Ochrobactrum Strains Research Articles

Genetic comparison of Brucella spp. and Ochrobactrum spp. erroneously included into the genus Brucella confirms separate genera

The facultative intracellular pathogen Brucella and the free-living bacteria Ochrobactrum are both α-proteobacteria and very close to each other. A group of researchers recently clustered Ochrobactrum strains into the genus Brucella according to a BLAST distance approach. Thus, we performed a deeper comparative genetic analysis for eleven Ochrobactrum strains and twelve different Brucella isolates to demonstrate important differences between these bacteria. In addition to the clear differences between Brucella and Ochrobactrum, like the differences in genes contents, and different genome sizes, the Brucella-specific gene bscp31 was not found in Ochrobactrum, as well as other important Brucella-specific proteins and virulence factors. Differences in antimicrobial resistance genes content and the presence or absence of plasmids were obvious between Brucella and Ochrobactrum spp. Genome alignment of Brucella spp. and Ochrobactrum spp. revealed a genome similarity of 85.7% maximum, whereas all analyzed Brucella spp. in this study had a similarity of 97.6-99.9%, and all compared Ochrobactrum spp. 82.6-98.0%. Because of these facts mentioned in this work, Brucella and Ochrobactrum should be considered separate genera.

A comparative study on chemical characterization and properties of surface active compounds from Gram-positive Bacillus and Gram-negative Ochrobactrum strains utilizing pure hydrocarbons and waste mineral lubricating oils.

Mineral lubricating oils are widely used in various industrial sectors for their applications in maintenance and functioning of machineries. However, indiscriminate dumping of these used oils have resulted in polluting the natural reservoirs which subsequently destroys ecological balance. Bacteria can emulsify or lower surface tension between phases of immiscible substrates and can acquire them as their carbon and energy sources. Such a phenomenon is mediated by production of extracellular polymers which can function as eminent surface active compounds based on their surfactant or emulsifying nature. The comparison between bacterial strains (Gram-positive Bacillus stratosphericus A15 and Gram-negative Ochrobactrum pseudintermedium C1) on utilization of pure straight chain hydrocarbons, waste mineral lubricating oils as sole carbon source and chemical characterization of the synthesized surface active compounds were studied. Characterization analysis by Ultraviolet Visible spectrophotometry, Fourier transform infrared spectroscopy, Nuclear Magnetic Resonance spectroscopy, Carbon-Hydrogen-Nitrogen analysis has given detailed structural elucidation of surface active compounds. The contrasting nature of bacterial strains in utilization of different hydrocarbons of waste mineral lubricating oils was observed in Gas Chromatography-Mass Spectroscopy analysis. The variation between both strains in utilization of hydrocarbons can be manifested in chemical structural differences and properties of the produced surface active compounds. Scanning Electron Microscopy has given detailed insight into the microstructural difference of the compounds. The utilization of lubricating oils can address waste disposal problem and offer an economical feasible approach for bacterial production of surface active compounds. Our results suggest that these surface active compounds can maneuver applications in environmental bioremediation and agriculture, pharmaceuticals and food as functional biomaterials.

Comparative Genomics of a Paddy Field Bacterial Isolate Ochrobactrum sp. CPD-03: Analysis of Chlorpyrifos Degradation Potential.

Ochrobactrum genus is known to catabolize aromatic compounds. This study reports a complete genome sequence of Ochrobactrum sp. CPD-03 (~4.6Mb of chromosomal features) responsible for chlorpyrifos (CP) isolated form a paddy field (20.3588° N, 85.8333° E) in Bhubaneswar, India. A comparative genomics approach was performed between CPD-03 and eight closely related genomes of other Ochrobactrum strains in order to deepen our knowledge, to establish its phylogenetic and functional relationships. The involvement of CP degrading genes indicated a versatile role of CPD-03 in additional field trails. This research would provide the genetic information for its use in natural environment for the depletion of organophosphorus (OP) compounds.

Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

The population structure of Ochrobactrum isolated from entomopathogenic nematodes indicates interactions with the symbiotic system

Entomopathogenic nematodes (EPNs) form specific mutualistic associations with bioluminescent enterobacteria. In Heterorhabditidis indica, Ochrobactrum spp. was identified beside the symbiont Photorhabdus luminescens but its involvement in the symbiotic association in the EPNs remains unclear. This study describe the population structure and the diversity in Ochrobactrum natural populations isolated from EPNs in the Caribbean basin in order to question the existence of EPN-specialized clones and to gain a better insight into Ochrobactrum-EPNs relationships. EPN-associated Ochrobactrum and Photorhabdus strains were characterized by multi-locus sequence typing, Pulsed-Field Gel Electrophoresis fingerprinting and phenotypic traits.Population study showed the absence of EPN-specialized clones in O. intermedium and O. anthropi but suggested the success of some particular lineages. A low level of genetic and genomic diversification of Ochrobactrum isolated from the natural population of Caribbean nematodes was observed comparatively to the diversity of human-associated Ochrobactrum strains. Correspondences between Ochrobactrum and P. luminescens PFGE clusters have been observed, particularly in the case of nematodes from Dominican Republic and Puerto Rico. O. intermedium and O. anthropi associated to EPNs formed less biofilm than human-associated strains.These results evoke interactions between Ochrobactrum and the EPN symbiotic system rather than transient contamination. The main hypothesis to investigate is a toxic/antitoxic relationship because of the ability of Ochrobactrum to resist to antimicrobial and toxic compounds produced by Photorhabdus.

Diversity of Ochrobactrum species in food animals, antibiotic resistance phenotypes and polymorphisms in the blaOCH gene.

Twenty-six lactose non-fermenting, oxidase, urease and citrate-positive Gram-negative rods, isolated from broiler chickens, pigs and cattle at slaughter, were subjected to the matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and 16S rDNA sequencing for identification. Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Ochrobactrum isolates resistant to third-generation cephalosporins were PCR-screened for the presence of the Ochrobactrum anthropi ampC gene (blaOCH). A 547-bp internal segment of blaOCH in the Ochrobactrum spp isolates was amplified with a newly designed primer set, and a phylogenetic reconstruction based on the complete amino acid sequence of blaOCH obtained from nine Ochrobactrum strains in our collection and 20 O. anthropi available in the GenBank was undertaken. All the Ochrobactrum isolates were resistant to the expanded-spectrum beta-lactams and streptomycin. None of the isolates was resistant to imipenem while 41.7% to 50.0% of them were resistant to fluoroquinolones. The blaOCH gene was detected in 16 (66.7%) and 20 (83.3%) of the 24 Ochrobactrum isolates (O. intermedium/O. tritici species), using primers designed for O. anthropi and the newly designed primer set, respectively. Six blaOCH variants grouped into two divergent clusters were identified. This is the first report of the complete nucleotide sequence of the blaOCH gene in non-antropi Ochrobactrum species.

Identification and antimicrobial susceptibilities of Ochrobactrum spp.

Ochrobactrum (O.) anthropi is an opportunistic emerging pathogen closely related to the genus Brucella. Identification and differentiation from brucellae and other Ochrobactrum spp. using routine biochemical test systems is not reliable due to the high phenotypic similarity. In this study, antibiotic susceptibilities of 103 Ochrobactrum isolates were determined using Etest™ for 19 clinically relevant antimicrobial agents. Ochrobactrum strains were highly resistant to β-lactam antibiotics, susceptible to ciprofloxacin, and 97.1% were susceptible to trimethoprim/sulfamethoxazole. It was also demonstrated that biochemical reaction profiles of the API ® and BD Phoenix™ 100 systems for identifying Ochrobactrum isolates can only be used on the genus level. Our in vitro data suggest that combinations of antimicrobial agents including ciprofloxacin and/or trimethoprim/sulfamethoxazole may be useful for empirical treatment of Ochrobactrum infections.

