Introdução: recente estudo brasileiro detectou a presença concomitante do Mycoplasma pneumoniae e Chlamydia pneumoniae em lesões ateromatosas coronárias estáveis e instáveis. O objetivo do presente estudo foi testar a associação entre títulos sorológicos de anticorpos anti-Chlamydia pneumoniae e anti-Mycoplasma pneumoniae e as Síndromes Isquêmicas Miocárdicas Instáveis. Métodos: foram incluídos de forma prospectiva, 138 pacientes divididos em 4 grupos: 34 pacientes com Síndrome Isquêmica Miocárdica Instável com supradesnível do segmento ST, 40 pacientes com Síndrome Isquêmica Miocárdica Instável sem supradesnível ST, 30 pacientes com aterosclerose crônica assintomática e 34 doadores de sangue sem doença coronária conhecida. Nos dois primeiros grupos, as amostras sorológicas foram colhidas durante o evento agudo e com seis meses de seguimento, enquanto nos outros dois (aterosclerose crônica e controle) as mesmas foram colhidas uma única vez. Em todas as amostras foram dosados anticorpos da classe IgG anti-Chlamydia pneumoniae e anti-Mycoplasma pneumoniae utilizando a técnica de imunoflorescência indireta.. Resultados: seis meses após a internação, os pacientes com síndrome isquêmica miocárdica instável com supradesnível ST apresentaram significativa redução dos títulos sorológicos, em relação às sorologias colhidas durante o evento coronário agudo, o que ocorreu tanto com a chlamydia (307,5+47,5 versus 650+115,7 p=0,0001) quanto com o mycoplasma (21,5+3,5 versus 36,5+5 p=0,0004). O grupo sem supradesnível ST não teve variação significativa dos níveis sorológicos em seis meses de seguimento (522,6+102,7 versus 576+84,1 p=0,27) para chlamydia e 27,6+5,8 versus 27,6 + 5,8 p >0,99 para mycoplasma. Foi realizada também uma comparação entre os níveis sorológicos de todos os grupos analisados, e observou-se que os grupos com síndrome isquêmica miocárdica instável (com e sem supra ST), tiveram valores sorológicos mais elevados do que os grupos aterosclerose crônica e controle, mas as diferenças não foram significativas. Os valores para chlamydia foram: 650+115,7 (com supra), 576+84 (sem supra), 373,3+65 (aterosclerose crônica) e 343,5+57,2 (controle), p=0,083; e os valores para mycoplasma foram: 36,5+5 (com supra), 27,6+5,8 (sem supra), 23+4,3 (aterosclerose crônica) e 26,7+3,3 (controle), p=0,171. Conclusões: o presente estudo demonstra associação entre títulos de anticorpos anti-Chlamydia pneumoniae e anti-Mycoplasma pneumoniae e a instabilização da placa coronária. Demonstra ainda a normalização dos mesmos títulos em um período de até seis meses, a partir do quadro agudo. / Objective of the study: to test the association of serum titers of anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae antibodies and Acute Coronary Syndrome (ACS). The patients were divided into 4 groups: ACS with ST-segment elevation, ACS without ST-segment elevation, chronic asymptomatic atherosclerosis and blood donors without known coronary disease. Serum samples were collected during the acute event and after six months of follow-up. Six months after the acute event, patients with ACS with St-segment elevation showed a significant decrease of serum titers, when compared to the other one. That\'s show the association between anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae antibody titers and acute coronary syndrome.

