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Lipid composition in the classification of Rhodococcus equi.

The fatty acid, menaquinone and polar lipid composition of representatives of Rhodococcus equi and related taxa were determined. All of the R. equi strains had major proportions of straight chain saturated, monounsaturated and 10-methyl branched fatty acids, dihydrogenated menaquinones with eight isoprene units as the predominant isoprenologue, and characteristic polar lipid patterns that contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and glycolipids including a "cord factor"-like compound that was most pronounced in fresh isolates. The mycolic acids of these strains fell within the range C24 to C48, had 0 to 4 double bonds and released major amounts of C14:0 esters on pyrolysis. These lipid data provide further evidence that R. equi strains form a distinct taxospecies within the genus Rhodococcus. The remaining strains also gave lipid profiles consistent with their assignment to the genus Rhodococcus. These organisms included strains identified as R. sputi.

Chromatography, Thin Layer↗

Sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens.

The nucleotide sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens, located on plasmid pREAT701, has been determined. The gene encodes a 19-kDa protein of 189 amino acids, with an Ala-rich leader signal sequence (SS). At least five SS peptidase cleavage sites were found in this region. The molecular diversity of 15-17-kDa antigens might be attributed to the multiple SS peptidase cleavage sites.

Amino Acid Sequence↗

Non-pulmonary Rhodococcus equi infections in patients with acquired immune deficiency syndrome (AIDS).

Rhodococcus equi, formerly known as Corynebacterium equi, was isolated repeatedly from the blood of two patients with the acquired immune deficiency syndrome (AIDS). Neither of the patients had pneumonia while they were bacteraemic, whereas pneumonia has been present in all previously reported cases of human infection with R equi. One of our patients had diarrhoea and the organism was isolated from a stool culture; the other patient had a large granulomatous soft tissue mass in his pelvis caused by R equi. Both isolates were resistant to penicillin and one produced a beta-lactamase. Both patients were treated with vancomycin but only one recovered.

Acquired Immunodeficiency Syndrome↗

A unique phosphatidylinositol bearing a novel branched-chain fatty acid from Rhodococcus equi binds to influenza virus hemagglutinin and inhibits the infection of cells.

From the aquatic bacterium Rhodococcus equi strain S(420), we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (PtdIns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this PtdIns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the PtdIns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virus-mediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and PtdIns, showing that the PtdIns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of PtdIns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.

Animals↗

Review of Corynebacterium (Rhodococcus) equi lung abscesses in foals: pathogenesis, diagnosis and treatment.

Corynebacterium (Rhodococcus) equi is becoming increasingly significant as a cause of bronchopneumonia and lung abscessation in foals. The organism can survive within macrophages and may thus escape normal pulmonary defence mechanisms, particularly in immunocompromised animals. The disease has hitherto been associated with mortality rates as high as 80 per cent, partly as a result of inappropriate therapy. The selection of lipid-soluble antibiotics capable of intracellular penetration is critical for the successful treatment of C equi lung abscesses. A combination of two such antibiotics, erythromycin (25 mg/kg three times daily) and rifampicin (5 mg/kg twice daily) has been used on foals since 1981. Most of these animals had radiographic evidence of extensive lung abscessation, and in all cases the presence of C equi was confirmed on culture of tracheal aspirates. The duration of therapy ranged from four to nine weeks. Mild gastritis and diarrhoea were occasionally noted, but never such as to require termination of the therapy. No other adverse side effects were encountered. The success rate, as judged by a return to normal of chest radiographs and plasma fibrinogen concentrations, has exceeded 80 per cent.

Actinomycetales Infections↗

Epidemiology of Rhodococcus equi infection in horses.

Current understanding of the epidemiology of Rhodococcus equi infection on horse farms is reviewed. Infection is widespread in herbivores and their environment, because herbivore manure supplies the simple organic acid substrates on which the organism thrives. There is a progressive development of infection in the soil on horse farms with prolonged use, because: (1) there is a continual supply of nutrients; (2) the organism multiplies progressively as temperatures rise; (3) the bacterium has a robust nature. While this aerobic organism fails to multiply in the largely anaerobic intestine of the adult horse, multiplication to very large numbers may occur in the intestine of a foal in its first 8-12 weeks of life. Farms used for foal breeding over many years may thus become particularly dangerous for foals. Areas for future study include the effectiveness of decontamination, manure-removal programs and dust reduction in reducing challenge to susceptible foals.

