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An unusual case of cavitary Rhodococcus equi pneumonia with endobronchial granulomas in congenital HIV infection is presented. The clinical features and radiological manifestations of pulmonary R. equi infection are discussed.
The review informs about substantial features of Rhodococcus equi with emphasis on the analysis of 115 as yet published and still expanding reports on the isolation of this zoopathogenic nocardioform actinomycete from man. Microbiological laboratories of human medicine have to learn not only how to identify R. equi but also recognize it as an opportunistic pathogen in particular in persons with the deficient immunity. R. equi is a gram-positive, encapsulated diphtheroid coccobacillus, partially acid fast. It grows well on common media, its colonies being after 48 hours characteristically mucoid, coalescing, irregular and mostly lightly pinkish. Biochemically it is little active, nevertheless it causes typical synergic haemolysis of erythrocytes influenced by staphylococcal beta-toxin. R. equi is found in soil and manure, especially in horse manure. It causes above all granulomatous pneumonia in young foals. In humans, it causes mostly pneumonia and lung abscess, more frequently in persons with immunity deficiency incl. AIDS, less often extrapulmonary abscesses, sepsis and wound infections. The disease are commonly chronic and recurrent. The ability of R. equi to persist in macrophages and destroy them is important in the pathogenesis of infection. In resistance to infection the cell-mediated immunity seems to be of major importance. The port of entry are the lungs, less often the alimentary tract or injured skin. About a third of the persons gives a history of contact with animals, manure or soil. The standard treatment is prolonged administration of a combination of rifampin and erythromycin. The isolation of R. equi is easy and if a laboratory suspects the presence of this microorganism, its identification is not difficult.
Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate). Virulent R. equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples. To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more. All of the PCR-negative and culture-positive samples were positive by the two methods. These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R. equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R. equi pneumonia in foals.
Increased recognition of Rhodococcus equi as a human pathogen has occurred since 1983, when the first review article summarized the world's literature of 12 cases. In this article, we present 12 cases from the University of Oklahoma Health Sciences Center and review 60 from the literature. Most cases occur in immunocompromised hosts and present as chronic cavitary pneumonias. Associated extrapulmonary disease is seen at diagnosis in 7% of patients with pneumonia, and relapse occurs at extrapulmonary sites in 13%, often without reappearance of pulmonary disease. Relapse may follow a course of antimicrobial therapy that is too brief, but can also occur during treatment. Infections also occur in the gastrointestinal tract, causing enteritis and regional adenitis with abscesses. Contaminated wounds may become infected. Isolated bacteremias may be a manifestation of latent infection recurring during a period of immune suppression. A common feature of human R. equi infection is delay in diagnosis. The insidious course of disease contributes to delay, as does failure to identify the organism. R. equi is easily cultured on nonselective media but commonly mistaken for a diphtheroid or occasionally for a mycobacterium based on acid-fast appearance. Form and duration of treatment are closely related to host immune status. Immunocompromised patients require prolonged or indefinite therapy with multiple antibiotics. Infections in immunocompetent hosts are easily treated with short courses of single agents. Infections related to contaminated wounds are treated primarily by irrigation and debridement. Infections in immunocompromised hosts are increasing in frequency largely due the AIDS epidemic. Infections in immunocompetent hosts, reported rarely before this series, may be underdiagnosed, perhaps because R. equi resembles common commensals and has limited virulence in this population. This report demonstrates that R. equi infections, including community-acquired pneumonias, occur in immunocompetent hosts.
Phagocytic uptake by cultured mouse macrophages (PD388D1) of a virulent strain (ATCC 33701) of Rhodococcus equi producing substantial cholesterol oxidase was accompanied by intracellular survival of the bacteria, and enzymatic oxidation of macrophage membrane cholesterol. A non-virulent strain (4219) lacking cholesterol oxidase was largely eliminated from the macrophages and did not bring about oxidation of membrane cholesterol. When R. equi 33701 was co-phagocytosed with Corynebacterium pseudotuberculosis there was a significant enhancement (10-fold) in the amount of oxidation product (4-cholesten-3-one) generated. R. equi and C. pseudotuberculosis are cooperative partners in the hemolysis of sheep erythrocytes, traceable to the cholesterol oxidase of the former, and phospholipase D of the latter. Results are discussed relative to the role of cooperative cytotoxins in damage to host tissue by bacterial pathogens.
