Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Mycobacterium tuberculosis is the etiological agent for tuberculosis, an infectious disease which is one of the leading causes of morbidity and mortality in developing countries. The emergence of drug resistant tuberculosis has negatively impacted the efficacy of current treatment regimens and threatens to undermine tuberculosis control programs worldwide. Rifampicin forms the backbone of the World Health Organization’s recommended treatment regimen for the treatment of drug susceptible tuberculosis. Resistance to rifampicin is caused by mutations in the 81 bp core region of the rpoB gene which encodes the β subunit of RNA polymerase. Numerous studies have shown that mutations at codons 531 and 526 are the most frequent in clinical isolates, yet little is known concerning the mechanistic effect of these mutations on the fidelity of RNA polymerase. In the present study, we aimed to determine the influence of rpoB mutations on the gene expression profile of M. tuberculosis cultured in vitro. To accomplish this, rifampicin resistant clinical isolates and spontaneous mutants (selected in vitro from H37Rv and a drug-sensitive clinical strain) harbouring rpoB H526Y and S531L mutations were subjected to whole genome sequencing and genome-wide transcriptional profiling. When comparing the transcription profile of H37Rv to the in vitro rpoB mutants, a large proportion of the differentially expressed genes were found to encode for proteins involved in intermediary metabolism and respiration; and cell wall and cell processes. The majority of these differentially expressed genes were downregulated. Prominent differential expression in the same functional categories was also evident when comparing the clinical isolates with these mutations; however, a greater number of genes were differentially expressed in this case. Furthermore, expression of genes that are part of the WhiB7 regulon were found to be upregulated in the rpoB526 mutants, and downregulated in the rpoB531 mutants. These findings indicate that both the position of the rpoB mutation, as well as the genetic background of the strain, play an important role in the gene expression profile of rpoB mutants. Surprisingly, transcriptional profiling of cultures that were exposed to the critical concentration of rifampicin for 24 hours did not exhibit significant differential gene expression. Whole genome sequencing, followed by bioinformatic analysis, revealed that the in vitro mutants harbour synonymous and non-synonymous single nucleotide polymorphisms in addition to the respective rpoB mutations. This suggests that the mycobacterial genome is constantly evolving, challenging previous assumptions of relatively static mycobacterial genomes. The findings from this research have provided novel insight into understanding the influence of resistance-conferring mutations on the biology of M. tuberculosis and have shown that further studies are urgently needed to better understand the complex physiology of this pathogen. This knowledge will be critical for the success of future drug development endeavours. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is die etiologiese agent vir tuberkulose, een van die grootste oorsake van morbiditeit en sterftes in ontwikkelende lande. Die verskyning van middelweerstandige tuberkulose het 'n negatiewe impak op die effektiwiteit van die huidige behandeling van tuberkulose en dreig om tuberkulose beheerprogramme wêreldwyd te ondermyn. Weerstandigheid teen rifampisien, een van die eerste-lyn anti-tuberkulose middels, word veroorsaak deur mutasies in die 81 bp kerngedeelte van die rpoB geen. Die mees algemene mutasies in kliniese isolate word gevind in kodons 531 en 526; alhoewel daar min inligting beskikbaar is oor die effek van hierdie mutasies op die funksie van RNS polimerase. Die doel van hierdie studie was om die effek van verskillende rpoB mutasies op die geen-uitdrukkingsprofiel van M. tuberculosis te bepaal. Rifampisien-weerstandige kliniese isolate en in vitro mutante (geselekteer vanaf H37Rv en 'n rifampisien-sensitiewe kliniese isolaat) met rpoB H526Y en S531L mutasies was vir hierdie doel