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Nitrilase-catalyzed production of pyrazinoic acid, an antimycobacterial agent, from cyanopyrazine by resting cells of Rhodococcus rhodochrous J1.

Using resting cells of Rhodococcus rhodochrous J1, in which a large amount of nitrilase is induced, a simple and efficient bioconversion process for the production of pyrazinoic acid, an antimycobacterial agent, through catalysis by a nitrilase was developed. The reaction conditions for production of pyrazinoic acid were optimized. Under optimum conditions, 3.5 M cyanopyrazine was converted to pyrazinoic acid, with a molar conversion yield of 100%. The highest yield achieved corresponded to 434 g of pyrazinoic acid per liter of reaction mixture. The synthesized pyrazinoic acid was isolated and identified physico-chemically.

Aminohydrolases↗

Rhodopeptins (Mer-N1033), novel cyclic tetrapeptides with antifungal activity from Rhodococcus sp. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

Five novel cyclic tetrapeptides, named rhodopeptin C1, C2, C3, C4 and B5, were isolated from a strain named Rhodococcus sp. Mer-N1033. They are a novel type of cyclic tetrapeptide composed of a beta-amino acid and three usual alpha-amino acids. Rhodopeptins show high in vitro antifungal activity against Candida albicans and Cryptococcus neoformans, whereas they show no activity against bacteria.

Animals↗

Temperature effects and substrate interactions during the aerobic biotransformation of BTEX mixtures by toluene-enriched consortia and Rhodococcus rhodochrous.

A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20 degrees C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X). Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35 degrees C. A consortium enriched at 35 degrees C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased. Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures. Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes. In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last. A pure culture isolated from the 35 degrees C-enriched consortium was identified as Rhodococcus rhodochrous. This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures. Additionally, R. rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources. Studies conducted with the 35 degrees C-enriched consortium and R. rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates. Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures. Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and noncompetitive inhibition kinetics. Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures. Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction.

Aerobiosis↗

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous.

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.

Amidohydrolases↗

Production of cholesterol oxidase by Rhodococcus equi No. 23 in a jar fermenter.

Rhodococcus equi No. 23 was grown in a batch fermenter. The effects of cultivation temperature, pH of the culture medium, aeration rate and agitation speed on the production of cholesterol oxidase (CholOx) by the test organism were examined. Results revealed that the cultivation temperature, the pH of the medium, the aeration rate and the agitation speed all affected the production of CholOx by R. equi No. 23. Adjusting the operation variables during the cultivation period increased the production of CholOx effectively and prevented the occurrence of overflow of foam during the fermentation period. A maximum CholOx activity of 0.34 unit/ml with a volumetric production rate of 0.011 unit/h per ml could be achieved in 30 h of cultivation at an aeration rate of 5.0 l/min, if the pH of the culture medium, the cultivation temperature and the agitation speed were controlled at 6.5, 39 degrees C and 200 rev. /min respectively during the first 24 h of cultivation, then shifted to 7.5, 37 degrees C and 300 rev./min respectively.

Bioreactors↗

Complement activation by mycoloyl glycolipids from Mycobacterium tuberculosis and Rhodococcus ruber.

In this study, we examined complement activation by mycoloyl glycolipids (MGL) such as trehalose-6,6'-dimycolate (TDM), often termed cord factor, and trehalose-6-monomycolate from Mycobacterium tuberculosis and Rhodococcus ruber, and also examined the effect of complement binding to MGL on phagocytosis by human monocytes. TDM and TMM, but not glucose mycolate, mannose mycolate or fructose mycolate which differ from TMM only in carbohydrate moiety, exhibited complement activation. TDM and TMM of M.tuberculosis exhibited stronger complement activation than those of R.ruber, the mycolic acids of which are much shorter than those of M.tuberculosis. Neither mycolic acids nor trehalose, which are products of TDM and TMM by hydrolytic cleavage, exhibited no complement activation. TDM activated complement through the alternative pathway, since supplementation with C4-deficient serum completely restored classical pathway-mediated hemolytic activity of complement which had been previously consumed by TDM. Next, we examined the effects of TDM and TMM on phagocytosis by human monocytes. Coating of heat-killed Staphylococcus aureus cells with TDM or TMM did not enhance their phagocytosis by monocytes, while, in the presence of complement, phagocytosis of these cells increased significantly. These findings suggest that TDM and TMM act as virulence factors that enhance the entry of mycobacteria into phagocytes via binding of C3 through activation of the alternative complement pathway.

Adjuvants, Immunologic↗

Isolation of rare opportunistic pathogens in Hungary: case report and short review of the literature. Rhodococcus equi.

