INTRODUCCIÓN: la NAC no complicada en el niño es una infección muy frecuente, cuya epidemiología se encuentra pobremente definida y cuyo diagnóstico microbiológico sigue siendo difícil, dificultando la elección del tratamiento antibiótico, especialmente cuando no puede distinguirse clínicamente entre neumonía típica y atípica.OBJETIVOS: describir la presentación clínico-epidemiológica de la NAC no complicada en el niño y buscar rasgos clínico-epidemiológicos predictivos de infección por M. pneumoniae.MÉTODOS: estudio prospectivo, descriptivo, de seguimiento de una cohorte, desde el 1 de Enero de 2000 hasta el 31 de Diciembre de 2001. Se incluyeron niños de edad > 1 mes, inmunocompetentes, diagnosticados de NAC no complicada en el Servicio de Urgencias de Pediatría del Hospital Vall d'Hebron, cuyos padres aceptaron el protocolo de estudio y seguimiento.RESULTADOS: Se estudiaron 166 pacientes, 54,2% varones, mediana de edad de 48 meses. La duración entre inicio de la sintomatología y diagnóstico tuvo una mediana de 3 días, y la mediana de la duración del tratamiento fue de 8 días. El 83,1% de los pacientes fueron tratados con amoxicilina-clavulánico y 7 días después del inicio del tratamiento el 98,2% de los pacientes estaban asintomáticos. Después de 2 semanas únicamente el 24,3% tenía imágenes pulmonares residuales. Se detectó etiología atípica en el 25,3% de los pacientes, siendo debida a M. pneumoniae en el 85,7%, C. pneumoniae en el 9,5% y coinfección en el 4,8%. La presentación clínico-radiológica no permitió un diagnóstico clínico de neumonía atípica vs no atípica y no se apreciaron diferencias en la evolución, habiendo sido tratados en su gran mayoría y en ambos grupos, con betalactámicos. El análisis de datos multivariable, con regresión logística demostró que la edad >5 años (RR 4,48, IC:2,14-9,39), la presencia de una cifra de neutrófilos<7000 (RR: 7,74, IC 3-20)y un valor de Proteina C Reactiva <7 mg/dL (RR3,99, IC 1,27-12,55), se asociaban a infección atípica.CONCLUSIONES: El recuento de neutrófilos, con un punto de corte de 7000, constituye una variable con capacidad predictiva en el diagnóstico diferencial de neumonía atípica, con una sensibilidad del 76,2%, especificidad de 83,9% y valor predictivo negativo de 91,2%. El 91,5% de nuestros pacientes recibieron un antibiótico beta-lactámico, no existiendo diferencias significativas en ambos grupos ni en cuanto a la clase de antibiótico ni en cuanto a la duración y vía del tratamiento. La evolución clínico-radiológica es buena, aún en ausencia de tratamiento específico, pues el 88,1% de las neumonías atípicas (37 pacientes), fueron tratados exclusivamente con beta-lactámicos. Se podría pues afirmar que el diagnóstico etiológico de la NAC no complicada no resulta imprescindible para su tratamiento y habría por tanto que valorar su costo-efectividad. No obstante sería interesante disponer de más estudios que permitieran elaborar conclusiones extrapolables a toda la población. / INTRODUCTION: Community-acquired childhood pneumonia (CAP) is a common infection but its precise epidemiology remains poorly defined and its treatment is complicated by the difficulty in microbiological diagnosis and the increasing incidence of antibiotic resistance among respiratory pathogens. OBJECTIVES: The purpose of this study is to present the main epidemiologic features of patients with CAP and to compare the clinical, biological, and radiologic features of presentation in atypical pneumonia and in other community-acquired pneumonia, to try to help in early diagnosis of atypical pneumonia. METHODS: Consecutive immunocompetent children with radiographically confirmed lower respiratory infection were enrolled and evaluated in the emergency department of a 400-bed university children's hospital, prospectively from January 2000 through December 2001. Clinical, radiologic and microbiological evaluations were performed at study entry, at 10-15 days, and at 4 weeks post-therapy. Mycoplasmal, chlamydial, Legionella and Coxiella serologic tests were performed on paired samples in order to identifye the infection by those organisms. Univariate and multivariate