Actinomycetales Infections↗

Polyacrylamide gel electrophoresis of whole-cell preparations of Rhodococcus equi.

The whole-cell proteins of ten strains of Rhodococcus equi isolated from horses, pigs, or humans, including the type strain ATCC 6939, were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein profiles of seven different capsular serotypes and the type strain were very similar when bacteria were cultured under the same conditions. Protein profiles were largely unaffected by incubation at two temperatures (30 degrees C, 37 degrees C) or times (12 h, 48 h). There were generally minor differences in protein profiles between strains grown in different media (brain heart infusion, nutrient, minca broths, tryptic soy-blood agar) with the marked exception of a prominent diffuse 17.5 kd protein which was expressed in nutrient broth. This protein was not produced by the type strain and was lost on repeated passage in vitro (50th, 100th passage) in two of three other strains examined.

Animals↗

Increased pulmonary activation of nuclear factor-kappaB (NF-kappaB) in foals inoculated with Rhodococcus equi is associated with increased expression of inflammatory cytokines.

Previous studies revealed that foals inoculated with virulent Rhodococcus equi had significantly higher pulmonary levels of interleukin-1beta, interleukin-12 p40, interferon-gamma, and tumor necrosis factor-alpha mRNA compared to foals inoculated with an avirulent plasmid-cured derivative. The purpose of this study was to determine if the increases in cytokine expression were associated with increased pulmonary activation of nuclear factor-kappaB (NF-kappaB). Electrophoretic mobility shift assays were performed on pulmonary nuclear protein extracted from foals treated with phosphate-buffered saline, or inoculated with either virulent or avirulent R. equi. NF-kappaB activation was increased in the nuclear extracts from foals inoculated with virulent R. equi at 14 days after inoculation when increased cytokine expression was also observed. Southwestern histochemistry revealed activated NF-kappaB in multinucleated giant cells that often contained bacteria. These results indicate that the cytokine response to R. equi is at least partially mediated by NF-kappaB activation.

Actinomycetales Infections↗

Characterization of virulence plasmids and serotyping of rhodococcus equi isolates from submaxillary lymph nodes of pigs in Hungary.

The plasmid types and serotypes of 164 Rhodococcus equi strains obtained from submaxillary lymph nodes of swine from different piggeries in 28 villages and towns located throughout the country were examined. The strains were tested by PCR for the presence of 15- to 17-kDa virulence-associated protein antigen (VapA) and 20-kDa virulence-associated protein antigen (VapB) genes. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphism characteristics. None of the 164 isolates contained the vapA gene, and 44 (26.8%) isolates were positive for the vapB gene, showing a product of the expected 827-bp size in the PCR amplification. The 44 isolates of intermediate virulence contained virulence plasmids that were identified as types 1 (3 isolates), 4 (1 isolate), 5 (36 isolates), 6 (1 isolate), and 7 (2 isolates) and as a new variant (1 isolate). On the basis of restriction digestion patterns of plasmid DNAs, we tentatively designated the variant as type 17. Use of the serotyping method of Prescott showed that 110 (67.1%) out of the 164 isolates were typeable and that serotype 2 predominated (83 isolates [50.6%]), followed by serotype 1 (26 strains [15.9%]). Only one isolate belonged to serotype 3. A total of 54 (32.9%) isolates were untypeable in Prescott's system. The prevalence of R. equi strains of intermediate virulence among the isolates that came from the submaxillary lymph nodes of swine in Hungary was lower than that seen with isolates obtained elsewhere.

Animals↗

Plasmid profiles of virulent Rhodococcus equi isolates from soil environment on horse-breeding farms in Hungary.

The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.

Actinomycetales Infections↗

Identification and epidemiological relationship of Rhodococcus equi isolated from cases of lymphadenitis in cattle.

The present study was designed to comparatively investigate 10 Rhodococcus equi isolates from cases of lymphadenitis in cattle. The isolates could be identified by cultural and biochemical properties. By serotyping the R. equi isolates 9 and 1, cultures could be classified as Nakazawa's serotypes 15 and 8, respectively. The isolates did not agglutinate rabbit erythrocytes, were uniformly susceptible to most of the antibiotics tested, did not contain plasmids nor expressed virulence-associated proteins and yielded identical patterns in protein fingerprinting. To further analyze the epidemiological relationships, the isolates were additionally subjected to DNA fingerprinting. This was performed by pulsed-field gel electrophoresis (PFGE) after digestion of the chromosomal DNA with the endonuclease AsnI. PFGE analysis of the chromosomal DNA revealed 4 DNA restriction groups with DNA pattern I with 7 isolates as predominant group and DNA pattern II to IV with one isolate, respectively. The present results indicate that a single R. equi clone belonging to Nakazawa's serotype 15 and according to PFGE to DNA restriction pattern I of the present investigation seems to be responsible for most of the cases of lymphadenitis of cattle described in this study.