geselekteer en gebruik om die heel genoom volgorde te bepaal en geenuitdrukking te kwantifiseer. Vergelyking van die H37Rv transkripsieprofiel met in vitro geselekteerde rpoB mutante het getoon dat 'n groot aantal gene wat differensieel uitgedruk is, vir proteïene kodeer wat betrokke is in selwand-prosesse en intermediêre metabolisme en respirasie. Die meerderheid van hierdie differensieel uitgedrukte gene was afgereguleer. ’n Soortgelyke verskynsel is ook waargeneem in kliniese isolate, met die verskil dat 'n groter aantal gene in hierdie geval differensieel-uitgedruk was. Gene wat deel vorm van die WhiB7 regulon is opgereguleer in die rpoB526 mutante, terwyl dit afgereguleer was in die rpoB531 mutante. Hierdie resultate is ’n baie sterk aanduiding dat beide die posisie van die rpoB mutasie, asook die genetiese agtergrond van die organisme, ’n belangrike rol speel in die uitdrukking van gene in rifampisin weerstandige M. tuberculosis. Kulture wat aan die kritiese konsentrasie van rifampisien vir 24 uur blootgestel is, het teenverwagting geen differensiële geen-uitdrukking getoon nie. Verder het die heel genoom volgorde bepaling van die in vitro mutante getoon dat sinonieme en nie-sinonieme enkel-nukleotied polimorfismes teenwoordig is in die onderskeie rpoB mutante. Dit dui daarop dat die mikobakteriële genoom voortdurend verander, moontlik nie so stadig as wat voorheen vermoed is nie. Die bevindinge van hierdie navorsing bied nuwe insigte om die invloed van mutasies wat middel-weerstandigheid veroorsaak op die biologie van M. tuberculosis te verstaan. Dit het ook getoon dat verdere studies dringend nodig is om die komplekse fisiologie van hierdie patogeen te verstaan. Hierdie kennis sal van kardinale belang wees vir die sukses van toekomstige ondernemings om nuwe anti-tuberkulose middels te ontwikkel.

Emerging rise in microbial drug resistance and the slow-growing characteristic of some intracellular pathogens such as MAP (Mycobacterium avium subspecies paratuberculosis) strongly urges the need for an effective approach for unconventional drug susceptibility testing. We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 10 MAP isolates in attempt to seek correlation with rpoB sequences. We determined that MAP strain 18 had an MIC ≥ 30 ug/ml and ≥ 5 ug/ml for RIF and RFB respectively, and a significant rpoB mutation C1367T, compared to an MIC of ≤ 1.0 ug/ml for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug's binding site; the beta subunit of RNA polymerase. Our in vitro induced mutation in MAP strain UCF5 resulted in the generation of a new resistant strain (UCF5-RIF16r) that possessed T1442C rpoB mutation and an MIC ≥ 30 ug/ml and ≥ 10 ug/ml for RIF and RFB respectively. The T1442C mutation resulted in a Leu481 to Pro481 amino acid change, consequently altering the beta subunit sequence. Sequencing the entire 3.5 kb rpoB in strains 18 and UCF5-RIF16r revealed no additional expressed nucleotide mutation. Of the 10 MAP strains analyzed, an additional one strain (UCF4) exhibited a slight increase in the MIC against RIF and RFB compared to the wild-type. Nucleotide sequencing of the rpoB gene revealed an A2284C mutation in strain UCF4 that occurred further downstream of the expected probable rpoB region and resulted in an amino acid alteration Asn762 to His762. The location of this mutation outside the binding site and its correlation with the minor increase in MIC suggests a possible secondary interaction between the drug and the beta subunit. We have provided three dimensional images through the utilization of PyMol Molecular-based Graphics to display a clear comparison of the mutations observed in the beta subunit for MAP strains 18, UCF5-RIF16r, and UCF4. We propose that these alterations may have caused a less stable interaction between RIF and the beta subunit, resulting in the observed increased in MIC. Furthermore, the change in amino acid sequence did not affect the viability for our RIF resistant strains. The data clearly illustrates that clinical and in vitro-induced MAP mutants with rpoB mutations result in resistance to RIF and RFB. Consequently, unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.

