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Rhodococcus equi and genetic susceptibility: assessing transferrin genotypes from paraffin-embedded tissues.

Rhodococcus equi is a bacterial pathogen, ubiquitous in the soil, that infects many foals and is lethal to some. Transferrin is an iron-binding protein that has bacteriostatic properties in the blood. Transferrin is also highly polymorphic in most species, with 15 variants identified for horses using biochemical methods, and may be responsible for variation in susceptibility to bacterial pathogens. The objectives of this study were 1) to compare biochemical typing with DNA typing of transferrin, 2) to determine if transferrin DNA from archival paraffin-embedded tissue samples can be recovered and typed with molecular methods, and 3) to determine if there was an association between foal death caused by R. equi and transferrin type. Comparing biochemical methods and DNA sequencing for 41 horses demonstrated correspondence between the typing methods. The allele frequency of archival paraffin-embedded tissue samples from 34 Thoroughbred foals that succumbed to R. equi showed an excess of the F allele and a deficiency of the D allele (P < 0.05). Year of collection (P > 0.8), age of foal (P > 0.3), and sex of foal (P > 0.6) were not statistically associated with transferrin type. The archival material was successfully transferrin typed using DNA sequencing, and there may be an association between foal death caused by R. equi and transferrin type.

Actinomycetales Infections↗

Rhodococcus equi pneumonia: case report and literature review.

OBJECTIVE: To present a case of Rhodococcus equi (RE) pneumonia and discuss its pathophysiology and treatment. CASE SUMMARY: An HIV-positive patient presented with pneumonia. A lung biopsy was performed after sputum and thoracentesis cultures failed to identify a pathogen. The lung biopsy revealed an unidentifiable, diphtheroid-like, gram-positive rod. A bronchoscopy performed five days after the lung biopsy produced the same diphtheroid-like, gram-positive rod. The patient was treated with several injectable antibiotics, but emergence of resistance to two of the antibiotics was suspected. Two weeks after the bacterial isolate was sent to a reference laboratory, it was identified as RE. The patient was discharged on oral antibiotics and experienced no recurrence of RE pneumonia. CONCLUSIONS: RE can be difficult to identify in the microbiology laboratory, or it may be assumed to be a colonizing diphtheroid. The isolation of difficult-to-identify, gram-positive rods, or diphtheroids, from a pulmonary source in a patient with decreased cell-mediated immunity should cause one to suspect RE. RE has been noted to develop resistance to beta-lactam antibiotics during therapy. A prolonged course of combination antibiotic therapy directed at the intracellular component of infection is necessary.

AIDS-Related Opportunistic Infections↗

Chronic pneumonia caused by Rhodococcus equi in a patient without impaired immunity.

A 48 year old, human immunodeficiency virus (HIV)-negative, immunocompetent male patient had a chronic progressive pulmonary infiltrate, without radiological cavitation, in the middle lobe of the right lung produced by Rhodococcus equi. He reported direct contact with a diseased dog. The patient was diagnosed by thoracotomy and treated by lobectomy. After 16 months of follow-up, the patient was asymptomatic and had neither recurrence nor immunological disturbances.

Actinomycetales Infections↗

Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration.

BACKGROUND: The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114. RESULTS: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation). The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration. CONCLUSIONS: The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.

Cholesterol Oxidase↗

Rhodococcus equi pneumonia in AIDS: high-resolution CT findings in five patients.

The aim of this study was to describe the high-resolution CT scan findings in five patients with AIDS and pulmonary infection due to Rhodococcus equi. The study included five patients with AIDS and proven R. equi infection. The CT scans were reviewed by two observers. The patients included four men and one woman ranging from 39 years to 49 years in age (mean 42 years). The findings included areas of consolidation (n=5) with single (n=1) or multiple cavitation (n=4), ground-glass opacities (n=5), centrilobular nodules (n=3), small centrilobular nodular opacities (n=3) and "tree in bud" opacities (n=3). None of the patients had pleural effusion or lymph node enlargement. The most common high-resolution CT manifestations of R. equi infection consist of areas of consolidation with cavitation, ground-glass opacities, nodules and a tree-in-bud pattern.

