Draft genome of Ochrobactrum intermedium strain M86 isolated from non-ulcer dyspeptic individual from India
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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.
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.
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.
KeywordsAmplify Fragment Length Polymorphism Draft Genome Sequence Personal Genome Machine Antral Biopsy Stomach Lumen
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 . 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 . 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 . Similarly, Gastrospirillum hominis, enterococcci  and staphylococci have been associated with gastric disorders . In some cases, they have been isolated from antral biopsies from patients with or without H. pylori colonization .
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.
Genomic DNA was isolated by PureLink®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 single-end 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 . Prediction and annotation of genes were done using RAST  server with SEED database and ISGA pipeline . The data were further validated using gene prediction tools such as Glimmer . 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  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
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.
Putative gene clusters responsible for survival of Ochrobactrum intermedium strain M86 in the acidic environment of stomach lumen
Comparisons of subsystem features between genome of Ochrobactrum intermedium strain M86 and Ochrobactrum intermedium LMG 3301 T
Number of CDS present in O. intermedium
Amino acids and derivatives
Cofactors, vitamins, prosthetic groups, pigments
Cell wall and capsule
Fatty acids, lipids, and isoprenoids
Nucleosides and nucleotides
Virulence, disease and defense
Motility and Chemotaxis
Regulation and cell signaling
Iron acquisition and metabolism
Metabolism of aromatic compounds
Cell division and cell cycle
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 . 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 prophage-predicting PHAST  Web server.
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 non-ulcer 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.
Girish Kulkarni acknowledges the financial support received by the Council of Scientific and Industrial Research. This work was supported by funding from the Department of Biotechnology, Government of India, and Microbial Culture Collection project. We thank Dr. Kamlesh Jangid, Dr. Om Prakash for extensive review and useful comments on the manuscript. We also thank two anonymous reviewers for useful comments on earlier versions of this manuscript.
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