Os complexos Mycobacterium chelonae M.abscessus e Mycobacterium fortuitum - M. peregrinum são compostos por espécies bacterianas de crescimento rápido e potencialmente patogênicas. Sua distribuição é ubíqua no ambiente, são resistentes a cloração da água e a sua replicação ocorre mesmo em condições de escassez de nutrientes. Estão envolvidos em casos de infecção pulmonar e extrapulmonar, e causam infecções em pacientes imunocomprometidos ou submetidos a rocedimentos cirúrgicos invasivos. Os objetivos do presente trabalho foram: confirmar através de testes fenotípicos e com as técnicas de PRA hsp65 e seqüenciamento do fragmento do rpoB, a identificação de micobactérias de crescimento rápido, incluídas nos complexos M. chelonae-M.abscessus e M. fortuitum-M.peregrinum. Foram incluídos no estudo os isolados provenientes de pacientes com dois ou mais isolamentos provenientes de sítio não estéril ou um isolamento de sítio estéril. O estudo de 38 isolados demonstrou que as provas fenotípicas disponíveis atualmente não permitem a identificação de todas as espécies de micobactérias de crescimento rápido já descritas na literatura. O PRA hsp65 possibilitou a identificação rápida e precisa de 63% das espécies de micobactérias e demonstrou um perfil compartilhado pelas espécies M. abscessus 2; M. bolletii 1 e M. massiliense 1. O seqüenciamento do gene rpoB confirmou a identificação das espécies citadas. Nossos resultados demonstraram que o PRA-hsp65 e o seqüenciamento do gene rpoB são ferramentas úteis para fornecer a identificação das espécies de micobactérias com mais acurácia. O uso dessas técnicas poderiam ser consideradas em laboratórios de referência para identificar Micobactérias de crescimento rápido uma vez que elas são patógenos emergentes implicados em surtos e isolados de pacientes em centros de referência para tratamento de tuberculose multirresistente. / Mycobacterium chelonae-M. abscessus and Mycobacterium fortuitum-M. peregrinum complexes are composed by bacterial species characterized by rapid grow and considered as potential pathogens. These microorganisms are ubiquitous in the environment, resistant to water treatment such as standard chlorination and are able to replicate even at poor nutrient conditions. They are related to lung and extralung infections in immune-compromised patients or those submitted to invasive surgical procedures. The aim of this study was to confirm the identification of Mycobacterium chelonae - M. abscessus and Mycobacterium fortuitum - M. peregrinum complexes, isolated from biological samples considering one or two repetitions if samples are originated from sterile or non-sterile site respectively. Phenotypic tests and molecular methods, PRA-hsp65 and rpoB gene sequencing, were applied to identify 38 strains previously isolated. Results demonstrated that available phenotypic tests did not allow the identification of all described fast growing Mycobacterium. The PRA of hsp65 gene confirmed the identification of 63% of the Mycobacterium studied, and demonstrated the band pattern shared by M. abscessus 2, M. bolletii and M. massiliensis. The rpoB gene sequencing confirmed the identification of the species cited. Our results demonstrated that PRA-hsp65 and rpoB gene sequencing are useful tools to provide a more accurate species identification of mycobacteria. The use of such techniques would be considered in reference laboratories to identify fast growing Mycobacterium species since they are considered emerging pathogens implicated in outbreaks and isolated from a patient in reference centers for treatment of multidrug resistant tuberculosis.

Os complexos Mycobacterium chelonae M.abscessus e Mycobacterium fortuitum - M. peregrinum são compostos por espécies bacterianas de crescimento rápido e potencialmente patogênicas. Sua distribuição é ubíqua no ambiente, são resistentes a cloração da água e a sua replicação ocorre mesmo em condições de escassez de nutrientes. Estão envolvidos em casos de infecção pulmonar e extrapulmonar, e causam infecções em pacientes imunocomprometidos ou submetidos a rocedimentos cirúrgicos invasivos. Os objetivos do presente trabalho foram: confirmar através de testes fenotípicos e com as técnicas de PRA hsp65 e seqüenciamento do fragmento do rpoB, a identificação de micobactérias de crescimento rápido, incluídas nos complexos M. chelonae-M.abscessus e M. fortuitum-M.peregrinum. Foram incluídos no estudo os isolados provenientes de pacientes com dois ou mais isolamentos provenientes de sítio não estéril ou um isolamento de sítio estéril. O estudo de 38 isolados demonstrou que as provas fenotípicas disponíveis atualmente não permitem a identificação de todas as espécies de micobactérias de crescimento rápido já descritas na literatura. O PRA hsp65 possibilitou a identificação rápida e precisa de 63% das espécies de micobactérias e demonstrou um perfil compartilhado pelas espécies M. abscessus 2; M. bolletii 1 e M. massiliense 1. O seqüenciamento do gene rpoB confirmou a identificação das espécies citadas. Nossos resultados demonstraram que o PRA-hsp65 e o seqüenciamento do gene rpoB são ferramentas úteis para fornecer a identificação das espécies