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Use of Rhodococcus equi virulence-associated protein for immunization of foals against R equi pneumonia.

OBJECTIVE: To evaluate use of the virulence-associated protein of Rhodococcus equi in immunizing foals against R equi pneumonia. ANIMALS: Eight (experimental group) and 6 (controls) mares with their foals. PROCEDURE: Virulence-associated protein extracted from R equi was used to prepare an acetone-precipitated. Triton X-extracted (APTX) antigen. After determination of the efficacy of passive immunization, in untreated foals or in foals given plasma from a horse vaccinated with APTX antigen or from a nonvaccinated horse, a field trial was done to evaluate the efficacy of vaccination of 8 mares, twice with APTX before parturition, and of their foals at ages 3 and 5 weeks; 6 mares and their foals served as unvaccinated controls. All 2-day-old foals were given plasma from local donor horses inoculated with a locally produced bacterin. Serum opsonizing activity produced by vaccination with APTX was determined. Passively immunized foals were challenge exposed with an aerosol of virulent R equi. Foals of the field trial were exposed to enzootic R equi infection. RESULTS: Inoculation with APTX resulted in high IgG antibody liters with opsonizing activity. Passive immunization of foals with plasma from an immunized horse enhanced bacterial clearance from the lungs, compared with that in foals not given plasma or given plasma without APTX antibodies. Vaccination of mares and foals exposed to natural infection resulted in development of R equi pneumonia in 4 of 8 vaccinated foals, but in only 1 of 6 unvaccinated foals. CONCLUSIONS: Vaccination with APTX antigen led to high-titer, opsonizing antibody. Plasma from a vaccinated horse appeared to enhance clearance of R equi from the lungs of foals. Paradoxically, vaccination of mares and their foals with APTX antigen did not protect foals and may have enhanced R equi pneumonia in the foals.

Actinomycetales Infections↗

Emergence of rifampin-resistant Rhodococcus equi in an infected foal.

To investigate the emergence of rifampin resistance in Rhodococcus equi strains isolated from foals and their environment in Japan, we compared the in vitro antimicrobial susceptibilities to rifampin of 640 isolates from 64 infected foals and 98 soil isolates from their horse-breeding farms. As a control, 39 human isolates from patients with and without AIDS were also tested for susceptibility to rifampin. All of the isolates showed rifampin sensitivity, except isolates from one infected foal and two patients with AIDS that showed rifampin resistance. To investigate the emergence of rifampin-resistant R. equi in the infected foal, which had received rifampin monotherapy for a month before euthanasia, 99 isolates of R. equi from the lesions and 20 isolates from the intestinal contents of the one foal with rifampin-resistant organisms were analyzed for rifampin susceptibilities, pathogenicities, and ribotypes. Of the 99 isolates from the lesions, all of which were virulent R. equi strains containing a virulence plasmid with a size of 85 or 90 kb, 90 (91%) isolates were rifampin resistant (MIC, > or = 12.5 microg/ml). On the other hand, of the 20 isolates from the intestinal contents, 11 (55%) isolates showed rifampin resistance (MIC, > or = 25 microg/ml), and 5 of them were avirulent R. equi strains. Among these 101 rifampin-resistant R. equi isolates with and without virulence plasmids characterized by ribotyping, 58 were type I, 20 were type II, 11 were type III, and 12 were type IV. These results demonstrated that at least eight different rifampin-resistant R. equi strains emerged concurrently and respectively from the different lesions and intestinal contents of the infected foal.

Actinomycetales Infections↗

Pulmonary malacoplakia associated with Rhodococcus equi infection in a patient with acquired immunodeficiency syndrome.