Genetic diversity and phylogenetic relationships of bacteria belonging to the Ochrobactrum– Brucella group by recA and 16S rRNA gene-based comparative sequence analysis

The genetic diversity and phylogenetic interrelationships among 106 Ochrobactrum strains ( O. anthropi: 72, O. intermedium: 22, O. tritici: 5, O. oryzae: 2, O. grignonense: 2, O. gallinifaecis: 1, O. lupini: 2), the type strains of the eight Brucella species and other closely related taxa were studied by recA and rrs gene (16S rRNA) comparative sequence analysis. Both markers correctly delineated the various Ochrobactrum species; however, resolution at the subspecies level was considerably higher in the recA gene-based approach. Phylogenetic analyses using neighbor-joining, parsimony, and maximum likelihood algorithms generated trees with similar topologies but the overall branching order, and also the order of the subclades, were not stable in either assay, which could be explained by generally high recA and rrs sequence similarities. Ochrobactrum and Pseudochrobactrum formed separate clades distinct from other Alphaproteobacteria with Bartonella, Agrobacterium, and Rhizobium as the closest relatives. O. gallinifaecis was the most distinct member, when compared to the type species O. anthropi, with rrs and recA similarities of 96.2% and 81.4%. Brucella species were indistinguishable, exhibiting high rrs and recA gene similarities of 98.6% and 85.5% compared with Ochrobactrum intermedium. At the protein level, all RecA sequences among the various Ochrobactrum species and between Ochrobactrum and Brucella were highly similar with only a few amino acid substitutions. O. anthropi and O. tritici were indistinguishable by means of their RecA proteins. A set of initially biochemically classified strains did not cluster within their assigned species and they either grouped within other known species or grouped as potential novel Ochrobactrum species. In further investigations, these strains were reclassified and described as novel species. In summary, Ochrobactrum is a highly diverse genus comprising several novel species. We recommend recA- in addition to rrs gene-analysis for correct species allocation and subtyping of novel Ochrobactrum isolates.

Isolation and Characterization of a Dichlorvos-Degrading Strain DDV-1 of Ochrobactrum sp.

ABSTRACT The objective of this research was to isolate a dichlorvos (2,2-dichlorovinyl dimethyl phosphate)-degrading strain of Ochrobactrum sp., and determine its effectiveness in remediation of a dichlorvos-contaminated soil. A dichlorvos-degrading bacterium (strain DDV-1) was successfully isolated and identified as an Ochrobactrum sp. based on its 16S rDNA sequence analysis. Strain DDV-1 was able to utilize dichlorvos as a sole carbon source, and the optimal pH and temperature for its cell growth and degradation were 7.0 and 30 °C, respectively. Also, the growth and degradation of strain DDV-1 showed the same response to dissolved oxygen. In addition, the soil degradation test indicated that in soil spiked with 100 mg L−1 or 500 mg L−1 dichlorvos and inoculated with 0.5% or 1.0% (v/v) strain DDV-1, complete degradation of dichlorvos could be achieved in 24 h. The present study showed that strain DDV-1 was a fast dichlorvos-degrading bacterium in soil. However, further research will be needed to clarify the degradation pathway and the properties of the key enzymes involved in its biodegradation.

Comparative sequence analysis of the internal transcribed spacer 1 of Ochrobactrum species

