Orientador: Luiz Carlos Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T11:46:58Z (GMT). No. of bitstreams: 1 Castro_IltonBarrosDaltrode_M.pdf: 2931991 bytes, checksum: c8a53e4f8a0a6d154ec91cd575ee9936 (MD5) Previous issue date: 2005 / Resumo: A migrastatina é um policetídeo que foi isolado da cultura de Streptomyces sp. MK929-43F1 em 2000 por Imoto e colaboradores e, posteriormente, isolado também da cultura de Streptomyces platensis NRRL 18993 por pesquisadores da Kosan Biosciences. Migrastatina apresenta um extraordinário efeito inibitório na migração de células tumorais, importantíssimo para o tratamento de metástese tumoral. Sua estereoquímica relativa e a configuração absoluta foram determinadas por análise cristalográfica de raios-X de um derivado. Migrastatina é uma macrolactona de 14 membros com uma cadeia lateral contendo anel de glutarimida. A molécula contém 5 centros estereogênicos e 3 ligações duplas. Nesse trabalho descreveremos uma rota sintética viavél para obtenção do fragmento C1-C13 da migrastatina. As etapas chaves para construção do fragmento C1- C13 são: a esterificação do ácido carboxílico 65 (fragmento C1-C6) com o álcool alílico 66 (fragmento C8-C13) e a reação de Nozaki-Hiyama-Kishi intramolecular. Os fragmentos foram construídos utilizando principalmente reações aldol syn seletiva e reações de Horner-Wadsworth-Emmons, além da utilização adequada de grupos protetores / Abstract: Migrastatin was isolated from a cultured broth of Streptomyces sp. MK929-43F1 in 2000 by Imoto and co-workers, as well as from a cultured broth of Streptomyces platensis NRRL 18993 by researchers from Kosan Biosciences. Migrastatin has a remarkable inhibitory effect on the migration of human tumor cells, very important to the tumor metastasis treatment. Its relative stereochemistry and absolute configurations were determined by X-ray analysis of a derivative. Migrastatin is a 14-membered lactone with a glutarimide side chain, 5 stereogenic centers and 3 double bonds. We wish to describe here an approach to the C1-C13 fragment of migrastatin. The key steps for the construction of the C1-C13 fragment of migrastatin are: a syn-aldol reaction to set up the C9 and C10 stereogenic centers, followed by a (Z)-seletive Horner-Wadsworth-Emmons reaction and esterification of carboxilic acid 65 (fragment C1-C6) with allylic alcohol 66 (fragment C8-C13) / Mestrado / Quimica Organica / Mestre em Química

Objectives: The discovery of new antibiotic targets is important to stem the increase in antibiotic resistance to most currently used antimicrobials. The bacterial ribosome is a major target for a large number of antibiotics that inhibit different aspects of translation. Most of these antimicrobial agents also inhibit ribosomal subunit formation as a second cellular target. Precise subunit assembly requires the activity of several distinct RNases for proper rRNA processing. The present work shows that the vanadyl ribonucleoside complex (VRC) inhibited RNases in Staphylococcus aureus involved in ribosomal subunit formation without an effect on translation. Methods: Methicillin-susceptible and -resistant strains of S. aureus were examined for the inhibitory effects of VRC on cell viability by colony counting. Protein synthesis rates were measured by isotopic methionine incorporation. Ribosome synthesis was measured by radiolabelled uridine incorporation into ribosomal subunits as displayed on sucrose gradients. Pulse and chase radiolabelling was used to measure subunit synthesis rates. RNA turnover was determined by a gel on a chip assay. Results: The rates of subunit synthesis and the amounts of both subunits were significantly reduced in the presence of the compound. Ribosomal RNA was degraded and cell viability was reduced as a consequence. VRC also stimulated the inhibitory effects of a macrolide and an aminoglycoside antibiotic on ribosome formation. Conclusions: Bacterial ribosomal subunit synthesis was specifically impaired in VRC-treated cells, with the rates and amounts of both subunits reduced. Cell viability was significantly reduced and rRNA turnover was stimulated.

No description available.

