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Cloning and sequence analysis of the gene encoding isocitrate lyase from Rhodococcus fascians.

An isocitrate lyase (Icl)-encoding gene (icl) from the Gram+ plant pathogen Rhodococcus fascians was identified serendipitously as part of a scrambled fragment after shotgun cloning in the promoter probe vector, pDP1. The Icl protein is 429 amino acids long (47.11 kDa) and has a predicted pI of 4.84; it is 54% similar to the Escherichia coli Icl and 24-27% to eukaryotic homologues. Comparison of the prokaryotic and eukaryotic Icl confirms the earlier proposal of Matsuoka and McFadden [J. Bacteriol. 143 (1988) 4528-4536] that the enzyme has enlarged during evolution.

Amino Acid Sequence↗

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↗

Identification and activity of two insertion sequence elements in Rhodococcus sp. strain IGTS8.

Two putative insertion sequence (IS) elements, IS1166 and IS1295, were identified on a plasmid present in Rhodococcus sp. IGTS8. Four copies of IS1166 were present in strain IGTS8: one copy on each of two separate plasmids and two copies on a third plasmid or in the chromosome. Of eight rhodococci tested, only R. zopfii contained a copy of an IS1166-like element. Two mutants of strain IGTS8 were isolated in which an additional copy of IS1166 was present, suggesting that at least one copy of this element may be able to transpose. IS1166 and IS1295 are new members of a family of IS elements which includes IS6120 from Mycobacterium smegmatis, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus and ISRm3 from Rhizobium meliloti. The 24-25-bp inverted repeats of these six elements are highly similar and, with the exception of IS1295, their transposases exhibit moderate identities. In particular, the predicted amino-acid sequence of the transposase from IS1166 is 86% identical to that from the known transposable element IS6120.

Amino Acid Sequence↗

Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl/chlorobiphenyl degradation by Rhodococcus sp. M5.

The nucleotide (nt) sequence of the bpdC1C2BADE genes which encode the first three enzymes in the biphenyl (BP) degradation pathway of Gram+ Rhodococcus sp. M5 (formerly Arthrobacter M5) was determined. Except for the ferredoxin component (BpdB) of the initial BP dioxygenase, the predicted amino acid (aa) sequences of the remaining proteins are found to be more closely related to the counterpart proteins (TodC1C2BADE) present in the toluene-degrader, Pseudomonas putida F1, than those of three BP-degrading pseudomonads. The cloned bpd genes were verified by their expression in the Escherichia coli T7 RNA polymerase/promoter system. In E. coli, BpdA was able to complement TodC1C2B in indigo biosynthesis, although the M5 native or cloned BP dioxygenase does not carry out this reaction.

Bacterial Proteins↗

Location and sequence analysis of a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase-encoding gene (bpdF) of the biphenyl/polychlorinated biphenyl degradation pathway in Rhodococcus sp. M5.

The 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase-encoding gene (bpdF) in the biphenyl (BP)/polychlorinated biphenyl (PCB)-degrading bacterium, Rhodococcus sp. M5 (M5), was found to be located within a 4.5-kb HindIII-BamHI genomic DNA that was 5.4 kb downstream from the bpdC1C2BADE gene cluster. The deduced amino acid (aa) sequence of bpdF revealed that the hydrolase contains 297 aa (32679 Da) that was verified by expression in the Escherichia coli T7 RNA polymerase/promoter system. Unlike previously known HOPD hydrolases, the aa sequence of BpdF appears unique. Interestingly, all HOPD hydrolases and related proteins from the phenol and toluene/xylene degradation pathways, were found to have a bias in the codon usage in the catalytic Ser within the conserved VGNS(M/F)GG motif.

Amino Acid Sequence↗

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↗

The pathogenesis of Rhodococcus equi pneumonia in foals.