Streptococcus pneumoniae is a highly diverse and adaptable opportunistic pathogen that can infect and colonise different niches within the human host to cause a wide range of invasive disease (sepsis and meningitis) and noninvasive disease (pneumonia, otitis media and sinusitis). The molecular mechanisms that contribute to the different patterns of pneumococcal infection remain largely unknown. This thesis aims to determine the physiological and proteomic responses that allow the pneumococcus to survive and adapt to invasive and non-invasive sites. The comparative proteomic analyses of clinical S. pneumoniae isolates recovered from blood cultures (classified as invasive site isolates) and mucosal surfaces such as sputum, skin and ear swabs (classified as non-invasive site isolates) was initiated. The pneumococci were grown in vitro under standard conditions and the total cellular bacterial proteins extracted and analysed using both gel based and non-gel based proteomic approaches. Analysis of the pneumococcal isolates by two-dimensional polyacrylamide gel electrophoresis (2DGE) revealed that a high degree of heterogeneity existed between the pneumococcal isolates particularly among isolates in the invasive site isolates. Differential patterns of protein synthesis were observed that discriminated the pneumococcal isolates according to their sites of isolation. These were proposed to be associated with the bacterial adaptation to invasive and non-invasive sites of infection. Mass spectrometry was used to identify selected significant (ANOVA, p < 0.05) protein spots, which were further categorised into functional groups by Gene Ontology analysis. An extension of the 2DGE data using an integrated approach comprising bioinformatics, surfome analysis and a shotgun proteomic workflow provided a comprehensive qualitative and quantitative analyses of the pneumococcal intracellular and cell-surface proteomes. Proteins potentially involved in pneumococcal niche-specific adaptation and surface proteins with potential for further investigation and inclusion in the pipeline of vaccine candidates were identified. Quantitative regulation of proteins involved in energy metabolism, genetic competence, stress response, surface adhesion and virulence were considered important for pneumococcal adaptation to invasive and non-invasive sites. The anatomical sites colonised by the pneumococcus vary in their V availability for iron. The 2DGE method was also used on selected pneumococcal isolates from the two sites of infection to define the proteome variability linked to the effect of iron starvation that may contribute to the different disease outcomes associated with pneumococcal infections. The iron restricted condition was generated by cation depletion of the growth medium using Chelex-100. Quantitative differences in protein abundance were demonstrated that correlated with pneumococcal adaptation to iron restriction. The identification of selected significant spots by liquid chromatography-mass spectrometry and systems biology analysis of the identified proteins contributed to the elucidation of the molecular mechanisms underlying pneumococcal survival under iron limitation. The expression/repression of proteins functionally associated with metal ion binding, oxidative stress response, translation and virulence mainly constituted the pneumococcal adaptive responses to growth under conditions of limited iron availability. The data presented in this thesis extended our understanding of the molecular events underlying pneumococcal physiological adaptation and provide the basis of future work in this area.

Introdução: recente estudo brasileiro detectou a presença concomitante do Mycoplasma pneumoniae e Chlamydia pneumoniae em lesões ateromatosas coronárias estáveis e instáveis. O objetivo do presente estudo foi testar a associação entre títulos sorológicos de anticorpos anti-Chlamydia pneumoniae e anti-Mycoplasma pneumoniae e as Síndromes Isquêmicas Miocárdicas Instáveis. Métodos: foram incluídos de forma prospectiva, 138 pacientes divididos em 4 grupos: 34 pacientes com Síndrome Isquêmica Miocárdica Instável com supradesnível do segmento ST, 40 pacientes com Síndrome Isquêmica Miocárdica Instável sem supradesnível ST, 30 pacientes com aterosclerose crônica assintomática e 34 doadores de sangue sem doença coronária conhecida. Nos dois primeiros grupos, as amostras sorológicas foram colhidas durante o evento agudo e com seis meses de seguimento, enquanto nos outros dois (aterosclerose crônica e controle) as mesmas foram colhidas uma única vez. Em todas as amostras foram dosados anticorpos da classe IgG anti-Chlamydia pneumoniae e anti-Mycoplasma pneumoniae utilizando a técnica de imunoflorescência indireta.. Resultados: seis meses após a internação, os pacientes com síndrome isquêmica miocárdica instável com supradesnível ST apresentaram significativa redução dos títulos sorológicos, em relação às sorologias colhidas durante o evento coronário agudo, o que ocorreu tanto com a chlamydia (307,5+47,5 versus 650+115,7 p=0,0001) quanto com o mycoplasma (21,5+3,5 versus 36,5+5 p=0,0004). O grupo sem supradesnível ST não teve variação significativa dos níveis sorológicos em seis meses de seguimento (522,6+102,7 versus 576+84,1 p=0,27) para chlamydia e 27,6+5,8 versus 27,6 + 5,8 p >0,99 para mycoplasma. Foi realizada também uma comparação entre os níveis sorológicos de todos os grupos analisados, e observou-se que os grupos com síndrome isquêmica miocárdica instável (com e sem supra ST), tiveram valores sorológicos mais elevados do que os grupos aterosclerose crônica e controle, mas as diferenças não foram significativas. Os valores para chlamydia foram: 650+115,7 (com supra), 576+84 (sem supra), 373,3+65 (aterosclerose crônica) e 343,5+57,2 (controle), p=0,083; e os valores para mycoplasma foram: 36,5+5 (com supra), 27,6+5,8 (sem supra), 23+4,3 (aterosclerose crônica) e 26,7+3,3 (controle), p=0,171. Conclusões: o presente estudo demonstra associação entre títulos de anticorpos anti-Chlamydia pneumoniae e anti-Mycoplasma pneumoniae e a instabilização da placa coronária. Demonstra ainda a normalização dos mesmos títulos em um período de até seis meses, a partir do quadro agudo. / Objective of the study: to test the association of serum titers of anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae antibodies and Acute Coronary Syndrome (ACS). The patients were divided into 4 groups: ACS with ST-segment elevation, ACS without ST-segment elevation, chronic asymptomatic atherosclerosis and blood donors without known coronary disease. Serum samples were collected during the acute event and after six months of follow-up. Six months after the acute event, patients with ACS with St-segment elevation showed a significant decrease of serum titers, when compared to the other one. That\'s show the association between anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae antibody titers and acute coronary syndrome.

