Το ριβόσωμα παρέχει την πλατφόρμα πάνω στη οποία το mRNA αναγνωρίζεται και αποκωδικοποιείται από τα μεταφορικά RNAs. Δρα καταλυτικά ως ριβοένζυμο και συνθέτει την αντίστοιχη αλληλουχία αμινοξέων. Σημαντικά λειτουργικό κομμάτι του ριβοσώματος αποτελεί το τούνελ εξόδου, το οποίο βρίσκεται στη μεγάλη υπομονάδα του ριβοσώματος και αποτελεί το κανάλι, μέσω του οποίου οι νεοσυντιθέμενες πεπτιδικές αλυσίδες εξέρχονται στο κυτταροδιάλυμα. Νεότερες ερμηνείες προσδίδουν στο τούνελ εξόδου έναν πιο ενεργό ρόλο, καταδεικνύοντας τη σπουδαιότητά του όχι μόνο ως διάδρομος εξόδου της πεπτιδικής αλυσίδας, αλλά και ως λειτουργική περιοχή του ριβοσώματος με σημαντικό ρόλο στη ρύθμιση της πρωτεϊνικής σύνθεσης. Το δίκτυο επικοινωνίας μεταξύ τούνελ και πεπτιδυλοτρανσφεράσης δεν έχει ακόμα χαρτογραφηθεί και μελετηθεί πλήρως, υπάρχουν όμως μέχρι στιγμής πολυπληθείς αναφορές που επιβεβαιώνουν ότι μέρος του αποτελούν κυρίως βάσεις του 23S rRNA, συστατικά του τοιχώματος της εισόδου στο τούνελ, και βάσεις ευρισκόμενες στο κέντρο της πεπτιδυλοτρανσφεράσης, συντηρημένες εξελεικτικά, γνωστές από την αλληλεπιδρασή τους με αναστολείς της πεπτιδυλοτρανσφεράσης και κυρίως με αναστολείς της οικογένειας των μακρολιδίων. Στην παρούσα διατριβή μελετήσαμε την αλληλεπίδραση των χαρακτηριστικών αυτών βάσεων που συγκροτούν το δίκτυο επικοινωνίας μεταξύ του τούνελ εξόδου και της πεπτιδυλοτρανσφεράσης με τη βοήθεια νέων μακρολιδίων τρίτης γενιάς, των κετολιδίων, που φέρουν επί πλέον και άτομα φθορίου, παρέχοντας έτσι στο μόριο μεγαλύτερη συγγένεια για φορτισμένες ομάδες. Σύμφωνα με τα αποτελέσματά μας, αποτελούν εξαιρετικά υποσχόμενους αντιμικροβιακούς θεραπευτικούς παράγοντες, και αναστέλλουν ισχυρά την μετάφραση. Παράλληλα καταλαμβάνουν αμοιβαία αποκλειόμενες θέσεις στο ριβόσωμα, στην είσοδο του τούνελ, αλληλεπιδρώντας με τις βάσεις Α2058, Α2059, Α2062 και Α752. Επειδή όλες οι παραπάνω βάσεις αποτελούν μέρος του δικτύου επικοινωνίας τούνελ-πεπτιδυλοτρανσφεράσης, θα μπορούσαμε να συμπληρώσουμε τον ήδη υπάρχοντα μηχανισμό δράσης των μακρολιδίων (drop-off, λόγω αύξησης της koff) με την επικοινωνία του τούνελ με την πεπτιδυλοτρανσφεράση, ώστε να απενεργοποιηθεί η τελευταία με τελική κατάληξη τη διακοπή της πρωτεϊνικής σύνθεσης και την επαγωγή του drop-off. Για την περαιτέρω μελέτη της αλληλεπίδρασης του τούνελ με την νεοσυντιθέμενη πεπτιδική αλυσίδα, σχεδιάσθηκαν νέα πεπτιδυλο παράγωγα της χλωραμφανικόλης, τα οποία προσομοιάζουν πεπτιδυλο-tRNAs προσδεδεμένα στην Α-θέση του ριβοσώματος, με την ολιγοπεπτιδική αλληλουχία να εκτείνεται βαθύτερα στο τούνελ, υιοθετώντας μία ανοικτή διαμόρφωση. Όπως διαπιστώσαμε με συναγωνιστικές μελέτες με ραδιενεργό ερυθρομυκίνη, τα συγκεκριμένα ανάλογα καταλαμβάνουν την Α-θέση στο κέντρο της πεπτιδυλοτρανσφεράσης, ενώ η συγγένεια του κάθε αναλόγου ως αναστολέα της λειτουργίας του ριβοσώματος, είναι απόλυτα ιδιοσυγκρατική, καθώς εξαρτάται από την αλληλουχία των αμινοξέων που συγκροτούν το πεπτίδιο. Μελετώντας το αποτύπωμα των ενώσεων αυτών στο ριβόσωμα, διαπιστώσαμε ότι ενώ η βάση της χλωραμφαινικόλης διατηρεί τη θέση της στη περιοχή Α, το πεπτίδιο εισέρχεται βαθειά στην είσοδο του τούνελ, αλληλεπιδρώντας με την βάση Α752. Συνεπώς τα πεπτιδυλο ανάλογα της χλωραμφαινικόλης, τα οποία συμπεριφέρονται ως ισχυροί αναστολείς της μετάφρασης, αποτελούν κατάλληλα εργαλεία μελέτης της αλληλεπίδρασης μεταξύ της νεοσυντιθέμενης πεπτιδικής αλληλουχίας και στοιχείων του τούνελ εξόδου του ριβοσώματος. / Ribosomes translate the genetic code into proteins in all living cells with extremely high efficiency, owing to their inherent flexibility and to their spectacular architecture. These large ribonucleoprotein particles synthesize proteins in all cells, using messenger RNA as the template, confirming that the ribosome is indeed a ribozyme, and aminoacyl-transfer RNAs as substrates. As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. New studies indicate that in some cases, the tunnel plays a more active role. Accumulated evidence had definitely concluded that ribosomal tunnel is not only a passive conduit for the nascent chains but a rather functionally important compartment, where specific peptide sequences establish direct interactions with the tunnel, talking back to the ribosome in order to regulate peptide synthesis, leading to translational stalling. In this study, we have investigated functional interactions between distinct locations of the ribosomal tunnel and specific residues of the peptidyltransferase, using new macrolides, known as ketolides, which carry also fluorine attached to the lactone ring either directly or indirectly. According to our results, the new antibiotics exhibited better antimicrobial activity, inhibiting strongly the translational apparatus and could be useful tools in the future for treatment of bacterial infections. Binding studies with radiolabeled erythromycin revealed that these drugs bound competitively with erythromycin at the large ribosomal subunit, with extremely low dissociation constants. In parallel, RNA footprinting indicated that the new ketolides occupy the main macrolide binding site in the ribosome, which is located at the entrance of the exit tunnel adjacent to the peptidyltransferase center. These drugs interact with A2058, A2059 and A2062, as well as their extended heteroaromatic alkyl-aryl side chain penetrates deeper in the tunnel protecting A752 of Helix 35, interacting with basepair A752-U2609. To explore further this interaction, we have synthesized new peptidyl conjugates of chloramphenicol, which resemble nascent peptidyl-tRNA chains bound to the A-site of the ribosome, with their peptide sequence located deeper within tunnel, adopting an extended configuration. According to our data, these compounds did indeed bind to the peptidyl transferase center, competing with radiolabelled- chloramphenicol for binding to the ribosome. The binding of each analog, as well as its inhibitory activity of ribosomal function, is absolutely idiosyncratic, depending on the sequence of amino acid of peptide moiety. Studying the footprinting pattern of these compounds on the ribosome, we confirmed that while the chloramphenicol base maintains its position on the A-site, the peptide moiety penetrates deeper in the entrance of the exit tunnel, interacting with nucleotide A752. Therefore, we believe that these chloramphenicol peptides could be useful tools for probing nascent polypeptide chain interaction with the ribosome.

