Impact of surface receptors TLR2, CR3, and FcγRIII on Rhodococcus equi phagocytosis and intracellular survival in macrophages.

The virulence-associated protein A (VapA) produced by virulent Rhodococcus equi allows it to replicate in macrophages and cause pneumonia in foals. It is unknown how VapA interacts with mammalian cell receptors, but intracellular replication of avirulent R. equi lacking vapA can be restored by supplementation with recombinant VapA (rVapA). Our objectives were to determine whether the absence of the surface receptors Toll-like receptor 2 (TLR2), complement receptor 3 (CR3), or Fc gamma receptor III (FcγRIII) impacts R. equi phagocytosis and intracellular replication in macrophages, and whether rVapA restoration of virulence in R. equi is dependent upon these receptors. Wild-type (WT) murine macrophages with TLR2, CR3, or FcγRIII blocked or knocked out (KO) were infected with virulent or avirulent R. equi, with or without rVapA supplementation. Quantitative bacterial culture and immunofluorescence imaging were performed. Phagocytosis of R. equi was not affected by blockade or KO of TLR2 or CR3. Intracellular replication of virulent R. equi was not affected by TLR2, CR3, or FcγRIII blockade or KO; however, avirulent R. equi replicated in TLR2-/- and CR3-/- macrophages but not in WT and FcγRIII-/-. rVapA supplementation did not affect avirulent R. equi phagocytosis but promoted intracellular replication in WT and all KO cells. By demonstrating that TLR2 and CR3 limit replication of avirulent but not virulent R. equi and that VapA-mediated virulence is independent of TLR2, CR3, or FcγRIII, our study provides novel insights into the role of these specific surface receptors in determining the entry and intracellular fate of R. equi.

Reinvestigation of the virulence of Rhodococcus equi isolates from patients with and without AIDS.

Rhodococcus equi emerged as a zoonotic pathogen of human immunodeficiency virus-infected patients over the last three decades. Two virulence plasmid types of R. equi, pVAPA and pVAPB associated with equine and porcine isolates, have been recognized, and more recently, pVAPN, a novel host-associated virulence plasmid in R. equi, was found in bovine and caprine isolates. We reinvestigated 39 previously reported isolates of R. equi from patients with and without acquired immunodeficiency syndrome (AIDS) by detecting vapA, vapB and vapN using PCR and plasmid profiling. After excluding one isolate that could not be cultured from frozen storage, eight isolates carried a virulence plasmid encoding vapA (pVAPA), 10 carried a virulence plasmid encoding vapB (pVAPB), seven carried a virulence plasmid encoding vapN (pVAPN) and 13 were negative for those genes. Of the 29 isolates from patients with AIDS, 7, 10 and 5 harboured pVAPA, pVAPB and pVAPN respectively. Among nine isolates from patients without AIDS, one and two harboured pVAPA and pVAPN respectively. This study demonstrated that pVAPN-positive R. equi existed in human isolates before 1994 and reaffirmed that equine-associated pVAPA-positive, porcine-associated pVAPB-positive and bovine- or caprine-associated pVAPN-positive R. equi are widely spread globally. Because domestic animals might be major sources of human infection, further research is needed to reveal the prevalence of pVAPN-positive R. equi infection in cattle and goats.

Pyogranulomatous enteritis and mesenteric lymphadenitis in an adult llama caused by Rhodococcus equi carrying virulence-associated protein A gene.

We report herein Rhodococcus equi infection in an 11-y-old, male llama with a history of diarrhea and endoparasitism. Postmortem examination revealed granulomatous and ulcerative enteritis, pyogranulomatous mesenteric lymphadenitis, fibrinosuppurative peritonitis, and granulomatous hepatitis. Intralesional macrophages were laden with gram-positive cocci. Bacteriology identified R. equi, and cultures tested positive for R. equi choE and vapA genes by PCR. This case expands the reported spectrum of lesions associated with R. equi infections in llamas from pyogranulomatous bronchopneumonia and peripheral lymphadenitis to pyogranulomatous mesenteric lymphadenitis and enteritis. We also link a R. equi that is carrying the virulent-associated protein gene VapA to clinical disease in New World camelids.

