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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↗
PubMed2007

Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103.

Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genesfurA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes.

Actinomycetales Infections↗
PubMed1993

[Detection of Rhodococcus equi antibodies in human serum].

The authors investigated in sera of patients with pneumonia the presence of antibodies against the equi factor Rhodococcus equi. For examination they used agar immunodiffusion and a test for neutralizing the equi factor. Some 110 sera of different donors were examined. In agar immunodiffusion they used the equi factor as antigen either in the separated form or equi factor produced by the growing strain of R. equi. The neutralization test was implemented on agar media with red blood cells sensitized with staphylococcal beta-haemolysin. In all serological tests 27 sera reacted positively only by precipitation 30 sera, while 53 sera did not contain antibodies against the equi factor. The authors discuss the impact of the assembled results and the possible participation of R. equi in human disease.

Actinomycetales Infections↗
PubMed2002

Growth and production of cholesterol oxidase by alginate-immobilized cells of Rhodococcus equi No. 23.

Rhodococcus equi No. 23 was immobilized in calcium alginate. No detrimental effect on the viability of the test organism was observed during the immobilization procedure. Approx. 98% of the cell population originally present in the alginate solution were immobilized in the gel beads. When the cells of an equal volume of the culture, obtained respectively at exponential phase (12 h preculture), late-exponential phase (20 h preculture) or stationary phase (36 h preculture) were immobilized, the gel beads prepared with the stationary-phase culture were found to contain the highest cell population [about 10(8) colony-forming units (CFU)/g of beads]. In addition, gel beads, prepared with late-exponential-phase culture, exhibited the highest production of cholesterol oxidase (CholOx) after 48 h of incubation. Increasing the bead mass from 3.5 to 14.0 g/100 ml of medium increased CholOx production. However, further increasing the bead mass resulted in a reduction of CholOx production. Furthermore, on the basis of a similar initial cell population, the alginate-immobilized cells of R. equi No. 23 produced a significantly higher amount of CholOx (P<0.05) than did the free cells.

Alginates↗
PubMed1994

Correlation between antibiotic resistance, phage-like particle presence, and virulence in Rhodococcus equi human isolates.

Rhodococcus equi is a gram-positive coccobacillus that appears to be emerging as a pulmonary pathogen in AIDS patients. In four human clinical isolates, two antibiotic resistance phenotypes were found to coexist: one beta-lactam resistant and the other beta-lactam susceptible. In vitro, beta-lactam-resistant mutants were obtained at a frequency of 1 x 10(-5) to 5 x 10(-5) from beta-lactam-susceptible strains on cephalothin-containing plates. Neither beta-lactamase nor plasmid DNA was detected in beta-lactam-resistant or -susceptible strains. The penicillin-binding protein patterns for the two types of strains were identical. Electron microscopy revealed that the beta-lactam-resistant strains possessed cell-surface-associated appendages and produced phage-like particles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein showed at least three additional bands of 42, 39, and 30 kDa found only in the beta-lactam-resistant strains. Testing for virulence in Swiss mice revealed that (i) phage-like-particle-producing strains had lower 50% lethal doses when injected intravenously in euthymic and nude mice than the non-phage-like-particle-producing strains did and (ii) intravenous inoculation of a sublethal dose (5 x 10(6) CFU) in nude mice led to chronic infection by the phage-like-particle-producing bacteria only. Finally, in vitro growth curves indicated that the phage-like-particle-producing strains possessed an ecological selection advantage. These results suggest that, among R. equi human isolates, the antibiotic resistance phenotype is associated with virulence and may be phage mediated.

AIDS-Related Opportunistic Infections↗
PubMed2003

Analysis of virulence plasmid gene expression of intra-macrophage and in vitro grown Rhodococcus equi ATCC 33701.