de micobactérias com mais acurácia. O uso dessas técnicas poderiam ser consideradas em laboratórios de referência para identificar Micobactérias de crescimento rápido uma vez que elas são patógenos emergentes implicados em surtos e isolados de pacientes em centros de referência para tratamento de tuberculose multirresistente. / Mycobacterium chelonae-M. abscessus and Mycobacterium fortuitum-M. peregrinum complexes are composed by bacterial species characterized by rapid grow and considered as potential pathogens. These microorganisms are ubiquitous in the environment, resistant to water treatment such as standard chlorination and are able to replicate even at poor nutrient conditions. They are related to lung and extralung infections in immune-compromised patients or those submitted to invasive surgical procedures. The aim of this study was to confirm the identification of Mycobacterium chelonae - M. abscessus and Mycobacterium fortuitum - M. peregrinum complexes, isolated from biological samples considering one or two repetitions if samples are originated from sterile or non-sterile site respectively. Phenotypic tests and molecular methods, PRA-hsp65 and rpoB gene sequencing, were applied to identify 38 strains previously isolated. Results demonstrated that available phenotypic tests did not allow the identification of all described fast growing Mycobacterium. The PRA of hsp65 gene confirmed the identification of 63% of the Mycobacterium studied, and demonstrated the band pattern shared by M. abscessus 2, M. bolletii and M. massiliensis. The rpoB gene sequencing confirmed the identification of the species cited. Our results demonstrated that PRA-hsp65 and rpoB gene sequencing are useful tools to provide a more accurate species identification of mycobacteria. The use of such techniques would be considered in reference laboratories to identify fast growing Mycobacterium species since they are considered emerging pathogens implicated in outbreaks and isolated from a patient in reference centers for treatment of multidrug resistant tuberculosis.

Made available in DSpace on 2014-07-29T15:30:37Z (GMT). No. of bitstreams: 1 Dissertacao_sueli.pdf: 1117407 bytes, checksum: d50c0b1dad4e594bc8cd5d3880aadcab (MD5) Previous issue date: 2010-03-11 / Multidrug-resistant tuberculosis is a challenge worldwide. Rapid diagnosis by molecular techniques can provide a more aggressive and appropriate initial therapy. This study aimed to analyze the molecular basis of resistance to isoniazid (INH) and rifampin (R) of Mycobacterium tuberculosis strains isolated from cases of human tuberculosis in Goiás and to genetically determine the causes of the observed resistances. Of the 4.607 cultures for mycobacteria processed in the period of September of 2005 and December of 2007, 24 isolates from 16 patients resistant to at least H and/or R were analyzed. We compared the results obtained by phenotypic tests with mutations in key genes responsible for the development of resistance to these drugs, the rpoB gene for isolates resistant to R and katG gene for strains resistant to H. Seventy one percent of the isolates were resistant to H, and the mutations involved with resistance observed in the katG gene were in codon 315 (41%). The most frequent mutations observed in the rpoB gene of the R resistant isolates (71%) were in codons 456 (76.5%) and 451 (17.6%). Our findings are similar to those reported in the literature. We conclude that the percentage of agreement between genotypic and phenotypic tests was 41% for H and 94% for R considering the number of isolates and 40% and 91%, respectively considering the number of patients. / A tuberculose multidroga resistente representa um desafio em escala mundial. O diagnóstico rápido através de técnicas moleculares é capaz de proporcionar uma terapêutica inicial mais agressiva e adequada. Este trabalho teve como objetivo analisar as bases moleculares da resistência à isoniazida (H) e rifampicina (R) de cepas de Mycobacterium tuberculosis isoladas de casos de tuberculose em Goiás e determinar geneticamente as causas destas resistências. Do total de 4.607 culturas para micobactérias realizadas no período de setembro 2005 a dezembro de 2007, foram analisados 24 isolados de 16 pacientes resistentes a H e/ou R. Os resultados obtidos dos testes fenotípicos de sensibilidade aos antimicrobianos foram comparados às mutações observadas nos principais genes responsáveis pelo desenvolvimento de resistência a estas drogas, gene rpoB para isolados resistentes à R e gene katG para os isolados resistentes à H. Dentre os 24 isolados, 71% eram fenotípicamente resistentes a H e as únicas mutações envolvidas com resistência foram observadas no códon 315 (41%). Dos isolados resistentes a R (71%), foram observadas mutações nos códons 456 (76,5%), 451 (17,6%) e 447 (5,9%). Nossos achados estão em concordância com as principais mutações observadas nos isolados resistentes a R e/ou H descritos na literatura. O percentual de concordância entre os testes fenotípicos e genotípicos foi de 41% para H e 94% para R considerando o número de isolados e de 40% e 91% respectivamente considerando-se o número de pacientes.