AIDS-Related Opportunistic Infections↗

Effect of sulfur sources on specific desulfurization activity of Rhodococcus erythropolis KA2-5-1 in exponential fed-batch culture.

The effects of sulfur sources on the desulfurization activity of Rhodococcus erythropolis KA2-5-1 were investigated by using an exponential fed-batch culture technique. The feed rate of a sulfur source was controlled independently of the feed rate of ethanol, which was used as a carbon and energy source. Among the sulfur sources examined were dibenzothiophene (DBT), ammonium sulfate, L-cysteine, L-methionine, and 2-amino-ethanesulfonic acid. When the fed-medium contained DBT as the sole sulfur source, KA2-5-1 cells showed a maximum desulfurization activity of approximately 130 mmol 2-HBP kg-cell(-1) h(-1). Similar levels of enzyme activity were also achieved with inexpensive ammonium sulfate by using the exponential fed-batch culture technique. In addition, higher levels of desulfurization activity were achieved by increasing the dosage of the DBT desulfurization (dsz) operon and dszD gene in R. erythropolis KA2-5-1. The recombinant strain showed a maximum desulfurization activity of approximately 250 mmol 2-HBP kg-cell(-1) h(-1) in the exponential fed-batch cultures.

Biodegradation, Environmental↗

Isolation and characterization of benzene-tolerant Rhodococcus opacus strains.

Twenty-two benzene-utilizing bacteria were isolated from soil samples. Among them, three isolates were highly tolerant to benzene. They grew on benzene when liquid benzene was added to the basal salt medium at 10--90% (v/v). Taxonomical analysis identified the benzene-tolerant isolates as Rhodococcus opacus. One of the benzene-tolerant isolates, designated B-4, could utilize many aromatic and aliphatic hydrocarbons including benzene, toluene, styrene, xylene, ethylbenzene, propylbenzene, n-octane and n-decane as sole sources of carbon and energy. Strain B-4 grew well in the presence of 10% (v/v) organic solvents that it was capable of using as growth substrates. Genetic analysis revealed the benzene dioxygenase pathway is involved in benzene catabolism in strain B-4. A deletion-insertion mutant defective in the benzene dioxygenase large and small subunits genes (bnz A 1 and bnz A 2) was as tolerant to organic solvents as the wild-type strain B-4, suggesting that utilization or degradation of organic solvents is not essential for the organic solvent tolerance of R. opacus B-4.

Benzene↗

Development of a genetic transformation system for benzene-tolerant Rhodococcus opacus strains.

Rhodococcus opacus B-4 and B-9 are tolerant to various organic solvents including benzene, toluene, ethylbenzene, xylenes and styrene, and are suitable bacterial hosts for the production of chemical products from hydrophobic substrates. A 4.4-kb endogenous plasmid (pKNR 01) was isolated from R. opacus B-4 and sequenced completely. Plasmid pKNR 01 encodes proteins that share similarity to replication proteins from the enteric bacterial and actinomycete theta-replication plasmids. A 7.4-kb chimeric plasmid, designated pKNR 01.1, was constructed by fusing XhoI-digested pKNR 01 and Escherichia coli vector pSTV 28. Plasmid pKNR 01.1 had the ability to replicate in B-4 and B-9. A protocol for transformation of B-9 by electroporation was optimized employing pKNR 01.1. Frequencies of 4.1 x 10(5) transformants per mug of plasmid DNA were obtained for B-9 cells, whereas B-4 harboring naturally occurring pKNR 01 was transformed at lower frequencies (approximately 1 x 10(4) transformants per mug of plasmid DNA). Deletion analysis of pKNR 01.1 showed that the 1.9-kb SphI-XhoI region containing the repA and rep B genes and the 0.6-kb region upstream of repA was essential for plasmid maintenance in R. opacus strains.

Benzene↗

Expression of bovine lactoferrin C-lobe in Rhodococcus erythropolis and its purification and characterization.

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 degrees C with shaking, and expression of the rBLF C-lobe was induced by adding 1 microg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.

Animals↗

Substrate specificity of regiospecific desaturation of aliphatic compounds by a mutant Rhodococcus strain.

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.

Fatty Acids↗

Production of isopropyl cis-6-hexadecenoate by regiospecific desaturation of isopropyl palmitate by a double mutant of a Rhodococcus strain.

