Xg. Pellets were treated with 2% cetrimide and incubated for 5 and 15 min for rapid and

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slow growers, respectively. After incubation sediments were washed twice with sterile water. 100μl of decontaminated suspension from each was inoculated on LJ medium. Slants were

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incubated at 370 C for 3 to 4 weeks. Primary colonies were subculture on LJ medium again. Each

AFB staining was performed

for phenotypic confirmation of

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morphology of colonies.

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sample was processed two times. Identification of growth was determined by growth rate and

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mycobacteria.

3.4 Genomic DNA isolation

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DNA was isolated from the mycobacterial growth in culture as described earlier (Somerville et al., 2005) with some modification. Growth from culture was collected in tube containing 400μl 1x Tris-EDTA (T.E.) buffer, pH 8.0, and was heated in water bath for 15 min at 95OC, followed by exposure of the sample to 80OC for 10 minutes. Lysozyme was added to each tube (final concentration, 1 mg/ml), followed by incubation at 37 OC for 2 hrs.

SDS (10%) (final

concentration, 1.1%) and proteinase k (final concentration, 1 mg/ml) were added, and the tube was vortexed

gently followed

by incubation at 60OC 8

for 60minutes. A mixture of

ACCEPTED MANUSCRIPT cetyltrimethylammoniumammonium bromide (CTAB) and 5M NaCl (final concentration, 0.6M) was added to each tube. The tube was again vortexed until the suspension turned milky and was incubated at 60o C for 20 min. Chloroform-isoamyl alcohol (24:1) (645μl) was added to each tube followed by vortexing and centrifugation in a micro centrifuge at 10,000 rpm for 10 min at

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room temperature.

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Aqueous phase was transferred into another clean centrifuge tube and 600μl of isopropanol was

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added for precipitation of DNA. Samples were incubated at -20o C for overnight. Sample was centrifuged at 10,000 rpm for 15min and supernatant was discarded. The pellet was washed with

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70% ethanol by centrifugation at 10,000 rpm for 15min. Supernatant was discarded and pellet

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was kept for drying at 37o C for 2-3 hrs. Pellet was finally suspended in 100μl distilled water and

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stored at -20o C for further downstream analysis.

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3.5 PCR amplification of mycobacteria using RLEP and 16S rRNA gene

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PCR amplification was performed using Veriti Thermal cycler (Applied Biosystems, Singapore). A total of 25μl of reaction volume was prepared containing 2μl of template DNA and primers at

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final concentration of 0.5μM (forward and reverse) and 1X Taq PCR master Mix (Qiagen).

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Mycobacterial specific universal primers were used (P1- AGAGTTTGATCCTGGCTCAG; P3 – CCTGCACCGCAAAAAGCTTTCC)

for

mycobacterial

TCGAACGGAAAGGTCTCTAAAAAATC; repetitive

element

species

and

(16S

rRNA-P2-

P3-CCTGCACCGCAAAAAGCTTTCC),

(RLEP-PS1TGCATGTCATGGCCTTGAGG

PS2

CACCGATACCAGCGGAAGAA) for M. leprae gene primer as described. (Jadhav et al., 2005, Turankar et al., 2015). The amplification was carried out in a thermal cycler at 95° C for 15 min for initial denaturation and followed by 37 cycles of denaturation (94°C for 2 min), annealing 9

ACCEPTED MANUSCRIPT (58°C for 2 min) and extension (72°C for 2 min) with a final extension at 72°C for 10 min. PCR product (227 bp for mycobacteria and 129 for M. leprae) containing amplified fragment of the target region and was electrophoresed in a 2% agarose gel (Sigma) using Tris-Borate-

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ethylenediaminetetraacetic acid (EDTA) buffer at 100 volts’ constant voltage.

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3.6 Data analysis

PCR amplicons were outsourced for sequencing from Eurofins Genomics India Pvt. Ltd. The

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Finch TV software Version 1.4.0 was used for chromatogram analysis which was developed by

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Geospiza research team. The chromatograms generated upon sequencing were subjected to BLAST analysis in NCBI GenBank and online 16S rRNA Based data bases. A stringent

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similarity index of 99–100% was kept with the type strain in GenBank.