Mycoplasma (M.) bovis est une bactérie pathogène des bovins, à l'origine de signes cliniques divers, comme des mammites, des arthrites, des otites et des bronchopneumonies, ces dernières étant majoritaires en France. Les mycoplasmoses à M. bovis ont un fort coût économique et leur contrôle impose une importante mobilisation sanitaire et un recours très fréquent à l'antibiothérapie. Peu de données étaient disponibles jusque récemment concernant le typage moléculaire et l'antibiosensibilité des souches françaises de M. bovis. Deux études antérieures à ce travail et réalisées au sein de l'UMR « Mycoplasmoses des ruminants » ont montré que les isolats cliniques de M. bovis collectés en France après 2000 appartiennent à un sous-type moléculaire majoritaire (ST2), très homogène et sont par ailleurs multirésistants à la plupart des familles antibiotiques à l'exception des fluoroquinolones. Ces résultats suggèrent la diffusion sur le territoire national d'un clone unique multirésistant. Le premier objectif de cette étude était de déterminer les mécanismes à la base de la perte de sensibilité aux antibiotiques des isolats français. Dans un deuxième temps, les liens entre les différents sous-types moléculaires, les profils d'antibiosensibilité, les maladies associées et le polymorphisme des gènes cibles des antibiotiques ont été investigués. Cette approche a été déployée pour trois familles d'antibiotiques utilisées en pratique vétérinaire: les macrolides, les tétracyclines et également les fluoroquinolones, quoique récemment classées comme molécules critiques. De façon générale, les mutations identifiées dans les cibles des antibiotiques expliquent à elles seules les phénotypes de résistance observés. Des mutations dans les ARNs ribosomaux, cibles des macrolides et des tétracyclines, ont été observées sur des isolats cliniques dès 1978 et sont devenues systématiques sur tous les isolats collectés après 2000 et appartenant au sous-type ST2 majoritaire. En ce qui concerne les fluoroquinolones, la faible augmentation des CMI (concentrations minimales inhibitrices) mesurée chez la plupart des isolats cliniques récents n'a pas été associée à des mutations des QRDR (« Quinolones Resistance-Determining Regions »). Par contre, des altérations cumulées de façon séquentielle dans ces QRDR, associées à une hausse des CMI, ont été mises en évidence lors d'expériences de sélection in vitro et majoritairement pour des souches appartenant à un sous-type récent minoritaire, ST3, apparemment plus variable et plus apte à fixer les mutations. En 2013, le premier isolat clinique présentant une CMI augmentée aux fluoroquinolones a été isolé: il appartient à ce sous-type ST3. L'ensemble des résultats obtenus montrent que les différents sous-types de M. bovis n'évoluent pas de la même façon vers la résistance. Ce constat ajouté à celui de la multirésistance des isolats récents (ST2 ou ST3) met en exergue l'intérêt de la surveillance (sous-typage et antibiosensibilité) et le suivi de l'évolution des isolats de M. bovis circulant en France. Ce suivi permettrait notamment d'anticiper une éventuelle émergence de la résistance aux fluoroquinolones / Mycoplasma (M.) bovis is a bacterial pathogen for cattle, responsible for various clinical signs, like mastitis, arthritis, otitis and respiratory diseases, the latter being the main syndrome present in France. Mycoplasmoses have a great economic impact and their control entails drastic sanitary measures and a frequent use of antibiotherapy. Few data was available until recently on the molecular subtyping and the antimicrobial susceptibility of the French strains of M. bovis. Two previous studies done in the UMR « Mycoplasmoses des ruminants » proved that clinical isolates collected in France after the year 2000 belonged to one major subtype (ST2), which is very homogeneous, and that they were multiresistant to the main antimicrobial families except fluoroquinolones. These results suggested the diffusion of one unique multiresistant clone on the national territory. The first aim of the present study was to decipher the molecular mechanisms underlying the loss of susceptibility to antimicrobials of the French strains. Secondly the links between the molecular subtypes, the antibiotics susceptibility profiles, the clinical origins and the polymorphisms of the target genes were assessed. This approach was used for 3 antimicrobial families currently used in veterinary medicine: macrolides, tetracyclines and fluoroquinolones, although recently classified as critical. Actually, the point mutations observed in the target genes of the antimicrobials accounted for the observed