Rhodococcus equi é um importante patógeno bacteriano em medicina veterinária, associado, sobretudo, a pneumonias piogranulomatosas em potros no primeiro semestre de vida. São descritos neste trabalho vinte casos de infecção por R. equi em potros recebidos para necropsia no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul entre janeiro de 1997 e janeiro de 2013. Os históricos clínicos obtidos com os veterinários apresentaram grande variabilidade e, mesmo os sinais clássicos de comprometimento respiratório e febre só foram vistos em metade dos casos. Os dados obtidos em uma visita a uma propriedade demonstram que a superpopulação de potros e a introdução de fêmeas no grupo de parição contribuíram para a ocorrência de um surto. Exames de necropsia e de histologia revelaram que pneumonia piogranulomatosa multifocal foi a forma de apresentação mais constante (dezenove casos), seguida por linfadenite piogranulomatosa (dez casos) e tiflocolite piogranulomatosa e ulcerativa (cinco casos). Três animais apresentaram osteomielite piogranuloamatosa, dos quais, dois em vértebras. Uveítes e polissinovites assépticas foram constatadas em três casos. Exame imuno-histoquímco anti-Rhodococcus equi revelou-se positivo em todos os pulmões com lesões, embora os linfonodos tenham sido positivos em apenas três das nove amostras testadas. O exame bacteriológico das amostras de necropsia foi positivo em quinze casos clínicos, assim como em uma amostra de solo da propriedade visitada. O exame de reação da polimerase em cadeia (PCR) revelou o gene de virulência VapA de R. equi em todos os isolados clínicos, mas não na amostra de solo. Adicionalmente os pulmões foram testados por imuno-histoquímica para Pneumocystis sp.e apresentaram marcação em treze dos vinte casos. / Rhodococcus equi is an important bacterial pathogen in veterinary medicine, especially associated with piogranulomatous pneumonia in foals under six months of age. Twenty cases of R. equi infection in foals received for necropsy at the Pathology Veterinary Sector (SPV) of the Federal University of Rio Grande do Sul (UFRGS) between January 1997 and January 2013 are described in this paper. Clinical history obtained with veterinary practitioners presented high variability, and even classical respiratory signs and fever were only observed in half of the cases. Data collected in an investigative visiting to a breeding farm showed that the foal superpopulation and the introduction of females to the parturition group contributed to the occurrence of an outbreak. Necropsy and histologic examinations revealed that multifocal piogranulomatous pneumonia was the most constant presentation (nineteen cases), followed by piogranulomatous lymphadenitis (ten cases) and piogranulomatous and ulcerative typhlocolitis (five cases). Three animals presented piogranulomatous osteomyelitis, two of them in vertebrae. Aseptic uveitis and polisynovitis were verified in three cases. Anti-Rhodococcus equi immunohistochemical examination stained positive in all lungs containing lesions, although lymphnodes have stained positive in only three of nine samples tested. Bacteriologic examination of the necropsy samples was positive in fifteen cases and in a soil sample from the visited breeding farm. Polymerase chain reaction (PCR) test revealed the VapA virulence factor of R.equi in all clinical isolates, but not in the soil sample. Additionally, the lungs were tested to the presence of Pneumocystis sp. by immunohistochemistry, and stained positive in thirteen of twenty cases.