Pulmonary malacoplakia associated with Rhodococcus equi, an opportunistic Gram-positive coccobacillus, is unusual. Although this patient is the ninth reported to have pulmonary malacoplakia, he is only the third patient with the acquired immunodeficiency syndrome who has been reported to have pulmonary malacoplakia associated with R equi infection. The patient was a 49-year-old man infected with the human immunodeficiency virus who on initial examination was found to have cavitary pneumonia in the right upper lobe of the lung. Histologic examination of an open-lung biopsy specimen revealed sheets of foamy intraalveolar macrophages that contained coccobacillary organisms and some "targetlike" cytoplasmic inclusions. Electron-microscopic studies showed Michaelis-Gutmann bodies, some of which contained residual bacterial fragments. Cultures of the lung tissue, urine, and blood yielded growth of Gram-positive coccobacilli subsequently identified as R equi. In immunocompromised patients, the possibility of malacoplakia should be considered in the differential diagnosis of diffuse pulmonary infiltrate composed of foamy intraalveolar macrophages.

Acquired Immunodeficiency Syndrome↗

Rhodococcus equi infection in HIV-positive subjects: a retrospective analysis of 24 cases.

Rhodococcus equi causes a rare infection in immunocompromised hosts. We describe 24 cases of infection in patients with AIDS-related complex (ARC)/acquired immunodeficiency syndrome (AIDS). Pneumonia was always the first manifestation of R. equi infection, but extrapulmonary involvement was also observed. The main sources of bacteria were sputum, bronchial washings and blood. The strains isolated were mainly susceptible to erythromycin, vancomycin, teicoplanin, rifampicin, imipenem and aminoglycosides. Initial treatment should involve an intravenously administered antibiotic combination therapy including imipenem or vancomycin or teicoplanin, followed by orally administered maintenance combination therapy. Drug combinations should be investigated for serum bactericidal activity in vitro. Surgery does not increase survival time and should only be performed in cases that do not respond to antibiotic treatment. Presumptive risks of infection (contact with horses or farm dust, or cohabiting with people affected by R. equi infection) were present in more than 50% of patients. This finding, and the frequency of bacteria in the sputum, are not sufficient proof of transmission between humans, but do suggest the need for respiratory isolation of patients affected by R. equi pneumonia.

AIDS-Related Complex↗

Partial genome sequencing of Rhodococcus equi ATCC 33701.

Preliminary analysis of a partial (30% coverage) genome sequence of Rhodococcus equi has revealed a number of important features. The most notable was the extent of the homology of genes identified with those of Mycobacterium tuberculosis. The similarities in the proportion of genes devoted to fatty acid degradation and to lipid biosynthesis was a striking but not surprising finding given the relatedness of these organisms and their success as intracellular pathogens. The rapid recent improvement in understanding of virulence in M. tuberculosis and other pathogenic mycobacteria has identified a large number of genes of putative or proven importance in virulence, homologs of many of which were also identified in R. equi. Although R. equi appears to have currently unique genes, and has important differences, its similarity to M. tuberculosis supports the need to understand the basis of virulence in this organism. The partial genome sequence will be a resource for workers interested in R. equi until such time as a full genome sequence has been characterized.

Aerobiosis↗

Rhodococcus equi infection in transplant recipients: case report and review of the literature.

BACKGROUND: Rhodococcus equi is an opportunistic pathogen that usually causes infection in immunocompromised hosts, mainly human immunodeficiency virus-positive patients, yet solid organ transplant recipients may be affected as well. Infections in this group of patients have not been sufficiently analyzed. METHODS: We report an R equi pneumonia in a heart transplant recipient and review another 11 cases. RESULTS: Infection appeared a mean of 49 months (range 1-180) after transplantation. Lung was primarily involved in 10 cases (83.3%). The remaining two cases presented with a paravertebral abscess and a purulent pericarditis. Invasive techniques were necessary to reach the diagnosis in nine cases. One patient healed with surgical resection of the lesion; the remaining 11 received antimicrobial agents. Six of them required additional surgical treatment. Three patients died. CONCLUSIONS: Clinicians should consider R equi when evaluating a solid organ recipient with an asymptomatic lung nodule. Microbiology laboratories should be alerted in these cases because it could be mistaken for a contaminant diphtheroid and will not respond to the standard empirical therapy.

Actinomycetales Infections↗

Immunogenecity of synthetic peptides representing linear B-cell epitopes of VapA of Rhodococcus equi.

