Multilocus sequence typing of Ochrobactrum spp. isolated from gastric niche.

The human stomach is colonized by diverse bacterial species. The presence of non-Helicobacter pylori bacteria in urease-positive biopsies of individuals has been reported. Bacteria belonging to the Ochrobactrum genus have been documented in the human gastric niche. The co-occurrence of Ochrobactrum spp. with H. pylori was previously reported in an antral biopsy of a non-ulcer dyspeptic (NUD) subject from Northern India. There is no information on the genetic diversity of Ochrobactrum spp. isolated from the gastric niche in the stomach. We aimed to study the species distribution and diversity of Ochrobactrum spp. with and without H. pylori in urease-positive biopsies across three different geographical regions in India. Sixty-two Ochrobactrum isolates recovered from patients with an upper gastric disorder (n=218) were subjected to molecular identification and multilocus sequence typing. H. pylori DNA was found in the majority of biopsies, which had a variable degree of Ochrobactrum spp present. Interestingly, some of the urease-positive biopsies only had Ochrobactrum without any H. pylori DNA. Based on phylogenetic analysis, the Ochrobactrum isolates were distributed into the O. intermedium, O. anthropi and O. oryzae groups. This indicates there are multiple species in the gastric niche irrespective of the presence or absence of H. pylori. Antibiotyping based on colistin and polymyxin B could differentiate between O. intermedium and O. anthropi without revealing the resistance-driven diversity. Considering the prevalence of multiple Ochrobactrum spp. in the human gastric niche, it is important to evaluate the commensal and/or pathogenic nature of non-H. pylori bacteria with respect to their geographical distribution, lifestyle and nutrition needs.

Mushroom tumor: a new disease on Flammulina velutipes caused by Ochrobactrum pseudogrignonense.

Mushroom tumor on Flammulina velutipes has become the main disease during the off-season cultivation of F. velutipes while the causal organism has remained unknown. The present study was aimed at identifying the pathogen confirming its pathogenisity following Koch's Postulates, characterizing it using morphological, physiological, biochemical and molecular features, and studying its current distribution. We determined that mushroom tumor is a new bacterial infection disease caused by Ochrobactrum pseudogrignonense. It produces tumor-like structures on the surface of the substrate, and inhibits the formation of primordia and fruiting of F. velutipes. The molecular studies showed that this new pathogen is closely related to Ochrobactrum based on 16S rRNA sequences. This is the first time that Ochrobactrum has been shown to be a pathogen of a mushroom.

Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains.

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301(T)) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.

Molecular mechanism of nicotine degradation by a newly isolated strain, Ochrobactrum sp. strain SJY1.

A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.

Antibacterial properties of silver nanoparticles synthesized by marine Ochrobactrum sp.

Metal nanoparticle synthesis is an interesting area in nanotechnology due to their remarkable optical, magnetic, electrical, catalytic and biomedical properties, but there needs to develop clean, non-toxic and environmental friendly methods for the synthesis and assembly of nanoparticles. Biological agents in the form of microbes have emerged up as efficient candidates for nanoparticle synthesis due to their extreme versatility to synthesize diverse nanoparticles with varying size and shape. In the present study, an eco favorable method for the biosynthesis of silver nanoparticles using marine bacterial isolate has been attempted. Very interestingly, molecular identification proved it as a strain of Ochrobactrum anhtropi. In addition, the isolate was found to have the potential to form silver nanoparticles intracellularly at room temperature within 24 h. The biosynthesized silver nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscope (TEM) and scanning electron microscope (SEM). The UV-visible spectrum of the aqueous medium containing silver nanoparticles showed a peak at 450 nm corresponding to the plasmon absorbance of silver nanoparticles. The SEM and TEM micrographs revealed that the synthesized silver nanoparticles were spherical in shape with a size range from 38 nm - 85 nm. The silver nanoparticles synthesized by the isolate were also used to explore its antibacterial potential against pathogens like Salmonella Typhi, Salmonella Paratyphi, Vibrio cholerae and Staphylococcus aureus.

Ochrobactrum daejeonense sp. nov., a nitrate-reducing bacterium isolated from sludge of a leachate treatment plant.