In dieser Arbeit wurde die plastidäre Genexpression hinsichtlich zweier Aspekte untersucht: der Cytokinineinfluss auf die plastidäre Transkription und ihrer Komponenten sowie eine in vivo-Charakterisierung von PrpoB-345, des Promotors des rpoB-Operons im Tabak. Cytokinine beeinflussen die Chloroplastenbiogenese und –funktion. Um den Einfluss von Cytokinin auf die plastidäre Genexpression zu untersuchen, wurden run-on-Transkriptionsassays und quantitative real-time RT-PCR von BA-behandelten seneszenten Tabakblättern und sieben-Tage-alten Arabidopsis- und Tabakpflanzen durchgeführt. Zeitreihenanalysen zeigten eine Aktivierung der plastidären Transkription in jungen Pflanzen und im seneszenten Tabak 2 h bzw. 3 h nach BA-Applikation. Abgeschnittene Blätter von Tabakmutanten mit konstitutiv reduziertem Cytokiningehalt antworteten bereits nach 30 min der Hormonbehandlung. Es gibt jedoch keinen eindeutigen Hinweis für eine direkte Korrelation zwischen der Expression der nukleär kodierten Phagentyp-RNA-Polymerasen und der BA-induzierten transkriptionellen Aktivierung der Plastidengene. Zusammengefasst, scheint die Antwort auf exogenes Cytokinin vom physiologischen Status der Chloroplasten, die von der Pflanzenart sowie vom endogenen Cytokiningehalt beeinflusst werden, abzuhängen. Plastidäre Gene höherer Pflanzen werden von mindestens zwei RNA-Polymerasen transkribiert: die plastidär kodierte RNA-Polymerase vom Bakterientyp (PEP) und die kernkodierte Phagentyp-RNA-Polymerase (NEP). NEP transkribiert das rpoB-Operon, das drei von vier Untereinheiten der PEP kodiert. Transkriptions- und Transkriptanalysen von rpoB-Promotor-Deletionsmutanten ergaben Hinweise auf mögliche Regulationsstellen der Kontrolle der rpoB-Transkription. Neben PrpoB-345 konnten zwei weitere Promotoren kartiert werden. Einer von ihnen ist ein putativer PEP-Promotor, der auf autoregulatorische Rückkopplungsmechanismen bei der PEP-Expression hindeutet. / In this study, plastid gene expression was analyzed focusing on two aspects: the effect of cytokinin on plastid gene transcription and its components, and the in vivo characterization of PrpoB-345, the promoter of the rpoB operon in tobacco. Cytokinins are involved in the control of chloroplast biogenesis and function. To study cytokinin effects on plastid gene expression, chloroplast run-on transcription and quantitative real-time RT-PCR from senescent tobacco leaves as well as Arabidopsis and tobacco seedlings after BA treatment were performed. Analyses of time series revealed that BA-induced changes in plastid gene expression are seemingly under circadian and homeostatic control. After 2 h and 3 h of incubation with cytokinin, a stimulation of chloroplast transcription could be observed in seedlings and senescent leaves, respectively. Detached leaves of tobacco mutants with reduced endogenous cytokinin content responded even faster to BA (30 min). There is no indication of direct correlation of the expression of nuclear-encoded plastid phage-type RNA-polymerases and the BA-induced transcriptional activation of plastid genes. In summary, the responsiveness to exogenous cytokinin depends on the physiological status of chloroplasts influenced by plant species and endogenous cytokinin pool. Plastid genes of higher plants are transcribed by at least two RNA polymerases: the plastid-encoded eubacterial-type RNA polymerase (PEP) and the nucleus-encoded phage-type RNA polymerase (NEP). NEP transcribes the rpoB operon encoding three of four subunits of PEP. Transcription and transcript analyses from rpoB promoter deletion mutants indicated putative regulatory sites of control of rpoB transcription which may also interact with (cytokinin-regulated) specificity factors. Beside PrpoB-345, two additional rpoB promoters could be mapped. One of them is a putative PEP promoter which may imply autoregulatory loops of PEP expression.

Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications. Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response. To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E. Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA. Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria.