The pathogenesis of Rhodococcus equi pneumonia in foals is reviewed. The main routes of infection are respiratory and alimentary. The latter is probably the chief route of exposure in all foals and probably leads to development of specific immunity. Susceptible foals, those whose maternal immunity wanes before generation of their own immune response, readily develop disease if exposed aerogenously to sufficient numbers of R. equi. Management and environmental circumstances have a major role to play in determining the magnitude of this challenge and, therefore, in the prevalence of the disease. Infection of a naive foal leads to severe, suppurative bronchopneumonia with suppurative lymphadenitis of regional nodes and, in approximately 50% of animals, to necrotizing enterocolitis. The foal is uniquely susceptible to R. equi pneumonia; comparable experimental infections do not produce progressive destructive pulmonary lesions in other animal species. In the naive foal lung, R. equi behaves as a facultative intracellular pathogen, avoiding destruction within the alveolar macrophage by inhibiting phagolysosome fusion and possibly by causing lysosomal degranulation. The role of putative virulence factors, such as equi factor, remains to be elucidated.

Actinomycetales Infections↗

The ecology of Rhodococcus equi and physicochemical influences on growth.

Growth of Rhodococcus equi was studied in vitro. Optimal growth occurred under aerobic conditions between pH 7.0 and 8.5, at 30 degrees C. R. equi survived better in a neutral soil (pH 7.3) than it did in two acid soils (pH less than 5.5). It grew substantially better in soils enriched with faeces than in soils alone. Simple organic acids in horse dung, especially acetate and propionate, appear to be important in supporting growth of R. equi in the environment. The ecology of R. equi can be best explained by an environmental cycle allowing its proliferation in dung, influenced by management, grazing behaviour and prevailing climatic conditions. Preventive measures should be aimed at reducing or avoiding focal areas of faecal contamination in the environment.

Animals↗

Rhodococcus equi in fecal and environmental samples from Kansas horse farms.

Horse farms in Kansas were surveyed for the incidence of Rhodococcus equi. Fecal specimens and soil or cobweb samples were collected from each farm and cultured on selective media. One control farm (with no history of R. equi infection), one farm which had an outbreak 3 and 4 years previously and 2 farms which had R. equi-infected foals that season were surveyed. In addition, fecal samples from 21 horses hospitalized in the Kansas State University Veterinary Hospital were cultured. There was no significant difference in the incidence of R. equi in fecal samples from the 2 farms with recent disease problems. The farm with a history of disease had a significantly higher percentage of positive fecal cultures than the 2 farms with a more recent history of disease. Neither ration composition nor sex of the horses appeared to affect the fecal culture results. Fecal samples from 2 birds on 1 farm were positive for R. equi.

Animals↗

Experimental infection of mice with Rhodococcus equi: differences in virulence between strains.

The growth kinetics in outbred mice of clinical and environmental isolates of Rhodococcus equi were followed by serial bacterial enumeration of organ homogenates. Clinical isolates multiplied until Day 4 before being progressively cleared, but could still be recovered from the liver at 3-4 weeks post-infection. Intravenous inoculation of clinical strains was associated with histopathological responses very similar to those elicited by intravenous infection with various facultative intracellular parasites. Whereas lesions in mice and foals at 7-9 days following respiratory infection are those of severe bronchopneumonia with massive consolidation, a week later the patterns of host response have diverged as the murine lesions resolve. The type strain, NCTC 1621 and 4-6 environmental isolates were eliminated without prior multiplication and these strains caused negligible lesions. The two environmental strains which behaved as the clinical strains were recovered from a stud with an R. equi problem. No association of colonial morphology of R. equi with virulence was apparent.

Actinomycetales Infections↗

The interaction of Rhodococcus equi and foal neutrophils in vitro.