O objetivo principal dessa tese foi avaliar se o tratamento com ceftriaxona e um macrolídeo leva a melhores desfechos quando comparada a monoterapia com ceftriaxona em pacientes hospitalizados com HIV/AIDS e pneumonia adquirida na comunidade (PAC). 227 adultos com HIV hospitalizados por uma suspeita de PAC foram randomizados numa proporção 1:1 para receber um dos dois regimes, ceftriaxona mais macrolídeo ou ceftriaxona mais placebo. Houve 2 exclusões após a randomização, um paciente retirou consentimento para uso de seus dados e outro paciente já havia sido incluído previamente no estudo, perfazendo um total de 225 pacientes analisados (112 receberam ceftriaxona mais placebo e 113 receberam ceftriaxona mais macrolídeo). Os pacientes tinham HIV há um longo tempo (período mediano de 10 anos) e a maioria não fazia uso regular de terapia antirretroviral. Somente 32/202 pacientes (16%) tinham carga viral menor que 50 cópias/mL e 146/202 (72%) tinham contagem de linfócitos T CD4+ menor que 200 células/mm³. A frequência do desfecho primário, letalidade durante a internação, não foi estatisticamente distinta entre os regimes estudados: 12/112 (11%) pacientes que receberam ceftriaxona mais placebo e 17/113 (15%) que receberam ceftriaxona mais macrolídeo foram a óbito durante a hospitalização (HR: 1.22, 95% CI: 0.57-2.59). Não foram encontradas diferenças entre os regimes para os desfechos secundários: letalidade em 14 dias (RR: 2.38, 95% CI: 0.87-6.53), uso de drogas vasoativas (OR: 1.18, 95% CI: 0.60-2.29) e ventilação mecânica (OR: 1.24, 95% CI: 0.64- 2.40). A etiologia das infecções pulmonares adquiridas na comunidade nesses pacientes com infecção pelo HIV também foi estudada e determinada prospectivamente. Essa investigação também buscou analisar a contribuição de diferentes métodos diagnósticos e o impacto de diferentes abordagens de investigação microbiológica. Além disso, os achados microbiológicos foram analisados levando em consideração a contagem de linfócitos T CD4+, gravidade da doença e a situação da vacina pneumocócica. 224 pacientes foram submetidos a investigação microbiológica estendida e 143 (64%) tiveram uma etiologia determinada. Por outro lado, a investigação microbiológica de rotina foi capaz de determinar o agente etiológico em 92 (41%) pacientes. Métodos baseados na reação em cadeia da polimerase foram essenciais para o diagnóstico de bactérias atípicas e vírus, além de melhorar a detecção de Pneumocystis jirovecii. Entre os 143 pacientes com uma etiologia determinada, Pneumocystis jirovecii foi o principal agente, detectado em 52 (36%) casos, seguido pelo Mycobacterium tuberculosis responsável por 28 (20%) casos. Streptococcus pneumoniae e Rhinovírus foram diagnosticados em 22 (15%) casos cada e Influenza em 15 (10%) casos. Entre as bactérias atípicas, Mycoplasma pneumoniae foi responsável por 12 (8%) e Chlamydophila pneumoniae por 7 (5%) casos. Infecções mistas ocorreram em 48 casos (34%). Streptococcus pneumoniae foi associado com maiores escores de gravidade, sem associação com o estado vacinal. A análise de agentes etiológicos baseada na contagem de linfócitos T CD4+ demonstrou que a etiologia da pneumonia nos pacientes que estavam gravemente imunossuprimidos (CD4+ < 200 células/mm³) foi similar aos que não estavam. Pneumocystis jirovecii foi o único agente mais frequente no primeiro grupo, um achado esperado levando em consideração os critérios diagnósticos empregados / The main purpose of this thesis was to evaluate if treatment with ceftriaxone and a macrolide improved patient outcome when compared with monotherapy with ceftriaxone in hospitalized patients with HIV/AIDS with community acquired pneumonia (CAP). 227 adult patients with HIV hospitalized due to suspected CAP were randomized to receive one of two regimens, ceftriaxone plus macrolide or ceftriaxone plus placebo, at a 1:1 proportion. We had 2 exclusions after randomization, one patient who withdrew consent for data inclusion and use and one that had previously been included, leaving a total of 225 patients to analyse (112 received ceftriaxone plus placebo and 113 received ceftriaxone plus macrolide). Patients had prolonged HIV infection, the median period was twelve years, and most of them did not make regular use of antiretroviral therapy. Only 32/202 patients (16%) had viral load below 50 copies/mL and 146/202 (72%) had a CD4+ T cell count below 200 cells/mm³. The frequency of the primary outcome, in-hospital mortality, was not statistically different between the studied regimens: 12/112 (11%) patients who received ceftriaxone plus placebo and 17/113 (15%) who received ceftriaxone plus macrolide died during hospitalization (HR: 1.22, 95% CI: 0.57-2.59). We did not find differences between the regimens for the secondary outcomes: mortality within 14 days (RR: 2.38, 95% CI: 0.87-6.53), need for vasoactive drug (OR: 1.18, 95% CI: 0.60-2.29) or mechanical ventilation (OR: 1.24, 95% CI: 0.64-2.40). The etiology of community-acquired pulmonary infections in these hospitalized patients with HIV was also studied and determined prospectively. This investigation also aimed to analyze the contribution of different diagnostic methods as well of the impact of different approaches to microbiological evaluation and to evaluate the microbiological findings in relation to the CD4+ T cell count, the severity of disease and pneumococcal vaccine status. 224 patients underwent the extended microbiological investigation of which 143 (64%) had an etiology determined. On the other hand, the microbiological routine investigation was able to determine the etiological agents in 92 (41%) patients. Polymerase chain reaction-based methods were essential for the diagnosis of atypical bacteria and viruses, besides contributing to ameliorate Pneumocystis jirovecii detection. Among the 143 patients with a determined etiology, Pneumocystis jirovecii was the main agent, detected in 52 (36%) cases and followed by Mycobacterium tuberculosis accounting for 28 (20%) cases. Streptococcus pneumoniae and Rhinovirus were diagnosed in 22 (15%) cases each and Influenza in 15 (10%) cases. Among atypical bacteria, Mycoplasma pneumoniae was responsible for 12 (8%) and Chlamydophila pneumoniae for 7 (5%) cases. Mixed infections occurred in 48 cases (34%). Streptococcus pneumoniae was associated with higher severity scores and not associated with vaccine status. Performing an analysis of causative agents based on CD4+ T cell count, we found that the etiology of pneumonia in those severely immunosuppressed (CD4+ < 200 cells/mm³) was similar to those who were not. Pneumocystis jirovecii is the only agent more frequent in the former group, an expected finding considering our diagnostic criteria

As micobactérias de crescimento rápido causam infecções pulmonares, infecções relacionadas a traumas cutâneos e aos cuidados com a saúde. Ha um número reduzido de opções terapêuticas para o tratamento dessas infecções e os dados nacionais sobre os determinantes genéticos dessa resistência são escassos. Duas classes de antimicrobianos importantes no tratamento são as fluorquinolonas e os macrolídeos. Enquanto a maioria dos isolados do Grupo M. chelonae-abscessus apresenta resistência intrínseca às fluorquinolonas, e são sensíveis aos macrolídeos, a maioria dos isolados do Grupo M. fortuitum é sensível às fluorquinolonas e expressa RNA-metilases que impedem a ação dos macrolídeos. Os determinantes da resistência às fluorquinolonas conhecidos em micobactérias são mutações no gene gyrA e para os macrolídeos é a expressão de genes erm, que codificam RNA-metilases. Nos dois casos os determinantes tem localização cromossômica. Outros determinantes de resistência podem ter localização plasmidial, a exemplo do gene aacA4\'-8, presente em plasmídio do grupo de incompatibilidade IncP, detectado em M. abscessus, que codifica resistência à kanamicina e tobramicina. Neste estudo foram avaliados: a correlação entre à susceptibilidade ao ciprofloxacino e moxifloxacino e presença de mutações nos genes gyrA e gyrB; a