resistance phenotypes. Some mutations in the ribosomal RNAs, targets of the macrolides and the tetracyclines, were observed in clinical isolates as soon as 1978 and they were generalized in all isolates collected after 2000 and belonging to the major subtype ST2. Concerning the fluoroquinolones, the slight increase in MIC (Minimum Inhibitory Concentration) observed in most of the recent isolates was not associated with mutations in the QRDR (Quinolone Resistance-Determining Regions). However alterations that were associated with increased MICs were highlighted and proved to be sequentially cumulated during experiments of in vitro selection under antimicrobials pressure. This was mainly true for strains belonging to a recent and uncommon subtype, ST3, which is apparently more variable and more able to fix the mutations. In 2013 the first clinical strain showing an increased MIC to fluoroquinolones was isolated and proved to belong to ST3. The whole results of this study showed that the different subtypes did not evolve with the same speed towards resistance. This fact, associated with the multiresistant phenotype of the recent isolates (ST2 or ST3), highlights the urge to monitor (subtyping and antimicrobial susceptibility profiles) and to follow-up the evolution of the isolates of M. bovis circulating in France in order to anticipate a potential emergence of the resistance to fluoroquinolones

La doxycycline est l’une des molécules recommandées par l’OMS en prophylaxie pour les voyageurs dans les zones d’endémie palustre, en particulier dans les zones de multirésistance. Une étude récente avait suggéré que les isolats de P. falciparum présentaient différents niveaux de sensibilité à la doxycycline et que l’augmentation du nombre de copies de deux gènes, pfmdt ou pftetQ, pouvait être associée à une baisse de sensibilité.Le premier objectif de ce travail a consisté à valider ce modèle à partir d’un nouvel échantillonnage d’isolats africains. Le second objectif était d’évaluer le nombre de copies de ces deux gènes sur des isolats originaires de Thaïlande. Le troisième objectif a consisté à rechercher d’autres sources de résistance en investiguant le polymorphisme des gènes codant l’ARN ribosomal plasmodial potentiellement impliqués dans la résistance in vitro à la doxycycline.Les résultats nous ont permis de confirmer que le nombre de copies des gènes pfmdt ou pftetQ pouvait être impliqué dans la résistance in vitro à la doxycycline en Afrique. Les résultats concernant les isolats Thaï n’ont pas permis de corréler le nombre de copies des gènes pfmdt et pftetQ au phénotype CI50. Ces éléments montrent que ce mécanisme de résistance seul est insuffisant pour expliquer la résistance à la doxycycline ; les résultats sont en faveur d’une résistance médiée par plusieurs gènes.La recherche de points de mutation sur le gène pfssrRNA codant pour la petite sous-unité ribosomale de l’ADN plasmodial n’a pas abouti. D’autres cibles moléculaires sont en cours d’étude pour expliquer les mécanismes de résistance de P. falciparum à la doxycycline. / Doxycycline is currently one of the recommended chemoprophylactic regimens for travellers visiting malaria-endemic, particularly in countries with a high prevalence of resistance to chloroquine and multiresistance. A previous study suggested that increased pfmdt or pftetQ copy number could be associated with a lower susceptibility to doxycycline.The first aim of this study was to validate the pre-established model involving these two molecular markers with other African isolates. The second was to evaluate these markers in P. falciparum isolates coming from a multiresistance area in Thaïland. The third was to investigate the eventual association between the polymorphism in genes encoding ribosomal rRNA and in vitro resistance to doxycycline.The results confirm that pfmdt or pftetQ copy numbers should be involved in in vitro susceptibility to doxycycline in African P. falciparum isolates. The results concerning the Thai isolates indicate that there is no correlation between the pfmdt and pftetQ genes copy numbers and the belonging to the high doxycycline IC50 phenotype; this implies that this mechanism of resistance is not enough by itself to explain resistance to doxycycline; it augurs that the resistance to doxycycline should be controlled by multiple genes, and that these genetic markers could be continent-dependent. The search for points of mutation in isolates from the different doxycycline IC50 phenotypic groups has not resulted with pfssrRNA. Other therapeutic targets are being considered to explain P. falciparum resistance to doxycycline.