Rhodococcus equi is a well-established pathogen in foal pneumonia and is increasingly recognized as a pathogen in immunocompromised humans. We have isolated a Gram-positive coccobacillus from 8 blood samples and lung tissues of a renal transplant patient. Colony morphology, growth in Lowenstein-Jensen medium, 21 biochemical reactions, the characteristic morphological cycle (coccus-rod-coccus) and the CAMP test established the R. equi diagnosis. Histological studies of 2 lung biopsy specimens revealed numerous microabscesses with aggregates of polymorphonuclear leukocytes surrounded by abundant foamy macrophages. Our isolates proved to be sensitive to majority of antibacterial drugs. The appropriate therapy (amoxicillin-clavulanate) proved to be effective, however six months later a relapse was observed. Data show that in spite of its rare occurrence, R. equi infection represents a diagnostic and therapeutic challenge. The taxonomical, epidemiological, clinico-pathological, diagnostic and therapeutic data of R. equi are discussed.

Actinomycetales Infections↗

[Adaptation of acrylamide producer Rhodococcus rhodochrous M8 to change in ammonium concentration in medium].

The mechanism of adaptation of the acrylamide producing strain Rhodococcus rhodochrous M8 to changes in ammonium concentrations in the medium was studied. An increase in the content of ammonium in the medium changed the activity of glutamine synthetase (GS) (EC 6.3.1.2) and glutamine dehydrogenase (GD) (EC 1.4.1.4), the enzymes of ammonium assimilation, as well as the activities of enzymes responsible for nitrile utilization: nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). This also caused inhibition of activation of GS induced by phosphodiesterase (EC 3.1.4.1). Increases in the activities of nitrile hydratase and amidase and resistance of these enzymes to ammonium were observed in mutant of R. rhodichrous resistant to phosphotricine, an inhibitor of GS. An important role of GS in the mechanism of adaptation is suggested.

Acrylamide↗

[Oxidation of organic compounds by Rhodococcus erythropolis 3/89 propanomonooxygenase].

The ability of propane monooxygenase of Rhodococcus erythropolis 3/89 to catalyze oxidation of higher liquid alkenes and aromatic hydrocarbons was studied. Optimal conditions of tetradecene epoxidation and benzene hydroxylation were found. Under these conditions, oxidation was shown to be accompanied by a 100% conversion of benzene to phenol.

Alkenes↗

[Growth of Rhodococcus opacus on mixtures of monohalogenated benzenes and phenols].

The growth of Rhodococcus opacus GM-14 on mixtures of 2-chloro- and 2-bromophenol, of 4-chloro, 4-bromo-, and 4-iodophenol, and of chloro-, bromo-, and iodobenzenes was accompanied by consumption of the substrates and excretion of halogen ions to the medium. During the growth on monochlorophenols, the substrates were consumed sequentially in the following order: 4-chloro-, 3-chloro-, and then 2-chlorophenol. Chlorine ions were excreted in a two-phase manner in amounts comprising 79% of the theoretical yield. The diauxic growth of R. opacus GM-14 can be explained by the existence in this bacterium of two modified metabolic pathways for the ortho-cleavage of halogenated pyrocatechols. The first pathway included 4-halogeno- or dihalogenopyrocatechols as intermediates, whereas the second pathway included 3-halogenopyrocatechols.

Benzene Derivatives↗

[A diagnostic medium for Arcanobacterium haemolyticum and other bacterial species reacting with hemolytic synergism to the equi-factor of Rhodococcus equi].

Colonies of Arcanobacterium haemolyticum on common blood agar can be easily overlooked. Therefore a diagnostic medium was developed, on which A. haemolyticum colonies produce a conspicuous zone of complete hemolysis. The medium under question is blood agar prepared from the Columbia Blood Agar Base and 5% washed sheep erythrocytes sensitised with equi factor (EF) of Rhodococcus equi. Optimally, 10 activity units (AU) of EF per 1 ml were used. EF was titrated on a non-nutrient medium consisting of agar Purified (Difco) and 5% washed sheep erythrocytes sensitized with beta-haemolysin (BL) of Staphylococcus aureus, 10 AU/ml. On the same medium, staphylococcal BL was titrated on the basis of its direct haemolytic effect. Despite the very distinct haemolytic reaction of the control strains evident on the diagnostic medium with EF, A. haemolyticum failed to grow from 2.597 throat swabs examined especially for the particular microbe during the a period of two years. However, A. haemolyticum was isolated on this medium twice from 223 swabs from wounds and skin lesions. The proposed medium makes also the rapid diagnosis of species Corynebacterium ulcerans, Dermatophilus congolensis and Listeria monocytogenes on the basis of haemolytic synergism possible.

Actinomycetales↗

Characterisation of Rhodococcus equi strains isolated from foals and from immunocompromised human patients.