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4. Results

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4.1 Ziehl-Neelsen and fluorescent staining method

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AFB were observed in all soil and water samples (Fig.1a). ZN staining was also performed from the growth in culture to confirm presence of mycobacterial species in these samples. Indirect

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fluorescent staining of mycobacterial smear from soil and water samples showed presence of fluorescent stained M. leprae (Fig 1b). 4.2 Mycobacterial growth on Lowenstein Jensen slant from environmental samples Nontuberculous mycobacteria isolate from Purulia Two hundred thirty soils and 160 water samples were processed for growth on LJ media, out of which 55(24%) isolates from soil and 51 (32%) isolates from water were obtained. Out of these 10

ACCEPTED MANUSCRIPT 106 isolates 23(42%) RGM were from soil and 25(49%) from water.

With regard to SGM

32(58%) were from soil and 26(51%) from water samples. Interestingly, slow grower isolates were obtained more in number from soil than that of water samples but rapid grower isolates were more from water than soil samples. (Table 1)

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4.3 Nontuberculous mycobacteria isolate from Champa

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One hundred and fifty soils and 90 water samples were processed for growth on LJ media, out of which 31(21%) isolates from soil and 26 (35%) isolates from water were obtained. From soil and

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water samples 11(35%) and 10(39%) RGM were respectively isolated. On the other hand, 20(65%) and 16(61%) SGM were isolated from soil and water respectively. Here also slow

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more from water than that of soil. (Table 1).

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growers were isolated more from soil compared to that of water and rapid grower’s isolates were

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4.4 PCR amplification of mycobacteria and sequencing A total 106 isolates were obtained from environmental samples. PCR amplification was

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performed using universal primer 16S rRNA gene of mycobacteria (Fig.2). Amplicons containing

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NTMs’ specific PCR products were sent to Eurofins Genomics India Pvt. Ltd. for commercial sequencing. Sequences were blasted on the National Center for Biotechnology Information (NCBI) nucleotide Blast http://blast.ncbi. nlm.nih.gov/Blast.cgi to confirm the sequences of the mycobacterial strains with the ones in the databases (Fig.3). Out of 106 isolates from Purulia, 23(42%) were RGM and included M. porcinum 1(4%) M. psychrotolerans 1(4%) first time isolated from soil Indian samples followed by isolation of M. vaccae 2(9%) M. smegmatis 3(13%), M. gilvum 5(22%). M. fortuitum was most common 11

ACCEPTED MANUSCRIPT speciesisolated from both soil 11(48%) and water 15(60%). Likewise, 25(49%) RGM species like M. smegmatis 6(24%), M. gilvum 4(16%) were isolated from water samples. On the other hand, 32(58%) SGMwere isolated from soil samples SGM. The species belonged to M. holsaticum 1(3%), M. yongonense 2(6%) which were first time isolated from soil samples of

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Purulia district of India followed by isolation of M. scrofulaceum 5(16%), M. parascrofulaceum 4(12%), M. szulgai 2(6%), M. europaeum 3(9%), M. simiae 3(9%). M. intracellulare species

in water samples such as M. scrofulaceum

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of SGM 26(51%) were also noted to be present

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was commonest SGM isolated from both soil 10(31%) and water 6(23%) samples. Other species

1(4`%) and some M. spp. 3(11%) (Table 2).

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5(19%), M. parascrofulaceum 5(19%), M. szulgai 2(7%), M. europaeum 4(15%), M. simiae

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Out of 57 isolates from Champa 11(35%) belonged to RGM which included M. asiaticum

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1(9%), M. arabiense 1(9%) and M. alsense 1(9%), first time isolated from soil samples of India in addition to isolation of M. smegmatis 1(9%), M. gilvum 1(9%). M. fortuitum was most

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common species isolated from both soil 6(54%) and water 6(60%) samples. Other species of

gilvum 2(20%).

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RGM 10(39%) which were isolated from water samples, are M. smegmatis 2(20%) and M.

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Similarly, 20(65%) SGM were isolated from soil samples. For the first time M. chimaera 2(10%), M. yongonense 1(5%), M.seoulense 1(5%), M. szulgai 1(5%), M. europaeum 2(10%), have been

isolated from soil samples in India. In addition, M. scrofulaceum 2 (10%), M.

parascrofulaceum 2(10%), M. spp3 (9%), were also isolated.

