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PubMed1991

Degradation of dioxane, tetrahydrofuran and other cyclic ethers by an environmental Rhodococcus strain.

By enrichment and isolation techniques bacterial strains with the capacity to grow on aliphatic cyclic ethers (dioxane, tetrahydrofuran, 1,3-dioxolane) have been isolated. Six strains that degrade tetrahydrofuran were classified as belonging to the genus Rhodococcus. One of two strains that degrade dioxane instead of or in combination with tetrahydrofuran was further characterized and a hypothetical catabolic pathway comprising an initial 2-hydroxylation and several oxidation steps is postulated.

Biodegradation, Environmental↗

PubMed1990

Aryl acylamidase from Rhodococcus erythropolis NCIB 12273.

A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around pH 8.0 and had a Km for acetaminophen of 0.11 mM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen.

Acetaminophen↗

PubMed1992

Cloning of DNA from a Rhodococcus strain conferring the ability to decolorize sulfonated azo dyes.

Azo dyes are recalcitrant pollutants. Two sulfonated azo dyes, Orange II and Amido black, are effectively decolorized by certain nocardioform strains of the genus Rhodococcus. A mutant of one of these strains was isolated which had lost azo-dye decolorizing ability and the strain was used to clone DNA conferring this ability, by screening a BclI library constructed from DNA of a decolorizing strain. The relevant genetic information was located on a 6.3-kb fragment of DNA.

Amido Black↗

PubMed1992

Sequence of a Rhodococcus gene encoding a protein with extensive homology to the mammalian propionyl-CoA carboxylase beta chain.

Sequence analysis of a 2-kilobase DNA fragment from Rhodococcus sp. NI86/21 revealed an open reading frame encoding a 476-amino-acid protein with striking homology to the rat and human propionyl-CoA carboxylase beta subunits. The nucleotide sequence of a corresponding prokaryotic gene has not yet been reported. Upstream, the C-terminal part of a putative beta-ketoacyl synthase was identified.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1992

Steroid-1-dehydrogenase of Rhodococcus erythropolis: purification and N-terminal amino acid sequence.

The inducible steroid-1-dehydrogenase from the bacterium Rhodococcus erythropolis IMET 7030 was purified to homogeneity using affinity chromatographic, electrophoretic, and ion exchange techniques. The spectrum of the pure enzyme is characterized by the associated FAD. The M(r) of the enzyme is 56,000. The amino acid composition and the sequence of the 13 N-terminal amino acids are given.

Amino Acid Sequence↗

PubMed2025

Nebulization of an mRNA-encoded monoclonal antibody for passive immunization of foals against Rhodococcus equi.

Inhalation of Rhodococcus equi causes severe pneumonia in humans and animals worldwide, most commonly affecting horse foals. The standard for preventing R. equi pneumonia in foals is transfusion of hyperimmune plasma, which is expensive and carries the risk of adverse effects. Our goal was to passively immunize foals against R. equi by nebulizing mRNA encoding an equine monoclonal antibody (mAb) against the virulence-associated protein A (VapA) directly into the lungs. VapA-specific memory B cells from an immunized horse were used to identify and select the sequence for an equine immunoglobulin (Ig)G1 mAb. In vitro-transcribed mRNA encoding this sequence expressed full-length, VapA-specific mAbs in vitro and safely and effectively produced intrapulmonary mAb in foals for at least 5 days following nebulization. These findings establish a platform to generate mRNA-encoded mAbs for immunotherapeutic and immunoprophylactic applications in horses and demonstrate the feasibility of delivering nebulized mRNA-mAb for intrapulmonary mAb expression in neonates.

Animals↗

PubMed1992

The plasmid-encoded chloramphenicol-resistance protein of Rhodococcus fascians is homologous to the transmembrane tetracycline efflux proteins.

The nucleotide sequence of the chloramphenicol-resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic-modifying enzyme. The amino acid sequence of ORF1 predicted a hydrophobic protein, with 12 putative membrane-spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5'-untranslated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol-resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.

Amino Acid Sequence↗

PubMed1992

Therapy of Rhodococcus equi disseminated infections in nude mice.

Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus increasingly reported as an opportunistic pathogen in human immunodeficiency virus-positive patients. However, the optimal drug regimen for treating R. equi pulmonary or systemic infections is not yet known. Therefore, a model of intravenously infected nude mice with disseminated infection was created to study the efficacy of antibiotics alone or in combination as determined by the reduction of bacterial CFU per gram in the lungs and spleen after 4 and 11 days of treatment. The studied antibiotics possessing low MICs against R. equi strains were amikacin, ciprofloxacin, erythromycin, imipenem, minocycline, rifampin, and vancomycin. Vancomycin, imipenem, and rifampin were the most effective agents in monotherapy. On the other hand, amikacin, ciprofloxacin, erythromycin, and minocycline alone were not active in this model. The most active drug combinations were those including vancomycin. No antibiotic-resistant mutants were selected in vivo with treatment involving any drugs used alone or in combination. Although the treatment recommended until now for R. equi infections is rifampin plus erythromycin, this study suggests that antibiotic combinations which include vancomycin may be the most effective in vivo.

Actinomycetales Infections↗

PubMed1992

Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2.

Two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2, were isolated from soil and river water by their abilities to utilize 2,4-dinitrophenol (0.5 mM) as the sole source of nitrogen. Although succinate was supplied as a carbon and energy source during selection, both isolates could utilize 2,4-dinitrophenol also as the sole source of carbon. Both strains metabolized 2,4-dinitrophenol under concomitant liberation of stoichiometric amounts of nitrite and 4,6-dinitrohexanoate as a minor dead-end metabolite.

2,4-Dinitrophenol↗

PubMed1992

Initial hydrogenation during catabolism of picric acid by Rhodococcus erythropolis HL 24-2.

Rhodococcus erythropolis HL 24-2, which was originally isolated as a 2,4-dinitrophenol-degrading bacterium, could also utilize picric acid as a nitrogen source after spontaneous mutation. During growth, the mutant HL PM-1 transiently accumulated an orange-red metabolite, which was identified as a hydride-Meisenheimer complex of picric acid. This complex was formed as the initial metabolite and further converted with concomitant liberation of nitrite. 2,4,6-Trinitrocyclohexanone was identified as a dead-end metabolite of the degradation of picric acid, indicating the addition of two hydride ions to picric acid.

Biodegradation, Environmental↗

PubMed1992

Role of T-lymphocyte subsets in Rhodococcus equi infection.

Rhodococcus equi, a facultative intracellular gram-positive bacterium, can induce life-threatening infections in immunocompromised patients, especially those with AIDS. We have studied the mechanism of acquired immunity to this pathogen in a murine model. Protective immunity was induced by live but not killed bacteria. Adoptive transfer of resistance was obtained with spleen cells but not immune serum from mice immunized intravenously 30 days earlier with live bacteria. In normal mice, an intravenous challenge of 5 x 10(6) CFU of R. equi was cleared from the spleen, liver, and lungs within 3 weeks, whereas athymic nude mice were unable to clear the bacteria. In vivo depletion with monoclonal antibodies showed that both CD4+ and CD8+ T-cell subsets participate in the clearance of bacteria and that CD8+ T cells play the major role.

Actinomycetales Infections↗

PubMed2026

Complete genome sequence of Rhodococcus qingshengii strain A3-8.

A chemostat culture was constructed with phenol and forest soil as an inoculum. We report the complete genome sequence of Rhodococcus qingshengii strain A3-8, which was isolated from the culture. The genome consists of a chromosome (6,436,695 bp) and a linear plasmid pA38 (257,365 bp).

Rhodococcus↗

PubMed1992

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↗

PubMed1992

Successful medical therapy of Rhodococcus equi pneumonia in a patient with HIV infection.

A 34-year-old HIV-infected man was successfully treated with antimicrobial therapy alone for Rhodococcus equi pneumonia and has survived longer than six months. In the current literature, only two of seven HIV-infected patients so treated have survived as long as six months. Based on our experience and the available literature, it seems reasonable to treat HIV-infected patients with R equi pneumonia who do not require surgical intervention with prolonged intravenous therapy followed by long-term oral therapy with at least two effective antibiotics. The optimal choice and duration of antibiotic therapy need to be determined.

AIDS-Related Opportunistic Infections↗

PubMed1992

Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase.