Polymorphonuclear neutrophil leukocytes (PMNL) from 8 healthy foals (2-14 weeks of age) and 2 foals with bacterial pneumonia were separated from whole blood using a 2 step Percoll gradient. Purified PMNL were tested for bactericidal function against Rhodococcus equi and Staphylococcus aureus in the presence of normal horse serum. The percentage uptake after a 15-min pre-incubation of PMNL and bacteria was also calculated. Ultrastructural examination of the interaction of R. equi and normal foal PMNL was performed after 15 min incubation. Results indicated that foal PMNL effectively phagocytose and destroy R. equi and S. aureus in the presence of normal horse serum. The mean percent uptake for R. equi was 99.3 +/- 0.4% and for S. aureus 99.9 +/- 0.1%. Further, 97.8 +/- 0.1% ingested R. equi and 98.4 +/- 0.1% ingested S. aureus were destroyed in the 15-min incubation period. Over the 3-h incubation, 91.9% of remaining R. equi were killed, but only 49.2 +/- 31.9% of S. aureus (P less than 0.01). Total bactericidal effect of foal PMNL, however, was 99.3 +/- 0.4% against R. equi and 99.9 +/- 0.1% against S. aureus. The percentage uptake and total bactericidal efficacy of neutrophils from sick foals was greater than 95%. Ultrastructural examination of the PMNL-R. equi interaction after 15 min incubation revealed phagocytosis of the bacteria and morphologic changes consistent with neutrophil degranulation. This study suggests that a defect in PMNL bactericidal capability is not likely to be a contributing factor in the pathogenesis of R. equi pneumonia in foals.

Actinomycetales Infections↗

Electron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages.

It has been suggested that R. equi causes pulmonary disease in foals by persisting within the lung as a facultative intracellular parasite of alveolar macrophages. This paper describes an ultrastructural study of the intracellular events after ingestion of R. equi by foal alveolar macrophages, in an attempt to determine the mechanism of intracellular survival of R. equi. Secondary lysosomes of alveolar macrophages recovered from foals by bronchoalveolar lavage were labelled with electron-dense ferritin, and the cells were challenged with either viable or formalin-killed R. equi. After 0-, 3-, 8- or 24-h incubation, the cells were fixed and processed for electron microscopy. There was no evidence of phagosome-lysosome fusion after ingestion of either viable or non-viable R. equi by foal alveolar macrophages. Rhodococcus equi persisted and multiplied within dilated phagosomes, which were often lined by elongate microvillous structures. After 24-h incubation, 75% of the ingested bacteria were still structurally intact. Macrophages with ingested viable R. equi were irreversibly damaged and released intracellular bacteria into the surrounding medium. These data confirm that R. equi is a facultative intracellular parasite of foal alveolar macrophages and is able to persist and multiply within the phagosome, apparently inhibiting phagosome-lysosome fusion by some as yet unknown mechanism.

Animals↗

Interaction of Rhodococcus equi with phagocytic cells from R. equi-exposed and non-exposed foals.

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.

Actinomycetales Infections↗

Humoral immune response of foals to experimental infection with Rhodococcus equi.

Humoral immune response to Rhodococcus equi in experimentally infected foals was studied with the enzyme-linked immunosorbent assay (ELISA) method. Class-specific antibodies were measured by ELISA in the sera of foals after intratracheal or oral inoculation with R. equi ATCC 6939 or T 48 and in the lung washings of a foal after intratracheal inoculation or of normal horses. After intratracheal or oral inoculation with R. equi, serum antibodies were first detected in immunoglobulin G (IgG) followed by IgM and IgA classes, but significant levels of IgM and IgA developed only in the foal infected intratracheally with R. equi T 48. Only the foal infected intratracheally with T 48 developed pneumonia. Anti-R. equi IgG and IgA antibodies appeared in lung washings of the intratracheally infected foal. There were differences in the antibody response to R. equi among the intratracheally infected foals, the orally infected foal and the naturally infected foal. These results suggest that the humoral immune response to R. equi may be affected by the type of R. equi strain and the route and extent of R. equi exposure.

Actinomycetales Infections↗

Rhodococcus equi pneumonia in 48 foals: response to antimicrobial therapy.

