Strain CD08_7 was isolated from the duodenal mucosa of a CD patient. The tissues samples from duodenal mucosa were recovered during endoscopy at the Postgraduate Institute of Medical Education and Research, Chandigarh, India. The samples were collected and aseptically transferred to CSIR-Institute of Microbial Technology, Chandigarh. The tissue was homogenized in sterile phosphate saline (PBS) and centrifuged at 4000 rpm for 2 min to remove debris. The supernatant was recovered and serially diluted with PBS and plated on to tryptic soy agar (TSA; HiMedia, India), incubated at 37°C in the presence of oxygen for 36 h. The individual colonies appearing in the plate were picked and plated on to fresh TSA medium which is a broad spectrum medium, further passaged for two times until separate colonies were obtained. The different pure isolates were selected on the basis of distinct cell shape and colony morphology, and these selected strains were identified by 16S rRNA gene sequencing. Antibiotic susceptibility tests for strain CD08_7 was performed by placing antibiotic disks (Icosa universal-2, HiMedia, India) on TSA plates seeded with suspensions of strain CD08_7. Characterization of the strain CD08_7 was performed according to the methods described by Kaur et al. (2016). Genomic DNA extraction and amplification was performed as previously described (Mayilraj et al., 2006). Identification of phylogenetic neighbors and the calculation of pairwise 16S rRNA gene sequence identity levels were achieved using the EzTaxon server (Kim et al., 2012) and alignments were carried out using Mega version 6.0 (Tamura et al., 2013). Phylogenetic trees were constructed using the neighbor-joining as well as maximum likelihood and maximum parsimony algorithms. Bootstrap analysis was performed to assess the confidence levels of the branching (Figure 1). The reference genomes of Nesterenkonia strains NP1^T, F, AN1, and JCM 19054 were obtained from the NCBI genome database and bear the following accession numbers CBLL00000000, AFRW00000000, JEMO00000000, and BAXI00000000, respectively.

A draft genome of strain CD08_7 was sequenced at C-CAMP¹ next-generation genomics facility, Bengaluru, India using an Illumina HiSeq 2 × 100 platform. Library preparation was performed at C-CAMP’s genomics facility using TruSeq^® DNA sample preparation kit (Illumina) as per manufacturer’s instructions. About 1 μg of pure genomic DNA was sonicated using Covaris shearing to obtain 300–400 bp fragment size. The resulting fragmented DNA was cleaned up using AMPure XP beads as described by the manufacturer. Fragmented DNA was subjected to a series of enzymatic reactions that repaired frayed ends, phosphorylated fragments, and added a single nucleotide ‘A’ overhang then ligated adaptors using TruSeq^® DNA sequencing kit following the protocol as described by the manufacturer. Sample clean-up was done using AMPure XP beads. After ligation-clean-up, ∼300–400 bp fragments were size selected on 2% agarose with SYBR Gold gel using TAE Buffer and cleaned using MinElute column, QIAGEN. PCR amplification of adaptor-ligated fragments was done and followed by a cleanup using AMPure XP beads. The prepared libraries were quantified and then validated for quality by running an aliquot on High Sensitivity Bio analyser Chip, Agilent. Assembly was carried out with CLC Bio Workbench v7.5.1 (CLC Bio, Denmark).

The genome annotation for strain CD08_7 was performed using RAST (Aziz et al., 2008; Overbeek et al., 2014; Brettin et al., 2015), which is an automated genome annotation server². Similarly, automated genome annotation for reference genomes of Nesterenkonia strains NP1^T, F, AN1, and JCM 19054 was also accomplished using RAST. Further, the ribosomal RNA genes in the genomes were identified using RNAmmer 1.2 (Lagesen et al., 2007). The tRNA and tmRNA genes were identified by ARAGON (Laslett and Canback, 2004). Insertion sequence [IS] elements were identified by the IS finder³ (Siguier et al., 2006). The RAST server provides a comprehensive platform for comparing two genomes after their annotation through the seed viewer. Homology search of the protein encoding genes is carried out at protein level using Gene Locator and Interpolated Markov ModelER (GLIMMER 2) against a set of protein families called FIGfams (Aziz et al., 2008). Two genes are considered homologous if they implement the same functional role and the region of similarity shared by them covers over 70% of each sequence. For further, comparative study of the genomes, the features of CD08_7 along with the other reference strains of Nesterenkonia were extracted from the RAST server on to an excel sheet and compared manually for presence of unique genes (presence of a gene homolog in strain CD08_7 but it’s absence in all other strains was considered as unique), potential pathogenicity determinants, genes involved in metabolic pathways related to virulence (comparative pathogenomics) and common genes among the strains as described previously (Nair et al., 2016).