Rhodococcus equi is a soil organism that infects macrophages of foals and immunocompromised humans. Virulence in foal isolates is tightly associated with an 80kb plasmid, which includes a pathogenicity island (PI) with a virulence-associated gene family, vap. A DNA microarray containing 66 of 69 putative open reading frames (ORFs) of the virulence plasmid was developed. Virulence plasmid gene expression of R. equi grown in macrophages or under different conditions in vitro was compared against in vitro growth at 30 degrees C, pH=7. When grown in macrophages, all seven vap family genes as well as six ORFs within, but not outside, the PI were induced. Cluster analysis of the gene expression matrix assembled from different growth conditions suggested that those genes that actively responded to environmental changes divided broadly into two groups. One group, orf1, 2, 5, 6-8, 12-15, 19, and 20 (which includes all the vap genes), was induced at 37 degrees C, mostly by low iron, and to a lesser extent by the synergy of low calcium and pH=5. The second group, orf3, 9, and 10, was induced at 37 degrees C by magnesium depletion (produced by EDTA treatment of growth medium). Temperature (37 degrees C) was the most important factor inducing gene expression for the both groups. Iron restriction led to down-regulation of Group II genes and magnesium restriction led to down-regulation of Group I genes. A putative consensus IdeR binding site was identified upstream of vapA, suggesting that vapA is a member of an IdeR regulon in R. equi. Expression of genes inside macrophages was most closely but not completely mimicked by growth of bacteria at 37 degrees C, pH=5, under conditions of restricted iron, calcium and magnesium; that is, similar to environmental factors found inside macrophages.

Animals↗
PubMed2002

Role of surgery in Rhodococcus equi pulmonary infections.

Rhodococcus equi (R. equi) is a soil-dwelling bacterium that is increasingly associated with pulmonary infections in immunocompromised patients. While antibiotic therapy remains the cornerstone of treatment, surgery combined with antibiotics has an important role in select cases. We report two cases of pulmonary R. equi in immunocompromised patients who were treated with antibiotics, followed by surgical resection of the infected tissues, and then long-term antibiotics. Both patients had excellent outcomes. We advocate surgical resection of grossly infected pulmonary tissue as an adjunct to antimicrobial therapy for treatment of this uncommon, but potentially lethal pathogen.

Abscess↗
PubMed2005

In vitro and intra-macrophage gene expression by Rhodococcus equi strain 103.

Rhodococcus equi is a facultative intracellular respiratory pathogen of foals that persists and multiplies within macrophages. In foals, virulence is associated with 80-90 kb plasmids, which include a pathogenicity island (PI) containing the virulence-associated protein (vap) gene family, but detailed understanding of the basis of virulence is still poor. A 60 spot-based DNA microarray was developed containing eight PI genes and 42 chromosomal putative virulence or virulence-associated genes selected from a recent partial genome sequence in order to study transcription of these genes by R. equi grown inside macrophages and under in vitro conditions thought to simulate those of macrophages. In addition to seven PI genes, nine chromosomal genes involved in fatty acid and lipid metabolism (choD, fadD13, fbpB), heme biosynthesis (hemE), iron utilization (mbtF), heat shock resistance and genes encoding chaperones (clpB, groEL), a sigma factor (sigK), and a transcriptional regulator (moxR) were significantly induced in R. equi growing inside macrophages. The pattern of R. equi chromosomal genes significantly transcribed inside macrophages largely differed from those transcribed under in vitro conditions (37 degrees C, pH 5.0 or 50mM H2O2 for 30 min). This study has identified genes, other than those of the virulence plasmid, the transcription of which is enhanced within equine macrophages. These genes should be investigated further to improve understanding of how this organism survives intracellularly.

Actinomycetales Infections↗
PubMed2006

VapI, a new member of the Rhodococcus equi Vap family.

Rhodococcus equi is a facultative intracellular bacterium which can cause bronchopneumonia in foals and AIDS patients. In this report we show that the ORF13-protein coded by the virulence associated plasmid of R. equi is clearly homologous to VapE. Nucleotide sequence analysis revealed frame shift mutations that shorten the sequence of the ORF13-protein. A theoretical extension of the sequence of ORF13 by the introduction of a single nucleotide yields a translated amino acid sequence that is highly homologous to VapE and other members of the Vap family. The data provided in this study indicate that the ORF13-protein is a novel member of the Vap family and is therefore designated VapI.

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↗
PubMed1999

Otomastoiditis caused by Rhodococcus equi in a patient with AIDS.