The cultural, morphological, biochemical, serological characteristics and antibiotic susceptibility of 25 Rhodococcus equi strains isolated from lungs and lung abscesses of pneumonic foals and 5 R. equi strains isolated from immuno-compromised human patients were examined. All R. equi strains showed common cultural, morphological and biochemical characteristics both with conventional tests and on the basis of their enzyme profile. The R. equi strains examined were resistant to penicillins with the exception of ampicillin, to sulphamethazine and several strains also to sulphamethoxazole-trimethoprim. All strains were susceptible to erythromycin and rifampicin. The strains isolated from humans showed somewhat higher rate of antibiotic resistance to penicillin, cefotaxime, kanamycin, streptomycin, lincomycin, and oxytetracycline. The overwhelming majority (96%) of the equine isolates belonged to serotype 1 in Prescott's serotyping system, while the human isolates could not be serotyped.

Animals↗

[The respiratory activity of Rhodococcus rhodochrous M8 cells producing nitrile-hydrolyzing enzymes].

The respiratory activity of Rhodococcus rhodochrous M8 cells containing nitrile hydratase and amidase was studied in the presence of nitriles and amides of carbonic acids. Culturing of cells with acrylonitrile and acrylamide yielding their maximum respiratory activity was studied. The optimum conditions for measurements and maintenance of respiratory activity were found. Curves for the linear concentration dependence of cell respiratory activity on 0.01-0.5 mM acrylonitrile, 0.025-1.0 mM acetonitrile, and 0.01-0.1 mM acrylamide were plotted. The selectivity of cell respiratory activity for some substrates was analyzed.

Acrylamides↗

[Role of cytology in the diagnosis of Rhodococcus equi infection].

A 59-year-old HIV-positive man developed necrotizing right-lobe pneumonia with abscess formation. Bronchial aspirates and bronchoalveolar lavage showed numerous inflammatory cells, mainly histiocytes and neutrophils, and intracellular and extracellular Gram- and Ziehl-Nielsen-positive cocci and coccobacilli. Rhodococcus equi was identified. The patient died one week after diagnosis and necropsy confirmed the presence of R. equi nectrotizing pneumonia.

Actinomycetales Infections↗

[The role of low-molecular-weight nitrogen compounds in the osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis].

Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool). The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media. It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria. The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R. erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values.

Amino Acids↗

Rhodococcus equi pleuropneumonia in an adult horse.

A 10-year-old warmblood gelding was evaluated for intermittent pyrexia, dullness, weight loss, and progressive respiratory disease. Multifocal necrotic pneumonia and pleuritis due to Rhodococcus equi infection was diagnosed. Case management is discussed, as well as factors that may have led to this rare cause of pleuropneumonia in an adult horse.

Actinomycetales Infections↗

[Biosensor of the reactor type based on Rhodococcus erythropolis HL TM-1 cells for determining 2,4-dinitrophenol].

A model of a reactor-type biosensor based on the Rhodococcus erythropolis HL PM-1 was developed for amperometric detection of 2,4-dinitrophenol (2,4-DNP). The effects of the matrix material (agar and calcium alginate gels, ceramic support, and cellulose powder) on the biosensor signal concentration dependence, detection time, and biosensor stability were studied. In case of bacterial cells immobilized on cellulose powder, the lower limit of 2,4-DNP detection was 20 microM and the time of single analysis, the biosensor recovery included, was 30-50 min. In the continuous detection mode, the biosensor response was maintained at a stable level without biosensor inactivation for ten days. The biosensor can be used as an element of a complex analytical system for detecting nitroaromatic compounds in samples.

2,4-Dinitrophenol↗

Survival of foals with experimentally induced Rhodococcus equi infection given either hyperimmune plasma containing R. equi antibody or normal equine plasma.

The purpose of this study was to determine if colostrum-deprived foals with experimentally induced Rhodococcus equi pneumonia have a decreased severity of the disease and decreased mortality rate when given hyperimmune (HI) R. equi antibody plasma (R. equi titer at least 100 % and virulence-associated protein A [VapA] at least 10000) prophylactically versus when given normal equine plasma (R. equi titer less than 20 % and VapA less than 160). Sixteen colostrum-deprived foals (R. equi titer less than 5 %) each received normal equine plasma in the first 24 hours of life (R. equi titer less than 20 %). At 14 days of age, six foals were given normal equine plasma and 10 foals were given HI plasma. All foals were subsequently infected intrabronchially with a pathogenic strain of R. equi (2.5 x 10 sup 8; organisms) at 21 days of age. Repeated physical examinations, weight measurements, complete blood cell counts, fibrinogen measurements, and thoracic radiographs (ventrodorsal and lateral) were performed to help determine the severity of the disease. Foals given HI plasma had significantly higher R. equi ELISA titers (42.4 %) than those given normal plasma (20.9 %) on the day of experimental infection. Mortality rates and severity of disease were statistically similar (P >.05) for the groups. Although none of the foals was treated with antibiotics, several with severe R. equi pneumonia recovered. Either HI or normal equine plasma administered to foals in the first few weeks of life caused no adverse effects and may be protective against R. equi, although the exact constituent responsible for protection is undetermined and requires further investigation.

Actinomycetales Infections↗