Case records of 48 foals with pneumonia due to Rhodococcus equi were reviewed. Twenty of the 48 foals survived and 28 died or were euthanized. There was no significant difference between the survivors and non-survivors in the age of onset of illness, duration of illness prior to admission, the mean white blood cell (WBC) count, or the mean plasma fibrinogen content. All foals had R. equi isolated from a tracheobronchial aspirate or lung specimens obtained at necropsy. All organisms were susceptible in vitro (Kirby-Bauer) to erythromycin and gentamicin. Susceptibilities to other drugs were: trimethoprim-sulfamethoxazole (88%), tetracycline (87%), chloramphenicol (83%); 97% were resistant to cephalothin and 83% to penicillin. Thirteen of the 20 surviving foals were treated with erythromycin and/or rifampin. A decline in mortality rate was observed with the introduction of the combination of erythromycin and rifampin. None of the 17 foals treated with penicillin and gentamicin survived. Chronic, active, non-septic synovitis was confirmed in 17 foals. These foals had joint distension with mild or no apparent lameness.

Actinomycetales Infections↗

Use of erythromycin-rifampin combination in treatment of Rhodococcus equi pneumonia.

The selection of lipid-soluble antibiotics capable of intracellular penetration is considered critical for the successful treatment of Rhodococcus equi pneumonia and lung abscesses in foals. Two such antibiotics: erythromycin (25 mg kg-1, three times daily) and rifampin (5 mg kg-1 twice daily) have been used in combination for this purpose at the University of Florida since 1981. Positive evidence of R. equi was present on culture of tracheal aspirates in 57 foals, most of which exhibited radiographic evidence of extensive lung abscessation. The duration of therapy ranged from 4 to 9 weeks. Mild diarrhea was sometimes noted, but was never severe enough to require the termination of therapy. No other adverse side effects were apparent. Judged by a return of chest radiographs and hematologic parameters to normal, 50 of the 57 foals were considered to have recovered from the disease; a success rate of 88%.

Actinomycetales Infections↗

Diagnosis of Rhodococcus equi infection in foals by the agar gel diffusion test with protein antigen.

A protein antigen that reacted in the agar gel diffusion (AGD) test and which had equi factor(s) activity, was partially purified from the culture supernatant of Rhodococcus equi by successive column chromatography on diethylaminoethyl cellulose and Sepharose 4B. Employing a standard foal serum, the concentration of this antigen was adjusted for the AGD test. Optimal dilutions of the antigen reacted in the AGD test with sera from foals naturally infected with serologically different R. equi. The antigen prepared was considered suitable for use in field surveys of R. equi infection. Accordingly, four groups of sera were tested: those from 18 foals diagnosed as being infected with R. equi, those from 54 control foals with culture-negative R. equi pneumonia, arthritis or cellulitis, those from 46 diseased foals suspected of having R. equi infection and those from 51 clinically normal foals. A positive precipitation reaction was observed with sera from 100% of the first group, 69.5% of the third group and 17.7% of the fourth group. A negative reaction was obtained with sera from 100% of the second group.

Actinomycetales Infections↗

Role of antibody to extracellular proteins of Rhodococcus equi in protection against R. equi pneumonia in foals.

Rhodococcus equi produces two exoenzymes (REE), a cholesterol oxidase in large amounts and a phospholipase C, which cause lysis of sheep red blood cells (SRBC) sensitized with Staphylococcus aureus beta toxin. Two immunization studies were done in foals to determine the role of antibody to REE in protection against R. equi pneumonia. In the first study, three foals (mean age 10 days) were vaccinated four times at 2-week intervals with over 1 million units of partially purified exoenzymes (PREE). In the second study, three foals (mean age 19 days) were administered plasma from an adult horse vaccinated with PREE. Relatively low titres (16-32) of neutralizing antibody were detected in the foals of the former group, and passive transfer of neutralizing antibody (titres 32-64) occurred in the latter. Following immunization, principal foals and an equal number of similarly aged nonimmunized foals were challenged by aerosol with 1 x 10(10) live R. equi per day for 5 consecutive days. No severe clinical pneumonia developed in either group and, with one exception, only minor and resolving lung abscesses developed in these foals. These studies showed that antibody response of foals to immunization with PREE was poor, antibody to PREE did not prevent foals from developing lung abscesses following experimental infection, and that foals even as young as 3 weeks of age may be largely refractory to aerosol challenge with virulent R. equi.

Actinomycetales Infections↗