susceptibilidade à claritromicina e presença de genes que codificam RNA-metilases; a presença de plasmídios do grupo de incompatibilidade IncP em diferentes espécies de micobactérias; a estabilidade e a transferência por conjugação do plasmídio pBRA-100; a relação clonal entre isolados que albergam plasmídios IncP e o perfil de susceptibilidade antimicrobiana de uma coleção de isolados da espécie M. abscessus. Cento e vinte e dois isolados dos Grupos M. abscessus e M. fortuitum foram analisados quanto às concentrações inibitórias mínimas, por microdiluição, utilizando-se o painel RAPMYCO&#174;. As sequências parciais dos genes gyrA e gyrB e a presença de genes que codificam RNA-metilases foram determinadas em 69 e 59 desses isolados, respectivamente. A presença de plasmídios do grupo IncP foi determinada por PCR e os produtos de amplificação tiveram suas sequencias caracterizadas. A estabilidade do plasmídio pBRA-100 em Escherichia coli TOP10&#174; foi avaliada realizando-se repiques sucessivos por 57 dias e semeadura em agar LB contendo kanamicina. A transferência horizontal foi avaliada por ensaio de conjugação com E. coli J53 resistente à azida sódica. Em M. abscessus não houve diferença entre as sequencias da região QRDR de GyrA e GyrB entre isolados sensíveis e resistentes ao ciprofloxacino. Em M. abscessus subsp. abscessus e M. abscessus subsp. bolletii todos os isolados testados apresentaram genes erm; entretanto não foi observada correlação entre a presença dos genes e resistência indutiva a claritromicina. O gene erm(45), descrito neste trabalho, foi detectado apenas em M. abscessus subsp. bolletii. Os ensaios de microdiluição evidenciaram que os antimicrobianos mais ativos contra M. abscessus subsp. abscessus foram amicacina (89%), tigeciclina (96%) e claritromicina (86%). Apenas nos dois isolados da espécie M. fortuitum resistentes ao ciprofloxacino foi detectada a substituição S83W na proteína GyrA. Trata-se de substituição ainda não descrita em micobactérias. Nao foi observada correlação entre substituições em GyrB e resistência ao ciprofloxacino ou moxifloxacino. O gene erm(46), descrito neste estudo, foi detectado em 65% dos isolados. Nos demais isolados foram detectados três outros genes: erm(47), erm(48) e erm(49), ainda nao descritos. Os plasmídios IncP foram detectados apenas em \"M. massiliense\" pertencentes a três grupos clonais distintos com índice de similaridade maior que 92%, quando analisados por ERIC-PCR. Houve sucesso na conjugação entre E. coli TOP-Myco e E. coli J53. Os ensaios de estabilidade evidenciaram que o plasmídio pBRA-100 é estável em E. coli com aproximadamente 100% da população bacteriana albergando o plasmídio após 57 subcultivos. / The rapidly growing mycobacteria cause lung infections, skin infections related to trauma and health care associated infections. There are a limited number of therapeutic options for treating these infections and national data on the genetic determinants of this resistance are scarce. Two major classes of antimicrobials used in treatment of these infections are macrolides and fluoroquinolones. While the majority of the isolates from M. chelonae-abscessus Group have intrinsic resistance to fluoroquinolones, and are sensitive to macrolides, most isolates from M. fortuitum Group are sensitive to fluoroquinolones and express RNA methylases that prevent the action of macrolides. The known fluoroquinolones resistance determinants in to mycobacteria are mutations in thegyrA gene and for macrolides the main determinant is the expression of erm genes which encode RNA methylases. In both cases the determinants are located at the chromosome. Other resistance determinants may have a plasmidial location, such as aacA4\'-8 gene, present in the pBRA100 plasmid detected in M. abscessus, which encodes resistance to kanamycin and tobramycin. This study evaluated the correlation between the susceptibility to ciprofloxacin and moxifloxacin and mutations in genes gyrA and gyrB; susceptibility to clarithromycin and the presence of RNA-methylases encodinggenes , the presence of plasmids of the incompatibility group IncP in different species mycobacteria; stability and conjugal transfer of plasmid pBRA-100; the clonal relationship between isolates harboring plasmids IncP and antimicrobial susceptibility profile of a collection of isolates of M. abscessus. One hundred and twenty-two isolates of M. chenolae-abscessus and M. fortuitum Groups were analyzed for their minimal inhibitory concentrations by microdilution, using the RAPMYCO&#174; Panel. The partial sequences of the genes gyrA and gyrB and the presence of genes encoding RNA methylases were determined in 69 and 59 isolates, respectively. The presence of IncP group plasmids was determined by PCR and the sequence of PCR products were characterized. The stability of the pBRA-100 plasmid in Escherichia coli TOP10&#174; was evaluated by performing successive subcultures for 57 days, and plating on LB agar containing kanamycin. The horizontal transfer was evaluated by conjugation with E. coli J53 a sodium azide resistant strain. In M. abscessus no differences between the sequences of the QRDR region of gyrA or gyrB among isolates susceptible and resistant to ciprofloxacin were observed. In M. abscessus subsp. abscessus and M. abscessus subsp. bolletii all isolates tested had erm genes; however there was no correlation between the presence of these genes and inducible resistance to clarithromycin. The erm(45), gene described in this paper, was only detected in M. abscessus subsp. bolletii. Microdilution tests showed that the most active antibiotics against M. abscessus subsp. abscessus were amikacin (89%), tigecycline (96%) and clarithromycin (86%). In only in two M. fortuitum isolates resistant to ciprofloxacin the amino acid substitution S83W was detected in GyrA. This substitution had not been described mycobacteria. No correlation was observed between substitutions in GyrB and resistance to ciprofloxacin or moxifloxacin. The erm(46) gene described in this study, was detected in 65% of the isolates. In the remaining isolates other three not yet described genes were detected: erm(47), erm(48) and erm(49). IncP plasmids were detected only in \"M. massiliense\" belonging to three clonal groups with at similarity index greater than 92% when analyzed by ERIC-PCR.We succeeded in conjugating E. coli TOP-Myco and E. coli J53. The stability tests showed that the plasmid pBRA-100 is stable in E. coli since approximately 100% of the bacterial population harbored the plasmid after 57 subcultures.

L'observation que nous avons faite d'une plus grande fréquence de souches de Staphylococcus aureus hypermutables isolées chez les patients atteints de mucoviscidose que chez un panel de souches témoins nous a amené à étudier les mécanismes de l'hypermutabilité chez cette espèce. L'analyse des gènes du système de réparation des mésappariements, mutS et mutL, a montré pour quatre souches cliniques une relation de cause à effet entre l'altération de ces gènes et leur phénotype hypermutable. Nous avons montré que ces gènes étaient co-transcrits chez S. aureus et que l'invalidation de l'un ou l'autre conduisait à un phénotype hypermutable. Nous avons mis au point un modèle pour l'étude de l'effet de ces gènes sur la recombinaison chez S. aureus, qui semble montrer que tous deux n'ont qu'un rôle limité dans le phénomène chez cette espèce. Cette propriété d'hypermutabilité a aussi un impact sur la résistance aux antibiotiques des souches. En effet, la résistance aux antibiotiques du groupe des macrolides est de plus en plus fréquente chez les souches de S. aureus isolées lors de la mucoviscidose, et nous avons montré qu'elle n'était pas due à des gènes de résistance portés par des plasmides ou des transposons mais, de façon très inhabituelle, à des mutations de la cible ribosomale des macrolides. Nous avons ainsi mis en évidence des mutations dans les gènes rrl (codant l'ARN ribosomal), rplD et rplV (codant respectivement les protéines ribosomales L4 et L22).

