Infection, Genetics and Evolution 72 (2019) 191–198

Contents lists available at ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Research Paper Association of non-tuberculous mycobacteria with leprae in environment of endemic regions in India T ⁎ Ravindra P. Turankara, , Vikram Singha, Hariom Guptaa, Vinay Kumar Pathaka, Madhvi Ahujaa, Itu Singha, Mallika Lavaniaa, Amit K. Dindab, Utpal Senguptaa a The Leprosy Mission Trust India, Stanley Browne Laboratory, TLM community Hospital Nand Nagari, New Delhi 110093, India b Department of Pathology, Institute/University: All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India

ARTICLE INFO ABSTRACT

Keywords: Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The pre- Mycobacterial species sent study was conducted to find out the presence of various species of NTM in leprosy endemic region along 16S rRNA gene target with Mycobacterium (M) leprae. Water and wet soil samples from the periphery of ponds used by the community Sequencing were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India. Samples were pro- FITC cessed and decontaminated followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction PGL-1 (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by sequencing M. leprae method. Indirect immune-fluorescent staining of M. leprae from soil was performed using M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by using MEGA-X 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 mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabiense and M. asiaticum from Indian environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out of all SGM, M. holsaticum, M. yongonense, M. seoulense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect immunofluorescent microcopy and PCR method from the same environmental samples. Phylogenetic tree was showing a close association between these NTMs and M. leprae in these samples. Several NTM species of pathogenic and nonpathogenic in nature 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.

1. Introduction to be important human pathogens and from the recent past reports suggest that there is an increasing trend in the incidence of NTM in- Nontuberculous mycobacteria (NTM), are ubiquitous environmental fections in human (Moore et al., 2010; Shojaei et al., 2011; Donohue opportunistic pathogens found in soil and water (Lavania et al., 2014; et al., 2015). NTMs have been classified according to their growth rate Parashar et al., 2004; Brook et al., 1984; Wang et al., 2004). Myco- and pigment formation (Runyon, 1959). Recently, major four groups of bacterium (M.) species are acid fast bacilli (AFB) and aerobic in nature human pathogens were recognized in mycobacterium genus such as 1. and include more than 196 different mycobacterial species (http:// M. tuberculosis complex, 2. M. leprae, 3. slow growing mycobacteria www.bacterio.net/mycobacterium.html, accessed on 21th April 2018; (SGM) and rapid growing mycobacteria (RGM) (Porvaznik et al., 2017). Euzbey, 1997). SGM included M. avium complex and are known to cause disseminated Several species of environmental mycobacterial species are known disease in immunodeficient subjects (Reichenbach et al., 2001;

Abbreviations: NTM, non-tuberculous mycobacteria; PCR, polymerase chain reaction; RGM, rapid growing mycobacteria; SGM, slow growing mycobacteria; PGL-1, phenol-glycolipid-1 antigen; AFB, acid fast bacilli; LJ, Lowenstein Jensen; ZN, Ziehl-Neelsen staining; FITC, fluorescein isothiocyanate; DNA, deoxyribonucleic acid; PBS, phosphate buffer saline; CTAB, cetyl trimethyl ammonium bromide; SDS, sodium dodecyl sulfate; NaOH, sodium hydroxide; Tris-EDTA, (T.E.) buffer; EDTA, ethylene diamine tetra acetic acid; NCBI, the National Center for Biotechnology Information ⁎ Corresponding author at: Stanley Browne Laboratory, TLM community Hospital Nand Nagari, Delhi 110093, India. E-mail addresses: [email protected], [email protected] (R.P. Turankar). https://doi.org/10.1016/j.meegid.2018.11.010 Received 29 May 2018; Received in revised form 31 August 2018; Accepted 11 November 2018 Available online 13 November 2018 1567-1348/ © 2018 Elsevier B.V. All rights reserved. R.P. Turankar et al. Infection, Genetics and Evolution 72 (2019) 191–198

