Ensaio imunoenzimático indireto (ELISA) para detecção de anticorpos anti-Rhodococcus equi em potros

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum

Introduction: Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics.Objective: This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi.Methods: The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification.Results: Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (R-f) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959 bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species.Conclusions: The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi. (C) 2012 Elsevier Editora All rights reserved.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cutaneous pyogranuloma in a cat caused by virulent Rhodococcus equi containing an 87 kb type I plasmid

A 2-year-old intact male domestic shorthaired cat presented with a chronic, nodular, ulcerated, cutaneous lesion on the right thoracic limb. Histological and cytological examination revealed a pyogranulomatous inflammation with basophilic organisms in the macrophages. A virulent form of Rhodococcus equi containing an 87 kb type I (VapA) virulence plasmid was identified from cultures of biopsy samples. This report describes the clinicopathological features, plasmid profile and virulence of this case of R equi infection.

Short Report: Identification of Virulence-Associated Plasmids in Rhodococcus equi in Humans with and without Acquired Immunodeficiency Syndrome in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Minimal inhibitory concentration of azithromycin in Rhodococcus equi strains isolated from foals

O perfil de sensibilidade microbiana e a concentração inibitória mínima (MIC) da azitromicina para 42 cepas de Rhodococcus equi isoladas de potros, no Brasil, e em uma cepa-controle, foi avaliado, respectivamente, pelos métodos de difusão com discos e E-test. A azitromicina apresentou 100% de efetividade in vitro para todas as cepas em ambos os testes. As cepas de R. equi apresentaram MIC90 para azitromicina em valores <1.5µg/ml. Este estudo mostra a alta efetividade da azitromicina em linhagens de R. equi isoladas no Brasil, sugerindo o uso dessa droga como alternativa na terapia da rodococose em potros.

Dimetilsulfóxido - DMSO no teste de sensibilidade microbiana in vitro em cepas de Rhodococcus equi isoladas de afecções pulmonares em potros

Comparou-se a sensibilidade microbiana in vitro de isolados de Rhodococcus equi pelo teste padrão de difusão com discos, com o modificado, pela adição de 5% de dimetilsulfóxido-DMSO. Observou-se aumento da sensibilidade do R. equi no teste com DMSO, frente a aminoglicosídeos (canamicina, amicacina, estreptomicina) e ao cloranfenicol, enquanto para a eritromicina e derivados ß-lactâmicos (penicilina G, cefalosporinas, amoxicilina, oxacilina), constatou-se redução da sensibilidade do agente.

Electron impact and chemical ionization mass spectral analyses of methyl esters species of free mycolic acid fraction from Rhodococcus lentifragmentus

The free mycolic acid fraction from Rhodococcus lentifragmentus was derivatized to methyl esters and further fractionated into saturated (F-0), monounsaturated (F-1) and diunsaturated (F-2) species using argentation-TLC. Methyl esters fractions F-0, F-1 and F-2, accounting for approximately 7.4%, 53.1% and 39.5%, respectively, were analyzed by electron impact (EI) and chemical ionization (CI) mass spectrometries. According to EI-MS, peaks observed for M(+)-18, that were prominent compared to those representing M(+)-32 and M(+)-(18 + 32), indicated that the carbon chain size ranged from C-36 to C-48. The pyrolytic cleavage of methyl mycolates (R(2)-CHOH-CH(R(1))-COOCH3), following the McLafferty rearrangement released fragment ions corresponding to, (a) the alpha-subunit, representing the fatty acid methyl ester (R(1)-CH2-COOCH3), methyl hexadecanoate, methyl tetradecanoate and methyl dodecanoate in decreasing order of relative intensity of peaks, and (b) the beta-subunit, representing the meroaldehyde moiety (R(2)-CHO). The saturated meroaldehyde species exhibited peaks representing meroaldehyde minus 18 mass units in which R(2) ranged from C19H39 to C31H63. The monunsaturated species exhibited peaks representing the meroaldehyde in which R(2) ranged from C19H37 to C31H61; peaks corresponding to meroaldehyde minus 18 mass units appeared only in the most abundant components, C29H57CHO, C27H53CHO, C25H49CHO and C31H61CHO, in a decreasing order of relative abundance. The diunsaturated species exhibited peaks essentially corresponding to meroaldehyde in which R(2) corresponded to C31H59 and C29H55; the latter displayed a relative intensity that was about one-half compared to that of the former. Fractions F-0, F-1 and F-2 showed a more intense pyrolytic fragmentation under CI-MS in contrast to results found under EI-MS. Therefore, peaks representing the alpha-subunit and the beta-subunit were more prominent than the ones representing the fragmentation of the hydrocarbon chain. Moreover, the beta-subunit of saturated species exhibited peaks corresponding to meroaldehyde plus hydrogen, and no dehydration of the beta-subunit occurred in this case. In turn, the beta-subunit of monounsaturated and diunsaturated species showed peaks representing both the meroaldehyde plus hydrogen and its dehydration product plus hydrogen. Thus, the presence of unsaturation in the meroaldehyde subunit of methyl mycolate facilitates appearance of dehydration fragment ions under chemical ionization procedure.