O objetivo principal dessa tese foi avaliar se o tratamento com ceftriaxona e um macrolídeo leva a melhores desfechos quando comparada a monoterapia com ceftriaxona em pacientes hospitalizados com HIV/AIDS e pneumonia adquirida na comunidade (PAC). 227 adultos com HIV hospitalizados por uma suspeita de PAC foram randomizados numa proporção 1:1 para receber um dos dois regimes, ceftriaxona mais macrolídeo ou ceftriaxona mais placebo. Houve 2 exclusões após a randomização, um paciente retirou consentimento para uso de seus dados e outro paciente já havia sido incluído previamente no estudo, perfazendo um total de 225 pacientes analisados (112 receberam ceftriaxona mais placebo e 113 receberam ceftriaxona mais macrolídeo). Os pacientes tinham HIV há um longo tempo (período mediano de 10 anos) e a maioria não fazia uso regular de terapia antirretroviral. Somente 32/202 pacientes (16%) tinham carga viral menor que 50 cópias/mL e 146/202 (72%) tinham contagem de linfócitos T CD4+ menor que 200 células/mm³. A frequência do desfecho primário, letalidade durante a internação, não foi estatisticamente distinta entre os regimes estudados: 12/112 (11%) pacientes que receberam ceftriaxona mais placebo e 17/113 (15%) que receberam ceftriaxona mais macrolídeo foram a óbito durante a hospitalização (HR: 1.22, 95% CI: 0.57-2.59). Não foram encontradas diferenças entre os regimes para os desfechos secundários: letalidade em 14 dias (RR: 2.38, 95% CI: 0.87-6.53), uso de drogas vasoativas (OR: 1.18, 95% CI: 0.60-2.29) e ventilação mecânica (OR: 1.24, 95% CI: 0.64- 2.40). A etiologia das infecções pulmonares adquiridas na comunidade nesses pacientes com infecção pelo HIV também foi estudada e determinada prospectivamente. Essa investigação também buscou analisar a contribuição de diferentes métodos diagnósticos e o impacto de diferentes abordagens de investigação microbiológica. Além disso, os achados microbiológicos foram analisados levando em consideração a contagem de linfócitos T CD4+, gravidade da doença e a situação da vacina pneumocócica. 224 pacientes foram submetidos a investigação microbiológica estendida e 143 (64%) tiveram uma etiologia determinada. Por outro lado, a investigação microbiológica de rotina foi capaz de determinar o agente etiológico em 92 (41%) pacientes. Métodos baseados na reação em cadeia da polimerase foram essenciais para o diagnóstico de bactérias atípicas e vírus, além de melhorar a detecção de Pneumocystis jirovecii. Entre os 143 pacientes com uma etiologia determinada, Pneumocystis jirovecii foi o principal agente, detectado em 52 (36%) casos, seguido pelo Mycobacterium tuberculosis responsável por 28 (20%) casos. Streptococcus pneumoniae e Rhinovírus foram diagnosticados em 22 (15%) casos cada e Influenza em 15 (10%) casos. Entre as bactérias atípicas, Mycoplasma pneumoniae foi responsável por 12 (8%) e Chlamydophila pneumoniae por 7 (5%) casos. Infecções mistas ocorreram em 48 casos (34%). Streptococcus pneumoniae foi associado com maiores escores de gravidade, sem associação com o estado vacinal. A análise de agentes etiológicos baseada na contagem de linfócitos T CD4+ demonstrou que a etiologia da pneumonia nos pacientes que estavam gravemente imunossuprimidos (CD4+ < 200 células/mm³) foi similar aos que não estavam. Pneumocystis jirovecii foi o único agente mais frequente no primeiro grupo, um achado esperado levando em consideração os critérios diagnósticos empregados / The main purpose of this thesis was to evaluate if treatment with ceftriaxone and a macrolide improved patient outcome when compared with monotherapy with ceftriaxone in hospitalized patients with HIV/AIDS with community acquired pneumonia (CAP). 227 adult patients with HIV hospitalized due to suspected CAP were randomized to receive one of two regimens, ceftriaxone plus macrolide or ceftriaxone plus placebo, at a 1:1 proportion. We had 2 exclusions after randomization, one patient who withdrew consent for data inclusion and use and one that had previously been included, leaving a total of 225 patients to analyse (112 received ceftriaxone plus placebo and 113 received ceftriaxone plus macrolide). Patients had prolonged HIV infection, the median period was twelve years, and most of them did not make regular use of antiretroviral therapy. Only 32/202 patients (16%) had viral load below 50 copies/mL and 146/202 (72%) had a CD4+ T cell count below 200 cells/mm³. The frequency of the primary outcome, in-hospital mortality, was not statistically different between the studied regimens: 12/112 (11%) patients who received ceftriaxone plus placebo and 17/113 (15%) who received ceftriaxone plus macrolide died during hospitalization (HR: 1.22, 95% CI: 0.57-2.59). We did not find differences between the regimens for the secondary outcomes: mortality within 14 days (RR: 2.38, 95% CI: 0.87-6.53), need for vasoactive drug (OR: 1.18, 95% CI: 0.60-2.29) or mechanical ventilation (OR: 1.24, 95% CI: 0.64-2.40). The etiology of community-acquired pulmonary infections in these hospitalized patients with HIV was also studied and determined prospectively. This investigation also aimed to analyze the contribution of different diagnostic methods as well of the impact of different approaches to microbiological evaluation and to evaluate the microbiological findings in relation to the CD4+ T cell count, the severity of disease and pneumococcal vaccine status. 224 patients underwent the extended microbiological investigation of which 143 (64%) had an etiology determined. On the other hand, the microbiological routine investigation was able to determine the etiological agents in 92 (41%) patients. Polymerase chain reaction-based methods were essential for the diagnosis of atypical bacteria and viruses, besides contributing to ameliorate Pneumocystis jirovecii detection. Among the 143 patients with a determined etiology, Pneumocystis jirovecii was the main agent, detected in 52 (36%) cases and followed by Mycobacterium tuberculosis accounting for 28 (20%) cases. Streptococcus pneumoniae and Rhinovirus were diagnosed in 22 (15%) cases each and Influenza in 15 (10%) cases. Among atypical bacteria, Mycoplasma pneumoniae was responsible for 12 (8%) and Chlamydophila pneumoniae for 7 (5%) cases. Mixed infections occurred in 48 cases (34%). Streptococcus pneumoniae was associated with higher severity scores and not associated with vaccine status. Performing an analysis of causative agents based on CD4+ T cell count, we found that the etiology of pneumonia in those severely immunosuppressed (CD4+ < 200 cells/mm³) was similar to those who were not. Pneumocystis jirovecii is the only agent more frequent in the former group, an expected finding considering our diagnostic criteria