Actinomycetales Infections↗

Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Humoral immune responses in 16 foals to virulence-associated 15- to 17-kDa antigens of Rhodococcus equi were studied during the first fourteen weeks of life on two horse-breeding farms with a persistent incidence of R. equi infection. Serum antibody levels specific for 15- to 17-kDa antigens were measured by enzyme-linked immunosorbent assay and Western immunoblotting. Immunoglobulin G (IgG) antibodies specific to 15- to 17-kDa antigens were detected by all the foals. R. equi was found in the feces of foals during week 1 of life, and the number of fecal R. equi rapidly increased to the highest level. Virulent R. equi were isolated from the feces of the foals at a high frequency and from their environmental soil on the farms. Evidence that serum antibody response to 15- to 17-kDa antigens of virulent R. equi occurred naturally in every foal in correlation with the quantitative changes of fecal R. equi during the first 1 to 3 months of life suggests that intestinal virulent R. equi might be the most important source of antigenic stimulation in foals from contaminated farms.

Actinomycetales Infections↗

The effect of experimental infection with Rhodococcus equi on immunodeficient mice.

To investigate the pathogenesis of respiratory lesions caused by the facultative intracellular pathogen, Rhodococcus equi, pulmonary clearance was compared in four groups of genetically defined mice, chosen for their specific deficits in immune and inflammatory responses. Complement-deficient A/J, immunodeficient nu/nu (nude), scid/scid.bg/bg (SCID/beige), C57BI/6J.bg/bg (beige) and normal Swiss mice (SW) received approximately 10(7) R. equi intranasally on day 0. Bacterial clearance was assessed in lung, liver and spleen on days 1, 4, 7 and 14. Pulmonary clearance was not significantly different between SW and A/J mice. Beige mice cleared R. equi more rapidly and completely than A/J and SW, indicating that deficits in phagocytic and NK cell function associated with the bg/bg gene did not compromise clearance. Pulmonary clearance in immunodeficient SCID/beige mice paralleled that of the SW and A/J mice initially but bacterial proliferation produced significant differences from SW mice at day 14. Nude mice were unable to clear R. equi from day 1, resulting in the death of two nude mice at day 11. Both SCID/beige and nude mice developed severe pyogranulomatous bronchopneumonia, whereas A/J and SW mice developed transient pulmonary lesions. Beige mice developed minimal lung lesions. Significant systemic bacterial proliferation occurred only in nude and SCID/beige mice. We conclude that deficiencies in complement components, phagocytic and NK cells do not impair the pulmonary clearance of R. equi but that a competent cellular immune system is required to prevent pneumonia and death. The difference in early phase pulmonary clearance in nude and SCID/beige mice indicates two phases are important for clearance. An acapsular mutant of R. equi was completely cleared from the lungs of SCID/beige mice suggesting an important role for the capsule in virulence for mice.

Actinomycetales Infections↗

Rhodococcus equi infection in HIV-positive patients: report of 5 cases and literature overview.

The importance of Rhodococcus equi infection, an uncommon human pathogen that almost exclusively affects immunocompromised hosts, has greatly increased following the advent of acquired immune deficiency syndrome (AIDS) epidemics. Until the present time, 38 cases of R. equi infection have been described in human immunodeficiency virus (HIV)-infected patients; we now report a further five personal cases. R. equi was acquired via respiratory exposure to animals in less than half of the patients, and caused invasive pulmonary infection (91%), bacteraemia and sometimes bloodstream dissemination. R. equi was easily cultured from sputum or blood, but its diagnosis was often difficult due to microbiological and clinical similarities with other pathogens. The persistence of the micro-organism inside macrophages and its high tissular load represent the major limitation to an effective treatment. Several antibiotics are active in vitro, but their efficacy in vivo depends on macrophage uptake and/or bactericidal activity. Treatment should start with at least two intravenous bactericidal antibiotics for 3-4 weeks, and then continue with oral therapy for a period of up to several months with at least two intracellularly active drugs. Surgical resection of the lesions may be beneficial in selected cases.