Made available in DSpace on 2014-08-20T14:37:58Z (GMT). No. of bitstreams: 1 dissertacao_sergio_jorge.pdf: 980348 bytes, checksum: 9e56cf9be902454868ace419c988ca33 (MD5) Previous issue date: 2009-07-20 / Leptospirosis is a zoonotic disease that occurs all over the world, particularly in developing countries, caused by bacteria of the genus Leptospira. Several marsupial species are considered susceptible to infection caused by a wide variety of Leptospira serovars, acting as reservoirs. In this work were collected serum and urine samples of 33 White-eared opossum, trapped within distinct locations of Capão do Leão and Pelotas cities, in the South of Brazil, aiming to detect antibodies and to isolate leptospires. Serum samples were screened against a panel of 58 Leptospira spp and Leptonema illini serovars using the microscopic agglutination test (MAT). To attempt isolation, urine samples from all animals were inoculated in culture medium.EMJH enriched with 10% of supplement Difco® One pathogenic serovar was isolated after two months of inoculation, and is first isolate the country for this animal species and was characterized by PCR using primers for 16S and Lipl32 genes. The technique of partial sequencing of the rpoB gene was used to identify the genomic specie. The isolated strain belongs to the specie L. borgpetersenii, which along with L .interrogans are the main cause disease in humans. The isolate, was tested on MAT using dog, cattle and human sera. Cut off titer of 50 was used for domestic animal sera and 25 for human and opossum sera. Both human and animal sera presented agglutination on MAT to isolated strain corresponding to 3.3% (2/60), 28.33% (17/60) and 1.67% (1/60) in human, dog and cattle sera, respectively. The opossum sera presented 36.36% (12/33) of reaction. These findings suggest a probable white-eared opossum role in the maintenance of pathogenic Leptospira on the environment since low titer sera and urine elimination were observed. These animals could be important reservoirs of Leptospira serovars for human and domestic animals. / A Leptospirose é uma antropozoonose de ocorrência mundial, particularmente nos países em desenvolvimento, causada por bactérias do gênero Leptospira. Várias espécies de marsupiais e didelfídeos são consideradas suscetíveis a infecção causada por uma grande variedade de sorovares patogênicos de Leptospira spp. tendo sido considerados possíveis hospedeiros deste agente. Neste trabalho foram coletadas amostras de soro e urina de 33 gambás-de-orelha-branca (Didelphis albiventris), capturados em diferentes regiões dos municípios do Capão do Leão e Pelotas, no sul do Brasil, com o objetivo de detectar anticorpos e isolar leptospiras. As amostras de soro foram testadas por aglutinação microscópica (MAT), utilizando 58 sorovares de Leptospira spp. e 1 de Leptonema. Para o isolamento, amostras de urina foram inoculadas em meio de cultura EMJH enriquecido com 10% de suplemento Difco®. Um sorovar patogênico foi obtido da urina, sendo o primeiro isolado do país para esta espécie, e foi caracterizado quanto ao gênero e patogenicidade por PCR utilizando primers para os genes 16S rDNA e Lipl32. A técnica de seqüenciamento parcial do gene rpoB foi utilizada para identificar a espécie genômica. A cepa isolada pertence à espécie L. borgpertersenii,, que juntamente com L. interrogans são as principais causadoras de doença em humanos. Anticorpos anti isolado de gambá foram avaliados em amostras sorológicas de 60 cães, 60 bovinos, 60 humanos e dos 33 gambás capturados através da MAT. Os soros humanos apresentaram reação de 3,3% (2/60), 28,33% (17/60) em soros caninos, 1,67% (1/60) em soros bovinos e 36,36% (12/33) em soros dos gambás capturados no estudo. Esses dados sugerem o envolvimento do gambá-de-orelha-branca na manutenção de leptospiras no ambiente, uma vez que estando infectados por sorovares patogênicos podem eliminar o agente pela urina e infectar direta ou indiretamente animais domésticos e conseqüentemente o homem.