Rhodococcus equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized hosts. We describe four immunocompromized patients who had serious R. equi infections with an overall mortality of 75%. The natural habitat of R. equi is soil, particularly soil contaminated with animal manure. Necrotizing pneumonia is the commonest form of infection but extrapulmonary infections, such as wound infections and subcutaneous abscess, have also been described in humans. R. equi is cultured easily in ordinary non-selective media. Large, smooth, irregular colonies appear within 48 hours. It is a facultative, intracellular, nonmotile, non-spore forming, gram-positive coccobacillus, which is weakly acid-fast staining and bears a similarity to diphtheroids. It forms a salmon-colored pigment usually after 48 hours incubation. A particular characteristic of this organism is that it undergoes synergistic hemolysis with some bacteria on sheep blood agar. R. equi may be misidentified as diphtheroids, Mycobacterium species, or Nocardia. In vitro R. equi is usually susceptible to erythromycin, ciprofloxacin, vancomycin, aminoglycosides, rifampin, imipenem and meropenem. The organism can be difficult to eradicate, making treatment challenging. Increased awareness of the infection may help with early diagnosis and timely treatment.
We report a case of Rhodococcus equi, an unusual pathogen, causing a right upper lobe lung abscess in a patient with Evan's syndrome (auto-immune haemolytic anaemia and thrombocytopenia) who was treated with high-dose corticosteroid therapy. The patient was treated successfully with clarithromycin, vancomycin, ciprofloxacin and imipenen which appear to be effective in combination for this unusual condition in which the treatment regimen has been controversial.
A DNA clone from Rhodococcus equi conferring low-level rifampin resistance through the ability to inactivate this antibiotic via its decomposition was identified. The iri (inactivation of rifampin) gene consisted of an open reading frame of 1,437 bp encoding a 479-amino-acid sequence strongly resembling those of monooxygenases acting upon phenolic compounds or involved in polyketide antibiotic synthesis. When expressed in Escherichia coli, the gene conferred resistance to a > 50-micrograms/ml concentration of the drug.
Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS)
We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.
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.
The condition of an electroporation method was re-evaluated for the introduction of foreign plasmid DNA into Rhodococcus equi. The method is based on an electroporation of the bacteria made competent by culturing in a broth containing glycine and by heat shock at 50 degrees C. Transformation of R. equi could be achieved with a chloramphenicol-resistant shuttle vector originating from Rhodococcus fascians at an efficiency of about 10(4) transformants/microgram DNA. The bacteria were also shown to become competent when they were incubated with a chemical transformation buffer prior to washing with an electroporation buffer.
OBJECTIVE: To identify farm characteristics as risk factors for the development of Rhodococcus equi pneumonia in foals. DESIGN: Prospective matched case-control study. ANIMALS: 2,764 foals on 64 equine breeding farms with 9,991 horses. PROCEDURE: During 1997, participating veterinarians completed paired data collection forms, 1 for a farm with > or = 1 foal with R equi pneumonia and 1 for an unaffected control farm. Matched data were compared by use of conditional logistic regression analysis. RESULTS: Farm characteristics found in bivariate analyses to be associated with increased risk for pneumonia caused by R equi in foals included > 200 farm acres, > or = 60 acres used in the husbandry of horses, > 160 horses, > or = 10 mares housed permanently on the farm (resident mares), > 17 foals, > 0.25 foals/acre, and the presence of transient mares (mares brought temporarily to the farm for breeding or foaling) and their foals. Affected farms were significantly more likely to be > 200 acres in size and have > or = 10 resident dam-foal pairs, whereas control farms were significantly more likely to have > or = 75% of their dam-foal pairs housed permanently on the farm. CONCLUSIONS AND CLINICAL RELEVANCE: Breeding farms with large acreage, a large number of mares and foals, high foal density, and a population of transient mares and foals are at high risk for foals developing pneumonia caused by R equi.
Although isolation of Rhodococcus equi from tracheobronchial aspirates is thought to be a definitive diagnosis of R. equi pneumonia in foals, virulence of isolates from the aspirates of infected foals remains obscure. In the present study, transtracheal aspirates were collected from thirty-one 1- to 6-month-old foals, which showed clinical signs of respiratory tract infection, and R. equi isolates were analyzed for the presence of virulence plasmids and virulence-associated antigens. Moreover, this method was compared with a serodiagnosis by an enzyme-linked immunosorbent assay (ELISA) to evaluate the sensitivity of the ELISA. Of the 31 foals, 21 revealed positive cultures for R. equi. Of the 21 foals, 20 (95%) had an ELISA OD value of 0.3 (positive limit of this test) or higher at the initial medical examination. All of the isolates from the aspirates were virulent R. equi, which contained virulence plasmids and expressed virulence-associated antigens. In the remaining 10 foals showing a negative culture for R. equi, 3 foals had positive ELISA titers. Six foals died during the treatment, and necropsy revealed that 5 of the 6 foals had R. equi infection characterized by large abscesses in the lungs, and 3 of the 5 foals also had intestinal lesions. All clinical isolates from the lesions of the foals were virulent R. equi. These results support the assumption that isolates from the transtracheal aspirates of infected foals are virulent R. equi and the sensitivity of ELISA might demonstrate a serodiagnostic value for early diagnosis of R. equi infection in foals.