analyses were performed to compare epidemiologic and demographic data and clinical, analytical, and radiologic features of presentation in the patients with atypical pneumonia and the patients with CAP by other bacterial etiology. RESULTS: 166 patients with CAP were studied, 54,2% male, mean age 48 months. The mean duration between onset of symptoms and diagnosis was 3 days, and the mean treatment duration was 8 days. 83,1% of the patients were treated with amoxicillin-clavulanate. Seven days after the start of therapy clinical symptoms were absent in 98,2% of patients and two weeks after radiologic infiltrates were only present in 24,3% of patients. Etiologic atypical agents were identified in 42 (25,3%) of 166 patients. Infection was attributed to M. pneumoniae in 85,7% (36 of 42), C. pneumoniae in 9,5% (4 of 42), and coinfections in 4,8% (2 of 42). 74,7% patients were diagnosed of CAP by other bacterial etiology . Clinical findings and chest radiographs did not distinguish patients with a defined atypical etiology from those without a known cause for pneumonia. There were no differences in the clinical responses of patients to the antimicrobial regimen. Multivariate logistic-regression analyses revealed that age> 5 years (odds ratio: 4,48; 95% confidence interval:2,14-9,39 ), the presence of neutrophiles<7000 (odds ratio:7,74 ; 95% confidence interval:3-20 ), and C Reactive Protein value < 7 mg/dL(odds ratio: 3,99 ; 95% confidence interval:1,27-12,55) were significantly associated with atypical pneumonia. CONCLUSION: Atypical etiology was identified in 25,3% of the evaluable patients and multivariate analysis demonstrated that only some independent factors were associated with atypical pneumonia. These factors as found in this study can help the evaluating physician to identify the atypical etiology.

La memòria que es presenta està composta per dos treballs d'investigació que tot i estar relacionats, formen cadascun un capítol independent. En la primera part del treball s'ha realitzat l'estudi del proteoma de Mycoplasma penetrans mitjançant electroforesis bidimensional i espectrometria de masses. S'ha generat un mapa de referència del proteoma d'aquest patogen humà, que compren els rangs de pH de 4 a 11 en tres finestres, de 4 a 7, de 6 a 9 i de 7 a 11NL (No lineal). Mitjançant tinció especifica s'ha obtingut el fosfoproteoma d'aquest organisme en el rang de pH de 4 a 7, i també s'han determinat els llocs específics de fosforilació de dos proteïnes, un transportador ABC i el Factor de elongació Tu. En la segona part del treball s'ha realitzat l'anàlisi del proteoma de Mycoplasma genitalium, ampliant la informació ja existent sobre el proteoma d'aquest patogen humà. S'ha analitzat el proteoma d'aquest organisme en fase de creixement estacionaria final utilitzant gradients estrets de pH. Per aquest micoplasma també s'ha generat una imatge del fosfoproteoma en el rang de pH de 4 a7, i s'ha pogut, també, determinar el lloc específic de fosforilació d'una proteïna implicada en l'adhesió d'aquest micoplasma, la proteïna P110. / La memoria que se presenta está formada por dos trabajos de investigación que todo y estar relacionados entre si, forman cada uno un capítulo independiente. En la primera parte del trabajo se ha realizado un estudio del proteoma de Mycoplasma penetrans mediante electroforesis bidimensional y espectrometría de masas. Se ha generado un mapa de referencia del proteoma de este patógeno humano, que comprende el rango de pH de 4 a 11 en tres ventanas, de 4 a 7 de 6 a 9 y de 7 a 11NL (no lineal). Mediante tinción específica se ha obtenido el fosfoproteoma de este organismo en el rango de pH de 4 a 7, y también se han determinado los sitios específicos de fosforilación de dos proteínas, un transportador ABC y el Factor de elongación Tu. En la segunda parte del trabajo se ha realizado un análisis del proteoma de Mycoplasma genitalium, ampliando la información ya existente sobre el proteoma de este patógeno humano. Se ha analizado el proteoma de este organismo en la fase de crecimiento estacionaria final utilizando gradientes estrechos de pH. Para este micoplasma también se ha