A infecção por Rhodococcus equi é a causa mais comum de pneumonia em potros durante os primeiros 6 meses de idade e as medidas utilizadas no controle da infecção são freqüentemente ineficientes. Os protocolos de tratamento são longos, de alto custo e nem sempre eficazes. A prevenção da rodococose através da imunização de potros com vacina eficaz e segura tem sido um desafio. Estudos recentes se baseiam na utilização da proteína A associada a virulência (VapA), que tem sido descrita como importante indutora da imunidade contra R. equi. Este estudo avaliou a resposta clínica e imunitária desenvolvida por potros imunizados com cepa atenuada de S. enterica Typhimurium capaz de expressar o antígeno VapA (grupo teste) ou apenas S. enterica Typhimurium sem o gene vapA (grupo controle), antes e após o desafio experimental. Duas etapas experimentais foram estabelecidas de acordo com a via de imunização: intranasal ou oral em até 12 horas após o nascimento e aos 14 dias de idade. Os animais do grupo teste e do grupo controle foram desafiados experimentalmente com cepa virulenta de R. equi 28 dias após o nascimento. Para acompanhar o desenvolvimento dos sinais clínicos, foram realizados exames clínicos, hemogramas e exames complementares de imagem. O padrão de resposta imune desenvolvida foi avaliada através da dosagem de munoglobulinas, mensuração da expressão de citocinas, ensaio de proliferação de linfócitos, isolamento de R. equi e perfil citológico do LTB. As manifestações clínicas foram observadas de maneira menos intensa nos animais do grupo teste da segunda etapa experimental e óbitos decorrentes da rodococose ocorreram apenas nos animais do grupo controle (2/3). Os animais do grupo teste apresentaram uma resposta de IgGb mais intensa quando comparada aos animais do grupo controle, porém não foi observado diferença estatística. A linfoproliferação e a expressão de citocinas de padrão Th1 foram superiores nos animais do grupo teste. Em contraste, os animais do grupo controle apresentaram uma resposta caracterizada pela maior expressão da citocina IL-4. A produção de IgA local foi mais significativa nos animais imunizados com Salmonella carreando vapA. Não foram observados efeitos tóxicos graves resultantes da imunização em nenhuma das etapas experimentais. A vacinação de potros neonatos com S. enterica Typhimurium expressando VapA foi considerada segura, capaz de modular eficientemente a resposta imune e aparentemente capaz de proteger contra a infecção experimental com R. equi. / Rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. Treatment protocols are long, expensive and do not always produce good results. Rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. Recent studies are based on the use of the virulence associated protein A (vapA) which has been described as an important inducer of immunity against R. equi. The present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of S. enterica Typhimurium expressing VapA antigen (test group) or S. enterica Typhimurium without the vapA gene (control group), previous to and following experimental challenge. Two experimental phases were established according to the immunization route: intranasal or oral vaccination up to 12 hrs following birth and at 14 days of age. The experimental and control groups were challenge on day 28 with a virulent stain of R. equi. Clinical examination, complete hemograms and image complementary exams were used to evaluate the development of clinical signs. Immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation essays, isolation of R. equi and cytological profiles of TBW. Clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group (2/3) and was due to R. equi pneumonia. The test group produced a more intense IgGb response when compared to controls however no statistical difference was observed. Lymphoproliferation and Th1 cytokine expression were higher in the test group. In contrast, controls produced an IL-4 response. Local IgA was significantly higher in animals immunized with Salmonella carrying vapA. Immunization protocols produced no severe toxic effect. The vaccination of neonatal foals with S. enterica Typhimurium expressing VapA was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental R.equi infection.

Rhodococcus equi é um importante patógeno bacteriano em medicina veterinária, associado, sobretudo, a pneumonias piogranulomatosas em potros no primeiro semestre de vida. São descritos neste trabalho vinte casos de infecção por R. equi em potros recebidos para necropsia no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul entre janeiro de 1997 e janeiro de 2013. Os históricos clínicos obtidos com os veterinários apresentaram grande variabilidade e, mesmo os sinais clássicos de comprometimento respiratório e febre só foram vistos em metade dos casos. Os dados obtidos em uma visita a uma propriedade demonstram que a superpopulação de potros e a introdução de fêmeas no grupo de parição contribuíram para a ocorrência de um surto. Exames de necropsia e de histologia revelaram que pneumonia piogranulomatosa multifocal foi a forma de apresentação mais constante (dezenove casos), seguida por linfadenite piogranulomatosa (dez casos) e tiflocolite piogranulomatosa e ulcerativa (cinco casos). Três animais apresentaram osteomielite piogranuloamatosa, dos quais, dois em vértebras. Uveítes e polissinovites assépticas foram constatadas em três casos. Exame imuno-histoquímco anti-Rhodococcus equi revelou-se positivo em todos os pulmões com lesões, embora os linfonodos tenham sido positivos em apenas três das nove amostras testadas. O exame bacteriológico das amostras de necropsia foi positivo em