Rhodococcus equi como causa de neumonía en el síndrome de inmunodeficiencia adquirida / Rhodococcus equi as a cause of pneumonia in the acquired immunodeficiency syndrome

RESUMEN Rhodococcus equi es un microorganismo emergente asociado a infecciones oportunistas en individuos inmunocomprometidos, especialmente en pacientes con infección por virus de inmunodeficiencia humana. Se desarrolló una búsqueda en la Biblioteca Virtual de Infomed, fueron revisados 215 trabajos científicos sin limitación de año y país, seleccionándose 55. El rhodococcus es un patógeno intracelular capaz de crecer y persistir dentro de los macrófagos que expresan en su superficie el receptor Mac-1 (CD11b/CC18), y posteriormente destruirlos. La manifestación clínica más frecuente es la neumonía de comienzo insidioso y en su evolución natural tiende a la cavitación. El diagnóstico se realiza mediante su identificación en cultivo de muestras de tejido afectado. Los hemocultivos son positivos en el 50% de los inmunodeprimidos En el diagnóstico radiográfico, los hallazgos más comunes referidos en la literatura científica son el compromiso lobar y la cavitación. La particular evolución que experimentan los pacientes con síndrome de inmunodeficiencia adquirida y neumonía por R. equi, obliga a implementar esquemas terapéuticos basados en antimicrobianos con actividad bactericida intracelular, administrados inicialmente por vía intravenosa y durante un tiempo prolongado e incluso la cirugía. La infección por R. equi es una complicación infrecuente en pacientes con síndrome de inmunodeficiencia adquirida, pero con una elevada tasa de letalidad, por lo que debe ser sospechado en pacientes que presenten una infección respiratoria de curso inhabitual. El diagnóstico precoz, el tratamiento antimicrobiano combinado y prolongado y el inicio de la Terapia Antiretroviral de Gran Actividad en forma temprana pueden mejorar la evolución y el pronóstico de estos pacientes.

ABSTRACT Rhodococcus equi is an emergent microorganism associated to opportunistic infections in immune-compromised individuals, especially in patients infected with the human immunodeficiency virus. A search was carried out in the Virtual Library of Infomed; 215 scientific works were reviewed without limits of publication years and countries. From them, 55 were chosen. Rhodococcus equi is an intracellular pathogen that is able to grow and live inside the macrophages expressing the Mac-1 (CD11b/CC18) receptor in the surface and destroying them later. The most common clinical manifestation is insidious beginning pneumonia, tending to cavitation in its natural evolution. The diagnosis is made through identification in culture of affected tissues samples. Blood cultures are positive in 50 % of the immune-depressed people. At the radiographic diagnosis, the most common findings referred to in the scientific literature are lobar compromise and cavitation. The particular evolution of the patients with acquired immune-deficiency syndrome and pneumonia due to Rhodococcus equi forces the implementation of therapeutic schemes based on antimicrobials with intracellular bactericide activity, administered firstly intravenously and during a long time, and even to perform the surgery. Rhodococcus equi infection is an infrequent complication in patients with acquired immunodeficiency syndrome, but having a high lethality rate, therefore it should be suspected in patients presenting a respiratory infection of unusual curse. The precocious diagnosis, combined and prolonged antimicrobial treatment and early beginning of the highly active antiretroviral therapy could improve the evolution and prognosis of these patients.

Absceso glúteo por Rhodococcus Equi: presentación atípica de enfermedad emergente / Gluteus abscess due to RhodococcusEqui: atypical presentation of emerging disease