L'objectif de cette thèse est d'appliquer la méthode d'identification bactérienne par spectrométrie de masse MALDI-TOF pour une utilisation en routine dans un laboratoire de microbiologie clinique. Dans un 1er temps et de manière prospective, nous avons évalué la performance et le coût-efficacité de l'identification bactérienne de routine par MALDI-TOF par rapport aux techniques conventionnelles d'identification phénotypique. Durant la période des 16 semaines d'étude, nous avons comparé la performance de la technique par MALDI-TOF aux techniques conventionnelles d'identification phénotypique comprenant la coloration de Gram, la galerie API ANA et le Vitek 2. En cas de résultats discordants entre ces deux techniques, l'identification était réalisée par biologie moléculaire. Nous avons montré que le MALDI-TOF est un moyen efficace et rentable pour l'identification des bactéries de routine. Le MALDI-TOF peut être utilisée en 1ère intention dans l'identification bactérienne avant l'ensemble de techniques phénotypiques. Dans un 2ème temps, nous avons évalué rétrospectivement la performance et le coût-efficacité de l'utilisation exclusive de MALDI-TOF en diagnostic bactériologique de routine en comparaison avec les techniques conventionnelles. En analysant les données des 11 dernières années, nous avons montré que le MALDI-TOF est efficace et tout à fait adaptée pour l'identification d'espèce bactérienne en routine. Nous avons également prouvé que MALDI-TOF est un outil puissant pour identifier les espèces bactériennes rarement impliquées dans les infections humaines. Cette technique pourrait être une alternative aux méthodes moléculaires dans le laboratoire clinique. / The objective of this thesis is to apply the method of bacterial identification by MALDI-TOF MS in daily practice in a routine clinical microbiological laboratory. Firstly, we prospectively evaluated the performance and the cost-effective of bacterial identification by MALDI-TOF in comparison with conventional phenotypic identification methods. During a 16-week study, we compared the performance of MALDI-TOF with conventional techniques of identification including Gram staining, API ANA identification strip and automated identification using the Vitek 2. The unmatched identifications between MALDI-TOF and conventional methods were resolved by molecular identification. In this study, we showed that MALDI-TOF was an effective tool and less expensive for the rapid identification of bacterial species in clinical microbiology laboratory. MALDI-TOF can be used in first intention for identification before Gram staining or other phenotypic identification techniques based on physicochemical properties of bacteria. Secondly, we retrospectively evaluated the performance and the cost-effectiveness of the exclusive use of MALDI-TOF in bacteriological diagnosis in comparison with conventional phenotypic identification. 11-year retrospective analysis of data showed that MALDI-TOF was efficient and completely adapted for the routine identification of bacterial species. We also showed that MALDI-TOF had capacity to identify bacterial species that were rarely involved in human diseases. This technique could be an alternative to molecular methods in the clinical laboratory.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Multidrug resistant tuberculosis (MDR-TB), defined as having resistance to at least the first-line drugs, isoniazid and rifampicin (RIF), is a global health problem. Mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, are implicated in RIF resistance - with the S531L and H526Y mutations occurring most frequently. The level of RIF resistance varies for strains with identical rpoB mutations, which suggests that other factors play a role in RIF resistance. Efflux has been implicated in determining the intrinsic level of RIF resistance. Increased expression of the multidrug efflux pump, Rv1258c, following RIF exposure was observed in some Mycobacterium tuberculosis MDR clinical isolates and H37Rv RIF mono-resistant mutants, but not others. The factors influencing the induction of Rv1258c are poorly understood. The aim of this study was to investigate the effects of rpoB mutations on expression of Rv1258c and whiB7, a transcriptional regulator of Rv1258c, in M. tuberculosis H37Rv in vitro generated RIF resistant mutants, in the absence and presence of RIF. The promoter region of M. tuberculosis H37Rv Rv1258c was cloned into a position upstream of a lacZ gene (encoding β-galactosidase) in multi-copy episomal and integrating vectors. Vector functioning and the effect of rpoB mutations on Rv1258c promoter activity were initially investigated in the non-pathogenic related species, Mycobacterium