generado una imagen del fosfoproteoma en el rango de pH de 4 a 7, y se ha podido determinar también, el sitio específico de fosforilación de una proteína implicada en la adhesión de este micoplasma, la proteína P110. / The report herein presented is composed of two research works that although they are related, they form an independent chapter. In the first part of this research the proteome of Mycoplasma penetrans has been analyzed using the combination of two-dimensional electrophoresis and mass spectrometry. A proteome reference map of this human pathogen has been constructed including the pH ranges of 4 to 11 in three windows, from 4 to 7, from 6 to 9 and from 7 to 11NL (Non lineal). With a specific phosphoprotein staining method, the phosphoproteome of this microorganism has been obtained in the pH range of 4 to 7. The specific phosphorylation sites have been characterized for two proteins, an ABC transporter and the Elongation Factor Tu. In the second part of this research, the proteome of Mycoplasma genitalium has been analyzed, extending the previous information of the proteome of this human pathogen. The proteome of this microorganism has been analyzed in the final stationary growth phase using narrow pH gradients. The phosphoproteome has been also analyzed for this microorganism using the specific phosphoprotein staining in the pH range of 4 to 7. The specific phosphorylation site has been characterized in a protein involved in adhesion, the protein P110.

En aquest treball, hem estudiat la motilitat de les cèl·lules de M. genitalium i hem desenvolupat un mètode que ens ha permès aïllar trenta mutants deficients en motilitat d'aquest microorganisme. La caracterització dels mutants aïllats, ens ha permès identificar cinc gens que redueixen la capacitat de desplaçament de les cèl·lules. / In this work, we analyzed the motility of Mycoplasma genitalium and we developed a straightforward procedure to isolate gliding deficient mutants of this microorganism. Characterization of the thirty mutants isolated, allowed us to identify five genes that are required for locomations.

Mikoplazmų tyrimus apsunkina genetinių įrankių trūkumas. Ne išimtis ir Mycoplasma gallinarum – platų šeimininkų ratą turinti silpno patogeniškumo bakterija. Šiame darbe aprašomas pirmųjų M. gallinarum skirtų vektorių, į kurių sudėtį įeina chromosominė replikacijos pradžios sritis (oriC), kūrimas. Be to, šio tyrimo metu pirmą kartą pademonstruota sėkminga šios mikoplazmų rūšies transformacija. Iš šešių sukonstruotų vektorių penki replikavosi M. gallinarum ląstelėse, o šeštasis nebuvo funkcionalus dėl AT turtingoje srityje pasroviui nuo antrosios DnaA dėžutės atsiradusios mutacijos. Kai kurie vektoriai, kultivuojant transformantus in vitro, integravosi į chromosomą. Homologinės rekombinacijos dažnis dar labiau padidėjo po transformacijos ilgą laiką gaivinant ląsteles augimo terpėje be antibiotiko. Sukurti oriC vektoriai galėtų būti naudojami genetiniams M. gallinarum tyrimams, ypač genams inaktyvuoti ir ekspresuoti, bei suteiktų daugiau žinių apie molekulines šios bakterijos savybes. / Genetic studies of mycoplasmas are limited by the lack of a replicable vector system. Mycoplasma gallinarum, an opportunistic pathogen having a wide range of hosts, is not the exception. This study describes the identification and cloning of the M. gallinarum origin of replication (oriC) in order to construct the first vectors for this species. Additionally, this report provides the first evidence of the successful transformation of M. gallinarum. Five out of six developed vectors have been functional. The replication of the sixth vector has not been supported due to the mutation in the AT rich region downstream the second DnaA box. During in vitro passages, some vectors have been integrated into the chromosome. The rate of chromosomal integration has been even further increased by the lack of antimicrobial selection pressure during cultivation. Hence developed oriC plasmids are useful for genetic studies, including inactivation or expression of selected genes, in M. gallinarum, and lead to a better understanding of its molecular biology.