As micobactérias de crescimento rápido causam infecções pulmonares, infecções relacionadas a traumas cutâneos e aos cuidados com a saúde. Ha um número reduzido de opções terapêuticas para o tratamento dessas infecções e os dados nacionais sobre os determinantes genéticos dessa resistência são escassos. Duas classes de antimicrobianos importantes no tratamento são as fluorquinolonas e os macrolídeos. Enquanto a maioria dos isolados do Grupo M. chelonae-abscessus apresenta resistência intrínseca às fluorquinolonas, e são sensíveis aos macrolídeos, a maioria dos isolados do Grupo M. fortuitum é sensível às fluorquinolonas e expressa RNA-metilases que impedem a ação dos macrolídeos. Os determinantes da resistência às fluorquinolonas conhecidos em micobactérias são mutações no gene gyrA e para os macrolídeos é a expressão de genes erm, que codificam RNA-metilases. Nos dois casos os determinantes tem localização cromossômica. Outros determinantes de resistência podem ter localização plasmidial, a exemplo do gene aacA4\'-8, presente em plasmídio do grupo de incompatibilidade IncP, detectado em M. abscessus, que codifica resistência à kanamicina e tobramicina. Neste estudo foram avaliados: a correlação entre à susceptibilidade ao ciprofloxacino e moxifloxacino e presença de mutações nos genes gyrA e gyrB; a susceptibilidade à claritromicina e presença de genes que codificam RNA-metilases; a presença de plasmídios do grupo de incompatibilidade IncP em diferentes espécies de micobactérias; a estabilidade e a transferência por conjugação do plasmídio pBRA-100; a relação clonal entre isolados que albergam plasmídios IncP e o perfil de susceptibilidade antimicrobiana de uma coleção de isolados da espécie M. abscessus. Cento e vinte e dois isolados dos Grupos M. abscessus e M. fortuitum foram analisados quanto às concentrações inibitórias mínimas, por microdiluição, utilizando-se o painel RAPMYCO&#174;. As sequências parciais dos genes gyrA e gyrB e a presença de genes que codificam RNA-metilases foram determinadas em 69 e 59 desses isolados, respectivamente. A presença de plasmídios do grupo IncP foi determinada por PCR e os produtos de amplificação tiveram suas sequencias caracterizadas. A estabilidade do plasmídio pBRA-100 em Escherichia coli TOP10&#174; foi avaliada realizando-se repiques sucessivos por 57 dias e semeadura em agar LB contendo kanamicina. A transferência horizontal foi avaliada por ensaio de conjugação com E. coli J53 resistente à azida sódica. Em M. abscessus não houve diferença entre as sequencias da região QRDR de GyrA e GyrB entre isolados sensíveis e resistentes ao ciprofloxacino. Em M. abscessus subsp. abscessus e M. abscessus subsp. bolletii todos os isolados testados apresentaram genes erm; entretanto não foi observada correlação entre a presença dos genes e resistência indutiva a claritromicina. O gene erm(45), descrito neste trabalho, foi detectado apenas em M. abscessus subsp. bolletii. Os ensaios de microdiluição evidenciaram que os antimicrobianos mais ativos contra M. abscessus subsp. abscessus foram amicacina (89%), tigeciclina (96%) e claritromicina (86%). Apenas nos dois isolados da espécie M. fortuitum resistentes ao ciprofloxacino foi detectada a substituição S83W na proteína GyrA. Trata-se de substituição ainda não descrita em micobactérias. Nao foi observada correlação entre substituições em GyrB e resistência ao ciprofloxacino ou moxifloxacino. O gene erm(46), descrito neste estudo, foi detectado em 65% dos isolados. Nos demais isolados foram detectados três outros genes: erm(47), erm(48) e erm(49), ainda nao descritos. Os plasmídios IncP foram detectados apenas em \"M. massiliense\" pertencentes a três grupos clonais distintos com índice de similaridade maior que 92%, quando analisados por ERIC-PCR. Houve sucesso na conjugação entre E. coli TOP-Myco e E. coli J53. Os ensaios de estabilidade evidenciaram que o plasmídio pBRA-100 é estável em E. coli com aproximadamente 100% da população bacteriana albergando o plasmídio após 57 subcultivos. / The rapidly growing mycobacteria cause lung infections, skin infections related to trauma and health care associated infections. There are a limited number of therapeutic options for treating these infections and national data on the genetic determinants of this resistance are scarce. Two major classes of antimicrobials used in treatment of these infections are macrolides and fluoroquinolones. While the majority of the isolates from M. chelonae-abscessus Group have intrinsic resistance to fluoroquinolones, and are sensitive to macrolides, most isolates from M. fortuitum Group are sensitive to fluoroquinolones and express RNA methylases that prevent the action of macrolides. The known fluoroquinolones resistance determinants in to mycobacteria are mutations in thegyrA gene and for macrolides the main determinant is the expression of erm genes which encode RNA methylases. In both cases the determinants are located at the chromosome. Other resistance determinants may have a plasmidial location, such as aacA4\'-8 gene, present in the pBRA100 plasmid detected in M. abscessus, which encodes resistance to kanamycin and tobramycin. This study evaluated the correlation between the susceptibility to ciprofloxacin and moxifloxacin and mutations in genes gyrA and gyrB; susceptibility to clarithromycin and the presence of RNA-methylases encodinggenes , the presence of plasmids of the incompatibility group IncP in different species mycobacteria; stability and conjugal transfer of plasmid pBRA-100; the clonal relationship between isolates harboring plasmids IncP and antimicrobial susceptibility profile of a collection of isolates of M. abscessus. One hundred and twenty-two isolates of M. chenolae-abscessus and M. fortuitum Groups were analyzed for their minimal inhibitory concentrations by microdilution, using the RAPMYCO&#174; Panel. The partial sequences of the genes gyrA and gyrB and the presence of genes encoding RNA methylases were determined in 69 and 59 isolates, respectively. The presence of IncP group plasmids was determined by PCR and the sequence of PCR products were characterized. The stability of the pBRA-100 plasmid in Escherichia coli TOP10&#174; was evaluated by performing successive subcultures for 57 days, and plating on LB agar containing kanamycin. The horizontal transfer was evaluated by conjugation with E. coli J53 a sodium azide resistant strain. In M. abscessus no differences between the sequences of the QRDR region of gyrA or gyrB among isolates susceptible and resistant to ciprofloxacin were observed. In M. abscessus subsp. abscessus and M. abscessus subsp. bolletii all isolates tested had erm genes; however there was no correlation between the presence of these genes and inducible resistance to clarithromycin. The erm(45), gene described in this paper, was only detected in M. abscessus subsp. bolletii. Microdilution tests showed that the most active antibiotics against M. abscessus subsp. abscessus were amikacin (89%), tigecycline (96%) and clarithromycin (86%). In only in two M. fortuitum isolates resistant to ciprofloxacin the amino acid substitution S83W was detected in GyrA. This substitution had not been described mycobacteria. No correlation was observed between substitutions in GyrB and resistance to ciprofloxacin or moxifloxacin. The erm(46) gene described in this study, was detected in 65% of the isolates. In the remaining isolates other three not yet described genes were detected: erm(47), erm(48) and erm(49). IncP plasmids were detected only in \"M. massiliense\" belonging to three clonal groups with at similarity index greater than 92% when analyzed by ERIC-PCR.We succeeded in conjugating E. coli TOP-Myco and E. coli J53. The stability tests showed that the plasmid pBRA-100 is stable in E. coli since approximately 100% of the bacterial population harbored the plasmid after 57 subcultures.