Rhodococcus equi is a well-recognized pathogen in veterinary medicine and a rare but well-documented cause of cavitary pneumonia in immunocompromised patients. Most cases of Rhodococcus equi infections in these patients involve the lungs. Otomastoiditis due to Rhodococcus equi is rare, and disseminated Rhodococcus equi with otomastoiditis has never been reported. We report a case of otomastoiditis with systemic dissemination due to Rhodococcus equi in a patient with AIDS.

AIDS-Related Opportunistic Infections↗
PubMed1994

Survival and replication of Rhodococcus equi in macrophages.

Rhodococcus equi is a facultative intracellular bacterium of macrophages that can cause serious pneumonia in both young horses and immunocompromised people. Essential to understanding rhodococcus pathogenesis is a quantitative documentation of the intracellular events that follow macrophage phagocytosis of the organism. By using a bacterial immunofluorescence staining assay, we verified the intracellular survival and replicative potential of R. equi in both murine peritoneal macrophages and equine alveolar macrophages in vitro. Following an initial lag period of 6 to 12 h, the intracellular numbers of R. equi begin to rise, often reaching macrophage-compromising levels by 48 h. A quantitative determination of bacterial growth by a novel image analysis cytometry technique confirmed our fluorescence microscopic results. By 48 h postinfection, bacterial numbers had increased by more than fivefold, and the majority of infected macrophages in the monolayer contained 10 or more bacteria per cell. The intracellular organisms were viable, as evidenced by the ability to incorporate radiolabeled uracil. The use of these techniques has identified differences in the in vitro replicative capacities of a virulent strain and an avirulent strain of R. equi. A clinical isolate of R. equi expressing a 17-kDa virulence-associated plasmid-encoded antigen was able to survive and replicate within macrophages, whereas an avirulent, non-plasmid-containing strain replicated poorly. These results suggest that plasmid-encoded bacterial virulence factors may contribute to the ability of R. equi to replicate within its host cell, the macrophage.

Animals↗
PubMed2002

Placentitis, fetal pneumonia, and abortion due to Rhodococcus equi infection in a Thoroughbred.

Rhodococcus equi is a rare cause of equine abortion. This report describes pyogranulomatous placentitis and fetal pneumonia in a case of abortion from a Thoroughbred mare. Numerous Gram-positive coccobacilli were noted histologically within macrophages in placental and pulmonary lesions. Rhodococcus equi was isolated in pure culture from the placenta, lung, liver, kidney, and stomach content. This is the first description of placentitis due to Rhodococcus equi infection in a horse.

Abortion, Veterinary↗
PubMed1991

Identification of 15- to 17-kilodalton antigens associated with virulent Rhodococcus equi.

Antigens of Rhodococcus equi were analyzed by immunoblotting with naturally infected foal sera. Immunoblots of whole-cell antigen preparations of clinical isolates of R. equi revealed that major protein bands with molecular masses of 15 to 17 kDa were present in all clinical isolates tested and all isolates virulent for mice. In contrast, the 15- to 17-kDa antigens were not identified by immunoblotting in ATCC 6939, a type strain of R. equi that was avirulent for mice. Whole-cell antigens of 102 environmental isolates were investigated by immunoblotting and the mouse pathogenicity test. Twenty-five of these isolates were demonstrated to contain the 15- to 17-kDa antigens by immunoblotting and were virulent for mice. The remaining 77 environmental isolates lacked the 15- to 17-kDa antigens and were avirulent for mice. These data suggest that the diffuse 15- to 17-kDa proteins are virulence-associated antigens with immunogenicity in foals and that they may be useful in marking virulent R. equi contamination in the environment of a horse-breeding farm.

Actinomycetales Infections↗
PubMed1989

The effect of immunosuppression on resistance to Rhodococcus equi in mice.

Rhodococcus equi, a natural pathogen of horses, produces lesions in mice following experimental infection. The effect of various immunosuppressing agents on the sequential development of these lesions has been assessed by measuring the growth of R. equi following intravenous or intranasal challenge and by histological examination. Cyclophosphamide treatment of mice, challenged intranasally, resulted in the development of lesions not unlike that seen in experimental and natural infection in foals. Cortisone acetate also impaired bacterial clearance from the lungs and affected the accumulation of mononuclear cells at infective foci. Most of the agents chosen to impair macrophage function failed to affect the resistance of mice to R. equi. Carbon, carrageenan and silica failed to alter significantly the growth kinetics of R. equi. Dextran sulphate depressed the rate of pulmonary clearance of organisms and affected the ability of animals to eliminate R. equi following rechallenge. Overall, these results support other evidence that cell mediated immunity is involved in host resistance to R. equi and that activated macrophages play a role in acquired immunity to this organism.

