Draft genome of Ochrobactrum intermedium strain M86 isolated from non-ulcer dyspeptic individual from India

Background Ochrobactrum intermedium is an emerging opportunistic pathogen of humans that is closely related to members of the genus Brucella. Earlier, we reported the case of an Indian subject with non-ulcer dyspeptic symptoms whose urease positive gastric biopsy revealed the presence of Helicobacter pylori along with non-Helicobacter like bacteria, eventually cultured and identified as O. intermedium strain M86. Results Here, we describe the unclosed draft genome of the strain M86 with a length of 5,188,688 bp and mean G+C content of 57.9%. We have also identified many putative gene clusters that might be responsible for its persistence in the gastric mucosa.Comparative analysis of genomic features of Ochrobactrum intermedium strain M86 and Ochrobactrum intermedium LMG 3301T was also done. Conclusions This paper attempts to gain whole-genome based insights into the putative gene determinants of O. intermedium for survival in the highly acidic stomach lumen environment .Identification of genes putatively involved in the various metabolic pathways may lead to a better understanding of the survival of O. intermdedium in acidic condition.


Background
The Alphaproteobacteria represent a biologically diverse group of bacteria with members like Brucella, Bartonella, Agrobacterium and Ochrobactrum that are capable of interacting with eukaryotic cells. Ochrobactrum intermedium is a Gram-negative, capsulating, aerobic bacilli belonging to the Alphaproteobacteria. It is the closest genetic relative of genus Brucella as evidenced by protein profiling, western blot, immunoelectrophoresis, amplified fragment length polymorphism, 16S rRNA gene and RecA gene sequence based studies [1]. Previous reports have suggested O. intermedium as an emerging pathogen in liver abscess post-liver transplantation and in the bladder cancer patient causing presumptive bacteremia [2,3]. But, clinical manifestations and diseases caused by O. intermedium are poorly characterised.
Several bacteria other than Helicobacter pylori have been detected earlier in gastric biopsies. The presence of O.
intermedium along with H. pylori was reported earlier in a subject from North-India diagnosed with non-ulcer dyspepsia [4]. A unique observation was the presence of severe fibrosis in the lamina propria of the gastric mucosa revealed during histological examination of the gastric antral biopsy. Whether this fibrosis was caused either partially or totally by O. intermedium was not clear. Other species of Ochrobactrum have also been associated along with H. pylori, e.g., O. anthropi with mild gastritis in squirrel monkeys [5]. Similarly, Gastrospirillum hominis [6], enterococcci [7] and staphylococci have been associated with gastric disorders [8]. In some cases, they have been isolated from antral biopsies from patients with or without H. pylori colonization [9].
Importantly, both H. pylori and O. intermedium produce urease, and thus the detection of H. pylori by urease test in the presence of Ochrobactrum may be confounded. The role of Ochrobactrum in gastric pathology remains uncertain and requires detailed pathologic, microbiological and genetic investigations in order to evaluate the link between H. pylori and O. intermedium in the gastric niche. This paper attempts to gain whole-genome based insights into the putative gene determinants of O. intermedium for survival in the highly acidic stomach lumen environment.

Genome sequencing
Genomic DNA was isolated by PureLink W Genomic DNA Kit. The draft genome sequence of strain M86 was determined by Ion Torrent Personal Genome Machine (PGM ™ ) sequencer using a 316 chip with 200-bp singleend shotgun sequencing. A total of 2,602,696 reads were obtained. PGM sequencing resulted in about 67X genome coverage with 148 contigs.

Assembly and annotation
The de novo approach was applied to finalize the unclosed draft genome using MIRA 3.4.0 version using default parameters [10]. Prediction and annotation of genes were done using RAST [11] server with SEED database and ISGA pipeline [12]. The data were further validated using gene prediction tools such as Glimmer [13]. Functional annotation was also performed by PGAAP using public database of National Centre for Biotechnology Information (NCBI). Prophages and putative phage like elements in the genome were identified using prophage-predicting PHAST [14] Web server. Regions identified algorithmically as "intact" by PHAST, as well as regions sharing a high degree of sequence similarity and conserved synteny with predicted "intact" prophages, were identified as prophages.