Resumen Rhodococcusequi, bacteria aislada principalmente en potros, se considera en la actualidad un patógeno zoonótico emergente en seres humanos, la mayoría inmunocomprometidos, coincidiendo con la epidemia de VIH y los avances en la medicina de trasplantes y la quimioterapia del cáncer, lo que justifica la revisión sobre su patogenia, características clínicas, diagnóstico y tratamiento. Más frecuente en hombres que en mujeres, el inicio de las infecciones es generalmente insidioso y los síntomas que presentan varían según el sitio de la infección, la enfermedad respiratoria, caracterizada por neumonía necrosante es la manifestación más común, y tiende a seguir un curso subagudo, mientras las personas inmunocompetentes son afectadas con baja frecuencia y suelen presentarse con enfermedad localizada. Las infecciones de la piel o partes blandas, incluyendo abscesos, son poco frecuentes incluso en huéspedes inmunocomprometidos y están usualmente relacionadas con contaminación ambiental. El laboratorio de microbiología juega un papel clave en la identificación del organismo, ya que este puede confundirse con flora normal de la piel. El caso que se presenta corresponde a una paciente adulta inmunocompetente, con infección de herida quirúrgica por R equiluego de una cirugía estética, requirió tratamiento antibiótico, múltiples lavados quirúrgicos y debridamientos, más drenaje de absceso, con mejoría del proceso infeccioso. Este caso sugiere la importancia clínica de la identificación precisa de patógenos inusuales, capaces de entorpecer la evolución normal de un proceso quirúrgico y de producir un alto costo de atención de salud y el alcance medidas preventivas relacionadas con la vigilancia de la salud.

Abstract Rhodococcusequi, a bacterium isolated mainly in foals, is currently considered an emerging zoonotic pathogen in humans, most of them immunocompromised, coinciding with the HIV epidemic and advances in transplant medicine and cancer chemotherapy, which justifies the review on its pathogenesis, clinical characteristics, diagnosis and treatment. More frequent in men than in women, the onset of infections is usually insidious and the symptoms they present vary according to the site of infection, the respiratory disease, characterized by necrotizing pneumonia is the most common manifestation, and tends to follow a subacute course , while immunocompetent people are affected with low frequency and usually have localized disease. Infections of the skin or soft tissues, including abscesses, are rare even in immunocompromised hosts and are usually related to environmental contamination. The microbiology laboratory plays a key role in the identification of the organism, since it can be confused with normal flora of the skin. The case presented corresponds to an adult immunocompetent patient, with surgical wound infection by R equiafter cosmetic surgery, required antibiotic treatment, multiple surgical washes and debridements, and abscess drainage, with improvement of the infectious process. This case suggests the clinical importance of the precise identification of unusual pathogens, capable of obstructing the normal evolution of a surgical process and of producing a high cost of health care and the scope of preventive measures related to health surveillance.

Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid.

Rhodococcus equi is a facultative intracellular bacterium of macrophages and is an important pathogen of animals and immunocompromised people wherein disease results in abcessation of the lungs and other sites. Prior work has shown that the presence of the major virulence determinant, VapA, encoded on the pVAPA-type plasmid, disrupts normal phagosome development and is essential for bacterial replication within macrophages. pVAPA- type plasmids are typical of R. equi strains derived from foals while strains from pigs carry plasmids of the pVAPB-type, lacking vapA, and those from humans harbor various types of plasmids including pVAPA and pVAPB. Through the creation and analysis of a series of gene deletion mutants, we found that vapK1 or vapK2 is required for optimal intracellular replication of an R. equi isolate carrying a pVAPB plasmid type. Complementation analysis of a ΔvapA R. equi strain with vapK1 or vapK2 showed the VapK proteins of the pVAPB-type plasmid could restore replication capacity to the macrophage growth-attenuated ΔvapA strain. Additionally, in contrast to the intracellular growth capabilities displayed by an equine R. equi transconjugant strain carrying a pVAPB-type plasmid, a transconjugant strain carrying a pVAPB-type plasmid deleted of vapK1 and vapK2 proved incapable of replication in equine macrophages. Cumulatively, these data indicate that VapK1 and K2 are functionally equivalent to VapA.

B-Cell Epitope Mapping Using a Library of Overlapping Synthetic Peptides in an Enzyme-Linked Immunosorbent Assay.

This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identified which corresponded to a 20 mer sequence of the VapA protein.

Rescue of an intracellular avirulent Rhodococcus equi replication defect by the extracellular addition of virulence-associated protein A.

Rhodococcus equi is a facultative intracellular bacterium that can escape from bactericidal mechanisms associated with phagocytosis. Virulence-associated protein A (VapA), encoded on a virulence-associated plasmid, is essential for intracellular survival in macrophages, but its function is not known. Here, we show that the extracellular addition of recombinant glutathione S-transferase (GST)-VapA fusion protein rescued the intracellular replication defect of a mutant lacking the vapA gene. Furthermore, the virulence-plasmid-cured strain could also multiply to nearly wild-type levels by the addition of GST-VapA. The present data suggest that VapA can alter the intraphagocytic environment, thereby affecting its suitability for the growth of R. equi.