Le ribosome est une machine biomoléculaire primordiale pour la survie de tout organisme du fait de son rôle central au sein de la synthèse protéique. La caractérisation des interactions avec ses nombreux partenaires est un élément crucial pour mieux comprendre les mécanismes de la traduction et de son inhibition chez les eucaryotes et procaryotes. Cette inhibition est d’ailleurs une stratégie utilisée par beaucoup d’antibiotiques ciblant le ribosome pour lutter contre les infections bactériennes. La compréhension de leur mode d’action est devenue une priorité mondiale pour faire face au problème de la résistance bactérienne. Chez les eucaryotes, une autre stratégie est employée par les virus pour bloquer et s’approprier la machinerie traductionnelle de l’hôte grâce à des structures d’ARN non codant (IRES) capables de recruter directement le ribosome. Bien que largement caractérisés, peu de données thermodynamiques et cinétiques sont disponibles concernant ces deux systèmes d’interaction avec le ribosome. Mon projet a pour vocation d’utiliser des approches biophysiques innovantes afin de compléter les études sur les interactions du ribosome d’E. coli avec les macrolides, et du ribosome de S. cerevisiae avec l’IRES intergénique du CrPV. / The ribosome is a biomolecular machine essential for the survival of any organism due to its central role in protein synthesis. The characterization of its interactions with its many partners is a crucial element in better understanding the mechanisms of translation and inhibition in eukaryotes and prokaryotes. Inhibition of translation is a strategy used by many ribosome-targeting antibiotics to fight bacterial infections. Understanding their mode of action has become a global priority in addressing the problem of bacterial resistance. In eukaryotes, another strategy is used by viruses to block and appropriate the host's translational machinery through non-coding RNA structures (IRES) capable of directly recruiting the ribosome. Although widely characterized, few thermodynamic and kinetic data are available for these two ribosome interaction systems. My project is intended to use innovative biophysical approaches in order to provide an original view of the interactions of the E. coli ribosome with macrolides, and of the S. cerevisiae ribosome with the intergenic IRES of the CrPV.

L’utilisation massive des antibiotiques contribue à leur accumulation dans les boues des stations d’épurations. L’application directe des boues est parmi les sources de dissémination des antibiotiques et des gènes de résistance aux antibiotiques. Le compostage et la méthanisation sont parmi les bioprocédés de traitement des boues qui permettent d’éliminer ou réduire les teneurs de certains antibiotiques. Dans ce travail, une boue primaire de la STEP de Marrakech a été contaminée par trois familles d’antibiotiques (macrolides, tétracyclines, fluoroquinolones) pour conduire 4 essais de compostage à différentes doses (dont un essai témoin) et un essai deméthanisation en mode semi-continu. Les résultats du compostage ont montré que l’augmentation des concentrations d’antibiotiques retarde la dégradation de la matière organique et affecte le ratioC/N. De même, la phase thermophile est perturbée, retardée et réduite dans le temps. Pour la méthanisation, une concentration unique et réaliste a été testée. Dans ces conditions, aucun effet sur la production du biogaz ou sur la dégradation de la matière organique n’a été observé. Afin de suivre la dissipation des trois familles d’antibiotiques utilisées au cours du compostage et de la méthanisation, une approche analytique basée sur l’extraction accélérée par solvant (ASE) suivie par l’application d’une méthode des ajouts dosés avant quantification par chromatographie liquide couplée à de la spectrométrie de masse en tandem (UPLC-MS/MS) a du être mise en point. Le compostage et la méthanisation permettent de réduire significativement les concentrations des molécules parents appartenant à la famille des macrolides et des tétracyclines. Par contre,l’élimination des fluoroquinolones est non-significative et ne dépasse pas 30%. Au cours du compostage, la dissipation des macrolides se fait en phase de stabilisation tandis que la phase de maturation est impliquée dans la dissipation des tétracyclines. Les concentrations encirprofloxacine (fluoroquinolone) semblent légèrement évoluer au cours du procédé probablement en raison d’une adsorption/désorption sur le co-substrat lignocellulosique utilisé. Concernant la méthanisation, l’élimination des macrolides et des tétracyclines est significative durant la stabilisation du procédé mais n’atteinds pas les rendements observés lors du compostage. Ladiminution des concentrations des molécules parents est probablement accompagnée par une biotransformation des antibiotiques sous forme de métabolites qui à ce stade ne sont pas connus.La question de la rémanence de certaines molécules comme les fluoroquinolones, interpelle quand au risque d’antibiorésistance. Ainsi, la valorisation des composts/digestats comme amendements organiques des sols dois à terme conduire