Objetivo: Avaliar mortalidade nas infecções por K. pneumoniae produtora de β- lactamase de espectro ampliado (KpESBL) em pacientes internados em unidades de terapia intensiva(UTI). Delineamento do estudo: Estudo de coorte histórico exposto-controlado, no período de janeiro de 2005 a outubro de 2007. Ambiente: UTIs do Hospital Nossa Senhora da Conceição (HNSC) em Porto Alegre /RS, Brasil. Pacientes: Adultos, maiores de 18 anos, internados nas UTIs e selecionados para completar 70 casos e 140 controles. Método: Os pacientes casos apresentavam infecção hospitalar por KpESBL e foram comparados aos pacientes controle que poderiam apresentar infecção por outro germe ou nenhuma infecção hospitalar (IH) na relação 1:2. Seguimento de 30 dias em ambos os grupos. As curvas de mortalidade foram realizadas pelo método de Kaplan-Meier. Resultados: Em ambos os grupos, o maior desfecho foi a cura. A taxa de mortalidade foi de 42,9% nos casos e 48,6% nos controles. O presente estudo não demonstrou diferença estatística na mortalidade entre os pacientes com infecção por KpESBL e o grupo controle, tanto na análise univariada (risco relativo ( RR)= 0,8 e intervalo de confiança (IC) de 95%, 0,52-1,23), como na regressão de Cox com fatores ajustados (RR= 0,86; IC95%= 0,54-1,35). Conclusões: Os pacientes com infecção por KpESBL não apresentaram maior mortalidade que os demais pacientes da UTI. Os pacientes com infecção por KpESBL que apresentaram infecção urinária e tiveram a terapia empírica adequada demonstraram menor mortalidade. / Objective: To evaluate mortality rate due to infections by extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) among patients in intensive care unit (ICU). Study design: Historical exposed-control cohort study conducted from January 2005 to October 2007. Setting: ICUs of Hospital Nossa Senhora da Conceição (HNSC) in Porto Alegre, Brazil. Patients: Adult patients older than 18 years hospitalized in ICU and enrolled up to 70 cases and 140 controls. Method: The patients in the case group, who had nosocomial infection by ESBL-Kp, were compared with control patients, who had infections by other organisms or no nosocomial infection (NI), at a 1:2 ratio. Both groups were followed up for 30 days. Mortality curves were estimated using the Kaplan-Meier method. Results: In both groups, the most frequent outcome was the cure. Mortality rate was 42.9% in the case group and 48.6% in the control group. There were no statistic differences in mortality between patients with ESBL-Kp infection and the control group in univariate analysis (Hazard ratio (HR = 0.8; 95% confidence interval( CI), 0.52 – 1.23) or in Cox regression with adjusted factors (HR = 0.86; 95%CI, 0.54 – 1.35). Conclusions: Patients with ESBL-Kp infection did not have higher mortality rates than the control patients in the ICUs. Patients with ESBL-Kp infection, urinary infection or who received adequate empirical therapy had lower mortality rates.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / En las últimas dos décadas, la especie bacteriana Klebsiella pneumoniae se ha convertido en uno de los principales focos de atención de las autoridades mundiales de salud, debido principalmente a la rápida aparición de cepas hipervirulentas y multiresistentes. La aparición de estas cepas estaría relacionada con el gran dinamismo del genoma de K. pneumoniae, promovido por una alta frecuencia de adquisición y pérdida de elementos genéticos móviles, entre los cuales las Islas Genómicas (GIs) jugarían un rol fundamental. Las GIs son segmentos de DNA que presentan un contenido GC distinto al del resto del cromosoma, y que pueden escindirse o integrarse en distintas posiciones de éste, normalmente en genes que codifican RNAs de transferencia (tDNAs). No obstante la importancia propuesta de las GIs en la evolución de K. pneumoniae, hasta ahora el número de GIs conocidas en esta especie es bajo, y poco se conoce respecto al uso de los tDNAs como sitios de integración de estos elementos en ésta y otras especies bacterianas. En este trabajo se caracterizó el set completo de tDNAs presentes en el cromosoma de 50 cepas de K. pneumoniae, identificando las GIs insertadas en dichos loci. Así, se identificaron y anotaron 97 clases de GIs, las que codifican sobre un 50% de proteínas con función desconocida. Además, se observó que solo un grupo reducido de tDNAs son