AIDS-Related Opportunistic Infections↗

Evidence for interfacial uptake in hexadecane degradation by Rhodococcus equi: the importance of cell flocculation.

The kinetics of hexadecane degradation were studied in four strains of Rhodococcus equi that did not produce biosurfactants. The aim was to analyse the characteristics of alkane uptake and their relevance to a mechanism of interfacial uptake. The kinetic studies involved continuous determination of degradation by electrolytic respirometry in a diphasic system where the hydrophobic phase was hexadecane or a solution of hexadecane in a non-toxic, non-biodegradable solvent, either 2,2,4,4,6,8,8-heptamethylnonane or silicone oil. The technique allowed large variations in interfacial area between the aqueous and hydrophobic phases. For the four strains, the kinetics obtained were reproducible and showed, in almost all cases, an initial short phase of exponential growth, followed by a long phase of linear growth. Specific growth rates during exponential growth varied amongst the strains from 0.11 to 0.20 h(-1) and were independent of interfacial area, in accordance with the very strong adsorption of bacterial cells at the interface of solvent and aqueous media. The degradation rates during linear growth did not increase with interfacial area but increased with efficiency of stirring. These characteristics can be explained by the formation of cellular flocs due to the hydrophobicity of the strains. These flocs were observed during growth on hexadecane in almost all conditions. In one case, with a non-flocculating culture, a kinetic pattern with a longer exponential phase, closer to that expected for simple interfacial uptake, was observed. The results show that strictly interfacial uptake, limited by floc formation (occurring at moderate and higher cell densities, and controlled by stirring efficiency) is a common pattern for growth on long-chain alkanes of micro-organisms that do not produce biosurfactants.

Alkanes↗

Cytologic appearance of Rhodococcus equi in bronchoalveolar lavage specimens. A case report.

Immunocompromised patients are at risk of developing infections caused by Rhodococcus equi, a gram-positive bacillus that can cause pneumonia in patients with acquired immunodeficiency syndrome. To the author's knowledge this is the first report describing the cytologic features of the infection in a patient who had a confirmatory tissue biopsy and positive culture. Infection with R equi may go unrecognized by pathologists unaware of its presentation and appearance in cytologic material.

Actinomycetales Infections↗

Rhodococcus equi--an increasingly recognized opportunistic pathogen. Report of 12 cases and review of 65 cases in the literature.

Rhodococcus equi, a gram-positive, weakly acid-fast coccobacillus, initially isolated from horses, is becoming increasingly recognized as an important pathogen for immunosuppressed human hosts since the first human case was reported in 1967. A review of the English medical literature yielded 53 cases. During the last 11 years, the microbiology laboratories of the authors isolated the organism from 12 patients. Of the total 65 cases, 60 occurred in immunosuppressed patients with HIV infection, malignant neoplasms, or chronic immunosuppressive therapy. The lung is the most common primary site of infection. Typically, the lesion is densely infiltrated by histiocytes with multiple microabscesses. Intracellular gram-positive coccobacilli are easily demonstrated. R equi grows well on routine non-selective media at 35 degrees C. Previously, many cases may have been missed because the organism resembles oropharyngeal commensal diphtheroids. Clinical information with gram and Kinyoun strains on fresh isolates is helpful in recognizing the possibility of R equi infection.

Acquired Immunodeficiency Syndrome↗

Pheno- and genotyping of Rhodococcus equi isolated from faeces of healthy horses and cattle.

The present study was designed to comparatively investigate 21 Rhodococcus equi isolates from the faeces of clinically healthy horses and cattle. The isolates were identified by cultural and biochemical properties and by PCR analysis. The latter, targeted to the gene coding for the 16S ribosomal RNA, revealed a species specific PCR product. The isolates were further characterised by serotyping with two typing systems, by haemagglutination tests and by plasmid and virulence protein profiling. Among the 21 cultures, four cultures contained plasmids, two of the four cultures expressed 15-17 kDa virulence proteins, no cultures contained 20 kDA virulence proteins. The 21 cultures were further analysed by DNA-fingerprinting. This was performed by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). The DNA-restriction patterns were different for most of the isolates indicating a clone heterogeneity among isolates from single farms. Serotyping, determination of virulence marker and PFGE analysis of R equi appeared to be useful for further characterisation of this species, possibly of importance for virulence estimation of single R equi isolates and for epidemiological studies.

Animals↗