Amino acid 65-78 of membrane protein VapA of the facultative intracellular Rhodococcus equi contained an immunodominant N-terminal B-cell epitope (N15Y peptide). Safety and immunogenecity of a synthetic peptide consisting of the amino acid 65-78 of VapA (peptide N15Y) were evaluated first in mice and in healthy adult horses. A single dose of a peptide-VapA vaccine induced and only in presence of adjuvant, specific IgG antibodies in sera of mice. After challenge with virulent R. equi 3 weeks after immunization, tissue clearance was more delayed in immunized mice than in control mice. An antibody-mediated response (restricted to IgG1 and IgG2b subclasses) predominated in vaccinated mice sera and no specific lymphocytes proliferation was observed. Next, a total of 15 mares were given systemic inoculation of N15Y peptide with IMS3012 ( n = 4 ) or IMS2211 ( n = 4 ) or ISA35 ( n = 4 ) or placebo ( n = 2). Serological responses to the peptide vaccine were found in all but not in placebo group. A significant increase of IgGb subclass in sera of vaccinated mare with N15Y peptide in presence of IMS3012 was observed in comparison to IMS2211 or ISA35 or control group. Moreover, INF-gamma, IL-2 and IL-10 mRNA expression increased more significantly in peripheral blood lymphocytes of IMS3012 group than in IMS2211 or ISA35 group. Interestingly, a significant decrease of IL-4 mRNA expression (undetectable level) was observed with all adjuvants. These results support the use of peptide N15Y in presence of IMS3012 adjuvant in future studies of protection of foals against R. equi.

Actinomycetales Infections↗

Restriction map of a virulence-associated plasmid of Rhodococcus equi.

The restriction cleavage map of a virulence-associated plasmid pREAT701 of Rhodococcus equi was constructed with EcoRI and HindIII by means of cloning the restriction fragments, cross Southern hybridization with each fragment, and hybridization with probes generated by modified inverse PCR. The genetic region responsible for expression of virulence-associated 15- to 17-kilodalton antigens was determined.

Blotting, Southern↗

Rhodococcus equi pneumonia in a patient with human immunodeficiency virus: case report and review.

Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus increasingly reported as an opportunistic pathogen in patients positive for human immunodeficiency virus (HIV). An HIV-positive man developed R. equi pneumonia and sepsis. He failed to improve despite surgical drainage of localized infection and many empiric antibiotics. Time-kill studies of R. equi isolated from the patient were performed against various antimicrobial agents to optimize therapy. Levofloxacin seemed to offer excellent in vitro bactericidal activity. Antagonism was observed with certain antibiotic combinations. Our anecdotal case report suggests that fluoroquinolones such as levofloxacin may offer superior efficacy to standard therapy in rhodococcal infections; their clinical utility deserves further investigation. In view of potential antagonism, prospective susceptibility testing for various drugs and drug combinations should be considered when clinically indicated.

AIDS-Related Opportunistic Infections↗

Rhodococcus equi infection: a cause of cavitary pulmonary disease in immuno-compromised patients.

Rhodococcus equi is a facultatively-aerobic, gram-positive and intracellular bacteria that causes pulmonary and disseminated infections in immuno-compromised patients. It is a common agent that causes pneumonia in farm animals and only rarely is transmitted to man. I report a case of pneumonia caused by this agent in a patient with AIDS who had been misdiagnosed as tuberculosis and treated as such for five months before the correct diagnosis was made. The diagnosis should be suspected on clinical and radiologic grounds as treatment is considerably different from other opportunistic pathogens in AIDS.

AIDS-Related Opportunistic Infections↗

Fite stain positivity in Rhodococcus equi: yet another acid-fast organism in respiratory cytology--a case report.