A Gram-reaction-negative, non-spore-forming, rod-shaped, aerobic bacterial strain, designated MJ11(T), was isolated from sludge of a leachate treatment plant in Daejeon, South Korea, and was characterized taxonomically by using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain MJ11(T) belonged to the family Brucellaceae, class Alphaproteobacteria, and was most closely related to Ochrobactrum ciceri Ca-34(T) (97.9 % sequence similarity) and Ochrobactrum pituitosum CCUG 50899(T) (96.4 %). Comparative sequence analyses of the additional phylogenetic marker genes dnaK, groEL and gyrB confirmed the affiliation of strain MJ11(T) to the genus Ochrobactrum. The G+C content of the genomic DNA of strain MJ11(T) was 59.3 mol%. The detection of a quinone system with ubiquinone Q-10 as the predominant respiratory lipoquinone, a fatty acid profile with C(18 : 1)ω7c (62.6 %) and C(19 : 0) cyclo ω8c (14.2 %) as the major components, a polar lipid profile with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol and unknown aminolipids AL1 and AL2 as major polar lipids and spermidine and putrescine as the predominant polyamines also supported the affiliation of strain MJ11(T) to the genus Ochrobactrum. The DNA-DNA relatedness between strain MJ11(T) and Ochrobactrum ciceri DSM 22292(T) was 29 ± 7 %, clearly showing that the isolate constitutes a new genospecies. Strain MJ11(T) could be clearly differentiated from its closest neighbours on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain MJ11(T) represents a novel species of the genus Ochrobactrum, for which the name Ochrobactrum daejeonense sp. nov. is proposed. The type strain is MJ11(T) ( = KCTC 22458(T) = JCM 16234(T)).

Characterization of a Cd(2+)-resistant strain of Ochrobactrum sp. isolated from slag disposal site of an iron and steel factory.

A cadmium-resistant bacterium designated as CdSP9 was isolated from the slag disposal site of IISCO, Burnpur, West Bengal, India. The isolate was identified as Ochrobactrum sp. on the basis of 16S rDNA sequence-based molecular phylogenetic approach and phenotypic characteristics. It is a Gram negative, short rod (0.5-1.0 micro), aerobic bacterium, growing well in LB medium between temperatures 10-42 degrees C, pH 6.0-9.0, and between 2 and 6% NaCl. The most preferred nitrogen and carbon sources for the strain are L: -proline, L: -lysine and fructose, maltose, respectively. Superoxide toxicity minimization by increased level of SOD activity also occurs in this bacterium. The heavy metal accumulation efficiency as determined by atomic absorption spectroscopy was found to be 0.214 mg/g of the dry weight at late log phase. The accumulation efficiency was directly proportional to the optimum growth conditions.

[Biodegradation of nicotine in tobacco extracts for making reconstituted tobacco by strain DN2].

The purpose of the study is to use O. intermedium DN2 to degrade nicotine in tobacco extracts for making reconstituted tobacco. Firstly, we studied the effects of various factors on degradation of nicotine in the extracts by strain DN2. When we added 0.1% yeast extract into the extracts, adjusted its pH value to 7.0 by ammonia solution, inoculated 15% cultures and maintained fermentation temperature of 30 degrees C, the degradation rate of nicotine by strain DN2 was the fastest. Furthmore, under these conditions, we studied the degradation rates of nicotine in three fed batches culture which carried out in a 30-L reactor, the result showed that the average degradation rate of nicotine by strain DN2 was 140.55 mg/L/h, which was much higher than that reported in other studies. These results indicated that strain DN2 may be useful for reducing nicotine content of reconstituted tobacco.

Brucellosis of the common vole (Microtus arvalis).

A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.

[Isolation and identification of a strain DN2 degrading nicotine].

A bacterial strain which was able to utilize nicotine as its sole carbon source was isolated from soil in which tobacco had grown at Sanming region in FUjian Province and named as DN2. Upon chemotaxonomic characterization and phylogenetic inference based 16S rDNA analysis, the strain DN2 was identified as a-proteobacteria, Ochrobactrum intermedium. For DN2 degrading nicotine, the optimal pH and temperature is 6.5, 30 degrees C respectively. It can tolerate high-concentration of nicotine up to 4000 mg/L in basal media. Using 500 mg/L nicotine as its sole carbon, the strain was able to degrade 15 mg/L of nicotine per liter per hour and reached its stationary phase in about 36 hours.

Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov.

The nodulation of legumes has for more than a century been considered an exclusive capacity of a group of microorganisms commonly known as rhizobia and belonging to the alpha-Proteobacteria. However, in the last 3 years four nonrhizobial species, belonging to alpha and beta subclasses of the Proteobacteria, have been described as legume-nodulating bacteria. In the present study, two fast-growing strains, LUP21 and LUP23, were isolated from nodules of Lupinus honoratus. The phylogenetic analysis based on the 16S and 23S rRNA gene sequences showed that the isolates belong to the genus Ochrobactrum. The strains were able to reinfect Lupinus plants. A plasmid profile analysis showed the presence of three plasmids. The nodD and nifH genes were located on these plasmids, and their sequences were obtained. These sequences showed a close resemblance to the nodD and nifH genes of rhizobial species, suggesting that the nodD and nifH genes carried by strain LUP21T were acquired by horizontal gene transfer. A polyphasic study including phenotypic, chemotaxonomic, and molecular features of the strains isolated in this study showed that they belong to a new species of the genus Ochrobactrum for which we propose the name Ochrobactrum lupini sp. nov. Strain LUP21T (LMG 20667T) is the type strain.