M. intracellulare was most

commonly isolated SGM from both soil 4(20%) and water 5(31%) samples. In water samples, 26(51%) SGM were noted as NTM species were M. scrofulaceum 2(12%), M. parascrofulaceum 3(19%), M. chimaera 2(12%), M. spp. 4(25%) (Table 2). 12

ACCEPTED MANUSCRIPT 4.5 PCR Amplification of Mycobacterium leprae DNA M. leprae DNA PCR was performed using RLEP and 16S rRNA gene and visualized on 2% agarose gel (Fig.4). We could detect 118 (31%) of M. leprae DNA from soil samples and 48(19%) from water samples. Further, we could detect viable M. leprae 16S rRNA, 53 (14%)

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from soil and 30(12%) from water samples.

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4.6 The neighbour-joining phylogenetic tree

The phylogenetic tree was constructed with different mycobacterial fasta sequences and distance

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percent identity matrix was calculated by MAFFT software. It was noted that M. leprae was in

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close association with M. smegmatis and M. holsaticum and M. yongonense (Fig.5).

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5. Discussion

Leprosy is endemic in certain pockets of India and the country houses 63% of world population

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(WHO, 2017). PR of leprosy at Purulia and Champa are much higher than the elimination figure

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of less than 1 per 10,000 and were recorded as 3.55/10,000 and 1.54/10,000 population respectively. (http://www.districthealthstat.com and http://rltrird.cg.gov.in). In our laboratory, we

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observed presence of viable M. leprae (RT-PCR) using 16S rRNA gene target from soil and water samples from these areas which were also reported earlier by our group (Turankar et al., 2012). Healthy individuals and leprosy patients in these villages

are using pond water for their

routine bathing and washing purposes and hence their environmental conditions or sanitation of the ponds are in a very poor state. In recent decades, NTM are also increasingly being recognized as pathogenic to human. Although the exact mechanism of pathogenesis of NTM in human infection is not known but these are causing localized or disseminated disease depending on their 13

ACCEPTED MANUSCRIPT local predisposition and degree of immune deficit in the host (Pinner, 1935; Wolinsky, 1979; Wallace et al., 1990). Paramasivan et al, (1985) described in his study from Chennai that M. avium intracellulare (MAI) to be the most frequently isolated species (22.6% of all NTM) followed by M. terrae (12.5%) and M. scrofulaceum (10.5%). Later, Kamla et al, (1994)

Further, Parasher et al,

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mycobacterium from the sputum samples of individuals in that area.

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demonstrated that MAI and M. scrofulaceum in water and dust and later isolated this

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(2004) have demonstrated the presence of M. fortuitum, M. avium, M. kansasii, M. terrae, and M. chelonae in water and M. avium, M. terrae and M. chelonae in soil samples of Agra region of

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Uttar Pradesh, India.

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The present study was conducted in two leprosy endemic districts of India and identified the

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presence of a variety of NTM species along with M. leprae from the same source of soil and water. We detected also presence of M. leprae by indirect immunofluorescent staining using

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FITC conjugated anti-rabbit IgG and rabbit polyclonal anti-PGL-1 antibody in these samples.

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(Fig.1b). and by RLEP gene PCR of M. leprae. It was observed that 31% water and 19% soil samples were positive for M. leprae DNA. Presence of viable M. leprae was observed in soil

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and water samples of endemic region which was also reported earlier by our group and others

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(Lavania et al., 2008; Turankar et al., 2012; 2016 Mohanty et al., 2016; Arraes et al., 2017). Similarly, in a recent study, (Baliga et al., 2018) has shown presence of M. tuberculosis complex and NTM in clinical samples by fluorescence in situ hybridization with DNA probes. Further, Forbes et al, (2018) also emphasized on the practice guidelines for identification of mycobacteria using culture and molecular methods. We earlier reported presence of RGM, M. gilvum in bathing places from Purulia region of West Bengal (Lavania et al., 2014). In continuation with this, the present study noted presence of a huge number of NTM isolates (SGM and RGM) in

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ACCEPTED MANUSCRIPT both soil and water samples from Purulia and Champa regions along with the presence of viable M. leprae. There are many factors that may affect recovery of mycobacteria from water and soil. These factors, including decontamination methods and climate conditions of the different regions, are pH of soil and water, temperature and presence of Ca and K ions in soil which play