Rhodococcus equi is an aerobic Gram-positive and acid-fast coccobacillus that may cause cavitary pneumonia in immunocompromised hosts such as HIV-infected patients. Numerous Grocott's methenamine silver (GMS)-positive organisms were initially noted on the direct smear; a minor number of acid-fast organisms were seen in the Thin-Prep slide. Since the abundant mucous material with the attached organisms seen in conventional smears may be lost in liquid-based preparations, more sensitive stains such as Fite, as well as a more diligent search for organisms, is needed. This case illustrates the importance of careful selection and evaluation of special stains in sputum specimens.

AIDS-Related Opportunistic Infections↗

Rhodococcus equi pneumonia: case report and literature review.

OBJECTIVE: To present a case of Rhodococcus equi (RE) pneumonia and discuss its pathophysiology and treatment. CASE SUMMARY: An HIV-positive patient presented with pneumonia. A lung biopsy was performed after sputum and thoracentesis cultures failed to identify a pathogen. The lung biopsy revealed an unidentifiable, diphtheroid-like, gram-positive rod. A bronchoscopy performed five days after the lung biopsy produced the same diphtheroid-like, gram-positive rod. The patient was treated with several injectable antibiotics, but emergence of resistance to two of the antibiotics was suspected. Two weeks after the bacterial isolate was sent to a reference laboratory, it was identified as RE. The patient was discharged on oral antibiotics and experienced no recurrence of RE pneumonia. CONCLUSIONS: RE can be difficult to identify in the microbiology laboratory, or it may be assumed to be a colonizing diphtheroid. The isolation of difficult-to-identify, gram-positive rods, or diphtheroids, from a pulmonary source in a patient with decreased cell-mediated immunity should cause one to suspect RE. RE has been noted to develop resistance to beta-lactam antibiotics during therapy. A prolonged course of combination antibiotic therapy directed at the intracellular component of infection is necessary.

AIDS-Related Opportunistic Infections↗

DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103.

The virulence plasmids of the equine virulent strains Rhodococcus equi ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (vap) genes, including vapA. These vap genes have extensive homology to vapA, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the vap genes. The vap genes were present as a cluster of three (vapA, vapC, and vapD), as a pair (vapE and vapF), or individually (vapG; vapH). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual vap genes. There was extensive homology among the C-terminal halves of all vap genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.

Amino Acid Sequence↗

Association with HeLa cells by Rhodococcus equi with and without the virulence plasmid.

HeLa cell association was examined in eight Rhodococcus equi isolates containing 80-85 kb plasmids and their plasmid negative derivatives, and in two other plasmid negative R. equi strains. Seven of the eight plasmid positive strains possessed an 85 kb plasmid and produced the virulence-associated protein (VapA) detected by monoclonal antibody staining in immunoblots; one of the eight had an 80 kb plasmid but did not produce VapA. Curing of the plasmids by repetitive subcultures at 42 degrees C in broth was confirmed by colony and DNA dot blot hybridization with a 7.5 kb BamHI-HindIII plasmid fragment probe, by attempted isolation of plasmid DNA, and by demonstration of lack of VapA. Most strains associated well with HeLa cells and no relationship was found with plasmid status and possession of VapA. Association with HeLa cells was significantly greater for strains with a dry colony type than for those with a mucoid colony, a result which correlated with hydrophobicity of the colonies. HeLa cell association does not correlate with the presence of the virulence plasmid in R. equi.

Actinomycetales Infections↗

Rhodococcus equi pneumonia and pulmonary malakoplakia in acquired immunodeficiency syndrome. Pathologic features.