Newport et al., 1996). RGM included species from M. abscessus complex 2.2. Sample collection which are known to cause chronic lung infections and skin and soft tissue infections. NTMs were isolated and identified from different parts Three hundred and eighty soils and 250 pond water samples were of India such as Chennai (Paramasivan et al., 1985; Kamala et al., collected from 100 leprosy patients' area. Ground soil was collected 1994), Maharashtra (Hardas and Jayaraman, 1984), Gujarat (Trivedi from the banks of bathing area of ponds by digging 2–5-in.-deep with et al., 1986), Bangalore (Chauhan, 1993), Amritsar (Aggarwal et al., the help of “hand trowel”. Water samples (50 ml) from the periphery of 1993), Agra (Parashar et al., 2009), Sevagram (Narang et al., 2009), but the pond of bathing area were collected in clean plastic containers and accurate picture of their geographical distribution is still not known. all samples were labeled with site code and the village name. The NTM caused diseases are not considered as serious diseases in most collected samples were transported to the laboratory at room tem- countries because of lacking evidence of human-to-human transmission perature (within 2 days) and stored at 4–8 °C till further use. and therefore, they are not considered as a public health problem. However, the organisms are ubiquitous in the environment, and sub- 3. Methods stantial evidence shows that the most common pulmonary, skin and subcutaneous infections and lymphadenitis can be caused by M. in- 3.1. Ziehl-Neelsen staining of environmental samples tracllulare (Falkinham et al., 2001), M. fortuitum (Petrini, 2006) and M. scrofulaceum (Wolinsky, 1995) respectively isolated from lining sub- Five grams of soil was dissolved in 50 ml distilled water and 50 ml urban water pipes. water samples were centrifuged at 400 ×g for 5 min. The supernatants M. leprae is a slow growing obligate intracellular pathogen, the were collected in 50 ml sterile tubes and centrifuged again at 8000 ×g causative agent of leprosy disease. It is an uncultivable mycobacterium for 15 min. Pellets were suspended in 0.1 ml of distilled water. Smears but it can grow in mouse foot pad and armadillo (Walsh et al., 1975, were prepared and subjected to Ziehl-Neelsen (ZN) staining for AFB 1981). Recently, several reports have shown isolation of M. leprae16S from all samples and were observed under binocular microscope rRNA from soil and water collected from the habitation of endemic (Olympus, Japan). regions of leprosy (Turankar et al., 2012, 2016; Lavania et al., 2008; Mohanty et al., 2016) indicating its viability and led us to find out the 3.2. Staining method for fluorescence microscopy role played by the environmental M. leprae in transmission of the dis- ease. Smears from environmental mycobacteria were subjected to fluor- The sugar epitopes of phenolic glycolipid eI (PGLeI), located on the escent staining and were observed under fluorescent microscope outermost surface of M. leprae act as a specific antigen and represent a (Olympus DP72). Smears were made on glass slides, kept at 37 °C for key molecule in penetration to nerve (Harboe et al., 2005) and recently 30 min for drying and fixing. The slides containing the fixed myco- PGL-1 has been shown to induce Schwann cells to liberate nitric oxide were washed twice with phosphate buffer saline (PBS). leading to demyelation and nerve damage (Madigan et al., 2017). In Primary rabbit polyclonal antibody against PGL-I (1:100) was added on contrast, the lipid core, called phenolphthiocerol dimycocerosates, is smears and incubated at 37 °C for 1 h in dark humidified chamber fol- conserved like other mycobacterial species and linked to different lowed by washing smears two times with PBS. FITC conjugated goat species-specific saccharide groups (Spencer and Brennan, 2011; Daffé anti-rabbit IgG antibody (Catalog No. F0382, Sigma-Aldrich, USA) and Laneelle, 1988). (1:10000) was added on smears and incubated at 37 °C for 1 h in dark Modern molecular biology methods are very useful for the identi- humidified chamber followed by rinsing smears two times with PBS. fication of NTMs using 16S rRNA gene target by PCR sequencing, The stained slides were observed immediately under oil immersion providing a faster and better ability to their accurate identification. It (X1000) in a fluorescent microscope (Olympus DP72 Japan). continues to serve as an important tool as an alternative to phenotypic identification methods. The direct sequencing of amplified deoxyr- 3.3. Growth of mycobacteria from environmental samples ibonucleic acid (DNA) from the 16S rRNA gene of Mycobacterium spe- cies is well described (Turenne et al., 2001). Environmental samples were processed and decontaminated as de- Considering the above our attempts have been made to isolate the scribed by the earlier published protocol (Parashar et al., 2004). Briefly, NTM species from the environment of leprosy endemic regions. environmental samples were treated with 3% sodium dodecyl sulfate Environmental M. leprae was detected by molecular method and im- (SDS), 4% sodium hydroxide (NaOH) and 2% cetrimide. Soil and water munofluorescent staining using PGL-1 antibody and PCR specific samples were centrifuged for 8000 ×g for 15 min at 4 °C. Pellets were primer 16S rRNA. Isolation of NTMs from pond water and soil by in vitro resuspended in 20 ml of treatment solution (3% SDS + 4% NaOH) and culture on Lowenstein Jensen (LJ) medium and further confirmed by divided into two parts: A and B. Part A was incubated for 15 min and B PCR containing specific primers of 16S rRNA gene targets followed by was incubated for 30 min at room temperature to obtain the growth of final confirmation by sequencing method. The neighbour-joining phy- rapid and slow growers, respectively. Both the suspensions were cen- logenetic tree was constructed using DNA sequencing of M. leprae and trifuged for 15 min after incubation at 8000 ×g. Pellets were treated different mycobacterial species by MEGA-X software. 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. 100 μl of decontaminated suspension from each was 2. Materials inoculated on LJ medium. Slants were incubated at 37 °C for 3 to 4 weeks. Primary colonies were subcultured on LJ medium again. Each 2.1. Site of samples collection sample was processed two times. Identification of growth was de- termined by growth rate and morphology of colonies. AFB staining was Prevalence rate (PR) of leprosy at Purulia was recorded as 3.55/ performed for phenotypic confirmation of mycobacteria. 10,000 in the year 2010 and that of Champa was recorded as 1.54/ 10,000 population. (http://www.districthealthstat.com and http:// 3.4. Genomic DNA isolation rltrird.cg.gov.in). We selected these sites because leprosy patients and their contacts are sharing the same pond and environment for their DNA was isolated from the mycobacterial growth in culture as de- daily routine activities of washing and bathing. scribed earlier (Somerville et al., 2005) with some modification. Growth from culture was collected in tube containing 400 μl 1× Tris- EDTA (T.E.) buffer, pH 8.0, and was heated in water bath for 15 min at