Human immunodeficiency virus (HIV) infection has reached epidemic proportions in South Africa. The availability of highly active anti-retroviral therapy (HAART) prolongs life in HIV-infected persons, who may subsequently present with chronic manifestations of HIV-infection. The respiratory morbidity attendant to HIV-infection, even in the presence of HAART is high, the aftermath of which is lung tissue destruction and bronchiectasis. As a consequence of the political decision not to offer HAART to HIV-infected children, a number of children in South Africa have been left with severe consequences of uncontrolled HIV-infection. Bronchiectasis is one of those and because children with this devastating condition were numerous in the Pretoria region, the author and her colleagues began a Chronic Lung Disease Clinic in that region. This prompted the idea of investigating both the epidemiological profiles of these children and an attempt to intervene with both standard bronchiectasis guideline care and the use of a form of therapy commonly employed in other forms of bronchiectasis. This thesis explores those ideas. Important new and novel findings that were consequent were; that bronchiectasis is diagnosed late in HIV-infected children at a mean age of 6.9 years. The predominant organisms cultured from the airways are Haemophilus influenzae and parainfluenzae in 49% of samples. Pseudomonas aeruginosa (PA), common in cystic fibrosis (CF)-bronchiectasis is an uncommon pathogen in HIV-related bronchiectasis; isolated in only 2% of specimens. Tuberculosis (TB), at least as reported, is a significant antecedent of bronchiectasis, reported in 48.5%of children. A further 21.2% of the patients had received more than two courses of anti-TB treatment. However, proof of TB infection has been lacking. Respiratory morbidity is significant with the mean forced expiratory flow in one second (FEV1) of 53%, in this cohort at the time of presentation. Thirty-six percent of all children were exposed to environmental tobacco smoke, although this was not correlated with disease severity or HIVdisease progression. There is elevation of immunoglobulins in HIV-related bronchiectasis, with a mean IgE of 79 kU/l. This was not, though, associated with HIV disease progression as previously described in adult studies, nor with the presence of allergic bronchopulmonary aspergillosis (ABPA). The elevation in IgE was also not associated with an elevation of T helper-2 mediated cytokines, confirming the lack of association with atopy. The predominant cytokine, identified is interleukin (IL)-8, both systemically and locally (in airway secretions). There was elevation of other T helper-1 driven cytokines, reflecting an ability to mediate adequate inflammatory responses, which was independent of the level of immunosuppression. With the presence of HAART, there was a decline in the pro-inflammatory cytokines over time, which may be attributed to the ongoing effect of HAART that ties in to, or goes beyond the restoration of T cell numbers. Soluble triggering receptor expressed on myeloid cells (sTREM), an innate immune marker, is elevated in children with HIV-related bronchiectasis when compared to a control group of children with cystic fibrosis-related bronchiectasis. sTREM is not associated with the presence of exacerbations and the level of immunosuppression. The use of an anti-inflammatory drug erythromycin also did not impact the sTREM values. There was also no relationship between sTREM and pro and antiinflammatory cytokines and chemokines. Fluorine-18-fluorodeoxyglucose positron emission tomography (18F-FDG PET) could not reliably predict the presence of pulmonary exacerbations. Its diagnostic value was limited to identifying disease activity in acute pneumonia. 18F-FDG PET also had no significant correlation with CRP, inflammatory cytokines or markers of HIV disease activity. In a randomised controlled trial of erythromycin, a cost-effective immunomodulatory drug, compared to placebo, erythromycin was ineffective in reducing the number of pulmonary exacerbations. Erythromycin also failed to demonstrate any effect on systemic and local pro- and anti-inflammatory cytokines/chemokines. With access to anti-retroviral therapy, airway clearance, nutritional rehabilitation and vigilant follow up there was an improvement in pulmonary function parameters and stability of the degree of bronchiectasis that we propose is probably in keeping with an organ system disease modifying effect that may be, an as yet, undefined and undescribed byproduct of HAART. / Thesis (PhD)--University of Pretoria, 2012. / Paediatrics and Child Health / unrestricted

<p><em>Streptococcus pneumoniae</em> (pneumokok) je&nbsp; jedan&nbsp; od&nbsp; vodećih&nbsp; uzroka morbiditeta i mortaliteta &scaron;irom sveta, kada su u pitanju infektivne bolesti. Pretežno izaziva infekcije gornjih respiratornih puteva (sinuzitis, otitis) i konjunktivitis. Vodeći je uzročnih vanbolničkih pneumonija, bakterijskog meningitisa i sepse. Lekovi izbora u terapiji pneumokoknih bolesti su beta laktamski antibiotici i makrolidi. Iako se makrolidni antibiotici uveliko koriste u lečenju pneumokoknih infekcija &scaron;irom sveta, porast rezistencije na makrolide&nbsp; bi&nbsp; mogao&nbsp; da&nbsp; kompromituje&nbsp; njihovu&nbsp; upotrebu. Rezistencija pneumokoka na makrolide je posredovana putem dva glavna mehanizma: modifikacija ciljnog mesta delovanja leka&nbsp; i aktivni efluks leka. Metilaciju 23S ribozomalne ribonukleinske kiseline (rRNK) obavlja enzim metilaza, čiju sintezu kodira<em> ermB</em> gen. Kod ovog tipa rezistencije dolazi do ukr&scaron;tene rezistencije na makrolide (M), linkozamide (L) i streptogramine B (Sb). Ovakav vid rezistencije se ispoljava kao MLS_b - fenotip i karakteri&scaron;e ga visok nivo rezistencije. Može se javiti kao konstitutivni (cMLS) i inducibilni (iMLS). Drugi mehanizam rezistencije na makrolide je aktivni efluks leka, kodiran od strane <em>mefA</em>&nbsp; gena. Efluks antibiotik a determini&scaron;e rezistenciju samo na 14-člane i 15-člane makrolide, bez ukr&scaron;tene rezistencije. Ispoljava se kao M-fenotip, a karakteri&scaron;e ga niži stepen rezistencije. Cilj ove studije je bio&nbsp; da&nbsp; se&nbsp; odredi u čestalost&nbsp; makrolidne&nbsp; rezistencije <em>Streptococcus pneumoniae</em> među invazivnim i neinvazivnim izolatima kod dece i odraslih, da se odrediti u čestalost korezistencije i multiple rezistencije kod makrolid rezistentnih sojeva&nbsp; <em>Streptococcus pneumoniae</em>, da se fenotipski odredi tip rezistencije na makrolide i da se ispita genska osnova makrolidne rezistencije (detektovati prisustvo <em>ermB</em> i <em>mefA</em> gena). Analizirani su podaci o 326 sojeva <em>Streptococcus pneumoniae</em> rezistentnih na makrolide (MRSP) sakupljenih &scaron;irom&nbsp; Srbije&nbsp; u&nbsp; periodu&nbsp; od&nbsp; januara&nbsp; 2010.&nbsp; do&nbsp; decembra&nbsp; 2012.&nbsp; godine. Sakupljeni&nbsp; MRSP&nbsp; izolati&nbsp; su&nbsp; transportovani&nbsp; u&nbsp; Nacionalnu&nbsp; referentnu laboratoriju za streptokok radi daljih ispitivanja. Identifikacija je vr&scaron;ena na osnovu mikroskopskih, kulturelnih i biohemijskih osobina. Konzervacija je vr&scaron;ena u moždano-srčanom bujonu sa 10% sadržajem glicerola na -80&deg;C. Dvostruki&nbsp; disk&nbsp; difuzioni&nbsp; test,&nbsp; kombinovani&nbsp; difuzion odilucioni&nbsp; test&nbsp; i automatizovani VITEK 2 sistem su kori&scaron;ćeni za određivanje fenotipova rezistencije na makrolide. Geni koji kodiraju rezistenciju na makrolide su detektovani PCR metodom. Ukupna rezistencija sojeva <em>S.pneumoniae</em> na makrolide u Srbiji je iznosila 34%. Sojevi <em>S.pneumoniae</em> rezistentni na makrolide su če&scaron;će bili izolovani kod dece (36%) u odnosu na odrasle (29%) osobe, i če&scaron;će su izolovani iz neinvazivnih (35,5%) u odnosu na invazivne (27,4%) materijale. Dominantan fenotip rezistencije na makrolide je bio MLS_b fenotip (78,5%). Konstitutivan MLS fenotip je bio zastupljen kod 73,9%, a inducibilan MLS kod 4,6% MRSP izolata. Potvrđena je udruženost <em>mefA</em>&nbsp; gena i M fenotipa; <em>ermB</em> gena i iMLS fenotipa, kao i <em>ermB</em> gena i cMLS fenotipa. Prisustvo oba ermB i mefA gena rezistencije je potvrđeno kod 43,9 % izolata. Svi izolati sa koji su imali oba gena rezistencije su ispoljili&nbsp; MLS_b fenotip.