Submission of genome sequence
The Ochrobactrum intermedium strain M86 whole genome shotgun (WGS) project was submitted to the GenBank and has the project accession AOGE00000000 and consists of sequences AOGE01000001-AOGE01000148

Quality assurance
The genomic DNA was isolated from pure bacterial isolate and was further confirmed with 16S rRNA gene sequencing. Bioinformatic assessment of potential contamination of the genomic library by allochthonous microorganisms was done using PGAAP and RAST annotation systems.  a mean read length of 155 bp. de-novo assembly using the MIRA assembler v3.4.0 [10] with default parameters yielded~67X coverage. A total of 148 contigs with >500 bp length were obtained. The unclosed draft genome sequence of strain M86 is of 5,188,688 bps and 5043 predicted coding DNA sequences (CDSs) and 66 RNA genes with mean G + C content of 57.9%. RAST server based annotation of the whole genome, showed the presence of 437 subsystems (related functional roles) [11]. Figure 1 describes the subsystem distribution of strain M86.

Initial findings
Putative gene clusters responsible for survival of Ochrobactrum intermedium strain M86 in the acidic environment of stomach lumen H. pylori have several genes for biosynthesis of cytosolic urease for its survival in the acidic environment of stomach lumen [15]. Genome of strain M86 contains urease gene cluster (see Figure 2): out of which, UreA, beta subunit, UreB gamma subunit, UreC, alpha subunit, are part of core Urease enzyme, While four accessory proteins: UreD, UreE, UreF and UreG play important role in Ni 2+ uptake and insertion into active site of apo-enzyme. Genetic relatedness of urease gene cluster with phylogeneticaly closely related bacteria is shown in Figure 2. A complete operon encoding the VirB gene involved in conjugative transfer is present in strain M86. Genes encoding osmotic stress, oxidative stress HPIIb, cold shock GrpE, heat shock DnaK, periplasmic stress DegQ and protection from reactive oxygen species, sod are found. Genes predicated to encode flagellar biosynthesis protein FlhA and FlhB has been identified in genome of strain M86 which are likely elementary to adaptation of new lifestyle. Enterobactin synthesis clusters of entA, entB1, entB2, entC, entD, entE,  Cell division and cell cycle 17 17 Potassium metabolism 15 16 Sulfur metabolism 28 31 entF, entG, entH genes were also observed in the genome sequence of strain M86 suggesting its ability of iron acquisition by siderophore production. Presence of membrane transport machinery with dominance of Dipeptidebinding ABC transporter, periplasmic substrate-binding component was detected in the genome of strain M86.It has been found that all the clinical isolates and the type strains of Ochrobactrum were highly resistant to all forms of ß-lactams except imipenem [16]. This resistance profile is consistent with the expression of the AmpC betalactamase characterized in O. anthropi [17,18]. The genome of strain M86 shows the presence of AmpC betalactamase gene which supports its resistance to ß-lactams antibiotic observed by Dharne et al. [4] . Table 1 shows the comparison of genomic features of Ochrobactrum intermedium strain M86 and Ochrobactrum intermedium LMG 3301 T .

Prediction of phage islands
The presence of prophage sequences may also allow some bacteria to acquire antibiotic resistance, to exist in new environmental niches, to improve adhesion or to become pathogenic [14]. In addition, phages play a crucial role in genome plasticity and chromosome remodelling. The intact phage of 32 kb in length together with phage-like proteins and hypothetical proteins were identified in the genome of strain M86 by prophagepredicting PHAST [14] Web server.

Future directions
The genomic properties of O. intermedium are poorly characterized and, as a consequence, their role in human health and disease remains unclear. Elucidation of the physiological properties and identification of genes putatively involved in the various metabolic pathways may lead to a better understanding of the survival of O. intermdedium in acidic condition. Further studies involving large scale genome sequencing and comparison of the O. intermedium strains isolated from several other nonulcer dyspeptic individuals will help us apprehend the genomic features of its survival in the acidic condition of the stomach. Comparative genomic analyses of O. intemedium strain M86 and other environmental isolates of genus Ochrobactrum will permit us to understand its mechanisms for adaptation to new environments.