192 R.P. Turankar et al. Infection, Genetics and Evolution 72 (2019) 191–198

95 °C, followed by exposure of the sample to 80 °C for 10 min. Lysozyme growth on LJ media, out of which 55(24%) isolates from soil and 51 was added to each tube (final concentration, 1 mg/ml), followed by (32%) isolates from water were obtained. Out of these 106 isolates incubation at 37 °C for 2 h. SDS (10%) (final concentration, 1.1%) and 23(42%) RGM were from soil and 25(49%) from water. With regard to proteinase k (final concentration, 1 mg/ml) were added, and the tube SGM 32(58%) were from soil and 26(51%) from water samples. was vortexed gently followed by incubation at 60 °C for 60 min. A Interestingly, slow grower isolates were obtained more in number from mixture of cetyltrimethylammoniumammonium bromide (CTAB) and soil than that of water samples but rapid grower isolates were more 5 M NaCl (final concentration, 0.6 M) was added to each tube. The tube from water than soil samples (Table 1). was again vortexed until the suspension turned milky and was in- cubated at 60 °C for 20 min. Chloroform-isoamyl alcohol (24:1) (645 μl) 4.2.2. Nontuberculous mycobacteria isolate from Champa was added to each tube followed by vortexing and centrifugation in a One hundred and fifty soils and 90 water samples were processed micro centrifuge at 10,000 rpm for 10 min at room temperature. for growth on LJ media, out of which 31(21%) isolates from soil and 26 Aqueous phase was transferred into another clean centrifuge tube (29%) isolates from water were obtained. From soil and water samples and 600 μl of isopropanol was added for precipitation of DNA. Samples 11(35%) and 10(39%) RGM were respectively isolated. On the other were incubated at -20 °C for overnight. Sample was centrifuged at hand, 20(65%) and 16(61%) SGM were isolated from soil and water 10,000 rpm for 15 min and supernatant was discarded. The pellet was respectively. Here also slow growers were isolated more from soil washed with 70% ethanol by centrifugation at 10,000 rpm for 15 min. compared to that of water and rapid grower's isolates were more from Supernatant was discarded and pellet was kept for drying at 37 °C for water than that of soil (Table 1). 2–3 h. Pellet was finally suspended in 100 μl distilled water and stored at -20 °C for further downstream analysis. 4.3. PCR amplification of mycobacteria and sequencing