Pulmonary infection with Rhodococcus equi is rare, and most cases are seen in immunocompromised patients, particularly those with acquired immunodeficiency syndrome. We describe the pathologic features in four cases of culture-positive R equi pneumonia occurring in patients infected with human immunodeficiency virus. All four patients had a solitary cavitary pulmonary mass that was resected (n = 3) or had undergone biopsy (n = 1). Pathologically, all specimens revealed sheets of histiocytes with abundant foamy to eosinophilic cytoplasm with numerous phagolysosomes that were positive for periodic acid-Schiff, Gomori methenamine silver, and Grocott stains. Occasional histiocytes contained Michaelis-Gutmann bodies, diagnostic of malakoplakia. The Michaelis-Gutmann bodies yielded positive results with periodic acid-Schiff, Gomori methenamine silver, Grocott, Giemsa, and von Kossa stains (three of three cases studied) and with alizarin red and Prussian blue stains (two of three cases studied). Many gram-positive coccobacilli within histiocytes and associated with neutrophils were found in one case. Ultrastructural study of one case showed histiocytes containing abundant phagolysosomes with degenerated bacterial components and Michaelis-Gutmann bodies. The latter had a targetoid appearance with variegated phagolysosome cores that were mineralized by deposition of electron-dense spicules surrounded by peripheral rings of granular and membranous material. Based on our observations and reports in the literature, there appears to be a more than coincidental association between pulmonary R equi infection, malakoplakia, and human immunodeficiency virus infection.

Acquired Immunodeficiency Syndrome↗

Rhodococcus equi: an animal and human pathogen.

Recent isolations of Rhodococcus equi from cavitatory pulmonary disease in patients with AIDS have aroused interest among medical microbiologists in this unusual organism. Earlier isolations from humans had also been in immunosuppressed patients following hemolymphatic tumors or renal transplantation. This organism has been recognized for many years as a cause of a serious pyogranulomatous pneumonia of young foals and is occasionally isolated from granulomatous lesions in several other species, in some cases following immunosuppression. The last decade has seen many advances in understanding of the epidemiology, pathogenesis, diagnosis, treatment, and immunity to infection in foals. The particular susceptibility of the foal is not understood but can be explained in part by a combination of heavy challenge through the respiratory route coinciding with declining maternally derived antibody in the absence of fully competent foal cellular immune mechanisms. R. equi is largely a soil organism but is widespread in the feces of herbivores. Its growth in soil is considerably improved by simple nutrients it obtains from herbivore manure. About one-third of human patients who have developed R. equi infections had contact in some way with herbivores or their manure. Others may have acquired infection from contact with soil or wild bird manure. R. equi is an intracellular parasite, which explains the typical pyogranulomatous nature of R. equi infections, the predisposition to infection in human patients with defective cell-mediated immune mechanisms, and the efficacy of antimicrobial drugs that penetrate phagocytic cells.

Acquired Immunodeficiency Syndrome↗

Rhodococcus equi can survive a phagolysosomal environment in macrophages by suppressing acidification of the phagolysosome.

Rhodococcus equi is one of the most important causes of pneumonia in foals and has emerged as a significant opportunistic pathogen of immunosuppressed hosts such as human immunodeficiency virus-infected patients. Virulent R. equi harbouring an 85 kb plasmid, but not the avirulent form lacking the plasmid, has the ability to survive in macrophages. However, the survival mechanism is not known. In the present study, morphological interactions were observed between virulent or plasmid-cured avirulent R. equi and phagolysosomes in murine macrophage-like J774.1 cells by immunocytological methods. The J774.1 cells phagocytosed virulent and avirulent bacteria to a similar extent, and both bacteria replicated in single membrane vacuoles at similar rates up to 6 h after infection. Thereafter, the virulent bacteria continued to grow, whereas the avirulent bacteria stopped growing. When the infected cells were stained with phagosomal and lysosomal markers and observed with a confocal fluorescence microscope, the majority of phagosomes containing these bacteria were fused with lysosomes. Neither R. equi organism has the ability to hinder phagosome-lysosome fusion. The acidity in phagolysosomes containing R. equi was examined by staining with LysoTracker Red DND-99, an acidotropic probe. The phagolysosomes containing virulent organisms were not acidic as compared with avirulent organisms. Over 90% of the phagolysosomes containing avirulent R. equi were stained with LysoTracker 6 h after infection, whereas less than 50% of those containing virulent R. equi were stained. Furthermore, when the supernatant obtained from a virulent R. equi culture was added to the cell cultures, the acidity of acidic compartments in macrophages was reduced. The authors conclude that some substance(s) produced by virulent R. equi suppress acidification in phagolysosomes, and help R. equi survival and replication in the bactericidal environment.

Amines↗