Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 6 (june 2021) : 642-646

Whole Genome Sequencing and Analysis of Mycobacteroides chelonae M77 Isolated from Cow Milk from the Hill State of Meghalaya, India

Esther Vise1, Sandeep Ghatak1, Akshay Garg2, Amarjeet Karam1, Chendu Bharat Prasad1, A. Arun Prince Milton1, Ingudam Shakuntala2, Kekungu Puro1, Raj kumari Sanjukta1, Arnab Sen1, Samir Das1,*
1Division of Animal Health, ICAR Research Complex for NEH Region, Umiam-793 103, Meghalaya, India.
2College of Veterinary and Animal Science, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut-250 110, Uttar Pradesh, India.
Cite article:- Vise Esther, Ghatak Sandeep, Garg Akshay, Karam Amarjeet, Prasad Bharat Chendu, Milton Prince Arun A., Shakuntala Ingudam, Puro Kekungu, Sanjukta kumari Raj, Sen Arnab, Das Samir (2021). Whole Genome Sequencing and Analysis of Mycobacteroides chelonae M77 Isolated from Cow Milk from the Hill State of Meghalaya, India . Indian Journal of Animal Research. 55(6): 642-646. doi: 10.18805/IJAR.B-4007.

ABSTRACT

Background: Mycobacteroides chelonae M77 isolated in a targeted Mycobacterial isolation study from bovine raw milk was whole genome sequenced (CP041150.1), followed by comparative genomics study for better understanding of the organism with special focus on its virulence and antibiotic resistance pattern. This is probably the first whole genome sequence (WGS) of M.cheloane submitted in NCBI database from India. 

Methods: The WGS has been carried out in Illumina Next Seq 500 platform and then assembled resulting in a genome of 5.12 MB. The assembled genome was annotated in RASTtk and viewed by SEED for its detailed analysis. Pathogen Finder 1.1 and Res Finder 3.1 soft wares were applied for knowing the isolate pathogenecity and antibiotic profiling respectively. Mauve analysis was carried out to know the genomic re-arrangements.

Result: Pathogen Finder 1.1 showed that M77 was non-pathogenic and Res Finder 3.1 server reveals it is susceptible to most of the antibiotics. Mauve analysis for alignment of isolate revealed no significant re-arrangement of genomic segment. The whole analysis showed that the genome of M77 is tight genome with not much variation in relation to previous complete genomes reported and is non-pathogenic and sensitive to most of the antibiotics.

INTRODUCTION

Mycobacteriodes chelonae (earlier known as Mycobacterium chelonae) is an opportunistic non tuberculous mycobacteria (NTM) present in environment and capable of causing infection to human. M.cheloane was first isolated from sea turtle tubercle nodule by Freidmann in 1903. Nowadays with increase reports of its isolation from various human infections it is being suspected as a potential zoonotic pathogen also (Falkinham, 2011). Identification of M. chelonae is done conventionally by culture and biochemical methods which are laborious and restricted to reference laboratories only as that of other Mycobacterial organism like Mycobacterium tuberculosis, M.bovis etc (Basit et al., 2015). The popularity of molecular and proteomics techniques like RFLP- hsp65, sequencing of hsp65 gene (Telenti et al., 1993), rpoB gene (Ade´kambi et al., 2003), 16sRNA (Pascual et al., 1995), MALDI-TOF (Genc et al., 2018) had given additional advantage to many laboratories other than reference also to confirm these isolates. Whole genome sequencing had also been now entered in the diagnostic laboratories for M.tuberculosis but restricted to developed countries at present (Zakham et al., 2019). WGS has been used for other Mycobacterial species for research and academic purpose in present time. With respect to M.chelonae, till date 47 WGS had been available in NCBI database including the present study. The decrease in the price of WGS is allowing researcher to carry WGS all over the globe which is helping in strengthening the global database. The WGS submitted from this study is also the first M.chelonae WGS submitted in NCBI database from India. The present study on WGS was carried out with an intention to know the in-cite of this organism with respect to pathogenicity, virulence, antibiotic resistance etc. It is a sequelae with our previous study which was oriented for isolation of mycobacterial flora from cow milk where we have documented a very high isolation rate for M.chelonae (Vise et al., 2017). To further assess its public health implications we have taken a representative M.chelonae isolate M77 and went for whole genome sequencing to decipher inside this organism so as to know whether this isolate is a potential pathogen to public or not.