quinze casos clínicos, assim como em uma amostra de solo da propriedade visitada. O exame de reação da polimerase em cadeia (PCR) revelou o gene de virulência VapA de R. equi em todos os isolados clínicos, mas não na amostra de solo. Adicionalmente os pulmões foram testados por imuno-histoquímica para Pneumocystis sp.e apresentaram marcação em treze dos vinte casos. / Rhodococcus equi is an important bacterial pathogen in veterinary medicine, especially associated with piogranulomatous pneumonia in foals under six months of age. Twenty cases of R. equi infection in foals received for necropsy at the Pathology Veterinary Sector (SPV) of the Federal University of Rio Grande do Sul (UFRGS) between January 1997 and January 2013 are described in this paper. Clinical history obtained with veterinary practitioners presented high variability, and even classical respiratory signs and fever were only observed in half of the cases. Data collected in an investigative visiting to a breeding farm showed that the foal superpopulation and the introduction of females to the parturition group contributed to the occurrence of an outbreak. Necropsy and histologic examinations revealed that multifocal piogranulomatous pneumonia was the most constant presentation (nineteen cases), followed by piogranulomatous lymphadenitis (ten cases) and piogranulomatous and ulcerative typhlocolitis (five cases). Three animals presented piogranulomatous osteomyelitis, two of them in vertebrae. Aseptic uveitis and polisynovitis were verified in three cases. Anti-Rhodococcus equi immunohistochemical examination stained positive in all lungs containing lesions, although lymphnodes have stained positive in only three of nine samples tested. Bacteriologic examination of the necropsy samples was positive in fifteen cases and in a soil sample from the visited breeding farm. Polymerase chain reaction (PCR) test revealed the VapA virulence factor of R.equi in all clinical isolates, but not in the soil sample. Additionally, the lungs were tested to the presence of Pneumocystis sp. by immunohistochemistry, and stained positive in thirteen of twenty cases.

Rhodococcus equi é um importante patógeno bacteriano em medicina veterinária, associado, sobretudo, a pneumonias piogranulomatosas em potros no primeiro semestre de vida. São descritos neste trabalho vinte casos de infecção por R. equi em potros recebidos para necropsia no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul entre janeiro de 1997 e janeiro de 2013. Os históricos clínicos obtidos com os veterinários apresentaram grande variabilidade e, mesmo os sinais clássicos de comprometimento respiratório e febre só foram vistos em metade dos casos. Os dados obtidos em uma visita a uma propriedade demonstram que a superpopulação de potros e a introdução de fêmeas no grupo de parição contribuíram para a ocorrência de um surto. Exames de necropsia e de histologia revelaram que pneumonia piogranulomatosa multifocal foi a forma de apresentação mais constante (dezenove casos), seguida por linfadenite piogranulomatosa (dez casos) e tiflocolite piogranulomatosa e ulcerativa (cinco casos). Três animais apresentaram osteomielite piogranuloamatosa, dos quais, dois em vértebras. Uveítes e polissinovites assépticas foram constatadas em três casos. Exame imuno-histoquímco anti-Rhodococcus equi revelou-se positivo em todos os pulmões com lesões, embora os linfonodos tenham sido positivos em apenas três das nove amostras testadas. O exame bacteriológico das amostras de necropsia foi positivo em quinze casos clínicos, assim como em uma amostra de solo da propriedade visitada. O exame de reação da polimerase em cadeia (PCR) revelou o gene de virulência VapA de R. equi em todos os isolados clínicos, mas não na amostra de solo. Adicionalmente os pulmões foram testados por imuno-histoquímica para Pneumocystis sp.e apresentaram marcação em treze dos vinte casos. / Rhodococcus equi is an important bacterial pathogen in veterinary medicine, especially associated with piogranulomatous pneumonia in foals under six months of age. Twenty cases of R. equi infection in foals received for necropsy at the Pathology Veterinary Sector (SPV) of the Federal University of Rio Grande do Sul (UFRGS) between January 1997 and January 2013 are described in this paper. Clinical history obtained with veterinary practitioners presented high variability, and even classical respiratory signs and fever were only observed in half of the cases. Data collected in an investigative visiting to a breeding farm showed that the foal superpopulation and the introduction of females to the parturition group contributed to the occurrence of an outbreak. Necropsy and histologic examinations revealed that multifocal piogranulomatous pneumonia was the most constant presentation (nineteen cases), followed by piogranulomatous lymphadenitis (ten cases) and piogranulomatous and ulcerative typhlocolitis (five cases). Three animals presented piogranulomatous osteomyelitis, two of them in vertebrae. Aseptic uveitis and polisynovitis were verified in three cases. Anti-Rhodococcus equi immunohistochemical examination stained positive in all lungs containing lesions, although lymphnodes have stained positive in only three of nine samples tested. Bacteriologic examination of the necropsy samples was positive in fifteen cases and in a soil sample from the visited breeding farm. Polymerase chain reaction (PCR) test revealed the VapA virulence factor of R.equi in all clinical isolates, but not in the soil sample. Additionally, the lungs were tested to the presence of Pneumocystis sp. by immunohistochemistry, and stained positive in thirteen of twenty cases.