à une réflexion concernant la réglementation qui inclus la présence de molécule de la classe des antibiotiques. / The intensive use of antibiotics for human purposes leads to their presence and accumulation inthe sludge produced from wastewater treatment plants. The direct application of sludge is amongthe sources of dissemination of antibiotics and antibiotics resistance genes. Composting andanaerobic digestion are some of the most used bioprocess for sludge treatment, and which allowthe removal/decrease of some antibiotics families. In this work, a primary sludge from thewastewater treatment plant of Marrakesh was spiked using 3 families of antibiotics (macrolides,tetracyclines, fluoroquinolones) to conduct (1) 4 composting experiments with variousconcentrations levels, and (2) an anaerobic digestion experiment in a semi-continuous mode.Composting results showed that the organic matter degradation was delayed and the C/N ratiowas affected by an increase of antibiotics concentrations. Likewise, the thermophilic stage wasdisturbed, the heat release was affected and the coming of the temperature maxima was delayed.In the other hand, one realistic concentration was used during the anaerobic digestion. In thiscondition, no effect was observed especially on the biogas production as well as the organicmatter degradation. To assess the fate of antibiotics during composting and anaerobic digestion,an analytical approach based on the accelerated solvent extraction followed by the standardaddition method and the UPLC-MS/MS was developed. Composting and anaerobic digestion leadto a significant removal of parent compounds belonging to the family of macrolides andtetracyclines. In contrast, the fluoroquinolones removal is not-significant and has not exceeded30%. During composting, the thermophilic stage was responsible on macrolides elimination. Incontrast, the maturation stage was more implicated on the removal of tetracyclines. Ciprofloxacin(fluoroquinolone) showed some fluctuations in concentrations. The sorption/desorption on palmrachis could probably explain the observed behavior of this molecule during composting. Duringthe anaerobic digestion, the removal of macrolides and tetracyclines was significant, within thestabilization, but still lower than the observed ones during composting. The decrease of parentcompounds of antibiotics is probably accompanied by a biotransformation of these compounds inunknown metabolites. The presence of recalcitrant compounds after the bioprocess could promotethe development of resistant bacteria including pathogens. In the future regulations, thevalorization of compost/digestate as an amendment of agricultural soil requires taking into accountthe presence of antibiotics.

Les principaux points de dispersion des composés pharmaceutiques dans l’environnement sont les stations d’épuration des eaux usées. A partir de l’analyse de la base de données constituée au cours de ce travail, les quantités de ces composés trouvées dans ces installations peuvent être directement liées à celles de médicaments consommés. La situation, quant à leur élimination de la phase liquide, est contrastée. Ainsi certaines hormones sont éliminées avec des rendements importants mais d’autres ne le sont pas du tout. Les produits radio-contrastants à base d’halogènes et notamment d’iode, soupçonnés d’être cancérogènes, sont majoritairement non biodégradables. Les effets d’antibiotiques sur les boues activées ont été plus particulièrement étudiés du fait de leur consommation, de leur présence dans les milieux aquatiques et de leur action spécifique sur les bactéries. Des expériences permettant d’évaluer la toxicité de ces principes actifs ont été conduites sur des boues activées en réacteur discontinu, avec un suivi de la morphologie des flocs par analyse d’images de microscopie optique. Il a été ainsi observé que, selon la concentration utilisée, l’érythromycine et la tylosine, macrolides largement utilisés respectivement en médecine humaine et vétérinaire, inhibent l’élimination de la pollution organique et dégradent l’ensemble de la biomasse. Ces antibiotiques ont également un effet néfaste sur le métabolisme de l’azote, qu’il s’agisse de l’ammonification, de la nitritation et de la nitratation / The main sources of dispersion of pharmaceutical substances in environment are wastewater treatment plants. Based on the analysis of the database built during this project, the amounts of pharmaceutical substances found in plants can be directly related to the amount consumed. Regarding their elimination from the liquid phase, the situation is very disparate. The elimination yield of hormones can be null or very large. Halogen-based X-ray contrast media (or AOX) are mainly non biodegradable. The effects of antibiotics on activated sludge have been especially investigated due to their consumption, presence in aquatic environment and specific action on bacteria. Batch tests have been performed to evaluate the toxicity of these active pharmaceutical ingredients on activated sludge. Sludge morphology was monitored by analysis of light microscopy images. Depending upon the applied dose, erythromycin and tylosin, two macrolides widely used for human and animal health care, inhibit the elimination of organic pollution and damage biomass. These antibiotics have a deleterious effect on ammonification, nitritation and nitratation