&nbsp; Istovremena&nbsp; neosetljivost&nbsp; na&nbsp; penicilin&nbsp; je bila zastupljena kod 16% MRSP sojeva. Visok nivo rezistencije na penicilin je imalo svega 5,8% MRSP izolata. Među MRSP sojevima je bio prisutan visok nivo&nbsp; rezistencije&nbsp; na&nbsp; tetraciklin&nbsp; (81,3%)&nbsp; i&nbsp; trimetoprim-sulfametoksazol (74,3%). Multirezistenti sojevi, koji su bili rezistentni na tetracikline i trimetoprim-sulfametoksazol su predstavljali dve trećine (66,1%) MRSP izolata.&nbsp; Zastupljenost&nbsp; udružene&nbsp; rezistencije&nbsp; MRSP&nbsp; na&nbsp; tetraciklin i trimetoprim-sulfametoksazol je bila veća kod sojeva sa MLS fenotipom (73,1%)&nbsp; u&nbsp; odnosu&nbsp; na&nbsp; sojeve&nbsp; sa&nbsp; M&nbsp; fenotipom&nbsp; (36,7%). Zastupljenost istovremene rezistencije na makrolide i druge antibiotike među kojima su penicilin, amoksicilin, cefotaksim, tetraciklin, trimetoprim-sulfametoksazol, kao&nbsp; i&nbsp; multirezistentnih&nbsp; sojeva&nbsp; je&nbsp; bila&nbsp; veća&nbsp; kod pedijatrijskih&nbsp; izolata pneumokoka&nbsp; u&nbsp; odnosu&nbsp; na&nbsp; sojeve&nbsp; dobijene&nbsp; kod&nbsp; odraslih.&nbsp; U čestalost istovremene rezistencije na makrolide i druge antibiotike među kojima su tetraciklin i ofloksacin je bila vi&scaron;e prisutna među neinvazivnim u odnosu na invazivne MRSP izolate. Invazivni MRSP izolati iz likvora su pokazivali veću rezistenciju na beta laktamske antibiotike u odnosu neinvazivne sojeve. MRSP sojevi su pokazali veoma visok nivo osetljivosti na levofloksacin (99,6), telitromicin (98,4%), cefotaksim (93,5%), i mipenem (97,3%). MRSP sojevi su u potpunosti bili osetljivi na vankomicin, linezolid, moksifloksacin, sparfloksacin, rifampicin&nbsp; i&nbsp; pristinamicin.&nbsp; Među&nbsp; invazivnim&nbsp; sojevima <em>S.pneumoniae</em> rezistentnim na makrolide je nađeno 12 različitih serotipova. Polovina izolata je pripadala serotipovima 19F (25%) i 14 (23%), dok su sledeći po učestalosti bili 6A (10,4%) i 23F (8,3%). Istovremena rezistencija na makrolide, penicilin, tetracikline i trimetoprim-sulfametoksazol je nađena kod serotipova 19F, 14 i 23F, dok su serotpovi 12F i 31 bili neosetljivi samo na makrolide. Na&scaron;e istraživanje predstavlja prvu detaljnu analizu fenotipskih i&nbsp; genotipskih&nbsp; osobina&nbsp; sojeva&nbsp; pneumokoka&nbsp; rezistentnih&nbsp; na&nbsp; makrolidne antibiotike u Srbiji. Dobijeni rezultati ukazuju na&nbsp; potrebu za aktivnim nadzorom nad pneumokoknim infekcijama u Srbiji.</p> / <p><em>Streptococcus pneumoniae</em> (pneumococcus) is one of the leading morbidity and&nbsp; mortality&nbsp; causes&nbsp; all&nbsp; over&nbsp; the&nbsp; world&nbsp; with&nbsp; respect&nbsp; to&nbsp; infectious&nbsp; diseases. <em>Streptococcus&nbsp; pneumoniae</em> is&nbsp; a&nbsp; leading&nbsp; cause&nbsp; of upper&nbsp; respiratory&nbsp; tract infections&nbsp; (&nbsp; sinusitis,&nbsp; otitis)&nbsp; and&nbsp; conjunctivitis. It&nbsp; is&nbsp; also&nbsp; the&nbsp; most&nbsp; common cause&nbsp; of&nbsp; community-acquired&nbsp; pneumonia, bacterial&nbsp; meningitis&nbsp; and&nbsp; sepsis. Beta lactam and&nbsp; macrolide antibiotics remained a first choice for empirical treatment of pneumococcal infections. Although macrolides are widely used for&nbsp;&nbsp; treatment&nbsp;&nbsp; of&nbsp;&nbsp; pneumococcal&nbsp;&nbsp; infections, an&nbsp;&nbsp; increase&nbsp;&nbsp; in&nbsp;&nbsp; macrolide resistance&nbsp; might compromise&nbsp; their use. Pneumococcal&nbsp; macrolide resistance is&nbsp; mediated&nbsp; by&nbsp; two&nbsp; major&nbsp; mechanisms:&nbsp; target&nbsp; site&nbsp; modification&nbsp; and&nbsp; active drug&nbsp; efflux.&nbsp; Methylation&nbsp; of&nbsp; the&nbsp; 23S&nbsp; ribosomal&nbsp; ribonucleic&nbsp; acid&nbsp; (rRNA)&nbsp; is performed&nbsp;&nbsp; by&nbsp;&nbsp; the&nbsp;&nbsp; enzyme&nbsp;&nbsp; methylase,&nbsp;&nbsp; encoded&nbsp;&nbsp; by&nbsp;&nbsp; the<em> ermB </em>gene. Modification&nbsp; of&nbsp; ribosomal&nbsp; targets&nbsp; leads&nbsp; to&nbsp; cross-resistance to&nbsp; macrolides (M),&nbsp; lincosamides&nbsp; (L)&nbsp; and&nbsp; streptogramins&nbsp; B&nbsp; (Sb). It&nbsp; is&nbsp; expressed&nbsp; as&nbsp; the MLS_b &ndash;phenotype,&nbsp; which&nbsp; confers&nbsp; a&nbsp; high-level&nbsp; resistance. This&nbsp; phenotype&nbsp; can&nbsp;&nbsp; be&nbsp;&nbsp; either&nbsp;&nbsp; constitutively&nbsp;&nbsp; (cMLS)&nbsp;&nbsp; or&nbsp;&nbsp; inducibly&nbsp;&nbsp; (iMLS). expressed. Another macrolide resistance mechanism is the active drug efflux, encoded by&nbsp; the <em>mefA&nbsp;</em> gene.&nbsp; The&nbsp; drug&nbsp; efflux&nbsp; confers&nbsp; resistance&nbsp; to&nbsp; 14-&nbsp; and&nbsp; 15-membered&nbsp; macrolides&nbsp; only,&nbsp; with&nbsp; no&nbsp; cross-resistance.&nbsp; It&nbsp; is&nbsp; expressed&nbsp; as&nbsp; the M-phenotype,&nbsp; which&nbsp; confers&nbsp; low-level&nbsp; resistance.&nbsp; The&nbsp; objective of&nbsp; this study&nbsp;&nbsp; was&nbsp;&nbsp; :&nbsp;&nbsp; 1) to&nbsp;&nbsp; examine&nbsp;&nbsp; the&nbsp;&nbsp; prevalence of&nbsp;&nbsp; macrolide&nbsp;&nbsp; resistant <em>Streptococcus&nbsp;&nbsp; pneumoniae </em>(MRSP) among&nbsp;&nbsp; invasive&nbsp;&nbsp; and&nbsp;&nbsp; noninvasive isolates in children and adults, 2) to examine the prevalence of coresistance and multiple-resistance among MRSP strains, 3) to examine the prevalence of&nbsp; macrolide&nbsp; resistant&nbsp; phenotypes,&nbsp; and&nbsp; 4)&nbsp; to&nbsp; examine&nbsp; the&nbsp; prevalence&nbsp; of macrolide&nbsp; resistant&nbsp; genotypes&nbsp; (detect&nbsp; the&nbsp; presence of&nbsp; the <em>ermB</em>&nbsp;&nbsp; and <em>mefA</em> gene).&nbsp; A&nbsp; total&nbsp; of&nbsp;&nbsp; 326&nbsp; MRSP&nbsp; strains&nbsp; were&nbsp; analyzed,&nbsp; which&nbsp; were&nbsp; collecte dall&nbsp; over&nbsp; Serbia&nbsp; in&nbsp; the&nbsp; period&nbsp; from&nbsp; January,&nbsp; 2010&nbsp; - December,&nbsp; 2012.&nbsp; The collected&nbsp;&nbsp; MRSP&nbsp;&nbsp; isolates&nbsp;&nbsp; were&nbsp;&nbsp; referred&nbsp;&nbsp; to&nbsp;&nbsp; the&nbsp;&nbsp; National&nbsp; Reference Laboratory&nbsp; for&nbsp; streptococci&nbsp; and&nbsp; pneumococci for&nbsp; further&nbsp; investigation. Identification based on microscopic, culture and biochemical features of the isolates. Conservation was performed in the brain-heart infusion broth with a&nbsp; 10%&nbsp; glycerol&nbsp; content&nbsp; at&nbsp; -80&deg;C.&nbsp; Macrolide&nbsp; resistance&nbsp; phenotypes&nbsp; were determined by a double disc diffusion test, combine d diffusion-dilution test and&nbsp;&nbsp; automatized&nbsp;&nbsp; VITEK&nbsp; 2 system. Macrolide&nbsp;&nbsp; resistance&nbsp;&nbsp; genes&nbsp;&nbsp; were&nbsp; determined by PCR. Overall, macrolide nonsusceptibility rate in Serbia was 34%.&nbsp; MRSP&nbsp; isolates&nbsp; were&nbsp; more&nbsp; prevale nt&nbsp; among&nbsp; children&nbsp; (36%)&nbsp; than adults&nbsp; (29%),&nbsp; and&nbsp; were&nbsp; more&nbsp; prevalent&nbsp; among&nbsp;&nbsp; noninvasive&nbsp; (35.5%)&nbsp; than invasive&nbsp; (27.4%)&nbsp; samples.&nbsp; Predominant&nbsp; macrolide&nbsp; resistance&nbsp; phenotype was&nbsp; the&nbsp; MLS b&nbsp; phenotype&nbsp; (78.5%),&nbsp; from&nbsp; which&nbsp; 73.9 %&nbsp; belonged&nbsp; to&nbsp; cMLS and&nbsp; 4.6%&nbsp; to&nbsp; iMLS&nbsp; phenotype.