Infecções pulmonares de potros jovens por Rhodococcus equi resultam em grave pneumonia levando à morte um grande número de animais todos os anos. Até o momento não há nenhuma vacina aceita globalmente para a prevenção da rodococose equina. O único tratamento preconizado é baseado em antibioticoterapia, porém os protocolos clínicos são longos, de alto custo, com efeitos colaterais e tem favorecido a seleção de cepas resistentes aos antibióticos. Diversas estratégias no desenvolvimento de uma vacina segura e eficiente contra a rodococose foram propostas, porém, não induziram um efeito protetor considerável. O principal fator de virulência do R. equi descrito e amplamente estudado é a proteína vapA, no entanto, outras proteínas localizadas na ilha de patogenicidade estão presentes em amostras de R. equi virulento extraídos de animais infectados. Trabalhos recentes têm demonstrado a presença do gene vapG em todas as cepas virulentas e este gene é altamente expresso quando a bactéria reside no interior de macrófagos, tornando-o um possível alvo vacinal. Nosso grupo já possui experiência prévia no uso de linhagem de Salmonella enterica Typhimurium atenuada carreando a proteína vapA. Baseado nos resultados positivos obtidos, foi construída uma linhagem atenuada de S. enterica Typhimurium 3987 expressando a proteína vapG. A administração desta linhagem em camundongos foi capaz de induzir proteção contra R. equi virulento. Os resultados observados foram a colonização e persistência da Salmonella nos órgãos alvo, a redução da carga bacteriana de R. equi, e a indução de um perfil imune protetor semelhante ao observado em animais adultos resistentes. Observou-se o aumento da produção de IL-12p70 e IFN-, alem da presença de níveis aumentados de IL-4 e a redução dos níveis de TNF-. Observou-se também o aumento na subpopulação de células T auxiliares (CD4+) com perfil de memória, além de aumento na população de linfócitos B totais quando comparado aos grupos controles. Este conjunto de resultados indica que a imunização com Salmonella enterica Typhimurium expressando a proteína vapG gera uma resposta imune celular eficiente tornando esta linhagem uma possível candidata a vetor vacinal. Alem disso, sugere que outros antígenos do R. equi podem ser / Pulmonary infections in young foals by Rhodococcus equi result in severe pneumonia, leading to death a large number of animals every year. There is no globally accepted vaccine for the prevention of equine rhodococosis so far. To date, the only acceptable treatment is based on antibiotics, but the clinical protocols are long lasting, expensive, having side effects and favoring the emergence of drug resistant strains. Several strategies for developing a safe and effective vaccine against rhodococosis have been proposed, however, none has induced a significant protective effect. The main virulence factor of R. equi described and extensively studied is the protein vapA, although other proteins encoded by genes in pathogenicity islands are detected in samples of virulent R. equi, isolated from infected animals. Recent works have demonstrated that the vapG gene is present in all virulent strains, and that this gene is highly expressed when bacteria reside within macrophages, making it a potential vaccine target. Our group has already been working with an attenuated Salmonella enterica Typhimurium strain expressing the VapA protein. Even though our results were very promising, we decided to construct a second vaccine strain expressing the VapG protein. Based on the positive results, it was constructed an attenuated strain of S. enterica Typhimurium 3987 expressing the vapG protein. Interestingly, the VapG-expressing strain induced protection against virulent R. equi in mouse model of infection. We could observe colonization and persistence of Salmonella vaccine cells in target organs, with reduction of bacterial loads of R. equi and induction of a protective immune profile similar to that seen in resistant adult animals. We could also observe increased production of IL-12p70 and IFN- in addition to the presence of increased levels of IL-4 and reduced levels of TNF-. Moreover, we detected an increase in the subpopulation of T helper cells (CD4), with a profile of memory, as well as in the population of B lymphocytes, when compared to control groups. This set of results shows that immunization with Salmonella enterica Typhimurium expressing the VapG protein raises an efficient cellular immune response, making this strain a potential candidate for vaccine vector. Furthermore, this work suggests that other R. equi antigens may be taken into account for vaccine construction, besides the VapA protein utilized in the majority of studies.