Master of Science / Department of Clinical Sciences / Daniel U. Thomson / Bovine Respiratory Disease (BRD) continues to be one of the largest animal health concerns in the cattle industry. BRD is a multifaceted group of pathogens, both viral and bacterial, that take advantage of an immune compromised calf to cause disease. This study took aim at comparing metaphylactic treatments for BRD in both the feedlot and pasture setting. In the feedlot study, heifers (n=579, 403.7 ± 27.4 lbs) from Southwest Texas were identified as being high risk for BRD and shipped to the Clayton Livestock Research Center in Clayton, NM. Cattle were randomly allocated within truck load lots into 18 to 20 head treatment pens (30 pens; 3 treatments; 10 reps). Cattle were given one of three metaphylactic treatments based on the randomly assigned treatment for their pen within a replicate. The three antibiotic treatments administered at initial processing were: 1) Tulathromycin (2.5 mg/kg), 2) Tilmicosin (13.3 mg/kg), and 3) Gamithromycin (6.0 mg/kg). Cattle were fed a typical commercial starter diet for the first 56-60 d with a step-up ration change at day 28. At the end of the feeding period, pens were weighed and body weights recorded. Dry Matter Intake, morbidity, and mortality were recorded by CLRC personnel daily. Cattle administered tulathromycin had higher daily gains than cattle administered gamithromycin by 0.29 lbs/d (P<.01) and tended (P=0.09) have higher daily gains than cattle that received tilmicosin by 0.18 lbs/d. Tulathromycin treated cattle tended (P = 0.12) to have improved feed efficiency compared to gamithromycin treated cattle. Cattle that received tulathromycin (5.2%) had lower morbidity rates (P < .02) than tilmicosin (14.6%) and gamithromycin (12.79%) treated cattle. There were no treatment differences in dry matter intake or mortality in cattle. For the wheat pasture study, heifers (n=120, 393.2 ± 28.6 lbs) from the same origin and risk were shipped to the CLRC and processed before being trailed to a nearby wheat pasture. Cattle were randomly assigned into three treatment groups (3 treatments, 40 reps), and were given one of three metaphylactic treatments. The three antibiotic treatments administered at initial processing were: 1) Tulathromycin (2.5 mg/kg), 2) Tilmicosin (13.3 mg/kg), and 3) Gamithromycin (6.0 mg/kg). Cattle were allowed to graze on wheat for 54 days with free-choice Hi-Pro mineral mixed with Lasalocid, an ionophore. After 54 days on wheat pasture, the cattle were trailed back to the CLRC facilities and final individual weights were recorded. Morbidity and mortality were recorded daily by CLRC personnel. No differences were identified for ADG (P=0.98), morbidity (P=0.46) or mortality (P=0.36) among the three treatment groups.

Streptococcus pneumoniae is a leading cause of pneumonia, bacteremia, meningitis, otitis media and sinusitis, and is responsible for significant morbidity and mortality worldwide. The burden of pneumococcal disease has been greatly impacted by the high prevalence of HIV, especially in developing countries. Macrolides are commonly used for the treatment of pneumococcal infections with the resulting effect of increasing resistance. Pneumococci develop resistance to macrolides predominantly by two mechanisms; target modification and drug efflux. Target modification occurs through the acquisition of an erm(B) gene (MLSB phenotype) or through ribosomal mutation, and drug efflux occurs through the acquisition of a mef(A) gene (M phenotype). Alternative protein synthesis-inhibiting antibiotics such as linezolid and telithromycin have been developed in response to the increasing level of antibiotic resistance. In this study, novel mechanisms of resistance to protein synthesis-inhibiting antibiotics, and the current prevalence and epidemiology of macrolide resistance in South Africa were investigated. Two clinical isolates of S. pneumoniae resistant to macrolides, linezolid and chloramphenicol were identified in the PROTEKT surveillance study and the ABCs program of the CDC. The isolates were found to each contain a 6 bp deletion, resulting in the deletion of two amino acids from a highly conserved region of ribosomal protein L4 (64PWRQ67 to 64P_Q67 and 67QKGT70 to 67Q_T70). The genes encoding the mutant ribosomal proteins transformed susceptible strain R6 to macrolide, linezolid and chloramphenicol resistance, proving that the ii deletions conferred the resistance on the isolates, and indicating that these antibiotics share a common binding site. Growth studies of the R6 transformants showed increased mass doubling times, suggesting that the L4 mutations were associated with a fitness cost, but the original strains showed evidence of fitness compensation. The L4 mutations in these isolates represent a novel mechanism of cross-resistance to macrolides, linezolid