The exact role of mycoplasma in feline ocular and respiratory disease is not yet understood. The results of previous studies are contradictory in this regard. There is some evidence to suggest that M. felis has a pathogenic role in such diseases, but it is inconclusive. / The aim of this study was to investigate the prevalence and anatomical distribution of mycoplasmas in a population of shelter cats, to determine which species were present, and establish the association of their presence with ocular or respiratory disease. / The prevalence of mycoplasma in the 110 cats examined was 71.8%, as determined by in vitro culture. Mycoplasma was most commonly isolated from the pharynx, followed by the bronchus and conjunctiva. In infected cats, mycoplasmas were likely to be isolated from multiple anatomical sites. / The polymerase chain reaction (PCR) was used to amplify part of the 16S rRNA gene, and the mutation scanning technique non-isotopic single-strand conformation polymorphism (SSCP) was utilised to delineate mycoplasma isolates based on nucleotide sequence variation. PCR-SSCP proved to be a useful method to screen large numbers of samples for variation and to group them according to species. / The species of mycoplasma identified by nucleotide sequencing were M. felis and M. gateae. It was not determined whether it was possible to differentiate between M. gateae and M. arginini based on SSCP profile results with the target DNA region used due to their almost identical nucleotide sequence. This group of M. gateae/M. arginini served as a useful non-pathogenic comparison group to M. felis. / There was no statistically significant difference between M. felis and the M. gateae/M. arginini group with respect to prevalence or anatomic distribution. There was no evidence of any association of mycoplasma with disease linked to any of the anatomic locations studied. / Mycoplasmas were isolated from the lower respiratory tract in 42.7% of cats. The isolation of mycoplasmas from the lower respiratory tract of healthy cats has been reported once, but this is the first report of M. felis being isolated from this location in healthy cats. This finding indicates that the isolation of mycoplasmas from the lower respiratory tract is not sufficient evidence to implicate a role in respiratory disease. / Mycoplasmas were not significantly involved in ocular or respiratory disease in the population of cats studied. More likely, they are commensal organisms in the conjunctiva, pharynx and bronchus. Whether they are capable of playing an opportunistic role in disease, or what conditions may facilitate such a role remains to be determined.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Missouri-Columbia, 2006. / "December 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Tierärztliche Hochsch., Diss., 2005--Hannover.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-graduação em Biotecnologia, Florianópolis, 2010 / Made available in DSpace on 2012-10-25T09:44:35Z (GMT). No. of bitstreams: 1 276928.pdf: 2942660 bytes, checksum: 0297aa2abae4ad6d7d216d59c57415d2 (MD5) / O Mycoplasma synoviae é um dos mais importantes patógenos de aves domésticas em todo o mundo, sendo o agente causador de doenças respiratórias, sinovites e doenças sistêmicas, e, por consequência, torna-se responsável por grandes perdas econômicas na produção avícola. Com a recente conclusão do sequenciamento do genoma do M. synoviae, deu-se novo impulso na realização de estudos pós-genômicos. O presente trabalho apresenta o mapa proteômico preliminar das proteínas citoplasmáticas do M. synoviae, desenvolvido utilizando a eletroforese bidimensional associada à espectrometria de massa. Os mapas proteômicos foram realizados em duas faixas de pH (pH 4 a 7 e pH 3 a 10), e em dois tamanhos de tiras de IPG (7 cm e 13 cm). As proteínas foram identificadas utilizando espectrometria de massa MALDI/TOF e a ferramenta de busca MASCOT. Baseado na sequência genômica um total de 42 sequências codificantes de DNA (CDSs) foram identificadas, sendo que as proteínas mais abundantes foram a chaperona DnaK, o fator de elongação Tu e a tiol peroxidase. De acordo com a classificação funcional, a maioria das proteínas identificadas pertence à classe de metabolismo (39%) e à classe de armazenamento e processamento de informação (29%). Finalmente, foi realizada uma análise imunológica, na qual identificamos duas proteínas antigênicas de M. synoviae reconhecidas pelo soro de animais imunizados com esse patógeno, sendo