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.

Orientador: Márcio Garcia Ribeiro / Banca: Antonio Carlos Paes / Banca: José Paes de Almeida Nogueira Pinto / Banca: Adolfo Carlos Barreto Santos / Banca: Rogério Giuffrida / Resumo: A linfadenite infecciosa em suínos representa uma das afecções mais preocupantes na criação de suínos em todo mundo, causada por patógenos de origem bacteriana, geralmente diagnosticada na linha de abate. Acarreta prejuízos econômicos com a condenação total ou parcial das carcaças, bem como reflexos em saúde pública, devido ao potencial zoonótico dos agentes causais. O presente estudo investigou a ocorrência e as principais espécies do gênero Mycobacterium, assim como marcadores de virulência plasmidial em linhagens de Rhodococcus equi (R. equi) isoladas de linfonodos e das fezes de suínos de abatedouros do interior do estado de São Paulo, com e sem linfadenite. Foram examinados 150 linfonodos (50 mesentéricos, 50 mediastínicos e 50 submandibulares) com lesões, 150linfonodos (50 mesentéricos, 50 mediastínicos e 50 submandibulares) sem lesões aparentes e 150 fezes de suínos, provenientes de animais destinados ao abate, em dois frigoríficos do interior do estado de São Paulo. As amostras de linfonodos e fezes foram submetidas ao cultivo microbiológico simultaneamente nos meios de ágar acrescido de sangue bovino (5%) desfibrinado, e meios seletivos de CAZ-NB, TCP e TVP para R. equi, e Stonebrink-Lesslie e Lowenstein-Jensen para micobactérias. As colônias sugestivas de R. equi e positivas no teste de CAMP, foram enviadas ao Japão para detecção de linhagens VapA ou VapB, associadas a virulência. As linhagens sugestivas no cultivo microbiano para o gênero Mycobacterium foram submetidas a caracterização de espécies por PCR pela técnica de PRA. Foram identificados nos linfonodos de suínos com lesões 48 (32,0%) linhagens de Mycobacterium spp.e 6 (4,0%) Rhodococcus equi. Nos linfonodos de suínos sem lesões foram identificados 11 (7,3%) isolados de Mycobacterium spp.e nenhuma linhagem de R. equi. Nas fezes foram identificadas 40 (26,6%) linhagens de Rhodococcus equi e ... / Abstract: The infectious lymphadenitis represents one of the most important diseases in pigs worldwide caused by bacterium, usually diagnosed on the slaughterhouses.The disease leads to economic losses due to total or partial condemnation of carcasses, as well as public health concern due to the zoonotic potential of microorganisms. The present study investigated the occurrence and the main species of the genus Mycobacterium as well as virulence markers of plasmid in Rhodococcus equi (R. equi) strains isolated from lymph nodes and feces from pigs of slaughterhouses in the state of São Paulo, with and without lymphadenitis. Were sampled 150 lymph nodes (50 mesenteric, 50 mediastinal and 50 submandibular) with lesions, 150 lymph nodes (50 mesenteric, 50 mediastinal and 50 submandibular) without visible lesions, and 150 feces from pigs of slaughterhouses of state of São Paulo, Brazil. The lymph nodes samples and feces were subjected to microbiological culture simultaneously indefibrinated bovine blood agar (5%), selective media of CAZ-NB, TCP, TVP for R. equi, and Stonebrink-Lesslie, and Lowenstein-Jensen for mycobacteria . The suggestive colonies of R. equi and positive to CAMP test were sent to Japan for evaluation of plasmid profile (VapA or VapB). The suggestive of Mycobacterium sp. were subjected to polymerase chain reaction (PCR) - based species identification using restriction enzyme pattern analysis (PRA). Among lymph nodes of pigs with lesions, 48 (32.0%) Mycobacterium spp. and 6 (4.0%) R. equi strains were identified. In the lymph nodes of pigs without lesions were identified 11 (7.3%) Mycobacterium spp. and none R. equi strain. From the fecal samples, 40 (26.6%) R. equi and 2 (1.3%) Mycobacterium spp. isolates were identified. From 48 Mycobcterium isolates from pigs with lesions, 37 (77.0%) were identified by PRA as M. avium type 1, and 11 (23.0%) M. avium type 2. Among limph nodes with lesions wer ... / Doutor

