Inaug.-Diss.--Rostock. / Lebenslauf. "Literatur-übersicht": 1. p. at end.

Inaug.-Diss.--Bern. / "Literatur": p. 26-27.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-02Bitstream added on 2014-06-13T18:44:31Z : No. of bitstreams: 1 silva_p_dr_arafcf.pdf: 2320520 bytes, checksum: 5b67cf713d666bdba286bd22e1e7c114 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As bactérias Gram-positivas que contêm ácidos micólicos na parede celular estão classificadas no grupo dos actinomicetos aeróbios ou bactérias corineformes e nocardioformes. Nesse grupo encontra-se o Rhodococcus equi, o qual é relevante à medicina veterinária e humana tal como as micobactérias, causando doença pulmonar que pode mimetizar casos de tuberculose. R. equi é considerado como agente patogênico em potros e tem emergido como oportunista em humanos, especialmente, associado à infecção pelo vírus da imunodeficiência humana. Assim como nos animais, a rodococose humana afeta principalmente, os pulmões, com características clínicas e patológicas, similares à tuberculose pulmonar, em pacientes imunocomprometidos ou não. A identificação de Rhodococcus equi pode ser realizada com base numa variedade de características, fenotípicas, genotípicas e técnicas cromatográficas. Morfologia das colônias, morfologia celular e resistência parcial ao álcool ácido são características chaves para a caracterização inicial. R. equi não oxida ou fermenta carboidratos e nem utiliza acetato, citrato e malonato, como única fonte de carbono, produz catalase, o fator equi (teste de CAMP) e lipase. Não produz amilase, b-galactosidase (ONPG), casease, DNase, esculinase, gelatinase, H2S, indol, lecitinase e oxidase. Demonstra comportamento variável para as provas de nitrato redutase, urease e redução do hipurato, decompõe a adenina, mas não hipoxantina, tirosina e xantina. O método molecular para a identificação de R. equi utiliza a PCR para amplificar um fragmento de 959 pares de base do gene choE, o qual codifica a enzima cholesterol oxidase (COX). Na identificação química, semelhante às espécies do gênero Mycobacterium, membros do gênero Rhodococcus contêm ácidos micólicos plausíveis de serem identificados pela cromatografia... / Gram-positive bacteria, containing mycolic acids in the cellular wall are classified in aerobic actinomycetes or corineform and nocardioform bacteria group. Rhodococcus equi is included in this group, and it is very important to the veterinary and human medicine. Rhodococcus equi is a well-recognized bacterial pathogen in veterinary medicine. First isolated from foals, it causes an important chronic granulomatous pneumonia and lung abscesses. The infection also occurs in humans, often following immunosuppression of various causes. The increased number of human cases reported recently is partly the result of the spread of AIDS but may also reflect the increasing awareness by medical laboratories of this opportunistic pathogen and their improved ability to identify it rather than to dismiss it as a contaminating micrococcus or diphtheroid. R. equi can be identified on the basis of a variety of conventional phenotypic characteristics including microscopic (Gram and acidfast staining) and macroscopic morphologies, growth requirements, metabolism of glucose, and phenotypic molecular characteristics including the presence of mycolic acid composition, which is detected by thin-layer chromatography. The colonial morphology of R. equi is diverse and consists of three major varieties: pale pink and slimy, coral and non-slimy, and pale yellow in color, non-slimy. Colorless colonial variants may also occur. R. equi is a non-motile gram-positive pleomorphic coccobacillus, varying from distinctly coccoid to bacillary depending on growth conditions. All of the rhodococci from clinical specimens are generally weakly acid fast when stained either by the modified Kinyoun method or by the Ziehl-Neelsen method. Regarding biochemical characteristics, the organism is generally biochemically unreactive. It fails to oxidize or ferment carbohydrates, neither uses sodium... (Complete abstract click electronic access below)

University, Diss., 2005--Würzburg.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2006. / Includes bibliographical references. Also available on the World Wide Web.