and chloramphenicol. A macrolide-resistant clinical isolate of S. pneumoniae with mutations in 23S rRNA showed a heterogeneous phenotype and genotype. A mutant gene encoding 23S rRNA from this isolate transformed susceptible strain R6 to resistance. Transformants displayed similar heterogeneity to the isolate. Culture of resistant strain R6 in the presence of antibiotic maintained resistance, however culture of the strain in the absence of antibiotic pressure resulted in a reversion to susceptibility. By DNA sequencing, gene conversion was shown to occur between the wild-type and mutant 23S rRNA alleles. Growth studies indicated that the resistant phenotype was associated with a fitness cost. Therefore, under antibiotic selective pressure alleles converted to the mutant form, and in the absence of selective pressure alleles reverted to wild-type, in order to regain fitness. Through gene conversion the pneumococcus has the ability to rapidly adapt to the environment, with implications for susceptibility testing and patient treatment. A rare clinical isolate of S. pneumoniae, highly resistant to telithromycin, was received from the Canadian Bacterial Surveillance Network and was investigated for the mechanism of resistance. The isolate was found to contain an erm(B) gene iii with a truncated control peptide, as well as a mutant ribosomal protein L4, containing a number of mutations. Transformation of susceptible strain PC13, containing a wild-type erm(B) gene, with the mutant erm(B) gene decreased the susceptibility of PC13 to telithromycin, but did not confer high-level resistance. Transformation of PC13 with the mutant L4 gene or a fragment of the L4 gene containing only the 69GTG71 to TPS mutation, conferred high-level resistance on PC13. In contrast, transformation of R6, which did not contain an erm(B) gene, with the L4 gene or L4 fragment only conferred reduced telithromycin susceptibility. High-level telithromycin resistance was therefore conferred by a combination of an erm(B) gene with a 69GTG71 to TPS mutation in a highly conserved region of ribosomal protein L4. The combination of mechanisms inhibited the binding of telithromycin to the ribosome, whereas neither mechanism individually was sufficient. A telithromycin-resistant clinical isolate of S. pneumoniae was received from the PROTEKT surveillance study and was investigated for the resistance mechanism. The isolate was found to contain a 136 bp deletion in the regulatory region of erm(B). This mutant gene was shown, by transformation studies, to confer resistance on susceptible strain PC13. Expression of erm(B) on the transcriptional level was quantified by real-time reverse transcription PCR. In the presence of erythromycin and telithromycin, erm(B) expression was significantly higher in the mutant PC13 strain than the wild-type strain. Growth studies showed that the mutant PC13 strain had a shorter lag phase than the wild-type strain in the presence of erythromycin. Telithromycin resistance was conferred by the mutant iv erm(B) gene that was expressed at a higher level than the wild-type gene, most likely resulting in higher ribosomal methylation levels sufficient to hinder telithromycin binding. Macrolide resistance in invasive pneumococcal disease in South Africa for the period 2000 to 2005 was investigated through a national laboratory-based surveillance system. Viable isolates (n=15982) collected during the six-year period were phenotypically characterised, by determination of MICs and serotyping. Two hundred and sixty random isolates from 2005 were genotypically screened for the presence of erm(B) and mef(A). Macrolide resistance increased significantly from 9% in 2000 to 14% in 2005. Resistant isolates were received from all provinces of South Africa, with Gauteng and the Western Cape having the highest incidence. Serotype 14 was the most common macrolide-resistant serotype and 96% of macrolide-resistant isolates in 2005 were serotypes included in the 7-valent pneumococcal conjugate vaccine and serotype 6A. Macrolide resistance was significantly higher in children <5 than in individuals 5 years and older. The majority of strains (75%) over the six-year period displayed the MLSB phenotype. Of the 260 strains genotypically screened, 57% were positive for erm(B), 27% were positive for mef(A), 15% contained both erm(B) and mef(A), and 1% were negative for both genes and were found to contain ribosomal mutations. Eighty percent of isolates containing both erm(B) and mef(A) were serotype 19F and were found to be clonal by PFGE and MLST. These multidrug-resistant isolates were related to the Taiwan19F-14 global clone. v Many protein synthesis-inhibiting antibiotics share overlapping binding sites on the large ribosomal subunit. Alterations in 23S rRNA and ribosomal proteins L4 and L22, within the binding pocket, confer resistance and often cross-resistance to many of these antibiotics. The ability of the pneumococcus to develop resistance and the global spread of resistant strains highlights the importance of monitoring resistance levels and understanding resistance mechanisms.