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PubMed1993

Involvement of interferon-gamma and tumor necrosis factor-alpha in host defense against Rhodococcus equi.

Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus increasingly reported as an opportunistic pathogen in AIDS patients. In vitro, splenic cells of noninfected euthymic mice produced tumor necrosis factor-alpha (TNF alpha) in greater amounts when incubated with live R. equi rather than with killed bacteria. In vivo, interferon-gamma (IFN-gamma) and TNF alpha serum levels of infected euthymic mice remained below the level of detectability. Treatment of infected nude mice, which developed chronic infection, with discontinuous injections of IFN-gamma, TNF alpha, or both did not decrease bacterial colony-forming units in liver, spleen, or lungs. However, treatment of infected euthymic mice, which cured a R. equi inoculum within 3 weeks, with anti-IFN-gamma or anti-TNF alpha antibodies (or both) significantly increased tissue colony counts. These data argue that, in this murine model, endogenous IFN-gamma and TNF alpha are involved in the cell-mediated immunologic response against R. equi infection.

Actinomycetales Infections↗
PubMed1994

Haemolytic and phospholipase C (PLC) activities of Rhodococcus equi.

Rhodococcus equi, an intracellular organism causing pneumonia and lung abscesses in foals, is generally thought to be non-haemolytic. In the present study, however, 13 of 14 representative isolates were found to be haemolytic when tested on agar media containing washed red blood cells rather than whole blood. Red cells of rabbits, dogs, horses and man were more sensitive to lysis than were those of ruminants. Two new enzymatic activities of the species were defined: a lecithinase and a phosphatidylinositol-specific phospholipase C (PI-PLC). As judged from tests for trypsin, temperature and ethanol sensitivity, the haemolytic activity was primarily dependent on PI-PLC though the participation of lecithinase seemed probable. The haemolytic activity of growing strains, but not of cell-free preparations, was partially inhibited by lecithin but enhanced by cholesterol; however, cholesterol oxidase (CO) activity, known to mediate cooperative lysis of RBC sensitized with sphingomyelin-specific phospholipases C or D of some other species, did not contribute to the direct haemolysis caused by R. equi as demonstrated here.

Actinomycetales Infections↗
PubMed1991

Association between a large plasmid and 15- to 17-kilodalton antigens in virulent Rhodococcus equi.

Rhodococcus equi strains showing 15- to 17-kDa antigens in immunoblots were found to be virulent in mice. To study the genes specific to these antigens in virulent R. equi, we compared plasmid profiles, immunoblot profiles, and murine pathogenicity profiles of 10 strains of R. equi. All the strains showing 15- to 17-kDa antigens contained a large plasmid of approximately 85 kbp and were virulent in mice; however, the remaining strains lacked both the antigens and the large plasmid and were avirulent in mice. Mutants of virulent strains ATCC 33701 and L1, which were cured of the large plasmid by repeated passage at 38 degrees C, lacked the 15- to 17-kDa antigens and showed a dramatic decrease in lethality in mice. These results suggested that the presence of an 85-kbp plasmid may be essential for virulence and expression of 15- to 17-kDa antigens of R. equi and offered support for earlier observations that freshly isolated strains of R. equi killed mice, whereas laboratory-adapted strains did not.

Actinomycetales Infections↗
PubMed1992

In-vitro antimicrobial susceptibility of Rhodococcus equi.

Rhodococcus equi is an intracellular facultative, Gram-positive cocco-bacillary organism of increasing importance as a pulmonary pathogen in HIV-positive patients. This study was carried out to evaluate the optimal antibiotic combinations for treating such infections. Four human R. equi isolates and one reference strain were tested for their susceptibilities to 36 antibiotics. In-vitro the most active antibiotics were amikacin, gentamicin, netilmicin, erythromycin, clarithromycin, roxithromycin, ciprofloxacin, sparfloxacin, rifampicin, vancomycin, teicoplanin, doxycycline, minocycline, imipenem, meropenem and trimethoprim/sulphamethoxazole. The only bactericidal antibiotics were the aminoglycosides, ciprofloxacin, sparfloxacin and vancomycin. As determined by FIC indices, four combinations were synergistic: rifampicin-erythromycin, rifampicin-minocycline, erythromycin-minocycline and imipenem-amikacin. However, no antibiotic combinations were synergistic with the time-kill kinetic method at achievable serum concentrations or at ten-fold and half-fold the MICs. Frequencies of selection of antibiotic-resistant mutants determined at concentrations of five- and ten-fold the MICs ranged from less than 1 x 10(-8) for erythromycin and trimethoprim/sulphamethoxazole to 5 x 10(-4) for amikacin. These results may be of help in selecting the antibiotics for treating infected HIV-positive patients.