ocupados como sitios de integración de GIs en esta especie, proponiendo y sometiendo a prueba posibles mecanismos o patrones que podrían explicar en parte este sesgo. Los resultados muestran que la dinámica de integración de GIs en K. pneumoniae es un área de investigación poco explorada, y que la evaluación del rol de las GIs en la hipervirulencia y resistencia a antibióticos de K. pneumoniae requerirá caracterizar una gran cantidad de proteínas codificadas en las mismas, cuya función se desconoce. / In the last two decades, the bacterial species Klebsiella pneumoniae has attracted the attention of global health authorities, mainly due to the dissemination of hypervirulent and multi-resistant strains, which are responsible for more severe infections and are more difficult to treat with the available antibiotics. The rapid development of these strains would be explained by the high dynamism of the K. pneumoniae genome, promoted by a high frequency of gain and loss of mobile genetic elements. Among them, genomic islands (GIs) were proposed to play a major role. GIs are DNA segments harboring a GC content that differs from the average for the host chromosome, and that can excise from and integrate into different locations, mainly into genes encoding transfer RNAs (tDNAs). In spite of the proposed role of GIs in K. pneumoniae evolution, a small number of these elements have been described in this species. Moreover, little is known regarding the usage of tDNAs as integration sites for these elements in bacteria. In this work we characterized the whole set of tDNAs typically present in the K. pneumoniae chromosome, searching for GIs integrated at each of them among 50 K. pneumoniae strains. This way, we identified a total of 97 classes of GIs, where more than 50% of the encoded genes have no predicted function. Additionally, we observed that only a small subset of tDNAs are used as integration sites for GIs in this species, and tested possible hypotheses explaining, at least partially, this bias in the selection of the integration site. Our results indicate that the dynamics of GIs integration in bacteria is an underexplored field, and that evaluating the impact of GIs over K. pneumoniae virulence and drug resistance will require efforts directed to address the functions of a high number of proteins encoded in their GIs.

Streptococcus pneumoniae is a gram-positive bacterium that can be found in both healthy carriers as well as in people that have developed disease. One of the major virulence factors of pneumococci is their polysaccharide capsule. Based on the capsule that surrounds the bacteria, pneumococci are divided into at least 90 different serotypes. Some serotypes seem to be more related to virulence than others. I have with comparative genome hybridization microarray technique, studied gene differences between 18 epidemiological well-characterised pneumococcal strains with different potential to cause disease. A microarray chip based on two sequenced pneumococcal genomes, R6 and TIGR4, has already been designed. According to Hierarchical clustering, both the serotype and the genetic type as assessed by MLST (sequence type or ST) seem to have impact on the relationship of clinical isolates. Most clinical isolates of the same serotype are clustered together except for serotype 14 isolates that seem to be more divergent. Further more the number of genes that are divergent between clinical isolates compared to R6 and TIGR4 differ from 65 to 289. Preliminary results indicate that although there is diversity among clinical isolates some are more closely related to each other then others. Absent genes seem to be evenly distributed among all 18 clinical isolates tested but hypothetical genes and genes for cell envelope are two groups of role categories that are absent to the largest extent in all isolates. According to results from microarray analysis, a gene region, spr0112-spr1015- is present in all type 9V isolates and absent in many isolates of serotype 14, 19F and 7F. These results have been confirmed by polymerase chain reaction (PCR). Conserved genes in a region around the capsule genes have been sequenced to identify marker genes for a capsulular switch between serotype 9V and 14. Preliminary results of the sequencing showed that as much as 750kb might have been transferred in the event of capsular switch.