Resumo: As bactérias Gram-positivas que contêm ácidos micólicos na parede celular estão classificadas no grupo dos actinomicetos aeróbios ou bactérias corineformes e nocardioformes. Nesse grupo encontra-se o Rhodococcus equi, o qual é relevante à medicina veterinária e humana tal como as micobactérias, causando doença pulmonar que pode mimetizar casos de tuberculose. R. equi é considerado como agente patogênico em potros e tem emergido como oportunista em humanos, especialmente, associado à infecção pelo vírus da imunodeficiência humana. Assim como nos animais, a rodococose humana afeta principalmente, os pulmões, com características clínicas e patológicas, similares à tuberculose pulmonar, em pacientes imunocomprometidos ou não. A identificação de Rhodococcus equi pode ser realizada com base numa variedade de características, fenotípicas, genotípicas e técnicas cromatográficas. Morfologia das colônias, morfologia celular e resistência parcial ao álcool ácido são características chaves para a caracterização inicial. R. equi não oxida ou fermenta carboidratos e nem utiliza acetato, citrato e malonato, como única fonte de carbono, produz catalase, o fator equi (teste de CAMP) e lipase. Não produz amilase, b-galactosidase (ONPG), casease, DNase, esculinase, gelatinase, H2S, indol, lecitinase e oxidase. Demonstra comportamento variável para as provas de nitrato redutase, urease e redução do hipurato, decompõe a adenina, mas não hipoxantina, tirosina e xantina. O método molecular para a identificação de R. equi utiliza a PCR para amplificar um fragmento de 959 pares de base do gene choE, o qual codifica a enzima cholesterol oxidase (COX). Na identificação química, semelhante às espécies do gênero Mycobacterium, membros do gênero Rhodococcus contêm ácidos micólicos plausíveis de serem identificados pela cromatografia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Gram-positive bacteria, containing mycolic acids in the cellular wall are classified in aerobic actinomycetes or corineform and nocardioform bacteria group. Rhodococcus equi is included in this group, and it is very important to the veterinary and human medicine. Rhodococcus equi is a well-recognized bacterial pathogen in veterinary medicine. First isolated from foals, it causes an important chronic granulomatous pneumonia and lung abscesses. The infection also occurs in humans, often following immunosuppression of various causes. The increased number of human cases reported recently is partly the result of the spread of AIDS but may also reflect the increasing awareness by medical laboratories of this opportunistic pathogen and their improved ability to identify it rather than to dismiss it as a contaminating "micrococcus" or "diphtheroid." R. equi can be identified on the basis of a variety of conventional phenotypic characteristics including microscopic (Gram and acidfast staining) and macroscopic morphologies, growth requirements, metabolism of glucose, and phenotypic molecular characteristics including the presence of mycolic acid composition, which is detected by thin-layer chromatography. The colonial morphology of R. equi is diverse and consists of three major varieties: pale pink and slimy, coral and non-slimy, and pale yellow in color, non-slimy. Colorless colonial variants may also occur. R. equi is a non-motile gram-positive pleomorphic coccobacillus, varying from distinctly coccoid to bacillary depending on growth conditions. All of the rhodococci from clinical specimens are generally weakly acid fast when stained either by the modified Kinyoun method or by the Ziehl-Neelsen method. Regarding biochemical characteristics, the organism is generally biochemically unreactive. It fails to oxidize or ferment carbohydrates, neither uses sodium... (Complete abstract click electronic access below) / Orientador: Clarice Queico Fujimura Leite / Coorientador: Sergio Roberto de Andrade Leite / Banca: Carmo Elias Andrade Melles / Banca: Rosilene Fressatti Cardoso / Banca: Carlos Henrique Gomes Martins / Banca: Antonio Carlos Pizzolitto / Doutor

Master of Science / Department of Clinical Science / Elizabeth Davis / Background: While there is a commercially-available vaccine for Streptococcus equi subsp. equi licensed for the intranasal route of administration, some equine practitioners are administering this vaccine orally despite a lack of evidence for its efficacy by this route of administration. Objectives: To compare systemic and local immune responses following intranasal or oral administration of the USDA-approved, live-attenuated Streptococcus equi subspecies equi vaccine (Pinnacle IN®, Zoetis, Florham Park, New Jersey). Study Design: Experimental, randomized clinical trial Methods: Eight healthy horses with low Streptococcus equi M protein (SeM) titers (<1:1600) were randomly assigned to an intranasal or oral two-vaccine series. SeM-specific serum immunoglobulins G (IgG) and A (IgA) and nasal secretion IgA were assessed using a commercially-available ELISA (Equine Diagnostic Solutions, LLC, Lexington, Kentucky) and a novel magnetic microsphere assay utilizing fluorescence. A general linear mixed models approach was used for statistical data analysis. Results: As expected, intranasal vaccinates showed substantial increases in both serum SeM-specific IgG and IgA levels post-vaccination (P=0.0006 and P=0.007, respectively). Oral vaccinates showed an increase in serum SeM-specific IgG post-vaccination (P=0.0150), though only one-third the magnitude of intranasal vaccinates. Oral vaccinates showed no evidence of change in SeM-specific IgA post-vaccination (P=0.15). Main Limitations: Changes in mucosal antibody responses were not identified in this study which may be related to small change in antibody response, timing of sample collection, or method of nasal secretion collection. Conclusions: Results indicate that intranasal or oral vaccine administration resulted in increased serum SeM-specific IgG, though the magnitude of response differed between routes.