The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis.

Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria.

Biofilm formation by Rhodococcus equi and putative association with macrolide resistance / Formação de biofilme por Rhodococcus equi e possível associação com resistência a macrolídeos

Rhodococcus equi is a facultative intracellular pathogen, which cause severe pyogranulomatous pneumonia in foals and tuberculosis-like lesions in humans. Its ability to form biofilm was described in strains isolated from chronic diseases associated to treatment failures in humans. This study aimed to verify the biofilm formation by 113 R. equi isolated from equine samples (clinical and fecal) using two different methods (biofilm-culturing with and without additional glucose and epifluorescence microscopy). We also aimed to determine the efficacy of azithromycin, clarithromycin and erythromycin on R. equi in established biofilm. We found 80.5% (26/41) and 63% (58/72) biofilm-positive isolates, in fecal and clinical samples, respectively. The additional glucose increased the biofilm formation by R. equi fecal samples, but not by clinical samples. The antimicrobials tested herein were not able to eradicate R. equi in biofilm even at higher concentrations. This is the first study showing the biofilm formation by R. equi isolated from equine samples. Our findings indicate that R. equi biofilm-producers may be more resistant to the antimicrobials evaluated. Further studies are warranted to test this hypothesis...

Rhodococcus equi é um patógeno intracelular facultativo, o qual causa pneumonia piogranulosa severa em potros e lesões semelhantes à tuberculose em humanos. A sua capacidade de formar biofilme foi descrita em cepas humanas, isoladas a partir de doenças crônicas associadas a falhas de tratamento. Este estudo teve como objetivo verificar a formação de biofilme por 113 cepas de R. equi, isoladas a partir de amostras de equinos (clínicas e fecais), utilizando-se dois diferentes métodos (biofilme em cultura - com e sem adição de glicose - e microscopia de epifluorescência). Além disso, buscou-se determinar a eficácia da azitromicina, claritromicina e eritromicina sobre biofilme consolidado de R. equi. Verificou-se 80,5% (26/41) e 63% dos isolados (58/72) positivos para formação de biofilme, em amostras fecais e clínicas, respectivamente. A adição de glicose amentou a formação de biofilme em amostras fecais, mas não em amostras clínicas. Os antimicrobianos aqui testados não foram capazes de erradicar R. equi em biofilme consolidado, mesmo em concentrações elevadas. Este é o primeiro estudo a demonstrar a formação de biofilme por cepas de R. equi isoladas a partir de amostras de equinos. Os resultados indicam que os isolados de R. equi produtores de biofilme podem ser mais resistentes aos antimicrobianos avaliados. Estudos adicionais são necessários para testar essa hipótese...

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.

A real-time impedance based method to assess Rhodococcus equi virulence.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains.

Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

Conjugal transfer of a virulence plasmid in the opportunistic intracellular actinomycete Rhodococcus equi.

Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ~81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.

The hydroxamate siderophore rhequichelin is required for virulence of the pathogenic actinomycete Rhodococcus equi.

We previously showed that the facultative intracellular pathogen Rhodococcus equi produces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that the rhbABCDE cluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. The rhbC gene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting of N(5)-formyl-N(5)-hydroxyornithine, serine, N(5)-hydroxyornithine, and N(5)-acyl-N(5)-hydroxyornithine. The other rhb genes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription of rhbC was upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion of rhbCD, rendering the resulting strain R. equi SID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype of R. equi SID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels of rhbC following infection and the inability of R. equi SID2 to grow within macrophages. Unlike the wild-type strain, R. equi SID2 was unable to replicate in vivo and was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenic Rhodococcus species also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution of R. equi virulence.

The steroid catabolic pathway of the intracellular pathogen Rhodococcus equi is important for pathogenesis and a target for vaccine development.

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and ß-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3'-propionic acid)-5α-hydroxy-7aß-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.