The strain designated as CD08_7 matched most of the phenotypic (cocci in shape, non-motile, negative for urease, indole and hydrogen sulfide production, negative for hydrolysis of gelatine and tween 80, nitrate reduction) and chemical [L-Lys-Gly-D-Asp peptidoglycan type, major menaquinones as MK-7 and MK-8 and major lipids as diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidyl inositol (PI)] characteristics of the genus Nesterenkonia and most of the features were matched with the species N. jeotagli (Yoon et al., 2006; negative for hydrogen sulfide and indole production, while positive for nitrate reduction, hydrolysis of starch, gelatine and tween 80 and for utilization of D-Glucose, D-Xylose, L-Arabinose, D-Cellobiose, D-Trehalose, Sucrose and Maltose; acid is produced from D-Galactose, D-Xylose and Mannitol, not from Lactose). 16S rRNA gene sequence showed that the strain CD08_7 correspond to the genus Nesterenkonia and is most closely related to N. jeotgali JG241^T (99.93% identity; 100% sequence completeness, one base difference of a total of 1444 bases) followed by N. sandarakina YIM 70009^T (99.79% identity: 99.8% sequence completeness, three bases difference of a total of 1459 bases), N. halotolerans YIM 7008^T (99.73% identity: 100% sequence completeness, four bases difference of a total of 1456 bases), N. lutea YIM 70081^T (99.38% identity :100% sequence completeness, nine bases difference of a total of 1456 bases) and N. xinjiangensis YIM 70097^T (97.05% identity:100% sequence completeness, 43 bases difference of a total of 1456 bases).

A combined phylogenetic tree (Neighbor joining, Maximum likelihood and Maximum parsimony) of strain CD08_7 was constructed using the 16S rRNA gene sequences of the closely related Nesterenkonia type strains and the reference strains NP1^T, JCM 19054, F and AN1. Strain CD08_7 formed a separate branch with N. jeotgali JG-241^T; likewise strain F clustered with N. halobia strain ATCC 21727^T and strain AN1 with N. halotolerans strain YIM 70084^T (Figure 1).

The draft genome of strain CD08_7 consisted of 2,925,195 bp with G+C content of 67.6 mol%, 2531 predicted CDSs, 379 sub-systems and 52 RNAs. The final assembly contained eight contigs with N₅₀ contig length of 731,296 bp and the largest contig assembled measured 813,259 bp. The genome size of Nesterenkonia sp. AN1 was the largest (3.0 Mb) among all genomes (ranging from 2.5 to 2.8 Mb). Highest genomic G+C content (71.4%) was of Nesterenkonia sp. strain F, followed by Nesterenkonia sp. strain AN1 (67.94%), N. jeotgali CD08_7 (67.6%), N. massiliensis strain NP1^T (63.0%), Nesterenkonia sp. strain JCM 19054 (61.1%). Other genome features of strain CD08_7 along with four reference genomes are shown in Table 1.

Genome comparison of N. jeotgali strain CD08_7 was carried out among the other four reference strain of genus Nesterenkonia. The analysis revealed various categories of genes, among which, (1) virulence, disease and defense; (2) phages, prophages, transposable elements and plasmids; (3) stress response; (4) membrane transport and (5) iron acquisition, were further studied because of their extreme importance in contributing pathogenicity. A total of 213 genes were present in the five genomes analyzed had potential to confer pathogenicity. It was surprising to find that strain AN1 (an environmental isolate) had 174 pathogenicity genes. Strain CD08_7 had the second highest number of such determinants (163), strain F had 115, strain JCM 19054 had 123 and strain NP1 had 149. Only 59 of these determinants in strain CD08_7 were common in all the genomes, which shows the functional diversity among species of genus Nesterenkonia. Two genes of phage and prophage origin were unique in the genome of strain CD08_7.