Anti-Bacterial Agents↗
PubMed1993

Monoclonal antibody specific to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Virulent Rhodococcus equi produces 15- to 17-kDa surface protein antigens. These antigens are used as markers to identify virulent R. equi isolates from foals and their environment by Western blot (immunoblot) analysis with naturally infected foal serum. In the present study, a monoclonal antibody (MAb; 10G5) was generated against the 15- to 17-kDa antigens excised from sodium dodecyl sulfate-polyacrylamide gels to develop sensitive and specific immunoblot assays for the identification of virulent R. equi. MAb 10G5 strongly reacted with R. equi ATCC 33701 and L1, which expressed 15- to 17-kDa antigens by Western blot, colony blot, and dot immunobinding assays, but it did not react with strains ATCC 33701P- and L1P-, which lacked the antigens. For identification of virulent R. equi, clinical and environmental isolates were tested by these assays with the MAb, and all virulent strains were successfully identified; these strains possessed virulence plasmids. These results suggest that the MAb is a useful reagent for the identification of virulent R. equi.

Animals↗
PubMed1996

Radiological findings in nine AIDS patients with Rhodococcus equi pneumonia.

Rhodococcus equi (R. equi) infections have been incidentally reported as a cause of pulmonary infection in severely immunocompromised hosts, including AIDS patients. Our purpose is to describe the radiological findings in nine AIDS patients with R. equi pneumonia assessed by bronchoalveolar lavage (BAL), biopsies, cultures of sputum, and hemocultures. All patients were examined by chest radiographs and contrast-medium-enhanced chest CT. Dense pulmonary consolidations with or without cavitations accounted for the most striking radiological patterns. Chest CT also revealed six mediastinal involvements, strongly mimicking a lymphoma. Two of them had multiple bilateral pulmonary nodular opacities. Pleural effusion was not identified. Although intensive therapies were administered, seven among nine patients died within few months. In an AIDS patient living in a rural area or exposed to horses and presenting these radiological patterns, the possibility of R. equi pneumonia should be considered in the differential diagnosis along with other infectious diseases or lymphomas.

AIDS-Related Opportunistic Infections↗
PubMed1995

Radiologic findings in two AIDS patients with Rhodococcus equi pneumonia.

Rhodococcus equi (R. equi) has been reported as an occasional cause of pulmonary infection in severely immunocompromised hosts, including AIDS patients. Our purpose is to describe the radiologic findings in two AIDS patients with R. equi pneumonia. Chest radiographs showed right-upper-lobe consolidation and cavitation in both patients. Chest CT confirmed upper mediastinal involvement and precarinal lymphadenopathy in both cases. Multiple lung nodules related to the bronchi were also identified in one patient. In an AIDS patient from a rural area or with exposure to horses, the possibility of R. equi infection should be considered when cavitary pneumonia is present, even if there is mediastinal involvement and/or lymphadenopathy, or if multiple lung nodules are also present.

AIDS-Related Opportunistic Infections↗
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↗
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↗
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↗
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↗
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↗
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↗
PubMed1996

Transfer of a CD4+ Th1 cell line to nude mice effects clearance of Rhodococcus equi from the lung.

Rhodococcus equi, and intracellular respiratory pathogen, causes sever e granulomatous pneumonia in humans with AIDS and in young horses. Pulmonary clearance of R. equi requires functional CD4+ T cells and gamma interferon (IFN-gamma) expression from bronchial lymph node cells. The purpose of this study was to investigate whether R. equi-specific CD4+ Th1 cells could effect clearance of R. equi from the lung. Adoptive transfer of a clearance of R. equi from the lungs. In contrast, mice transfused with a R. equi-specific CD4+ Th2 cell line expressed interleukin-4 but not IFN-gamma mRNA, failed to clear pulmonary infection, and developed granulomas in the lung. Control mice, which did not receive cells, did not produce IFN-gamma or interleukin-4 and developed small pulmonary granulomas. These results clearly show that a Th1 response is sufficient to effect pulmonary clearance of R. equi.

Animals↗