The vapA co-expressed virulence plasmid gene vcgB (orf10) of the intracellular actinomycete Rhodococcus equi.

The virulence plasmid of the pathogenic actinomycete Rhodococcus equi is essential for proliferation of this pathogen in macrophages and the development of disease. The pathogenicity island of this plasmid encodes a family of virulence-associated proteins (Vap), one of which (VapA) is a virulence factor. This paper describes the vcgAB operon (vapA co-expressed gene), located upstream of the vapA operon. Transcription of the vcgAB operon gave rise to transcripts with a half-life similar to those determined for other virulence plasmid genes (1.8 min). Transcription started at a promoter similar to the vapA promoter, and proceeded through an inefficient terminator into the downstream vcgC gene. In addition, vcgC is also transcribed from a promoter downstream of vcgB. The vcgAB and vapA operons were coordinately regulated by temperature and pH in a synergistic manner. The latter parameter only affected transcription at higher growth temperatures, indicating that temperature is the dominant regulatory signal. Transcription of the vcgAB operon increased 10-fold during the late exponential and stationary growth phases. Transcription was also upregulated during the initial hours following phagocytosis by phagocytic cells. In contrast to vcgA and vcgC, the vcgB gene is conserved in the porcine VapB-encoding plasmid, as well as in pathogenic mycobacteria. The coordinated regulation of vcgB and vapA, transcription of vcgB following phagocytosis and conservation of vcgB in pathogenic mycobacteria indicate a role for vcgB and the vcg genes in the virulence of R. equi.

Imaging and pathological findings of AIDS complicated by pulmonary Rhodococcus equi infection.

BACKGROUND: Rhodococcus equi (R. equi) infection commonly occurs in grazing areas, especially in patients with AIDS or with T-lymphocyte immuno-deficiencies. Literature reviews revealed that cases radiologically and pathologically diagnosed of AIDS complicated by R. equi infection are rare. This study aimed to investigate the imaging features and pathological basis of AIDS complicated by pulmonary R. equi infection. METHODS: A total of 13 cases of AIDS complicated by pulmonary R. equi infection were retrospectively analyzed based on their imaging, bacterial culture and pathological data, including 10 cases by chest CT scanning and X-ray radiology and 3 cases by only X-ray radiology. All 13 cases were definitely diagnosed by bacterial culture, including one by CT-guided pulmonary puncture with following H&E staining and periodic acid-Schiff (PAS) staining for diagnostic biopsy and another one by bronchial biopsy with following H&E staining and PAS staining for pathological diagnosis. The imaging findings and the pathological findings of AIDS complicated by pulmonary R. equi infection were compared and evaluated. RESULTS: Totally 9 subjects (70%) had radiological demonstrations of central ball liked high density shadows in unilateral pulmonary hilus areas; 10 (77%), cavities and liquefied levels; 3 (23%), pleural effussion. The foci were found in pulmonary inner zone in 10 subjects (77%) and in pulmonary outer zone in one subject (7%). The pathological findings included intra-alveolar hemorrhage, lymphocyte infiltration and granulation tissue proliferation, which were in line with the pathological process of necrotic pneumonia. After 8-month follow-up of anti-R. equi therapy of these 13 cases, 9 cases had obviously decreased or shrunk pulmonary cavities, one died, one missed follow-up, one completely absorbed foci and one did not receive reexaminations. CONCLUSIONS: The radiological demonstrations of AIDS complicated by pulmonary R. equi infection are central ball liked high density areas in unilateral pulmonary hilus area, parenchymal changes, secondary cavities, ground glass liked changes in the lung fields, nodules and treeinbuds sign, which are characteristic rather than specific.

Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells.

Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues.

Pulmonary rhodococcosis in a cat.

Feline Rhodococcus equi infection is rare, despite the bacteria is widespread in the environment. R equi infection is typically observed in equine species but the infection has also been reported in dogs, cats and other domestic animals. There are a few reports regarding pulmonary R equi infection in cats and the disease appears to be limited to the skin and the subcutaneous tissue. This report describes the pathological, microbiological and the virulence features associated with an acute necrosuppurative pneumonia in a cat. To the best of our knowledge, this is the first report of feline pulmonary R equi infection in Italy.