The soil-dwelling gram-positive coccobacillus Rhodococcus equi is a well-known veterinary pathogen and emerging human pathogen. Although Rhodococcus infection is primarily associated with pyogranulomatous pneumonia in foals, these bacteria can also infect other animal species including humans. R. equi pathogenicity is mediated by the conjugative virulence plasmid (pVAP) which promotes intracellular proliferation in host macrophages. Currently R. equi is endemic in horse breeding farms worldwide. No R. equi vaccine is available and both treatment and prophylaxis rely on the administration of a prolonged course with a combination of macrolide antibiotics (typically erythromycin) and rifampicin. These antimicrobials were introduced in the therapy against R. equi in the 1980s and multiresistance has now emerged among foal isolates, increasing the risk of zoonotic transmission. In this thesis, the role of conjugal extrachromosomal replicons in the host adaptation of R. equi was explored. A previous epidemiological study indicated that different variants of the pVAP virulence plasmid are associated with different animal hosts. This PhD project provides experimental confirmation of a R. equi plasmid-driven host tropism. In vivo and in vitro competition assays were performed using a set of isogenic strains only different in the virulence plasmid type, in adapted (horse) and non-adapted (mouse) model species. The data obtained in the horse model provides clear evidence of a significant negative selection of the non-equine virulence plasmids both at a cell and at the animal level, while no selection was observed in the non-adapted mouse model. Furthermore, this project characterized the determinant responsible for macrolide resistance in R. equi, a novel erm methylase gene, erm(46). The erm(46) determinant was shown to be transferable between strains by conjugation and herein the underlying mechanism and how erm(46) becomes stabilized in R. equi is described. PacBio SMRT-sequencing based analysis revealed that the erm(46) gene is carried in a self-replicating conjugative plasmid of about 80 kb, that we designated pRErm46. The conjugation machinery of pRErm46 was hypothesized to be responsible for bringing the erm(46) determinant into R. equi. However, some erythromycin resistant isolates lack pRErm46 but erm(46) transfer is still observed. This reflects the observation that erm(46) is present in a mobile element that, upon acquisition with the pRErm46 replicon, transposes at a high frequency and to multiple locations of the host genome. If the erm(46) mobile element transposes to the chromosome, no further transfer of the resistance is observed at a detectable frequency in the absence of pRErm46. On the other hand, if the erm(46) element transposes to the R. equi virulence plasmid, the erm(46) determinant co-opts the pVAPA conjugal transfer machinery and gets transferred at the same high frequency as the virulence plasmid (10-2). This constitutes a unique example of efficient co-transfer, in the same genetic vehicle, of virulence and antimicrobial determinants, two key niche-adaptive traits required for within-host survival of bacterial pathogen.

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).