Evaluating the Effect of Azole Antifungal Agents on the Stress Response and Nanomechanical Surface Properties of Ochrobactrum anthropi Aspcl2.2.

Azole antifungal molecules are broadly used as active ingredients in various products, such as pharmaceuticals and pesticides. This promotes their release into the natural environment. The detailed mechanism of their influence on the biotic components of natural ecosystems remains unexplored. Our research aimed to examine the response of Ochrobactrum anthropi AspCl2.2 to the presence of four azole antifungal agents (clotrimazole, fluconazole, climbazole, epoxiconazole). The experiments performed include analysis of the cell metabolic activity, cell membrane permeability, total glutathione level and activity of glutathione S-transferases. These studies allowed for the evaluation of the cells' oxidative stress response to the presence of azole antifungals. Moreover, changes in the nanomechanical surface properties, including adhesive and elastic features of the cells, were investigated using atomic force microscopy (AFM) and spectrophotometric methods. The results indicate that the azoles promote bacterial oxidative stress. The strongest differences were noted for the cells cultivated with fluconazole. The least toxic effect has been attributed to climbazole. AFM observations unraveled molecular details of bacterial cell texture, structure and surface nanomechanical properties. Antifungals promote the nanoscale modification of the bacterial cell wall. The results presented provided a significant insight into the strategies used by environmental bacterial cells to survive exposures to toxic azole antifungal agents.

Temperature-dependent regulation of the Ochrobactrum anthropi proteome.

Ochrobactrum anthropi is a Gram-negative rod belonging to the Brucellaceae family, able to colonize a variety of environments, and actually reported as a human opportunistic pathogen. Despite its low virulence, the bacterium causes a growing number of hospital-acquired infections mainly, but not exclusively, in immunocompromised patients. The aim of this study was to obtain an overview of the global proteome changes occurring in O. anthropi in response to different growth temperatures, in order to achieve a major understanding of the mechanisms by which the bacterium adapts to different habitats and to identify some potential virulence factors. Combined quantitative mass spectrometry-based proteomics and bioinformatics approaches were carried out on two O. anthropi strains grown at temperatures miming soil/plants habitat (25°C) and human host environment (37°C), respectively. Proteomic analysis led to the identification of over 150 differentially expressed proteins in both strains, out of over 1200 total protein identifications. Among them, proteins responsible for heat shock response (DnaK, GrpE), motility (FliC, FlgG, FlgE), and putative virulence factors (TolB) were identified. The study represents the first quantitative proteomic analysis of O. anthropi performed by high-resolution quantitative mass spectrometry.

[Research progress of Ochrobactrum anthropic--A review].

Ochrobactrum anthropi is a gram-negative bacillus, usually known as an opportunistic pathogen. Mostly its infection is related with systemic or local lower immunity, and manifested as bacteremia, meningitis, purulent infection. Recently, along with expanded infection, it has become an important human pathogen. The prevention, clinical diagnose and treatment become complicated because varied clinical symptoms increased antibiotic resistance and cross immune reaction with others pathogens. In this review, we summarized the biological characteristics, differential diagnosis, immunity, resistance and genomic characteristics of Ochrobactrum anthropi, to provide reference for prevention, control and treatment management of this disease.

Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO-111 against blister blight disease of tea.

AIMS: The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition. METHODS AND RESULTS: A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO-075, BMO-111 and BMO-147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36-h-old culture of BMO-111 (1 × 10(8) colony-forming units ml(-1) ) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO-111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO-111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses. CONCLUSIONS: It could be concluded that the biocontrol agent O. anthropi BMO-111 was effective against blister blight disease of tea. SIGNIFICANCE AND IMPACT OF THE STUDY: Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.

Integrated control of blister blight disease in tea using the biocontrol agent Ochrobactrum anthropi strain BMO-111 with chemical fungicides.

AIMS: The present study was aimed to evaluate the integration of Ochrobactrum anthropi BMO-111 and chemical fungicides (copper oxychloride and hexaconazole) against blister blight disease of tea. METHODS AND RESULTS: Application of the liquid culture of O. anthropi BMO-111 (36-h-old culture broth) was found to be effective in combined sprays with individual chemical fungicides (copper oxychloride and hexaconazole). Spray application of O. anthropi BMO-111 to tea bushes improved the biochemical parameters such as the levels of chlorophyll, polyphenols, and catechins in the harvestable tea shoots. Moreover, in the microplot and large scale trials, the integrated treatment of every two O. anthropi BMO-111 sprays followed by a single fungicides spray was found to be more efficient than the stand alone O. anthropi BMO-111 or chemicals sprays. Further, pathogenicity study employing Swiss albino mice showed no mortality in the test animals when challenged with O. anthropi BMO-111 through oral, intravenous and intranasal routes. CONCLUSIONS: The field trials clearly established that O. anthropi BMO-111 has capability to reduce incidence in integrated management of blister blight disease of tea and safe to use in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that O. anthropi BMO-111 can be used as an agricultural input in the integrated crop protection systems.