Rhodococcus equi is a soil-dwelling facultative intracellular pathogen that can infect many mammals, including humans. R. equi is most well known for its ability to cause severe pyogranulomatous disease in foals, primarily involving the lungs although other body systems may also be affected. The disease is endemic on many horse-breeding farms worldwide and poses a severe threat to the horse breeding industry because there is no vaccine available. Current prophylaxis is based on systematic preventative treatments with macrolides combined with rifampicin, which are also used to treat clinical cases of the disease in foals. In this thesis I have used a combination of wet laboratory and bioinformatic approaches to identify the molecular basis of emerging combined resistance to macrolides and rifampicin in R. equi foal isolates from the USA. The genomes of a selection of resistant and susceptible strains from across the USA were sequenced and assembled. Resistance genes were systematically searched by reciprocal best-match BLASTP comparisons to known antibiotic resistance determinants. This led to the discovery of a novel erythromycin ribosomal methylase (erm) gene, erm(46), in all resistant strains. Complementation analysis in a susceptible R. equi strain showed that erm(46) was sufficient to confer resistance to all macrolides, lincosamides, and streptogramin B. The erm(46) gene is carried by an integrative conjugative element (ICE) which is transferable between R. equi strains. The ICE is formed by two distinct parts, a class I integron associated with an IS6100 sequence and the erm(46) determinant carried by a sub-element which contains putative actinobacterial conjugative translocase apparatus and a transposase/integrase. All resistant strains also carry the same non synonymous point mutation in rpoB conferring rifampicin resistance. Thus, these strains are carrying double resistance to the most commonly used antibiotics to treat R. equi worldwide. Phylogenetic analysis based on the core genome demonstrated that all resistant strains are clonal. This indicates that although conjugal acquisition of the erm(46) conjugative element may occur at a high frequency, the need for the concurrent presence of a second rpoB mutation for survival in the macrolide and rifampicin dominated farm environment has effectively selected for the spread of a single clone. In the second section of this work, we sequenced a further 20 R. equi genomes from difference sources (equine, porcine, bovine, human), including representatives of each of the seven major genogroups previously defined in our laboratory based on pulsed field gel electrophoresis. I have used the newly acquired genetic information to study the genome of R. equi and analyse its diversity within and outwith its species group. This enabled us to explore the pan genome and define that R. equi is a genetically well-defined bacterial species. Our results provide definitive evidence that resolves the current dispute over R. equi classification, specifically they do not support the recent proposal (based on classical polyphasic bacterial taxonomical methods) that R. equi should be transferred to a new genus. Our core-genome phylogenomic analyses unambiguously show that the genus Rhodocococcus is monophyletic and that R. equi forms a clade together with the most recently described related environmental species R. defluvii that radiates from within the genus. Together with other shared biological and genetic characteristics, namely the unique niche-adaptive mechanism based on evolutionarily related extrachromosomal replicons, R. equi should be conseidered a bona fide member of the genus Rhodococcus. We also confirm that Rhodococcus spp. and Nocardia spp. are sufficiently distinct to warrant them belonging to different genera. In conclusion, this work used whole genome sequencing to characterize the molecular basis underlying the emergence and clonal spread of multi-resistant R. equi in horse breeding farms in the USA. This work also highlights the limitations of classical taxonomical approaches in bacterial systematics, and illustrates the importance of incorporating modern phylogenomic approaches to understand the evolutionary relationships between bacterial strains and their accurate taxonomic position.

Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).

Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).