Resting cells of a double mutant noted as KSM-MT66, derived from Rhodococcus sp. strain KSM-B-3 by UV irradiation, were found to cis-desaturate isopropyl hexadecanoate, yielding isopropyl cis-6-hexadecenoate. Addition of sodium glutamate (1.0%), Mg SO4 (2 mM), and thiamine (2 mM) increased the productivity of the unsaturated product in phosphate buffer. Optimal temperature and pH for the reaction were around 26 degrees C and 7, respectively. Under the optimized conditions, more than 50 g/l of isopropyl cis-6-hexadecenoate was produced after a 3-day incubation by resting cells of the mutant. Thus, cis-6-hexadecenoic acid, the main component of human sebaceous lipids, can be manufactured economically by the rhodococcal bioconversion.

Amino Acids↗

Degradation of car engine base oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A.

Two microorganisms (NDKK48 and NDKY76A) that degrade long-chain cyclic alkanes (c-alkanes) were isolated from soil samples. Strains NDKK48 and NDKY76A were identified as Rhodococcus sp. and Gordonia sp., respectively. Both strains used not only normal alkane (n-alkane) but also c-alkane as a sole carbon and energy source, and the strains degraded more than 27% of car engine base oil (1% addition).

Alkanes↗

Biphenyl-inducible promoters in a polychlorinated biphenyl-degrading bacterium, Rhodococcus sp. RHA1.

Five transcriptional promoters of biphenyl-degradation genes in Rhodococcus sp. RHA1 were characterized. We newly identified the etbA4 promoter region, which was located adjacent upstream from a ferredoxin reductase gene, etbA4 and a dihydrodiol dehydrogenase gene, bphB2. The etbA4 promoter activity was determined in RHA1 using a promoter probe vector with a luxAB luciferase reporter gene, and was induced by a variety of aromatic compounds as well as the bphA1, ebdA1, etbA1, and etbD1 promoters. All these promoters were induced by aromatic compounds in a closely related heterologous host, R. erythropolis IAM1399 in the presence of RHA1 bphST genes, suggesting that these five promoters are under the control of bphST-coding two-component regulatory system. Sequence comparison of the bphA1 promoter with the ebdA1 and etbA1 promoters, whose transcription starts were determined by primer extension analysis, revealed a consensus sequence centering 42-bp upstream from the transcription start. This consensus was also conserved in the etbA4 and etbD1 promoters, and deletions of the bphA1 promoter affecting the consensus impaired inducible promoter activity. These results suggest that this consensus plays a role in transcription induction and/or the promotion of biphenyl degradation genes in RHA1.

Biodegradation, Environmental↗

Multiplicity of 2,3-dihydroxybiphenyl dioxygenase genes in the Gram-positive polychlorinated biphenyl degrading bacterium Rhodococcus rhodochrous K37.

Rhodococcus rhodochrous K37, a Gram-positive bacterium grown under alkaline conditions, was isolated for its ability to metabolize PCBs. Analysis revealed that it has eight genes encoding extradiol dioxygenase, which has 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and these genes were designated bphC1 to bphC8. According to the classification of extradiol dioxygenases [Eltis, L. D., and Bolin, J. T., J. Bacteriol., 178, 5930-5937 (1996)], BphC3 and BphC6 belong to the type II enzyme group. The other six BphCs were classified as members of the type I extradiol dioxygenase group. BphC4 and BphC8 were classified into a new subfamily of type I, family 3. Two linear plasmids, 200 kb and 270 kb in size, were found in K37, and the bphC6 and bphC8 genes were located in the 200 kb linear plasmid. Northern hybridization analysis revealed that the bphC1, bphC2, and bphC7 genes were induced in the presence of testosterone, the bphC6 gene was induced by fluorene, and the bphC8 gene was induced by biphenyl. All eight BphC products exhibited much higher substrate activity for 2,3-dihydroxybiphenyl than for catechol, 3-methylcatechol, or 4-methylcatechol.

Blotting, Northern↗

Mutational study on alphaGln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771.