Study on biodegradation kinetics of di-2-ethylhexyl phthalate by newly isolated halotolerant Ochrobactrum anthropi strain L1-W.

OBJECTIVE: Di-2-ethylhexyl phthalate (DEHP) pollution is one of the major environmental concerns all over the world. This research aimed at studying the biodegradation kinetics of DEHP by a newly isolated bacterial strain. Water and sediment samples were collected from Wuhan South Lake and potent bacterial isolates were screened for DEHP degradation, characterized by biochemical, physiological, morphological and 16S rDNA gene sequencing, and optimized under suitable pH, temperature, NaCl and DEHP concentrations. DEHP and its metabolites were quantified by High Performance Liquid Chromatography and their degradation kinetics were studied. RESULTS: The newly isolated bacterium was identified as Ochrobactrum anthropi strain L1-W with 99.63% similarity to Ochrobactrum anthropi ATCC 49188. It was capable of utilizing DEHP as the carbon source. The optimum growth temperature, pH, DEHP and NaCl concentration for the strain L1-W were 30 °C, 6, 400 mg/L and 10 g/L respectively. Strain L1-W was capable of degrading almost all (98.7%) of DEHP when the initial concentration was 200 mg/L within a period of 72 h. Besides, it was also found capable of degrading five other phthalates, thus making it a possible candidate for bioremediation of phthalates in the environmental settings.

Evaluation of Ochrobactrum anthropi TRS-2 and its talc based formulation for enhancement of growth of tea plants and management of brown root rot disease.

AIM: To evaluate Ochrobactrum anthropi TRS-2 isolated from tea rhizosphere and its talc based formulation for growth promotion and management of brown root rot disease of tea. METHODS AND RESULTS: Ochrobactrum anthropi TRS-2, isolated from tea rhizosphere could solubilize phosphate, produce siderophore and IAA in vitro and also exhibited antifungal activity against six test pathogens. Application of an aqueous suspension of O. anthropi to the rhizosphere of nursery grown tea seedlings of five varieties of tea (TV-18, T-17, HV-39, S-449, UP-3 and) led to enhanced growth of the treated plants, as evidenced by increase in height, in the number of shoots and number of leaves per shoot. Treatment with O. anthropi also decreased brown root rot of tea, caused by Phellinus noxius. Multifold increase in activities of chitinase, beta-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in tea plants was observed on application of O. anthropi to soil followed by inoculation with P. noxius. A concomitant increase in accumulation of phenolics was also obtained. Further, talc based formulation of O. anthropi was prepared and its survival determined every month up to a period of 12 months. Ochrobactrum anthropi could survive in the formulation up to a period of 9 months with a concentration of 7.0 log(10) CFU g(-1), after which there was a decline. Talc formulation was as effective as aqueous suspensions in both plant growth promotion and disease suppression. CONCLUSION: Ochrobactrum anthropi, either in aqueous suspension or as talc formulation induced growth of tea plants and suppressed brown root rot disease. It induced defense responses in tea plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochrobactrum anthropi and its talc based formulation can be considered as an addition to available plant growth promoting rhizobacteria (PGPR) currently being used for field application. The present study offers a scope of utilizing this bacterium for growth promotion and disease management which would help in reduction of the use of chemicals in tea plantations.

Isolation of the exoelectrogenic bacterium Ochrobactrum anthropi YZ-1 by using a U-tube microbial fuel cell.

Exoelectrogenic bacteria have potential for many different biotechnology applications due to their ability to transfer electrons outside the cell to insoluble electron acceptors, such as metal oxides or the anodes of microbial fuel cells (MFCs). Very few exoelectrogens have been directly isolated from MFCs, and all of these organisms have been obtained by techniques that potentially restrict the diversity of exoelectrogenic bacteria. A special U-tube-shaped MFC was therefore developed to enrich exoelectrogenic bacteria with isolation based on dilution-to-extinction methods. Using this device, we obtained a pure culture identified as Ochrobactrum anthropi YZ-1 based on 16S rRNA gene sequencing and physiological and biochemical characterization. Strain YZ-1 was unable to respire using hydrous Fe(III) oxide but produced 89 mW/m(2) using acetate as the electron donor in the U-tube MFC. Strain YZ-1 produced current using a wide range of substrates, including acetate, lactate, propionate, butyrate, glucose, sucrose, cellobiose, glycerol, and ethanol. Like another exoelectrogenic bacterium (Pseudomonas aeruginosa), O. anthropi is an opportunistic pathogen, suggesting that electrogenesis should be explored as a characteristic that confers advantages to these types of pathogenic bacteria. Further applications of this new U-tube MFC system should provide a method for obtaining additional exoelectrogenic microorganisms that do not necessarily require metal oxides for cell respiration.