MATERIALS AND METHODS

Place of research
 
The work was carried from March 2018 to November 2019 at Mycobacteriology Laboratory of Division of Animal Health, ICAR-Research Complex for North Eastern Hill Region, Meghalaya.
 
Bacterial strain
 
The laboratory available isolate Mycobacteriodes chelonae M77 was already confirmed in the laboratory through culture growth characteristics, acid fast staining morphology, PCR assays directed to hsp65 and IS6110 genes; hsp65-restriction enzyme analysis; hsp65 sequencing followed by BLAST in our previous studies. The isolate M.cheolane M77 was sub-cultured in Lowenstein jensen media with incubation at 37°C for 7 days in multiple copy so as to get ample colony required for DNA isolation. The handling of organism was carried out in biosafety level III cabinet following BSL II practices.
 
DNA isolation
 
DNA isolation was done using Cetyltrimethylammonium bromide (CTAB) lysis method (Murray and Thompson, 1980) with suitable modification. We earlier followed the DNA isolation by DNeasy Blood and Tissue, Qiagen, USA kit but because of fewer yields, we opted for conventional CTAB method. We also skipped the heat killing step of 80°C as previous experience shows that this step is enhancing the shearing of genomic DNA. The yield was checked for its quality and quantity with agarose gel electrophoresis (0.8%) and NanoDrop respectively. We have carried out the genomic DNA isolation in four replicates and then immediately sent to the service provided, Eurofins India in refrigerated condition by fastest mode.
 
Whole genome sequencing
 
The WGS service provider Eurofins rechecked the quality and quantity of DNA by nanodrop and 0.8% agarose gel electrophoresis. They checked the isolate identity by 16sDNA gene sequencing using Sanger sequencing technique. Then they proceeded for WGS in illumina Next Seq 500 platform from one of the four DNA replicate. 2x150 Next Seq 500 shotgun library was prepared in illumina Tru Seq Nano DNA library preparation kit. The PCR enriched library was analyzed in 4200 Tapestation system (Agilent technologies). The high quality pair end sequence generated was assembled de-novo by SPAdes genome assembler (Bankevich et al., 2012) and later refined by reference based scaffolding via Conti Guater2 (Galardini et al., 2011) using the closest strain of M.chelonae subsp. gwanakae MOTT36W (NZ_CP031516.1) and by SOAPdenovo2 Gapcloser (Luo et al., 2012).
 
Bioinformatics analysis of M77 isolate
 
gene prediction from the final assembled genome was performed by using RASTtk (Brettin, 2015). G+C % was calculated by ANI (Yoon et al., 2017) through ezbiocloud online platform (https://www.ezbiocloud.net/tools/ani). ChunLab’s online Average Nucleotide Identity (ANI) calculator using OrthoANIu algorithm was used to calculate distance between M77 with M.chelonae CCUG47445 (NZ_CP007220.1) and M.chelonae MOTT36W (NZ_CP031 516.1) (https://www.ezbiocloud.net/tools/ani).
 
Comparative genomics study was carried out with Mauve analysis of isolate M77 with M.chelonae MOTT36W (NZ_CP031516.1) and M.chelonae CCUG47445 (NZ_CP 007220.1) (Darling et al., 2004). ResFinder-3.1 Server was used to find acquired antimicrobial resistance in M77G isolate which can identify the acquired resistance gene or chromosomal mutations (Zankari et al., 2012). Pathogen finder 1.1 (centre for genomics epidemiology) – a web server based detection of bacterial pathogenicity (http://cge.cbs.dtu.dk/services/PathogenFinder) was used to predict the pathogenicity of the M77 isolate (Cosentino et al., 2013). The analyzed and checked WGS of M77 was submitted to NCBI WGS database. The phylogenetic analysis of M77 was carried with other 46 whole genome database of M.chelonae available in the NCBI and was observed in NCBI itself.