Species-specific primers for the polymerase chain reaction (PCR) for the detection of Rhodococcus equi were developed. These primers were based on unique DNA fragments produced from R. equi reference strains and field isolates. Following random amplification of polymorphic DNA from R. equi and R. rhodochrous with a set of 40 arbitrary 10-base pair (bp) primers, a pair of species-specific primers was designed to detect a unique 700-bp fragment of R. equi chromosomal DNA. This PCR product was limited to R. equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria.
OBJECTIVE: To identify farm characteristics and management practices associated with development of Rhodococcus equi pneumonia in foals. DESIGN: Prospective case-control study. ANIMALS: 5230 foals on 138 breeding farms with 9136 horses. PROCEDURE: During 2003, participating veterinarians provided data from 1 or 2 farms with > or =1 foal with R equi pneumonia and unaffected farms. Data from affected and unaffected farms were compared by use of logistic regression analysis. RESULTS: A number of variables relating to farm size and desirable management practices were significantly associated with increased odds of farms being affected with R equi pneumonia. By use of multivariate logistic regression, affected farms were determined significantly more likely to have raised Thoroughbreds, housed > or =15 foals, used concrete floors in foaling stalls, and tested foals for passive transfer of immunity than unaffected farms. These results remained significant even after accounting for exposure of foals to other breeding farms during the first month of life. Conclusions and Clinical Relevance-Breeding farms with large acreage and a large number of mares and foals have greater odds of being affected by R equi pneumonia. Clinical relevance of associations with Thoroughbred breed and concrete flooring in foaling stalls remains uncertain. Desirable management factors commonly used on farms were not effective for controlling or preventing development of R equi pneumonia. This finding indicates a need to focus on host factors that influence disease development.
In order to improve the isolation rate of Rhodococcus equi from animals and soil, the efficacy of four previously described selective media (CAZ-NB, M3T, NANAT and TINSDALE) and that of four other media (NC, PNP, TCP and TVP) composed by us was compared and evaluated. Two selective plating media proved to be the best for the isolation of R. equi from contaminated samples. One of them was CAZ-NB containing ceftazidime, novobiocin and cycloheximide, while the other was the newly composed TCP containing trimethoprim, cefoperazone, polymyxin B, cycloheximide and potassium tellurite as selective components. These two media allowed the growth of at least 62-72% of R. equi present in the artificially contaminated samples, and the inhibition of unwanted contaminant bacteria and fungi was satisfactory with both media. TCP medium proved to be superior to CAZ-NB since the colony morphology of R. equi was much more characteristic (shiny, smooth, black colonies 3-5 mm in diameter) on it, and it inhibited the unwanted contaminant bacterial and fungal flora more effectively, especially in the case of faecal and soil samples. Therefore, TCP is recommended as a new, highly selective plating medium for the isolation of R. equi from contaminated samples.
The interaction of Rhodococcus equi with alveolar macrophages from adult horses, foals experimentally exposed to R. equi (sensitized foals) and non-exposed foals was studied using in vitro bactericidal assays, cytochemical staining and transmission electron microscopy. It was demonstrated that R. equi is a facultative intracellular parasite, able to survive and multiply within the alveolar macrophages of the host by interfering with phagosome-lysosome fusion. Opsonization of R. equi with antibody against capsular components was associated with increased phagosome-lysosome fusion and significantly enhanced (P less than 0.05) killing of the organism by alveolar macrophages from non-exposed foals. Macrophages from non-exposed foals were able to ingest the non-opsonized organism, but unable to kill greater than 65% of the infective dose by 6 h post-exposure. Alveolar macrophages from sensitized foals behaved as adult macrophages, able to kill greater than 95% of the infective dose by 6 h. Lymphocyte factors, derived by in vitro incubation of sensitized peripheral blood lymphocytes with R. equi surface antigens, enhanced macrophage bactericidal activity. Macrophages from non-exposed foals incubated in the presence of the lymphocyte factors had a 50% increase in killing of R. equi, while sensitized macrophages incubated with lymphocyte factors had a greater than 100% increase in killing capacity.