3.5. PCR amplification of mycobacteria using RLEP and 16S rRNA gene A total 106 isolates were obtained from environmental samples. PCR amplification was performed using universal primer 16S rRNA PCR amplification was performed using Veriti Thermal cycler gene of mycobacteria (Fig.2). Amplicons containing NTMs' specific PCR (Applied Biosystems, Singapore). A total of 25 μl of reaction volume products were sent to Eurofins Genomics India Pvt. Ltd. for commercial was prepared containing 2 μl of template DNA and primers at final sequencing. Sequences were blasted on the National Center for Bio- concentration of 0.5 μM (forward and reverse) and 1× Taq PCR master technology Information (NCBI) nucleotide Blast http://blast.ncbi. Mix (Qiagen). Mycobacterial specific universal primers were used (P1- nlm.nih.gov/Blast.cgi to confirm the sequences of the mycobacterial AGAGTTTGATCCTGGCTCAG; P3 – CCTGCACCGCAAAAAGCTTTCC) strains with the ones in the databases (Fig.3). for mycobacterial species and (16S rRNA-P2-TCGAACGGAAAGGTCTC Out of 106 isolates from Purulia, 23(42%) were RGM and included TAAAAAATC; P3-CCTGCACCGCAAAAAGCTTTCC), repetitive element M. porcinum 1(4%) M. psychrotolerans 1(4%) first time isolated from soil (RLEP-PS1TGCATGTCATGGCCTTGAGG PS2 CACCGATACCAGCGGAA of Indian samples followed by isolation of M. vaccae 2(9%) M. smegmatis GAA) for M. leprae gene primer as described. (Jadhav et al., 2005; 3(13%), M. gilvum 5(22%). M. fortuitum was most common species Turankar et al., 2015). The amplification was carried out in a thermal isolated from both soil 11(48%) and water 15(60%). Likewise, 25(49%) cycler at 95 °C for 15 min for initial denaturation and followed by RGM species like M. smegmatis 6(24%), M. gilvum 4(16%) were isolated 37 cycles of denaturation (94 °C for 2 min), annealing (58 °C for 2 min) from water samples. and extension (72 °C for 2 min) with a final extension at 72 °C for On the other hand, 32(58%) SGM isolated from soil samples. The 10 min. PCR product (227 bp for mycobacteria and 129 for M. leprae) species belonged to M. holsaticum 1(3%), M. yongonense 2(6%) which containing amplified fragment of the target region and was electro- were first time isolated from soil samples of Purulia district of India phoresed in a 2% agarose gel (Sigma) using Tris-Borate-ethylenedia- followed by isolation of M. scrofulaceum 5(16%), M. parascrofulaceum minetetraacetic acid (EDTA) buffer at 100 V' constant voltage. 4(12%), M. szulgai 2(6%), M. europaeum 3(9%), M. simiae 3(9%). M. intracellulare species was commonest SGM isolated from both soil 3.6. Data analysis 10(31%) and water 6(23%) samples. Other species of SGM 26(51%) were also noted to be present in water samples such as M. scrofulaceum PCR amplicons were outsourced for sequencing from Eurofins 5(19%), M. parascrofulaceum 5(19%), M. szulgai 2(7%), M. europaeum Genomics India Pvt. Ltd. The Finch TV software Version 1.4.0 was used 4(15%), M. simiae 1(4`%) and M. spp. 3(11%) (Table 2). for chromatogram analysis which was developed by Geospiza research Out of 57 isolates from Champa 11(35%) belonged to RGM which team. The chromatograms generated upon sequencing were subjected included M. asiaticum 1(9%), M. arabiense 1(9%) and M. alsense 1(9%), to BLAST analysis in NCBI GenBank and online 16S rRNA Based data first time isolated from soil samples of India in addition to isolation of bases. A stringent similarity index of 99–100% was kept with the type M. smegmatis 1(9%), M. gilvum 1(9%). M. fortuitum was most common strain in GenBank. species isolated from both soil 6(45%) and water 6(60%) samples. Other species of RGM 10(39%) which were isolated from water sam- 4. Results ples, such as M. smegmatis 2(20%) and M. gilvum 2(20%). Similarly, 20(65%) SGM were isolated from soil samples. For the 4.1. Ziehl-Neelsen and fluorescent staining method 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 AFB were observed in all soil and water samples (Fig.1a). ZN samples in India. In addition, M. scrofulaceum 2 (10%), M. para- staining was also performed from the growth in culture to confirm scrofulaceum 2(10%), M. spp 3 (9%), were also isolated. M. intracellulare presence of mycobacterial species in these samples. Indirect fluorescent was most commonly isolated SGM from both soil 4(20%) and water staining of mycobacterial smear from soil and water samples showed 5(31%) samples. In water samples, 26(51%) SGM noted as NTM species presence of fluorescent stained M. leprae (Fig. 1b). were M. scrofulaceum 2(12%), M. parascrofulaceum 3(19%), M. chimaera 2(12%), M. spp. 4(25%) (Table 2). 4.2. Mycobacterial growth on Lowenstein Jensen slant from environmental samples 4.4. PCR amplification of DNA

4.2.1. Nontuberculous mycobacteria isolate from Purulia M. leprae DNA PCR was performed using RLEP and 16S rRNA gene Two hundred thirty soils and 160 water samples were processed for and visualized on 2% agarose gel (Fig.4). We could detect 118 (31%) of

193 R.P. Turankar et al. Infection, Genetics and Evolution 72 (2019) 191–198

Fig. 1. a- Ziehl-Neelsen staining of Mycobacterium species from environmental samples (Pink colour rod shaped mycobacteria) (X1000). b- Fluorescent staining of Mycobacterium leprae from environmental samples (X1000). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 4.5. The neighbour-joining phylogenetic tree Non-tuberculous mycobacteria isolated from environmental samples from Purulia and Champa. The phylogenetic tree was constructed with different mycobacterial

Sample No. of No. of Rapid growing Slow growing fasta sequences and distance percent identity matrix was calculated by samples isolates mycobacteria mycobacteria MEGA-X software. It was noted that M. leprae was in close association obtained (RGM) (%) (SGM) (%) with M. simiae and M. smegmatis (Fig.5).

Purulia district of West Bengal Soil 230 55 (24%) 23 (42%) 32 (58%) 5. Discussion Water 160 51 (32%) 25 (49%) 26 (51%) Champa district of Chhattisgarh Leprosy is endemic in certain pockets of India and the country Soil 150 31 (21%) 11 (35%) 20 (65%) houses 63% of world population (WHO, 2017). PR of leprosy at Purulia Water 90 26 (29%) 10 (39%) 16 (61%) and Champa are much higher than the elimination figure of less than 1 per 10,000 and were recorded as 3.55/10,000 and 1.54/10,000 popu- lation respectively. (http://www.districthealthstat.com and http:// rltrird.cg.gov.in). In our laboratory, we 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 sanita- tion 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 de- pending on their local predisposition and degree of immune deficit in the host (Pinner, 1935; Wolinsky, 1979; Wallace Jr. et al., 1990). Paramasivan et al. (1985) described in his study from Chennai that M. fi Fig. 2. PCR Ampli cation of mycobacterial species using 16S rRNA gene target. avium intracellulare (MAI) to be the most frequently isolated species (22.6% of all NTM) followed by M. terrae (12.5%) and M. scrofulaceum M. leprae DNA from soil samples and 48(19%) from water samples. (10.5%). Later, Kamala et al. (1994) demonstrated that MAI and M. Further, we could detect viable M. leprae 16S rRNA, 53 (14%) from soil scrofulaceum in water and dust and later isolated this mycobacterium and 30(12%) from water samples. from the sputum samples of individuals in that area. Further, Parashar et al. (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 Uttar Pradesh, India. The present study was conducted in two leprosy endemic districts of

194 R.P. Turankar et al. Infection, Genetics and Evolution 72 (2019) 191–198

Fig. 3. Mycobacteria species confirmation by sequencing Method.