RESULTS AND DISCUSSION

Mycobacteriodes chelonae M77 used in the study was available in the laboratory and it was already characterized with acid fast, hsp65 positive, IS6110 negative, hsp-65 RE pattern corresponding to M.chelonae and hsp65 sequence BLAST resulting as M.chelonae (NCBI accession-KX232660.1). The DNA purity for WGS came around 1.85 to 1.87 in nanodrop OD A260/A280 which was optimum as it was required to be between 1.8 to 2. All the 4 replicates were having around 5 microgram of DNA in total as desired by service provider. The QC analysis by service provider was also same for genomic DNA and hence they proceeded with one replicate having DNA concentration of 113.1 ng/µl in Nanodrop and 101.0 ng/µl in Qubit reading. The isolation of Mycobacterial DNA is always a challenge with requirement of biosafety cabinets, optimum lysis of its cell wall (because of thick layer of mycolic acid). The isolation for optimum quantity and quality DNA for WGS was itself a big basic hurdle. Our laboratory use to prefer commercial kit of Qiagen Tissue and blood DNA kit for Mycobacterial DNA for routine biotechnological work of polymerase chain reaction (PCR), PCR- restriction enzyme digestion etc. Most of the other commercial DNA isolation kit intended for bacterial DNA isolation use to fail for Mycobacterial DNA isolation. We have first tried with this Qiagen kit but because of its low yield and later it failed to qualify the QC report at the service provider; we adopted the basic CTAB method with suitable modification. The first heat killed step was skipped to minimize shearing and degradation of DNA following measures to minimize aerosolization. Nevertheless, when the isolate is from clinical samples such as tubercle nodule, it is strongly recommended to follow the heat kill step. DNA isolation is always a limiting factor for mycobacterial research (Basit et al., 2015), even most of the commercial kits need to be modified for mycobacerial DNA isolation atleast for the initial lysis step (Fell et al., 2016; Radomski et al., 2013). The CTAB method practiced for Mycobacterial DNA isolation still stood good and in our case also, it fulfilled the requirement for WGS.

The 16s DNA analysis of the M77 isolate at service provider showed BLAST result of 99% to M.chelonae with M.chelonae MOTT36W being the first hit. The WGS yielded paired end read of 12,342,508 reads with 3,667,933,277 total bases giving a total data of 3.67 Gb. de-novo genome assembly gives 941 scaffolds, 5,626,111assembly size, average size of scaffolds as 5,979; N50 value of 276,036 with a maximum size of scaffold 1,047,708 and a minimum of 212. Genome finishing was carried out through SPAdes and with reference guided assembly was done taking the closest match whole genome of M.chelonae MOTT36W through Contiguator 2 and later the gap were reduced by SOAPdenovo2Gapcloser on illumina data. Finally, the assembled genome of M.chelonae M77 came to be 5,179,244.
 
The assembled WGS was further analyzed further and its G+C % through ANI came to be 64.03% which reveals the basic Mycobacterial family properties of high G:C%. OrthoANIu value between M77G and M.chelonae CCUG47445 (NZ_CP007220.1) came to be 95.64% and to be considered as same species the value should come more than 95% and here it proved to be same species.  OrthoANIu value (%) between M77 and M.chelonae_MOTT (NZ_CP031516.1) gave 97.57% and here it again proved to be same species. 
 
Protein coding and RNA genes were identified in the M77 using this assembled genome using the gene annotation pipeline RAST followed by SEED Viewer which showed  5,170 genes with 5,119 protein (CDS) and 51 RNA.  Out of 5,119 CDS, 1719 were in subsystem and 3400 were not in sub-system. Number of SEED subsystem (functional role) were 397 and feature category of subsystem is around 27.
 