The internal 16S/23S rDNA ( rrs/ rrl) internal spacer region 1 (ITS1) of 54 Ochrobactrum strains and close relatives was analysed. Separation of ITS1 containing PCR products by gel-electrophoresis, DGGE, cloning and sequencing revealed ITS1 length and sequence heterogeneity. We found up to 5 different allelic ITS1 stretches within a single strain ( Ochrobactrum intermedium LMG 3301 T), and 2–3 different ITS1 alleles in O. tritici. Within ITS1, ITS1c, being part of the conserved double-stranded rrn processing stem dsPS1, produced the most reliable segment tree. The overall ITS1, ITS1c and rrs phylogenetic tree topologies were generally consistent, but there was evidence for horizontal rrn (segment) transfer in O. tritici LMG 2134 (formerly O. anthropi). Good correlations were found between ITS1, ITS1c and rrs sequence similarity and DNA–DNA hybridization values indicating that phylogenetic analysis of ITS1 and ITS1c both can be used to preliminarily deduce the phylogenetic affiliation if HGT was excluded. Strains sharing > 9 6.1 9 % ITS1c ( > 9 5.1 1 % ITS1) similarity fell within a species, and ⩽ 6 8.4 2 % ITS1c ( ⩽ 7 0.3 3 % ITS1) similarity outside a genus. Both ITS1 and ITS1c analysis resolved microdiversity more profoundly than rrs analysis and revealed clades (genomovars) within O. anthropi that were also produced in rep cluster analysis. There was no evidence for habitat-specific ITS1 genomovars within Ochrobactrum species. Diversity of Ochrobactrum was higher in soil than at the rhizoplane below and at the species level. Isolates from soil contained only 1 rrn type whereas isolates from human clinical, animal and rhizoplane specimens could contain more.

Genotyping of Ochrobactrum spp. by AFLP analysis.

AFLP was used to analyze the genetic diversity among Ochrobactrum strains. AFLP patterns showed a great genomic variability that separated the samples into three distinct clusters. Ochrobactrum intermedium was found to be closely related to Brucella abortus S99.

A novel symbiotic nitrogen-fixing member of the Ochrobactrum clade isolated from root nodules of Acacia mangium.

Ten strains of root nodule bacteria were isolated from the nodules of Acacia mangium grown in the Philippines and Thailand. Partial sequences (approx. 300 bp) of the 16S rRNA gene of each isolate were analyzed. The nucleotide sequences of strain DASA 35030 indicated high homology (>99%) with members of the genus Ochrobactrum in Brucellaceae, although the sequences of other isolates were homologous to those of two distinct genera Bradyrhizobium and Rhizobium. The strain DASA 35030 was strongly suggested to be a strain of Ochrobactrum by full length sequences of the 16S rRNA gene, fatty acids composition, G+C contents of the DNA, and other physiological characteristics. Strain DASA 35030 induced root nodules on A. mangium, A. albida and Paraserianthes falcataria. The nodules formed by strain DASA 35030 fixed nitrogen and the morphology of the nodules is the same as those of nodules formed by the other isolates. This is the first report that the strain of Ochrobactrum possesses complete symbiotic ability with Acacia.

Tracking the fate of petroleum hydrocarbons: Chin, B. H., McKelvey, J. A., Tyler, T. R., Calisti, L. J., Kozbelt, S. J. & Sullivan, L. J. (1980). Absorption, distribution, and excretion of ethylbenzene, ethylcyclohexane, and methylethylbenzene isomers in rats. Bull. envir. Contam. Toxicol.24, 477

The objective of this research was to isolate a dichlorvos (2,2-dichlorovinyl dimethyl phosphate)-degrading strain of Ochrobactrum sp., and determine its effectiveness in remediation of a dichlorvos-contaminated soil. A dichlorvos-degrading bacterium (strain DDV-1) was successfully isolated and identified as an Ochrobactrum sp. based on its 16S rDNA sequence analysis. Strain DDV-1 was able to utilize dichlorvos as a sole carbon source, and the optimal pH and temperature for its cell growth and degradation were 7.0 and 30 °C, respectively. Also, the growth and degradation of strain DDV-1 showed the same response to dissolved oxygen. In addition, the soil degradation test indicated that in soil spiked with 100 mg L−1 or 500 mg L−1 dichlorvos and inoculated with 0.5% or 1.0% (v/v) strain DDV-1, complete degradation of dichlorvos could be achieved in 24 h. The present study showed that strain DDV-1 was a fast dichlorvos-degrading bacterium in soil. However, further research will be needed to clarify the degradation pathway and the properties of the key enzymes involved in its biodegradation.