India and identified the presence of a variety of NTM species along with antibody in these samples. (Fig.1b). and by RLEP gene PCR of M. leprae. M. leprae from the same source of soil and water. We detected also It was observed that 31% water and 19% soil samples were positive for presence of M. leprae by indirect immunofluorescent staining using M. leprae DNA. Presence of viable M. leprae was observed in soil and FITC conjugated anti-rabbit IgG and rabbit polyclonal anti-PGL-1 water samples of endemic region which was also reported earlier by our

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

Runyon classification of NTM Purulia region Soil (32) Water (26) Champa region Soil (20) Water (16)

Type I M.simiae 3 (9%) 1 (4%) M. simiae 1 (5%) Photochromogen (slow growers) M. holsaticum 1 (3%)

Type II M. europaeum 3 (9%) 4 (15%) M. parascrofulaceum 2 (10%) 3 (19%) Scotochromogen (slow growers) M. scrofulaceum 5 (16%) 5 (19%) M. szulgai 1 (5%) M. parascrofulaceum 4 (12%) 5 (19%) M. europaeum 2 (10%) M. szulgai 2 (6%) 2 (7%) M. scrofulaceum 2 (10%) 2 (12%) M. spp. 2 (6%) 3 (11%) M. spp. 3 (9%) 4 (25%) M. seoulense, 1 (5%) M. chimaera 2 (10%) 2 (12%) 2%

Type III M. yongonense 2 (6%) M. intracellulare 4 (20%) 5 (31%) Non-chromogen (slow growers) M. intracellulare 10 (31%) 6 (23%) M. yongonense 1 (5%)

Rapid growers −23 −25 −11 −10 Type IV M. fortuitum 11 (48%) 15 (60%) M. fortuitum 6 (45%) 6 (60%) Rapid growers M. smegmatis 3 (13%) 6 (24%) M. smegmatis 1 (9%) 2 (20%) M. gilvum 5 (22%) 4 (16%) M. gilvum 1 (9%) 2 (20%) M. porcinum 1 (4%) M. asiaticum, 1 (9%) M. psychrotolerans 1 (4%) M. arabiense, 1 (9%) M. vaccae 2 (9%) M. alsense 1 (9%)

195 R.P. Turankar et al. Infection, Genetics and Evolution 72 (2019) 191–198

neutral pH. In contrast, as soil contain lesser concentration of oxygen than surface water with low pH and lower temperature it might be promoting the growth of SGM. M. fortuitum was the most common NTM which was isolated from soil (50%) and water (60%). NTM were con- firmed by PCR method using 16S rRNA gene followed by sequence analysis of 16S rRNA allowing their rapid classification of prokaryotes using universally distributed mycobacterial species (Woese, 1987). RGM such as M. porcinum, M. psychrotolerans, M. vaccae were iso- lated for the first time from leprosy endemic soil of India. M. vaccae was originally isolated from soil in an area of Uganda and has been reported to play an immunomodulating role for the host susceptibility to leprosy. M. psychrotolerans, an RGM, first isolated from pond water near aur- anium mine in Spain (Trujillo et al., 2004) has also been isolated in the present study. M. porcinum is the causative agent of submandibular Fig. 4. PCR amplification of RLEP gene region of Mycobacterium leprae obtained lymphadenitis in swine and having similar characteristics of M. for- from environmental samples from Purulia (WB). tuitum but it is di fferent at species level (Wallace Jr et al., 2004). M. yongonense causing pulmonary disease and M. holsaticum causing var- group and others (Lavania et al., 2008; Turankar et al., 2012, 2016; ious extra-pulmonary diseases were reported from Saudi Arabia Mohanty et al., 2016; Arraes et al., 2017). Similarly, in a recent study, (Varghese et al., 2017). Other NTMs were M. scrofulaceum, M. para- (Baliga et al., 2018) has shown presence of M. tuberculosis complex and scrofulaceum, M. szulgai, M. europaeum, M. simiae isolated in the present fl NTM in clinical samples by uorescence in situ hybridization with DNA study are pathogenic in nature and were reported earlier in clinical probes. Further, Forbes et al. (2018) also emphasized on the practice specimens. In the present study, M. europaeum which was reported fi guidelines for identi cation of mycobacteria using culture and mole- earlier from clinical samples in Europe (Tortoli et al., 2011) has been cular methods. We earlier reported presence of RGM, M. gilvum in isolated from the soil samples. bathing places from Purulia region of West Bengal (Lavania et al., Similarly, in water samples noted, M. fortuitum was the most 2014). In continuation with this, the present study noted presence of a common RGM and other species were M. smegmatis, M. gilvum. On the huge number of NTM isolates (SGM and RGM) in both soil and water other hand, M. intracellulare was the most common SGM and other samples from Purulia and Champa regions along with the presence of species were M. scrofulaceum, M. parascrofulaceum, M. szulgai, M. ff viable M. leprae. There are many factors that may a ect recovery of europaeum, M. simiae and M. spp were noted. M. fortuitum was first mycobacteria from water and soil. These factors, including deconta- characterized in 1991, is known for respiratory infections, a spectrum ff mination methods and climate conditions of the di erent regions, and of soft tissue and skeletal infections, bacteraemia and disseminated pH of soil and water, temperature and presence of Ca and K ions in soil disease in humans. M. szulgai is a slow-growing unusual pathogen for which play important role for the growth of mycobacteria. Soils with humans. Pulmonary disease is the most common manifestation of M. neutral pH have been shown to yield high frequency of NTM (Rahbar szulgai infection with clinical and radiological resemblance to tu- et al., 2010). The present study found more RGM in water than soil and berculosis (Marks et al., 1972). this may be due to higher multiplication rate of RGM in surface water Larger number (65%) NTM species were obtained from soils of which contain high oxygen concentration at 37 °C temperature with Champa region as compared to that 58% isolates of Purulia region.