Rhodococcus ATCC 39484 produced a nitrilase when induced with isovaleronitrile. The enzyme was obtainable pure in milligram amounts, had a subunit Mr of 40 kDa, and demonstrated a substrate-induced activation related to aggregation of subunits to form a 560-kDa complex. The enzyme had a broad substrate specificity, had a pH optimum of 7.5, was stable up to 40 degrees C, and had one disulfide bridge and two free cysteine residues, one of which appeared to be catalytically essential. The N-terminal sequence was determined and found to have 78.3% homology, in a 23-residue overlap, with Klebsiella ozaenae nitrilase. The enzyme was inhibited competitively by benzylamine and benzaldehyde and irreversibly by benzyl bromide. However, benzyl bromide was shown to be nonspecific, causing multiple alkylation. Acid quenching of enzyme-substrate mixtures allowed for the detection of covalent enzyme-substrate complexes using mass spectrometry. The covalent intermediate is suggested to be either a thioimidate or an acylenzyme and a reaction mechanism consistent with this observation and also the inhibitor results is proposed. The rate of breakdown of the covalent intermediates was found to be rate limiting even for substrates with undetectable rates of hydrolysis or those with very slow rates of intermediate formation. For phenylacetonitrile, a poor substrate, in addition to acid, approximately 2% of the product was the corresponding amide. This result suggests that a tetrahedral intermediate is formed which, for selected substrates, can break down anomalously to produce amide in place of the normal acid product. Under the conditions used in this study all other substrates tested were converted to acid.

Amino Acid Sequence↗

PubMed1992

Rhodococcus equi cavitary pneumonia in HIV-infected patients: an unsuspected opportunistic pathogen.

Two patients seropositive for human immunodeficiency virus (HIV) and with no previous acquired immunodeficiency syndrome-defining conditions developed cavitary pneumonia and pleural disease caused by Rhodococcus equi. R. equi was isolated from these patients' sputum and lung biopsy specimens, respectively, but the microorganism was initially considered to be a contaminant (patient 1) or misidentified as a nontuberculous mycobacterium (patient 2). The R. equi infection was fatal in one patient, who died after 4 months without specific antimicrobial therapy; the second patient was unresponsive to combination therapy with various antimicrobial agents. R. equi may cause life-threatening infections in HIV-infected patients. Microbiology laboratories should be cognizant of the need to exclude R. equi as a cause of infection in highly immunosuppressed patients.

AIDS-Related Opportunistic Infections↗

PubMedDate not supplied

[Rhodococcus equi infections in AIDS: personal cases].

We report on the occurrence of an uncommon pathogen, Rhodococcus equi (R.e.) in HIV infection. In the period february 1990-July 1991 we performed the diagnosis of R.e. infection (lung, lung-central nervous system) in two infected patients, aged 27 and 31 years, both males, drug addicts, classified as CDC IV. In the first of them an cavitating right bronchopneumonia (bpm) was diagnosed as of tuberculous origin, on the ground of the microscopic demonstration of an acid-fast, gram-positive strain in b.a.l.. A standard antituberculous regimen was uneffective and R.e. was eventually isolated in three blood culture taken shortly before the exitus and at post-mortem examination of the lungs. In the second patient a subclavian right bpm developed without microbiological evidence of any common pathogen. A third-generation cephalosporin course was only partially successful (clinical amelioration, incomplete clearing of radiological findings). After 3 months the patient relapsed and an aseptic meningitis developed. Cultures of blood and csf yielded a R.e. strain; post-mortem examination revealed a diffuse, cavitating bpm. Our personal experience underlines the difficulty in recognizing R.e. infections in compromised host, because of similarity to other entities (i.e. tuberculosis) and the absence of pathognomonic hints.

AIDS-Related Opportunistic Infections↗

PubMed1992

Effect of cosubstrates on the dechlorination of selected chlorophenolic compounds by Rhodococcus erythropolis 1CP.

Effects of aromatic and nonaromatic cosubstrates on the rate of 2,4-dichlorophenol degradation by R. erythropolis 1CP were studied under growth and nongrowth conditions. Glucose and maltose were found to accelerate 2,4-dichlorophenol (initial conc. 50 mg/l) dechlorination from 11 days to 24 and 20 h, respectively. The stimulating effect was observed within the range of glucose concentration of 0.5-2.0 g/l in the medium. Ascorbic acid at a concentration of 10-70 mg/l as a nongrowth substrate also increased the decomposition of 2,4-dichlorophenol, though at a lower degree than glucose. Experiments with washed cells showed the limiting stage of chlorophenol degradation by R. erythropolis 1CP to be, possibly, aromatic ring dechlorination after its hydroxylation. As an additional source of electrons, glucose introduced into suspensions of washed cells grown on 3-hydroxybenzoate made it possible to eliminate the bottleneck in 2,4-dichlorophenol metabolism by significantly accelerating dechlorination of 3,5-dichlorocatechol. Based on the experimental and literature data, the enzyme systems involved in 2,4-dichlorophenol degradation by the culture R. erythropolis 1CP are assumed to be nonspecific.

Biodegradation, Environmental↗