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important role for the growth of mycobacteria. Soils with neutral pH have been shown to yield

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high frequency of NTM (Rahbar et al., 2010). The present study found more RGM in water than

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soil and this may be due to higher multiplication rate of RGM in surface water which contain high oxygen concentration at 370 C temperature with neutral pH. In contrast, as soil contain lesser

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concentration of oxygen than surface water with low pH and lower temperature it might be

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promoting the growth of SGM. M. fortuitum was the most common NTM which was isolated from soil (50%) and water (60%). NTM were confirmed by PCR method using 16S rRNA gene

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followed by sequence analysis of 16S rRNA allowing their rapid classification of prokaryotes

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using universally distributed mycobacterial species (Woese, 1987).

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RGM such as M. porcinum, M. psychrotolerans, M. vaccaewere isolated for the first time from leprosy endemic soil of India. M. vaccae was originally isolated from soil in an area of Uganda

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and has been reported to play an immunomodulating role for the host susceptibility to leprosy.

(Trujillo

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M. psychrotolerans, an RGM, first isolated from pond water near auranium mine in Spain et al., 2004) has also been isolated in the present study. M. porcinum is the causative

agent of submandibular lymphadenitis in swine and having similar characteristics of M. fortuitum but it is different at species level (Wallace et al., 2004). M. yongonense causing pulmonary disease and M. holsaticum causing various extra-pulmonary diseases were reported from Saudi Arabia (Varghese et al., 2017). Other NTMs were M. scrofulaceum, M. parascrofulaceum, M. szulgai, M. europaeum, M. simiae isolated in the present study are

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ACCEPTED MANUSCRIPT pathogenic in nature and were reported earlier in clinical specimens.

In the present study, M.

europaeum which was reported earlier from clinical samples in Europe (Tortoli et al., 2011) has been isolated from the soil samples. Similarly, in water samples, M. fortuitum was the most common RGM, Other species were M.

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smegmatis, M. gilvum. M. intracellulare was the most common SGM and other species were M. scrofulaceum, M. parascrofulaceum, M. szulgai, M. europaeum, M. simiae and some M. spp. M.

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fortuitum was first characterized in 1991, is known for respiratory infections, a spectrum of soft

slow-growing

unusual pathogen for humans.

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tissue and skeletal infections, bacteraemia and disseminated disease in humans. M. szulgai is a Pulmonary disease is the most common

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manifestation of M. szulgai infection with clinical and radiological resemblance to tuberculosis

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(Marks et al., 1972).

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Larger number (65%) NTM species were obtained from soils of Champa region as compared to that 58% isolates of Purulia region. NTM species M. asiaticum, M. arabiense, M. alsense and M.

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seoulense from Champa region were isolated for the first time in India. Phylogenetic analysis

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showed that M. leprae was having close association with RGM, M. smegmatis and SGM, M.

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simiae, M. holsaticum and M. yongonense which are known to be pathogenic to humans.

6. Conclusion,

The present study showed presence of more number of SGM species in soil compared to water and conversely water samples showed presence more number of RGM species compared to the soil samples. Several pathogenic NTM were first time isolated from leprosy endemic area which are known to cause a variety of infections to human along with M. leprae. Environmental 16

ACCEPTED MANUSCRIPT mycobacterial species may sensitize differently in different population and may pre-sensitize the population for development of a proper acquired immunity or susceptibility to tuberculosis or leprosy (Rook and Stanford, 1981). Whether such a role is being played by these NTMs in these endemic regions is difficult to explain from the present study. In conclusion, identification of

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different species of NTMs by molecular PCR sequencing method is thus urgently needed for

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epidemiological and clinical purposes for adequate treatment and management of patients. A

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future study will have to be carried out to understand the role of these NTMs in susceptibility to

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leprosy. Competing interests

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All authors declare that there are no competing interests.

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Acknowledgements

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We acknowledge the financial support rendered by Effect: Hope (The leprosy Mission Canada) and infrastructural support granted by the host institution - The Leprosy Mission Trust India to

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carry out this research work at Stanley Browne Research Laboratory – The Leprosy Mission

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Community Hospital, Shahdara – New Delhi. We also acknowledge Dr. Mary Verghis, Director - TLM India, Dr.Joydeepa Darlong Head, Knowledge and management TLM - India for their guidance and encouragement. We also wish to acknowledge support of the Superintendent and staff of TLM Hospital, Purulia. We are likewise grateful to Mr Manish Gardia and Mr.Atul Roy for assisting in the sample collection.