Reporter genes for real-time in vive monitoring of Ochrobactrum anthropi infection.

Despite the increasing interest in Ochrobactrum anthropi as an emerging nosocomial pathogen resistant to most commonly used antimicrobials, relatively little is known about the pathogenesis and factors contributing to its virulence. Also, many aspects of interaction between Ochrobactrum spp. and their hosts remain unclear. The ability to monitor O. anthropi infection in the host will facilitate our understanding of the pathogenic mechanisms and will lead to better choices of antimicrobial or additional therapeutic strategies. We have demonstrated the ability to stably express three reporter genes (green fluorescence protein GFP, red fluorescence protein RFP and luciferase Lux) and track the infection in a J774A.1 murine macrophage cell line as well as in the BALB/c mouse. Our results suggest that these reporter genes should improve genetic studies in O. anthropi, particularly those aimed at understanding pathogenesis, virulence factors and host interaction.

Peritonitis in type 2 diabetes mellitus due to Ochrobactrum anthropi complicating automated peritoneal dialysis.

Epidemiology data predict that by the year 2025, diabetes will affect about 380 million people worldwide with a significant increase in patients with chronic renal disease progressing to hemodialysis. Diabetes-related peripheral vascular disease is a major risk factor for vascular access failure in patients on extracorporeal hemodialysis. Although peritoneal dialysis is a valid option for diabetics, peritonitis is still a main complication for these patients. We report the case of a 71-year-old type 2 diabetes patient treated by subcutaneous insulin, undergoing automated peritoneal dialysis (APD) who developed peritonitis and bloodstream infection by Ochrobactrum anthropi (O. anthropi). The patient was initially shifted to continuous ambulatory peritoneal dialysis (CAPD) and treated with intraperitoneal cefotaxime and gentamicin. According to antibiogram, cefotaxime was discontinued but lasting gentamicin. Within 48 h from admission, clear peritoneal effluent was observed with reduction in white blood cells count from 580/mm³ 77.9% neutrophils to less than 10/mm³. Prompt regression of infection without catheter removal and no relapse after over 7-month follow-up allowed supposing that O. anthropi did not colonized peritoneal catheter. O. anthropi is an emerging cause of nosocomial infection in immunocompromised patients. Cases of such infection in patients undergoing CAPD and hemodialysis have been already described. However, this is the first reported case of O. anthropi in a patient undergoing APD.

Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions.

Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573-579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51 kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24 kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553-559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.

Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida).

Bacteria naturally associated with the symbiont Photorhabdus luminescens subsp. akhurstii were isolated from the entomopathogenic nematode Heterorhabditis indica. Bacterial isolates distinct from P. luminescens subsp. akhurstii were obtained from 33% of the samples. Fourteen bacterial isolates, from nematodes collected from three different Caribbean islands, were characterized by conventional phenotypic tests, restriction fragment length polymorphism and sequence analyses of PCR-amplified 16S rRNA genes (16S rDNAs). Isolates were grouped into three genotypes, each one being associated with one Caribbean island. Phenotypic characteristics and 16S rDNA analysis showed that the Photorhabdus-associated bacteria were closely related to Ochrobactrum anthropi for the group from Guadeloupe, and to Ochrobactrum intermedium for the two groups from the Dominican Republic and Puerto Rico. No pathogenicity of the Ochrobactrum spp. to the insects Galleria mellonella and Spodoptera littoralis (Lepidoptera) was detected. Since Ochrobactrum spp. are considered as human opportunist pathogens, the mass production of entomopathogenic nematodes for biological control requires strict vigilance.

Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance.

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortus LPS, but not of Escherichia coli LPS, into Ochrobactrum OM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropi and E. coli-O. anthropi LPS hybrid aggregates. Although Ochrobactrum was sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity. Ochrobactrum and B. abortus LPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, Ochrobactrum LPS, but not B. abortus LPS, contained galacturonic acid. B. abortus and Ochrobactrum smooth LPS aggregates had similar size and zeta potential (-12 to -15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrum smooth LPS while remaining negative (-5 mV) for B. abortus smooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrum and Brucella share a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution of B. abortus to pathogenicity.

Endocarditis infecciosa por Ochrobactrum anthropi / Infective endocarditis due to Ochrobactrum anthropi

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