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme having post-translationally modified cysteine ligands, alphaCys112-SO2H and alphaCys114-SOH. We replaced alphaGln90, which is conserved in all known NHases and involved in the hydrogen-bond network around the catalytic center, with glutamic acid or asparagine. The kcat of alphaQ90E and alphaQ90N mutants decreased to 24% and 5% that of wild type respectively, but the effect of mutations on Km was not very significant. In both mutants, the alphaCys114-SOH modification appeared to be responsible for the catalysis as in native NHase. We crystallized the nitrosylated alphaQ90N mutant and determined its structure at a resolution of 1.43 A. The structure was basically identical to that of native nitrosylated NHase except for the mutated site and its vicinity. The structural difference between native and alphaQ90N mutant NHases suggested the importance of the hydrogen bond networks between alphaGln90 and the iron center for the catalytic activity.

Crystallography, X-Ray↗

Immunohistochemical observations on pneumonic lesions caused by Rhodococcus equi in foals.

An immunohistochemical analysis of Rhodococcus equi-induced pneumonia in 10 foals was performed by biotin-streptavidin system. The detection of R. equi was more sensitive in immuno-stain using anti-R. equi serum than in Gram's stain. This bacteria also reacted to anti-BCG serum. Lysozyme and alpha 1-antitrypsin were detectable in macrophages. A particularly intense staining was observed in association with intracellular bacteria. Though a degree of reaction for alpha 1-antichymotrypsin was very low in comparison with lysozyme and alpha 1-antitrypsin, it was also demonstrated in macrophages ingesting R. equi. These bacteria were almost intact under an electron microscope. Therefore, the surface components of R. equi may play important roles of protection from intracellular enzymes of macrophages. The cells containing intracytoplasmic IgM, IgG or IgA were a few in number and scattered predominantly around the pneumonic lesion. It is considered that the bactericidal activity by immunoglobulins may be weak in comparison with phagocytosis by macrophages.

Actinomycetales Infections↗

Serological survey of Rhodococcus equi infection in horses in Hokkaido.

Serological survey of Rhodococcus equi infection in horses in Hokkaido was performed using ELISA. Of 2,879 horse sera, 318 (11.0%) gave antibody-positive (OD greater than or equal to 0.3) reactions. The antibody-positive rate of female was significantly higher (p less than 0.01) than that of male, and no statistical difference between Anglo-Arab and thoroughbred was detected in the antibody-positive rate. The maximum antibody-positive rate (27.1%) was shown at 14 years of age. The antibody-positive rates on the 160 farms were found to vary widely from 0 to 78.9%. A significant difference (p less than 0.01) in the antibody-positive rate was detected among horse farms. It was elucidated that 100 (62.5%) out of 160 horse farms had an antibody-positive horse. These results indicate that R. equi was widespread on horse farms, and the level of environmental contamination with R. equi differed among horse farms.

Actinomycetales Infections↗

Evaluation of a monoclonal antibody-based colony blot test for rapid identification of virulent Rhodococcus equi.

We recently generated a monoclonal antibody immunoglobulin G1 (MAb 10G5), which can recognize 15- to 17-kDa antigens, virulence-associated antigens of Rhodococcus equi, and developed a colony blot enzyme-linked immunosorbent assay with MAb 10G5 for the rapid identification of virulent R. equi. In this epidemiologic study, we evaluated the results of the colony blot test in the identification of virulent isolates of R. equi from feces of horses and soil and compared them with those from a conventional procedure (plasmid profiles of isolates by agarose gel electrophoresis). Environmental isolates (778 isolates from feces of foals, 170 isolates from feces of dams, and 1,267 isolates from soil on horse-breeding farms in Hokkaido) were tested by the colony immunoblot test, and 238 of the 778 isolates, 6 of the 170 isolates, and 85 of the 1,267 isolates showed positive signals. Positive isolates were then analyzed for the presence of virulence plasmid DNA, and 235 (98.7%) of the 238 isolates from foals, 6 (100%) of the 6 isolates from dams, and 75 (88.2%) of the 85 isolates from soil showed the presence of virulence plasmids. On the other hand, 50 isolates from each source, which were randomly selected from the isolates that showed negative signals by colony immunoblot, did not contain virulence plasmids. These results demonstrated that the colony blot test that uses a monoclonal antibody specific for virulence-associated antigens is a rapid and reliable test for the identification of virulent R. equi.

Actinomycetales Infections↗