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Table: 1. Non-tuberculous mycobacteria isolated from environmental samples from Purulia and Champa. Rapid growing mycobacteria (RGM) (% )

Slow growing mycobacteria (SGM) (% )

Purulia District of West Bangal Soil 230 55 (24%) Water 160 51 (32%)

23 (42%) 25 (49%)

32 (58%) 26 (51%)

Champa District of Chhattisgarh Soil 150 31 (21%) Water 90 26 (35%)

11 (35%) 10 (39%)

20 (65%) 16 (61%)

Sample

No. of samples

No. of isolates obtained

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ACCEPTED MANUSCRIPT Table: 2. Comparison of Non-tuberculous mycobacteria isolated from Purulia and Champa.

Champa region

1 (4%)

M. simiae

M.simiae

3 (9%)

Photochromogen (slow growers)

M. holsaticum

1 (3%)

Type II

M. europaeum

3 (9%)

4 (15%)

M. scrofulaceum

5 (16%)

5 (19%)

Scotochromogen (slow growers)

M. parascrofulaceum M. szulgai

M. yongonense

Non-chromogen (slow growers)

M. intracellulare

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Rapid growers

M. fortuitum M. smegmatis M. gilvum M. Porcinum M. Psychrotolerans M. vaccae

M. parascrofulaceum

2 (10%)

2 (7%) 3 (11%)

M. scrofulaceum M. spp. M. seoulense, M. chimaera

2 (10%) 2 (10%) 3 (9%) 1 (5%) 2 (10%)

6 (23%)

-23 11 (48%) 3 (13%) 5 (22%) 1 (4%) 1 (4%) 2 (9%)

-25 15 (60%) 6 (24%) 4 (16%)

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3 (19%)

M. szulgai

1 (5%)

10 (31%)

Water (16)

1 (5%)

M. europaeum

2 (6%)

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Type III

Rapid growers Type IV

4 (12%) 2 (6%) 2 (6%)

Soil (20)

5 (19%)

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M. spp.

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Type I

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Soil (32)

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Water (26)

Purulia region

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of

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Runyon Classification NTM

2 (12%) 4 (25%) 2 (12%)

M. intracellulare

4 (20%)

M. yongonense

1 (5%)

5 (31%)

M. fortuitum M. smegmatis M. gilvum M. asiaticum, M. arabiense, M. alsense M. seoulense

-11 6 (45%) 1 (9%) 1 (9%) 1 (9%) 1 (9%) 1 (9%) 1 (9%)

-10 6 (60%) 2 (20%) 2 (20%)

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Figure 1 a- Ziehl-Neelsen staining of Mycobacterium species from environmental samples (Pink

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colour rod shaped mycobacteria) (X400). b- Fluorescent staining of Mycobacterium leprae from

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environmental samples (X400).

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Figure 2 PCR Amplification of mycobacterial species using 16S rRNA gene target.

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Figure 3 Mycobacteria species confirmation by sequencing Method.

environmental samples from Purulia (WB).

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Figure 4 PCR amplification of RLEP gene region of Mycobacterium leprae obtained from

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associated in leprosy endemic area

   

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

For the first time a large number of NTM species have been isolated on LJ medium from environmental samples of leprosy endemic regions. It was found that slow growing species of mycobacteria are present in more number in the environmental soil as compared to water. On the other hand, rapid growing mycobacteria species were more in number in water samples which are known to cause a variety of infections to human. In both soil and water samples M. leprae has been found to be present along with NTM species. Mycobacterial species were confirmed by PCR sequencing method using universal primer of 16S rRNA gene target. Environmental M. leprae was detected by M. leprae specific primer 16S rRNA and RLEP gene PCR method and also by immunofluorescent staining using PGL-1 antibody. The neighbour-joining phylogenetic tree was constructed using DNA sequencing of M. leprae and different mycobacterial species by using software MAFFT.

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

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Highlights: 

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Figure 5 Phylogenetic tree of Non-tuberculous mycobacteria and Mycobacterium leprae

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5