Molecular epidemiology of virulent Rhodococcus equi from foals in Brazil: virulence plasmids of 85-kb type I, 87-kb type I, and a new variant, 87-kb type III

We investigated the prevalence of virulent Rhodococcus equi in clinical isolates from 41 foals (19 sporadic and seven endemic cases) in Brazil between 1991 and 2003. of the 41 virulent isolates, six contained an 85-kb type I plasmid, 33 contained an 87-kb type I plasmid, both of which have been found in isolates from the Americas, and the remaining two contained a new variant, which did not display the EcoRI, EcoT221 and BamHI digestion patterns of the 11 representative plasmids already reported (85-kb types I-IV; 87-kb types I and II; 90-kb types I-V). We tentatively designated the new variant as the '87-kb type III' plasmid, because its BamHI digestion pattern is similar to that of the 87-kb type I plasmid. This is the first report of the molecular epidemiology surveillance of virulent R. equi in clinical isolates from Brazilian foals. (C) 2004 Elsevier Ltd. All rights reserved.

Virulence of Rhodococcus equi isolated from cats and dogs

Nine cat isolates and nine dog isolates of Rhodococcus equi from clinical material were investigated for the presence of the virulence-associated antigens (VapA and VapB) and virulence plasmids. Five of the cat isolates and one dog isolate were VapA positive and contained an 85-kb type I or an 87-kb type I plasmid. The remaining 12 isolates were avirulent R. equi strains and contained no virulence plasmids.

Immune Response to the Rhodococcus equi infection in high and low antibody-producing mice (selection IV-A)

Rhodococcus equi is a Gram-positive, facultative intracellular bacterium which infects macrophages and causes rhodococcal pneumonia and enteritis in foals. Recently, this agent has been recognized as an opportunistic pathogen for immunocompromised humans. Several murine experimental models have been used to study R. equi infection. High (H IV-A) and Low (L IV-A) antibody (Ab)-producers mice were obtained by bi-directional genetic selections for their ability to produce antibodies against sheep and human erythrocytes (Selection IV-A). These lines maintain their phenotypes of high and low responders also for other antigens than those of selection (multispeciflc effect). A higher macrophage activity in L IV-A mice has been described for several intracellular infectious agents, which could be responsible for their intense macrophage antigens (Ag)-handling and low Ab production. Due to these differences, L IV-A mice were found to exhibit a better performance to trigger an effective immune response towards intracellular pathogens. The objective of this work was to characterize the immune response of Selection IV-A against R. equi. H IV-A and L IV-A mice were infected with 2.0 × 10 6 CFU of ATCC 33701 +R. equi by intravenous route. With regards to bacterial clearance and survival assays, L IV-A mice were more resistant than H IV-A mice to virulent R. equi. L IV-A mice presented a higher hydrogen peroxide (H 2O 2) and nitric oxide (NO) endogenous production by splenic macrophages than H IV-A mice. L IV-A expressed the most intense cellular response, available by the Delayed-Type Hypersensitivity (DTH) reaction, which activated macrophages and produced more H 2O 2 and NO. The three times higher specific antibodies titres in H IV-A indicated that Selection IV-A maintained the multispecific effect and the polygenic control of humoral and cellular responses also to R. equi.