The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment.

Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.

Macroamphiphilic cell envelope components of Rhodococcus equi and closely related bacteria.

Recent progress towards an understanding of the architecture of the mycobacterial cell envelope (P.J. Brennan and H. Nikaido, Annual Review of Biochemistry 64 (1995) 29-63) provides a model with features more generally applicable to cell envelope organisation in other mycolic acid-containing bacteria. Using this archetype, a model for the organisation of the rhodococcal cell envelope is presented here, with particular reference to cell envelope composition in Rhodococcus equi. The likelihood that mycolic acids bound to the cell wall arabinogalactan contribute to the formation of a distinct outer lipid layer is emphasised. Furthermore, the model incorporates recent work which has characterised rhodococcal macroamphiphiles (lipoglycans and lipoproteins), including the VapA virulence-associated lipoproteins of R. equi.

Pulmonary malacoplakia associated with Rhodococcus equi infection in an AIDS patient. Report of a case with diagnosis by fine needle aspiration.

BACKGROUND: Malacoplakia is an uncommon, peculiar inflammatory disease with few cytologic descriptions. It is seen mostly in the lower urinary tract, with rare occurrences in the lungs. Escherichia coli is the most common underlying etiologic agent, with only a few cases reported with Rhodococcus equi infection. CASE: A 36-year-old female with AIDS presented with dysphagia, dry cough and fever. Physical examination revealed diffuse bronchi and wheezing. Chest roentgenography showed multiple cavitary lesions bilaterally in the lungs. Fine needle aspiration (FNA) of one of these lesions revealed clusters of granular histiocytes with numerous intracytoplasmic and extracytoplasmic Michaelis-Gutman bodies. Also noted were numerous coccobacillary bacteria, which, on culture, were identified as R equi. Ultrastructural findings are also presented. CONCLUSION: The case illustrates the cytologic findings of malacoplakia in a rare pulmonary lesion associated with R equi infection in an AIDS patient. It reiterates the diagnostic role of FNA in dealing with a nonneoplastic entity for timely and definitive treatment and follow-up.

Immunogenicity of an electron beam inactivated Rhodococcus equi vaccine in neonatal foals.

Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.

Induction of proinflammatory cytokines in human lung epithelial cells during Rhodococcus equi infection.

Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

Growth and production of cholesterol oxidase by alginate-immobilized cells of Rhodococcus equi No. 23.

Rhodococcus equi No. 23 was immobilized in calcium alginate. No detrimental effect on the viability of the test organism was observed during the immobilization procedure. Approx. 98% of the cell population originally present in the alginate solution were immobilized in the gel beads. When the cells of an equal volume of the culture, obtained respectively at exponential phase (12 h preculture), late-exponential phase (20 h preculture) or stationary phase (36 h preculture) were immobilized, the gel beads prepared with the stationary-phase culture were found to contain the highest cell population [about 10(8) colony-forming units (CFU)/g of beads]. In addition, gel beads, prepared with late-exponential-phase culture, exhibited the highest production of cholesterol oxidase (CholOx) after 48 h of incubation. Increasing the bead mass from 3.5 to 14.0 g/100 ml of medium increased CholOx production. However, further increasing the bead mass resulted in a reduction of CholOx production. Furthermore, on the basis of a similar initial cell population, the alginate-immobilized cells of R. equi No. 23 produced a significantly higher amount of CholOx (P<0.05) than did the free cells.

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.