Fig. 5. Phylogenetic tree of Non-tuberculous mycobacteria and Mycobacterium leprae.

196 R.P. Turankar et al. Infection, Genetics and Evolution 72 (2019) 191–198

NTM species M. asiaticum, M. arabiense, M. alsense and M. seoulense from Forbes, B.A., Hall, G.S., Miller, M.B., Novak, S.M., Rowlinson, M.C., Salfinger, M., Champa region were isolated for the first time in India. Phylogenetic Somoskövi, A., Warshauer, D.M., Wilson, M.L., 2018. Practice guidelines for clinical microbiology laboratories: mycobacteria. Clin. Microbiol. Rev. 31, e00038–e00047. analysis showed that M. leprae was having close association with RGM, Harboe, M., Aseffa, A., Leekassa, R., 2005. Challenges presented by nerve damage in M. smegmatis and SGM, M. simiae, which are known to be pathogenic to leprosy. Lepr. Rev. 76, 5–13. humans. Hardas, U.D., Jayaraman, V.S., 1984. Differential identification of mycobacteria. Ind. J. Tub. 31, 11–12. Jadhav, R.s, Kamble, R.R., Shinde, V.S., Edward, S., Edward, V.K., 2005. Use of reverse 6. Conclusion transcription polymerase chain reaction for the detection of Mycobacterium leprae in the slit-skin smears of leprosy patients. Ind. J. Lepr. 77, 116–127. The present study showed presence of more number of SGM species Kamala, T., Paramasivan, C.N., Herbert, D., Venkatesan, P., Prabhakar, R., 1994. Evaluation of procedures for isolation mycobacteria from soil and water. Appl. in soil compared to water and conversely water samples showed pre- Environ. Microbial. 60, 1021–1024. sence more number of RGM species compared to the soil samples. Lavania, M., Katoch, K., Katoch, V.M., et al., 2008. Detection of viable Mycobacterium Several pathogenic NTM were first time isolated from leprosy endemic leprae in soil samples: insights into possible sources of transmission of leprosy. Infect. Genet. Evol. 8, 627–631. area which are known to cause a variety of infections to human along Lavania, M., Turankar, R.P., Singh, I., Nigam, A., Sengupta, U., 2014. Detection of with M. leprae. Environmental mycobacterial species may sensitize first time from the bathing water of leprosy patient from – differently in different population and may pre-sensitize the population Purulia, West Bengal. Int. J. Mycobacteriol. (4), 286 289. Madigan, C.A., Cambier, C.J., Kelly-Scumpia, K.M., et al., 2017. A macrophage response for development of a proper acquired immunity or susceptibility to to mycobacterium leprae phenolic glycolipid initiates nerve damage in leprosy. Cell tuberculosis or leprosy (Rook and Stanford, 1981). Whether such a role 170, 973–985. is being played by these NTMs in these endemic regions is difficult to Marks, J., Jenkins, P.A., Tsukamura, M., 1972. Mycobacterium szulgai a new pathogen. – fi ff Tubercle 53, 210 214. explain from the present study. In conclusion, identi cation of di erent Mohanty, P.S., Naaz, F., Katara, D., Misba, L., Kumar, D., Dwivedi, D.K., Tiwari, A.K., species of NTMs by molecular PCR sequencing method is thus urgently Chauhan, D.S., Bansal, A.K., Tripathy, S.P., Katoch, K., 2016. Viability of needed for epidemiological and clinical purposes for adequate treat- Mycobacterium leprae in the environment and its role in leprosy dissemination. Ind. J. Dermatol. Venereol. Leprol. 82, 23–27. ment and management of patients. A future study will have to be car- Moore, J.E., Kruijshaar, M.E., Ormerod, L.P., Drobniewski, F., Abubakar, I., 2010. ried out to understand the role of these NTMs in susceptibility to le- Increasing reports of non-tuberculous mycobacteria in England, Wales and Northern prosy. Ireland, 1995-2006. BMC Public Health 10, 612. Narang, R., Narang, P., Mendiratta, D.K., 2009. Isolation and identification of non- tuberculous mycobacteria from water and soil in central India. Indian J. Med. Competing interests Microbiol. 27, 247–250. Newport, M.J., Huxley, C.M., Huston, S., Hawrylowicz, C.M., Oostra, B.A., Williamson, All authors declare that there are no competing interests. R., et al., 1996. A mutation in the interferon- gamma-receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335, 1941–1949. Paramasivan, C.N., Govindan, D., Prabhakar, R., Somasundaram, P.R., Subbammal, S., Acknowledgements Tripathy, S., 1985. Species level identification of non-tuberculous mycobacteria from south Indian BCG trial area during 1981. Tubercle 66, 69. fi ff Parashar, D., Chauhan, D.S., Sharma, V.D., Chauhan, A., Chauhan, S.V.S., Katoch, V.M., We acknowledge the nancial support rendered by E ect: Hope 2004. Optimization of procedures for isolation of mycobacteria from soil and water (The leprosy Mission Canada), grant number 205 and infrastructural samples obtained in northern India. Appl. Environ. Microbiol. 70, 3751–3753. support granted by the host institution - The Leprosy Mission Trust Parashar, D., Das, R., Chauhan, D.S., Sharma, V.D., Lavania, M., Yadav, V.S., Chauhan, S.V., Katoch, V.M., 2009. Identification of environmental mycobacteria isolated from India to carry out this research work at Stanley Browne Research Agra, north India by conventional & molecular approaches. Indian. J. Med. Res. 129, Laboratory – The Leprosy Mission Community Hospital, Shahdara – 424–431. New Delhi. We also acknowledge Dr. Mary Verghis, Director - TLM Petrini, B., 2006. Non-tuberculous mycobacterial infections. Scand. J. Infect. Dis. 38, 246–255. India, Dr.Joydeepa Darlong Head, Knowledge and management TLM - Pinner, M., 1935. Atypical acid-fast microorganisms. Am. Rev.Tuberc. 