elas a frutose-bifosfato aldolase e a proteína hipotética MS53_0025. O mapa proteômico obtido será útil como referência para outras análises de expressão protéica em M. synoviae. / Mycoplasma synoviae is a major pathogen of poultry throughout the world, being the causative agent of respiratory diseases, synovitis and systemic diseases, and consequently, it is responsible for great economic losses in poultry production. Recently the sequencing of M. synoviae genome was concluded and this accomplishment has stimulated pos-genomic studies This study presents the preliminary proteomic map of citoplasmatic proteins of M. synoviae, constructed using two-dimensional gel electrophoresis in combination with mass spectrometry. The proteomic maps were produced in two pH ranges (pH 3-10 and pH 4-7), and using two different IPG strips lengths (7 cm and 13 cm). Proteins were identified using MALDI/TOF mass spectrometry and the search engine MASCOT. Based on the genome sequence of M. synoviae, a total of 42 different coding DNA sequences (CDSs) were identified. The most prominent proteins were the molecular chaperone DnaK, the elongation factor Tu and thiol peroxidase. According to the functional classification, the majority of the identified proteins belong to the class of metabolism (39%) and the class of information storage and processing (29%). Finally, we performed an immunological analysis which identified two antigenic proteins from M. synoviae recognized by sera from animals immunized with this pathogen. The proteome map obtained will be useful as reference for further analysis of protein expression in M. synoviae.

Mycoplasma hyopneumoniae é a bactéria causadora da pneumonia micoplásmica suína, que afeta o rebanho suíno em nível mundial. Durante o processo de infecção, o sistema imune de organismos afetados produz espécies reativas de oxigênio (ERO), conhecidas por causarem uma variedade de lesões celulares, como uma das estratégias para neutralização do patógeno. Embora a presença de enzimas antioxidantes clássicas seja esperada em M. hyopneumoniae, genes importantes relacionados à proteção contra ERO como superóxido-dismutase, catalases e glutationa-peroxidase não foram identificados por homologia na anotação de seqüências genômicas. Entre os poucos genes de M. hyopneumoniae codificando proteínas possivelmente envolvidas na supressão da resposta do hospedeiro através da produção de ERO, foi identificado um codificando uma peroxirredoxina. Análises de seqüência e filogenéticas mostram que a peroxirredoxina de M. hyopneumoniae (MhPrx) está relacionada com a subfamília das peroxirredoxinas 2-Cys atípica, embora ela tenha apenas uma cisteína em sua seqüência. A seqüência de DNA codificadora (CDS) da MhPrx foi clonada e expressa em Escherichia coli para a produção de uma MhPrx recombinante (rMhPrx), a qual foi purificada e usada para a imunização de camundongos para produzir um anti-soro policlonal anti-MhPx. Este anti-soro foi utilizado para investigar extratos protéicos de M. hyopneumoniae e demonstrou a expressão de MhPrx nas três cepas testas (J, 7422 e 7448). A atividade da rMhPrx foi determinada com uso de um sistema de oxidação catalisado por metais, que mostrou que esta enzima pode proteger moléculas de DNA do dano causado por ERO e deve ter uma função essencial durante a infecção. / Mycoplasma hyopneumoniae is the causative agent of the porcine enzootic pneumonia, which affects swineherds worldwide causing heavy economical losses. In the infection process, the host generates reactive oxygen species (ROS) as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected in M. hyopneumoniae, important genes directly related to protection against ROS, like superoxide-dismutase, catalases and glutathione-peroxidase were not identified by sequence homology in the genome sequence annotation. Among the few identified M. hyopneumoniae genes coding for proteins possibly involved with suppression of the host ROS-mediated response, one coding for a peroxiredoxin was recognized. Sequence and phylogenetic analysis indicate that M. hyopneumoniae peroxiredoxin (MhPrx) is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed in Escherichia coli to produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. This antiserum was used to probe M. hyopneumoniae extracts and demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). A metal catalyzed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROSmediated damage and may play an essential role during infection.