smegmatis mc2155 rpoB mutants and subsequently in M. tuberculosis by doing β-galactosidase assays. qRT-PCR was done to investigate the effects of rpoB mutations on native Rv1258c and whiB7 gene expression. Episomal and integrating vectors were functional and the integrating vector system was used for subsequent β-galactosidase assays in M. tuberculosis. Rv1258c promoter activity in the S531L mutant was approximately 1.5 times less and in the H526Y mutant 1.5 times higher than that of the wild-type in M. smegmatis. Similarly, Rv1258c promoter activity in the S531L mutant was approximately half and in the H526Y mutant approximately double that of the wild-type in M. tuberculosis. A similar trend in Rv1258c and whiB7 expression to those observed using β-galactosidase assays were observed when investigating the native Rv1258c and whiB7 gene transcript levels compared to the wild-type using qRT-PCR, although differences were not significant. Exposure of the M. smegmatis and M. tuberculosis rpoB mutants to sub-inhibitory levels of RIF did not affect Rv1258c promoter activity. Mutations in rpoB had a marginal effect on Rv1258c and whiB7 transcript levels, but showed the same trend as that seen for Rv1258c promoter activity. It remains to be determined whether these differences are biologically significant. When considering efflux pumps as new targets for treatment, possible differences in efflux pumps expression due to different rpoB mutations should be considered. / AFRIKAANSE OPSOMMING: Multi-middel weerstandige tuberkulose (MDR-TB) word gedefinieer as weerstandigheid tot ten minste rifampisien (RIF) en isoniasied, wat deel van die eerstelyn anti-tuberkulose behandeling vorm. Mutasies in die rpoB geen, wat die β-subeenheid van die RNA polimerase enkodeer, word geassosieer met RIF weerstandigheid. S531L en H526Y rpoB mutasies kom die algemeenste voor. RIF weerstandigheids vlakke verskil egter tussen isolate met identiese rpoB mutasies, wat impliseer dat ander faktore ook 'n rol in RIF weerstandigheid speel. 'n Toename in transkripsie van die Rv1258c geen, wat 'n multi-middel effluks pomp enkodeer, is waargeneem met blootstelling aan RIF, slegs in sommige M. tuberculosis H37Rv RIF mono-weerstandige mutante and MDR kliniese isolate, maar nie in ander nie. Die faktore wat die induksie van die Rv1258c effluks pomp beïnvloed is nie goed nagevors nie. Die studie ondersoek die effek van die rpoB mutasies op die uitdrukking van die Rv1258c en whiB7,'n transkripsionele regulator van Rv1258c, gene in M. tuberculosis H37Rv in vitro gegenereerde RIF weerstandige mutante, in die teenwoordigheid en afwesigheid van RIF. Die promotor area van die M. tuberculosis H37Rv Rv1258c geen is in 'n posisie stroomop van 'n lacZ geen, wat vir β-galaktosidase enkodeer, in multi-kopie episomale en integreerende vektors ingekloneer. Die funksionaliteit van die vektor en effek van rpoB mutasies op Rv1258c promotor aktiwiteit is ondersoek in die naverwante nie-patogeniese spesies, M. smegmatis en daarna in M. tuberculosis deur β-galaktosidase essais te doen. qRT-PCR is gedoen om die effek van rpoB mutasies op die vlak van transkripsie van die natuurlike Rv1258c geen en die whiB7 geen te bestudeer. Beide die episomale en integreerende vektors was funksioneel en daar is besluit om die integreerende vektor vir daaropeenvolgende β-galaktosidase essais in M. tuberculosis te gebruik. Rv1258c promotor aktiwiteit van die S531L mutant was ongeveer 1.5 keer minder as en die van die H526Y mutant 1.5 keer hoër as die van die ongemuteerde bakterië in M. smegmatis. Soortgelyk was die Rv1258c promoter aktiwiteit van die S531L mutant ongeveer die helfde van en die van H526Y mutant ongeveer dubbel die van die ongemuteerde bakterië in M. tuberculosis 'n Soortgelyke neiging in die vlakke van Rv1258c en whiB7 transkripte van die natuurlike geen is gedurende qRT-PCR waargeneem alhoewel die verskille nie beduidend was nie. Blootstelling aan sub-inhibitoriese konsentrasies van RIF het geen effek op Rv1258c uitdrukking in die M. smegmatis of M. tuberculosis rpoB mutante gehad nie. Die rpoB mutasies het net 'n effense effek op Rv1258c en whiB7 transkrip vlakke in M. tuberculosis rpoB mutante, maar transkrip vlakke het 'n soortgelyke neiging as die Rv1258c promoter aktiwiteit getoon. Of die waargenome verskille biologies betekenisvol is, moet nog bepaal word. Indien effluks pompe as teikens vir bahandeling gebruik sou word, moet in ag geneem word dat effluks pompe moontlik verskillend uitgedruk word in verskillende rpoB mutante. / The DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, Stellenbosch University / DAAD-NRF in Country Scholarship and Ernst and Ethel Eriksen Trust / Harry Crossley Foundation