32, 424–445. India for their guidance and encouragement. We also wish to ac- Porvaznik, I., Solovič, I., Mokrý, J., 2017. Non-tuberculous mycobacteria: classification, knowledge support of the Superintendent and staff of TLM Hospital, diagnostics, and therapy. In: Pokorski, M. (Ed.), Respiratory Treatment and – Purulia. We are likewise grateful to Mr. Manish Gardia and Mr.Atul Roy Prevention. Adv Exp Med Biol. 944. pp. 19 25. Rahbar, M., Lamei, A., Babazadeh, H., Yavari, S.A., 2010. Isolation of rapid growing for assisting in the sample collection. mycobacteria from soil and water in Iran. Afr. J. Biotechnol. 9, 3618–3621. Reichenbach, J., Rosenzweig, S., Doffi nger, R., Dupuis, S., Holland, S.M., Casanova, J.L., fi References 2001. Mycobacterial diseases in primary immunode ciencies. Curr.Opin. Allergy. Clin. Immunol. 1, 503–511. Rook, G.A.W., Stanford, J.L., 1981. The heterogeneity of immune response to myco- Aggarwal, M., Jindal, N., Arora, R., Aggarwal, N.P., Arora, S., 1993. Non-tuberculous bacterial and the relevance of the common antigens to their pathogenicity. Ann. mycobacteria: the changing scenario at Amritsar. Indian J. Tuberc. 40, 25–27. Immunol. (Paris) 132D, 155–164. Arraes, M.L.B.M., Holanda, M.V., Lima, L.N.G.C., Sabadia, J.A.B., Duarte, C.R., Almeida, Runyon, E.H., 1959. Anonymous mycobacteria in pulmonary disease. Med. Clin. North R.L.F., Kendall, C., Kerr, L.R.S., Frota, C.C., 2017. Natural environmental water Am. 43, 273–290. sources in endemic regions of northeastern Brazil are potential reservoirs of viable Shojaei, H., Heidarieh, P., Hashemi, A., Feizabadi, M. M., Daei, Naser, A., 2011. Species Mycobacterium leprae. Mem. Inst. Oswaldo Cruz 112, 805–811. identification of neglected nontuberculous mycobacteria in a developing country. Baliga, S., Murphy, C., Sharon, L., Shenoy, S., Biranthabail, D., Weltman, H., Miller, S., Jpn. J. Infect. Dis. 64, 265–271. Ramasamy, R., Shah, J., 2018. Rapid method for detecting and differentiating Somerville, W., Thibert, L., Schwartzman, K., Behr, M.A., 2005. Extraction of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by Mycobacterium tuberculosis DNA: a question of containment. J. Clin. Microbiol. 43, fluorescence in situ hybridization with DNA probes. Int. J. Infect. Dis. https://doi. 2996–2997. org/10.1016/j.ijid.2018.07.011. Spencer, J.S., Brennan, P.J., 2011. The role of Mycobacterium leprae phenolic glycolipid I Brook, R.W., Parker, B. C., Gruft, H., Falkinham, J, O., 1984. Epidemiology of infections (PGL-I) in serodiagnosis and in the pathogenesis of leprosy. Lepr. Rev. 82, 344–357. by nontuberculous mycobacteria Numbers in eastern United States soils and corre- Tortoli, E., Böttger, E.C., Fabio, A., Falsen, E., Gitti, Z., Grottola, A., Klenk, H.P., Mannino, lation with soil characteristics. Am. Rev. Resp. Dis. 130, 630–633. Mariottini, A., Messinò, M., Pecorari, M., Rumpianesi, 2011. Mycobacterium euro- Chauhan, M.M., 1993. Nontuberculous mycobacteria isolated from an epidemiological paeum sp. nov., a species related to the survey in rural population of Bangalore district. Indian J. Tuberc. 40, 195–197. complex. Int. J. Syst. Evol. Microbiol. 61, 1606–1611. Daffé, M., Laneelle, M,.A., 1988. Distribution of phthiocerol diester, phenolic mycosides Trivedi, S.S., Desai, S.G., Trivedi, S.B., 1986. Non-tuberculous lung mycobacteriosis in and related compounds in mycobacteria. J. Gen. Microbiol. 134, 2049–2055. Gujarat. Ind. J. Tub. 33, 175–178. Donohue, M.J., Mistry, J.H., Donohue, J.M., O'Connell, K., King, D., Byran, J., Covert, T., Trujillo, M.E., Velázquez, E., Kroppenstedt, R.M., Schumann, P., Rivas, R., Mateos, P.F., Pfaller, S., 2015. Increased frequency of nontuberculous mycobacteria detection at Martínez-Molina, E., 2004. Mycobacterium psychrotolerans sp. Nov. isolated from pond potable water taps within the United States. Environ. Sci. Technol. 49, 6127–6133. water near a uranium mine. Int. J. Syst. Evol. Microbiol. 54, 1459–1463. Euzbey, J.P., 1997. List of bacterial names with standing in nomenclature genus Turankar, R.P., Lavania, M., Singh, M., et al., 2012. Dynamics of Mycobacterium leprae- Mycobacterium. Int. J. Syst. Bacteriol. 47, 590–592. Available: from. http://www. transmission in environmental context: deciphering the role of environment as a bacterio.net/mycobacterium.html (accessed 26.04.18). potential reservoir. Infect. Genet. Evol. 12, 121–126. Falkinham, J.O.I.I.I., Norton, C.D., LeChevallier, M.W., 2001. Factors influencing num- Turankar, R.P., Pandey, S., Lavania, M., Singh, I., Nigam, A., Darlong, J., Darlong, F., bers of Mycobacterium avium, Mycobacterium intracellulare and other mycobacteria in Sengupta, U., 2015. Comparative evaluation of PCR amplification of RLEP, 16S rRNA, drinking water distribution systems. Appl. Environ. Microbiol. 67, 1225–1231. rpoT and Sod a gene targets for detection of M. leprae DNA from clinical and