Actinomycetales Infections↗
PubMed1991

Severe Rhodococcus equi pneumonia: case report and literature review.

Rhodococcus equi is an aerobic, gram-positive, non-motile pleomorphic bacillus infecting immunocompromised patients. Forty-nine cases of Rhodococcus equi infection have been reported, mainly in patients infected with the human immunodeficiency virus (HIV). A case in which Rhodococcus equi caused severe pulmonary infection, the most common presentation, is described. Clinically, patients have symptoms of pneumonia with hemoptysis as a prominent feature. X-ray will often show a cavitating upper-lobe infiltrate, resembling infection with mycobacteria. Rhodococcus equi is easily cultured from blood or sputum on standard media, but is frequently regarded as a contaminant. Mortality from Rhodococcus equi pneumonia is high (25%) and early surgical intervention has been recommended. Based on this review, the benefit of surgery seems dubious, whereas good results have been obtained using long-term antibiotic treatment with erythromycin plus rifampicin, or vancomycin in combination with either of these antibiotics.

Acquired Immunodeficiency Syndrome↗
PubMed1992

[Pulmonary infection caused by Rhodococcus equi in a renal transplant recipient].

Rhodococcus equi is a primarily pathogen in animals and it has only rarely been seen in immunocompromised humans. We describe our experience with an HIV-negative patient with a functioning renal graft under cyclosporin-azathioprine-prednisone therapy. The patient died after a two months recurrent multifocal pneumonia. The cultures from sputum and blood were negatives but R. equi was isolated from lungs in the necropsy. Most of the 41 cases we had found in the literature had AIDS or HIV infection. Six cases had a kidney graft under azathioprine-prednisone therapy. Rhodococcus equi infection may be missed in some instances due to incomplete or improper identification of the organism, that grows well in aerobic media but is usually regarded as a component of normal flora or as a contaminant. The ability of R. equi to persist in and eventually to destroy macrophages is the basis of its pathogenicity. It also explains the clinical resistance to antibiotics without intracellular activity. Rhodococcus equi infection must be suspected in immunocompromised patients with recurrent pneumonia. Correct identification and combined therapy with lipophilic antibiotics that penetrate the macrophages are necessary to prevent the high mortality of this infection.

Actinomycetales Infections↗
PubMed1996

Attempts to find phenotypic markers of the virulence plasmid of Rhodococcus equi.

Four isolates of Rhodococcus equi, from pneumonic foals, and containing the 85 kb virulence plasmid, a porcine isolate containing an 80 kb plasmid, and their plasmid cured derivatives, were examined for 239 phenotypic properties in an attempt to find characters other than the virulence-associated protein (VapA) which might be encoded by the virulence plasmid in organisms grown at 37 degrees C. Tests chosen included those which have previously given variable results for R. equi isolates, since such variability might be attributed to plasmid curing, and characteristics which have been described as properties of plasmids of Rhodococcus species other than R. equi. Tests included cadmium resistance, Congo red binding, resistance to 26 antibiotics, conventional clinical microbiological tests, utilization of 95 different carbon sources, enzymatic activities in API ZYM, fluorogenic assays for exo- and endopeptidase, glycosidase activities, and testosterone degradation. Apart from production of VapA by foal isolates, no phenotypic property was identified in the plasmid-positive isolates. Phenotypic characteristics of R. equi that have not been described before, and might be useful in identification were: metabolism of N-acetyl-beta D-glucopyranoside, alpha- and beta-hydroxybutyric, alpha-ketobutyric and N-acetyl-glutamic acids, of methylpyruvate, heptanoate, nonanoate and stearate esters; exopeptidase activity against alanine-alanine-tyrosine, alanine-phenylalanine-lysine, glycine-arginine, lysine-alanine, and valine-glycine-alanine; endopeptidase activity against arginine and methionine; and hydrolysis of bis-phosphate ester.

Amino Acids↗