Introduction: Mycoplasma pneumoniae(M. pneumoniae) causes 10% to 30% of community-acquired pneumonia (CAP). The commonly used first-line antibiotic macrolide (ML) against respiratory tract infection may lead to the increase of ML-resistant M. pneumoniaeinfection. To resolve the problem, a rapid and accurate method for detection of ML-resistant M. pneumoniaeis necessary for treatment adjustment. Aims: The study aims to (1) develop a rapid method for diagnosis of ML-resistance of M. pneumoniaedirectly from clinical specimens; and (2) investigate the prevalence of M. pneumoniaeand ML-resistant M. pneumoniae. Methods: The M. pneumoniaeqPCR results of 689 respiratory tract samples from Queen Mary Hospital collected during April 2010 to May of 2012 were analyzed. Positive nucleic acid from M. pneumoniaeqPCR samples were tested with SimpleProbe real-time PCR coupled to melting curve analysis (SimpleProbe PCR), pyrosequencing and 23S rRNA gene sequencing(23S sequencing) for detection of ML-resistance. Results: A total of 111 samples (16.11%) in 689respiratory tract samples were found M. pneumoniaepositive by qPCR. Of 111, 96 positive nucleic acids were available for this study. Overall, 29 (30.21%, n=96) of ML-resistant M. pneumoniaewere found. 23S sequencing identified 28 mutants (29.17%) and 62 wild–type (64.58%), while 6 (6.25%) of them are failed to be identified. Pyrosequencing identified 28 mutants (29.17%) and 63 wild–type (65.63%), while 5 (5.21%) of them are failed to be identified. The SimpleProbe PCR identified 29 mutants (30.21%) and 65 wild–type (67.71%), while 2 (2.08%) of them are failed to be identified. All ML-resistant M. pneumoniaepositives were found to have A2063G mutation either by 23S sequencing or pyrosequencing. Conclusion: From this study, SimpleProbe PCR is the most sensitive and simple to perform. Therefore, it is highly recommended to be included in the routine testing with positive M. pneumoniaesamples for diagnosis of ML-resistant strain. 23S sequencing or pyrosequencing is recommended to use as a confirmatory test if necessary. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Introduction: Mycoplasma pneumoniae (M. pneumoniae) has been a major cause of community-acquired pneumonia (CAP), accounting for about 10-30% of the cases. Previously, a local study revealed that more than 60% of clinical isolates of M. pneumoniae exerted A2063G mutation, which confers a high level of macrolide drug resistance and results in treatment failure. While A2063G is the only mutation identified locally, a rapid diagnostic assay for detection of this single point mutation is urgently needed for switching the drug of choice. Aims: This study aims to develop a rapid PCR assay for detection of A2063G mutation of M. pneumoniae isolates for our locality, to compare with other commercially available assays and to further confirm the prevalence of A2063G mutation in macrolide-resistance M. pneumoniae (MRMP) in Hong Kong. Methods: A total of 110 respiratory tract samples were collected from 102 patients in Hong Kong Sanatorium and Hospital during April 2013 to April 2014. They were analyzed by an in-house hybridization-probe real-time PCR assay coupled with melting curve analysis to detect the presence of M. pneumoniae and the target A2063G point mutation. Results were compared with a commercial real-time PCR assay and the A2063G point mutation was further confirmed by 23S rRNA gene sequencing. The limit of detection (LOD), mutation threshold determination and cross reactivity of the in-house assay were also evaluated. Results: Over 40% (47/110) of the respiratory tract samples were tested positive for M. pneumoniae by the in-house assay and 36.2% (17/47) of the positive samples exerted A2063G mutation. The limit of detection was 500 copies/ml as evaluated using external quality control samples. Twenty well-characterized clinical isolates of M. pneumoniae were used to evaluate the A2063G mutation threshold. The mutation threshold for A2063G mutant detection was above 60%. This assay did not show any cross-reactivity with common clinical isolates from the respiratory tract samples. Conclusion: In this study, an in-house real-time PCR assay was evaluated and demonstrated its great potential as a rapid clinical diagnostic tool. The assay was highly sensitive and specific in detecting M. pneumoniae and its A2063G mutation from clinical samples in Hong Kong. The results were almost concordant to the current routine testing, with the advantage of lower cost and shorter turnaround time for rapid detection. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Bibliography: leaves 250-227. / 251 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The genetic loci encoding capsular polysaccharide synthesis have been characterized for all members of streptococcus pneumoniae serogroup. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999?

Thesis (M. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 217-261) Also available in print.