197 R.P. Turankar et al. Infection, Genetics and Evolution 72 (2019) 191–198

environmental samples. Int. J. Mycobacteriol. 4, 54–59. Walsh, G.P., Storrs, E.E., Burchfield, H.P., et al., 1975. Leprosy-like disease occurring Turankar, R.P., Lavania, M., Singh, M., Sengupta, U., Siva Sai, K., Jadhav, R.S., 2016. naturally in armadillos. J. Reticuloendotheliol. Soc. 18, 347s–351. Presence of viable Mycobacterium leprae in environmental specimens around houses Walsh, G.P., Meyers, W.M., Binford, C.H., et al., 1981. Leprosy – a zoonosis. Lepr. Rev. 52, of leprosy patients. Indian J. Med. Microbiol. 34, 315–321. 77–83. Turenne, C.Y., Tschetter, L., Wolfe, J., Kabani, A., 2001. Necessity of quality controlled Wang, Y., Ogawa, M., Fukuda, K., Miyamoto, H., Taniguchi, H., 2004. Isolation and 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium identification of Mycobacteria from soil at an illegal dumping site and landfills in species. J. Clin. Microbiol. 39, 3637–3648. Japan. Microbiol. Immunol. 50, 513–524. Varghese, B., Enani, M., Shoukri, M., Althawadi, S., Aljohani, S., Al- Hajoj, S., 2017. WHO, 2017.Global Leprosy Update 2017. Avaiable from http://www.who.int/wer. 92, Emergence of rare species of nontuberculous mycobacteria as potential pathogens in 501–520. (Accessed on 21.04.18). Saudi Arabian Clinical setting. PLoS Negl. Trop. Dis. 11, e0005288. Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51, 221–227. Wallace Jr., R.J., Brown-Elliott, B.A., Wilson, R.W., et al., 2004. Clinical and laboratory Wolinsky, E., 1979. Nontuberculous mycobacteria and associated disease. Am. Rev. features of Mycobacterium porcinum. J. Clin. Microbiol. 42, 5689–5697. Respir. Dis. 119, 10759. Wallace Jr., R.J., O'Brein, R., Glassroth, J., Raleigh, J., Dutta, A., 1990. Diagnosis and Wolinsky, E., 1995. Mycobacterial lymphadenitis in children: a prospective study of 105 treatment of disease caused by nontuberculous mycobacteria. Am. Rev. Respir. Dis. nontuberculous cases with long-term follow-up. Clin. Infect. Dis. 20, 954–963. 142, 940–953.

198