In our previous findings we found a dominant M.chelonae from milk in this region, which we presumed a real threat to public health. There were no better way with us in limited setting to access its pathogencity for these isolates, hence, we decided to go ahead for whole genome analysis for a representative isolate to decipher more inside its pathogenecity. Through the pathogen finder 1.1, the M77 was categorized as non-pathogenic with a value of 0.324. We analyzed the reference strain M.chelonae CCUG47445 (NZ_CP007220.1) also in the server which was isolated from turtle tubercle gave a value of 0.581 which is greater than 0.5 corresponding to a pathogen.  We analyzed some more mycobacterial genome to this software but could not make a clear difference between pathogenic and non-pathogenic for some of the mycobacterial isolates (Table 1). We found that it miss to identify the some known pathogen and categorized them as non-pathogen viz., M.chelonae subsp. chelonae DSM 43804, M. abscessus G153 and M. abscessus G220. It also identified the vaccine strain M. tuberculosis variant bovis BCG str. Pasteur as pathogenic. Hence, though this software may hold good for distinguishing between pathogenic and non-pathogenic for other bacterial species as claimed by authors (Cosentino et al., 2013) but for mycobacterial study it can gave only a fair idea but cannot tell with confidence. Taking account of this limitation we can only say that the M77 isolate comes under the non-pathogenic category by this software but require further confirmations.
 

Table 1: Application of representative Mycobacterial isolates on pathogenfinder1.1.


 
Res Finder-3.1 server result showed that the isolate M77G doesn’t posses resistance genes for any of the following antimicrobials, Aminoglycoside, Beta-lactam, Colistin, Fluoroquinolone, Fosfomycin, Fusidic Acid, Glycopeptide, MLS - Macrolide, Lincosamide and Streptogramin B, Nitroimidazole, Oxazolidinone, Phenicol, Rifampicin, Sulphonamide, Tetracycline and Trimethoprim. The Res Finder-3.1 could tell that the isolate M77 is antibiotic sensitive but here also the database is not complete for the first line of TB drugs. Though for second line of drugs and for drugs pertaining to NTM it still hold promise but without the basic four TB drugs rifamcin, isoniazid, ethambutal and pyrazinamide, the analysis is still restricted.
 
Mauve analysis was used for alignment of whole genome of M77 isolate with M.chelonae MOTT36w-495 and M.chelonae CCUG 47445 concluded that in the M77 genome there is no significant re-arrangement of genomic segment.
 
WGS assembled data for M.chelonae M77 was successfully submitted with the Accession number - CP041150.1 in NCBI. The phylogenetic graph for all WGS available in the NCBI (47 assembled M.chelonae genome including M77) have been analyzed and M77 came closest to strain 96-1724 (isolated from leg abscess from Belgium), strain- D16R10 (surface water, Germany), ATCC 19237 (gastric lavage, no geographical details available) and D16R9 (tap water, Germany).

CONCLUSION

Whole genome analysis has been used for various comparative genomics studies for knowing the in-details of an isolate in term of its identification, origin, pathogenecity, antibiotic profile and other parameters. The ever increasing availability of user friendly bioinformatics tools both free and paid versions have led researchers to use them in their respective fields. With regard to Mycobacteriology, WGS had already entered in diagnostics for tuberculosis patients but is limited to high end laboratories and developed countries. The mycobacterial organisms are tough to handle and require special biosafety facility. The pathogenecity studies of mycobacterial organisms are even more tougher and requires animal isolator facility for handling live infection studies on animal models. Such facilities are not only costly but repeated handling of such pathogenic organism is also not advisable as it increases the risk for handler and laboratory staffs. The molecular era paved many new ways for studying targeted virulence genes of microorganisms but for mycobacterial organism, the virulence factors or genes are not so well documented and then for NTMs the reports are meager. The WGS and availability of newer bioinformatics gave few solutions to enter in this difficult research area. In our earlier study also we had isolated increased number of M. chelonae and we want to know the actual public health threat from these isolates. Therefore, we planned to do a representative WGS to infer into various aspects of pathogenicity and antibiotic resistance which ultimately gave us a fair idea that the isolate is clean and possess minimal threat to public.

ACKNOWLEDGEMENT

The authors are thankful to Director Public Health ICAR RC NEH for giving permission and fund for conducting the research (IXX05508).

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