In this category around 30 genes were present in strain CD08_7, when compared to strain NP1^T which had 35, AN1 and JCM 19054^T had 33 followed by strain F which had 29 genes. Two further subcategories were studied under the category Virulence, disease and defense, for their probable contribution in pathogenesis: genes involved in resistance to antibiotics and toxic compounds and genes involved in invasion and intracellular resistance (Figure 2).

Resistance to antibiotics and toxic compounds is a primary feature which highlights the organisms as possibly virulent. There are a total of 29 genes present in this subcategory, 19 of which were present in strain CD08_7 whereas 22 in AN1 and NP1^T. Strain JCM 19054 had 20 of these determinants in its genome, followed by Strain F that had 17 genes of these determinants in its genome for this subcategory (Figure 3). Strain CD08_7 is resistant to Nalidixic acid (10 μg) and sensitive to all other antibiotics of Icosa universal-2 panel, which is in accordance with the presence of mutant genes DNA gyrase subunit B (gyrB) and DNA gyrase subunit A (gyrA). Nalidixic acid inhibits the bacterial growth by damaging DNA replication process after blocking the action of above enzymes (Sugino et al., 1977). Mutations in these genes are supposed to cause resistance in microorganisms due to structural change in the active site of these enzymes.

Mycobacterium has SSU and LSU ribosomal proteins which contribute in the development of tuberculosis (Nair et al., 2016). This category highlights the presence of these determinants described in Mycobacterium as a characteristic for invading the host and disease progression. Four subsystems were present in strain CD08_7, strain AN1, strain JCM 19054, and strain F whereas strain NP1^T had one gene of an additional subsystem. These results show that strain CD08_7 has the potential to be invasive (Figure 4).

Several pathogenic bacteria have evolved mechanisms to counteract the host mediated degradation via endocytic pathway, modulating the membrane transport proteins is one such mechanism through which the bacteria are able to use a T3SS (type III secretion system) or T4SS (type IV secretion system) to translocate their proteins across the membrane, where they manipulate the host proteins residing in the cytoplasm. Other bacteria have developed efflux pumps to keep intracellular concentrations of antibiotics low (Alix et al., 2011). Strain AN1 had 58 transport genes in its genome, followed by strains CD08_7 and NP1^T with 51, strain F with 43 and strain JCM 19054 with 37. Strain AN1 had some additional transport genes of subcategories, protein secretion system type II (3), ABC transporters (2), TRAP transporters (3) and copper transporters (1) that were absent in CD08_7 (Figure 5).

Stress response is activated in bacteria when they encounter unfavorable conditions in the host environment. The response mechanism involves adaptation to new conditions by regulation of several molecular pathways that control transcription, translation and post-translation modifications (Rukhsana et al., 1996). Therefore, bacteria having stress response genes in their genomes are able to cope with the oxidative stress produced by human immune cells and osmotic stress due to host osmolytes (Rothe et al., 2012; Paiva and Bozza, 2014). In this category 67 genes were present in strain CD08_7 and AN1, while strain F had 60, strain NP1^T had 56 and JCM 19054 had 41 (Figure 6).

The genes encoding phages, transposable elements and prophages contribute to variability and thus pathogenic success by promoting recombination in bacterial genomes. Strain AN1 contains nine genes in this category, thus strain CD08_7 was second with five followed by strain JCM 19054 which had only two and strains F and NP1^T had none (Figure 9). Although strain CD08_7 had the highest number of genes (4) in sub-category phages and prophages, it lacked genes for phage replication. The only other strain possessing phage-related genes (just two for replication) was JCM 19054 (Figure 10).

It has been shown that iron is essential for growth in bacteria. Within the host bacteria and host cells compete for iron. Bacteria have developed strong mechanisms to capture iron from the host like receptors for eukaryotic iron binding proteins and siderophores, which have high affinity for iron. Therefore, iron acquisition capability is considered as an important measure of pathogenicity associated with any bacterial strain (Baron, 1996). Among the strains analyzed, CD08_7 comprises the highest number of genes related to iron acquisition (10) like strains AN1 and JCM 19054, whereas strains NP1^T and F had 7 genes (Figure 11). Some of the most important genes in this category were homologs of Streptococcus Siderophore Aerobactin, Petrobactin-mediated iron uptake system and iron acquisition (Figure 12).