&nbsp; All&nbsp; the&nbsp; strains&nbsp; assigne d&nbsp; to&nbsp; the&nbsp; MLS_b phenotype harbored<em> ermB</em> gene, while all the strains with M phenotype had the mefA gene.&nbsp; The&nbsp; presence&nbsp; of&nbsp; both ermB and mefA resistance&nbsp; genes&nbsp; was confirmed&nbsp; in&nbsp; 43.9&nbsp; %&nbsp; of&nbsp; isolates. All&nbsp; the&nbsp; isolates&nbsp; which&nbsp; harbored&nbsp; both resistance genes expressed the MLS_b phenotype. Among macrolide resistant strains,&nbsp; penicillin&nbsp; nonsusceptiblility&nbsp; was&nbsp; observed&nbsp;&nbsp; in&nbsp; 16% .&nbsp; A&nbsp; high&nbsp; level resistance was confirmed in 5. 8% of MRSP isolates. MRSP strains showed high&nbsp; resistance rates to tetracyclin&nbsp; (81.3%) and&nbsp; trimethoprim-sulfamethoxazole&nbsp; (74.3%).&nbsp; Multiresistant&nbsp; strains,&nbsp; resistant&nbsp; to&nbsp; tetracyclines and&nbsp; trimethoprim-sulfamethoxazole,&nbsp; made&nbsp; two&nbsp; thirds&nbsp; (66.1&nbsp; %)&nbsp; of&nbsp; MRSP isolates.&nbsp; Among&nbsp; MRSP,&nbsp; co-resistance&nbsp; to&nbsp; tetracycline&nbsp; and&nbsp; trimethoprim-sulfamethoxazole&nbsp; was&nbsp; more&nbsp; prevalent&nbsp; among&nbsp; MLS&nbsp; phenotypes&nbsp; (73.1%) than&nbsp; M&nbsp; phenotypes&nbsp; (36.7%).&nbsp; Co-resistance&nbsp; strains&nbsp; to&nbsp; macrolides&nbsp; and&nbsp; other antibiotics including&nbsp;&nbsp;&nbsp; penicillin,&nbsp;&nbsp; amoxicillin,&nbsp;&nbsp;&nbsp; cefotaxime,&nbsp; tetracyclin, trimethoprim-sulfamethoxazole and multiresistant&nbsp; strains&nbsp; were more prevalent among children than adult. Coresistance to macrolides and other antibiotics&nbsp; including&nbsp; tetracycline&nbsp; and&nbsp; ofloxacin&nbsp; was&nbsp; more&nbsp; prevalent&nbsp; among noninvasive&nbsp;&nbsp; than&nbsp;&nbsp; invasive&nbsp;&nbsp; strains.&nbsp;&nbsp; Invasive&nbsp;&nbsp; MRSP&nbsp;&nbsp; isolates&nbsp;&nbsp; from&nbsp;&nbsp; the cerebrospinal fluid showed a higher resistance rate to beta lactam antibiotics than&nbsp; noninvasive&nbsp; strains.&nbsp; MRSP&nbsp; strains&nbsp; had&nbsp; a&nbsp; high&nbsp; susceptibility&nbsp; rates&nbsp; to levofloxacin&nbsp;&nbsp; (99.6),&nbsp;&nbsp; telithromycin&nbsp;&nbsp; (98.4%),&nbsp;&nbsp; cefotak sime&nbsp;&nbsp; (93.5%)&nbsp;&nbsp; and imipenem&nbsp; (97.3%).&nbsp; MRSP&nbsp; strains&nbsp; were&nbsp; fully&nbsp; susceptible&nbsp; to&nbsp; vancomycin, linezolid, moxifloxacin, sparfloxacin, rifampicin a nd pristinamycin. Among macrolide&nbsp;&nbsp; resistant <em>S.pneumoniae</em> strains,&nbsp;&nbsp; 12 different&nbsp;&nbsp; serotypes&nbsp;&nbsp; were identified.&nbsp; One&nbsp; half&nbsp; of&nbsp; these&nbsp; isolates&nbsp; belonged&nbsp; to the&nbsp; 19F&nbsp; (27.1%)&nbsp; and&nbsp; 14 (22.&nbsp; 9%)&nbsp; serotype,&nbsp; followed&nbsp; in&nbsp; frequency&nbsp; by&nbsp; the&nbsp; 6A&nbsp; (10.41%)&nbsp; and&nbsp; 23F (8.3%)&nbsp; serotype .&nbsp; Multiresistant&nbsp; strains&nbsp; (macrolides,&nbsp; penicillin,&nbsp; tetracyclines and&nbsp; trimethoprim-sulfamethoxazole)&nbsp; belonged&nbsp; to&nbsp; serotypes 19F,&nbsp; 14&nbsp; and 23F, while the 12F and 31 serotype were resistant to macrolides only. This in vestigation&nbsp;&nbsp; represents&nbsp;&nbsp; the&nbsp;&nbsp; first&nbsp;&nbsp; detailed&nbsp;&nbsp; analysis of&nbsp;&nbsp; phenotypes&nbsp;&nbsp; and genotypes&nbsp; of&nbsp; macrolide&nbsp; resistant&nbsp; pneumococcal&nbsp; strains&nbsp; in&nbsp; Serbia.&nbsp; The obtained&nbsp; results suggest&nbsp; the need for an active surveillance&nbsp; of pneumococcal infections in Serbia.</p>

<p>Uvod: U pro&scaron;losti žučne kiseline su uglavnom razmatrane sa stanovi&scaron;ta njihove funkcije koju obavljaju u crevima jer posreduju u varenju masti i apsorpciji liposolubilnih vitamina. Nedavne studije potvrđuju da žučne kiseline ne igraju samo ulogu u varenju masti, nego se pona&scaron;aju i kao signalni molekuli koji stupaju u interakciju sa raznim receptorima uključujući nuklearne receptore i receptore vezane za G-proteine. Kao amfipatični molekuli one su sposobne da reaguju sa fosfolipidima ćelijskih membrana i da pobolj&scaron;avaju prolazak lekova kroz njih. Stoga se žučne kiseline razmatraju kao promoteri u bukalnim, okularnim i nazalnim farmaceutskim formulacijama. Cilj: Svrha ovog istraživanja je bila da se ispitaju žučne kiseline i njihovi okso derivati kao jedinjenja koja utiču na propustljivost ćelijskih membrana i prolazak lekova do ciljnih tkiva. Materijal i metod: Interakcije makrolidnih antibiotika i žučnih kiselina su ispitivane uz pomoć NMR difuzionih merenja i relaksacije paramagnetičnim jonima. Retencioni parametri odabranih žučnih kiselina su dobijeni kori&scaron;ćenjem hromatografije na normalnim fazama i evaluisani su primenom pet različitih softvera. In vivo ekaperimenti su sprovedeni na 126 eksperimentalnih životinja koje su bile podeljene u 21 grupu. Rezultati: Vezivanje žučnih kiselina za micele je indikovano razlikama u hemijskom pomeranju makrolida i pro&scaron;irenju signala kao posledica redukovane mobilnosti unutar micela. Dodatak micela žučnih kiselina povećava solubilizaciju makrolida za faktor približno 2-3. Sprovedena korelaciona analiza pokazala je značajnu zavisnost između faktora retencije i intestinalne apsorpcije, prodora u MDCK epitelne ćelije, permeabilnost kroz kožu, logBB i PPB%. Putem implementacije in vivo eksperimentalnog dela pokazano je da žučne kiseline utiču na prolazak makrolida u tkivo mozga, bubrega i jetre. Zaključak: Ispitivane žučne kiseline pokazuju dobre farmakokinetske karakteristike i olak&scaron;avaju prolazak makrolida kroz različite ćelijske membrane.</p> / <p>Introduction: In the past, bile acids were mostly considered to function in the intestine where they play a role in digestion of fats and mediate absorption of fat-soluble vitamins. Recent studies confirm that bile acids not only facilitate solubilization of fats but behave as signal molecules that interact with various receptors including nuclear receptors and G protein-coupled receptors. As amphipathic molecules they are able to interact with phospholipids of cells membranes and enhance drugs permeation. Thus, bile acids are considered as drug promoters in buccal, ocular, nasal, and transdermal dosage forms. Purpose: The purpose of this research was to investigate bile acids and its oxo derivatives as enhancers in drug permeability. Three research methods to evaluate the characteristics of bile acids and its properties were used. Material and method: The interaction between macrolide antibiotics and bile acids was investigated by NMR chemical-shift titration, self-diffusion measurements and paramagnetic relaxation enhancements. Retention parameters of selected bile acids are acquired by normal-phase thin layer chromatography and evaluated using five different softwares. In vivo experiments were conducted on 126 animals which were divided in 21 groups. Results: Binding bile acids to the micelles is indicated by differences in the chemical shift of the macrolides and line broadening as a consequence of reduced mobility in the micelle. Addition of bile micelles increases the solubility of macrolide antibiotics by a factor of approximately 2&ndash;3. Examined correlation analysis confirmed significant dependence between retention factor and intestinal absorption, MDCK epithelial cells, skin permeability, logBB and PPB%. Through the implementation of in vivo experiments it is shown that bile acids promote penetration of macrolides in brain tissue, kidney and liver. Conclusion: Investigated bile acids showed good pharmacokinetic properties and facilitate in macrolides permeation through various membranes.</p>

Protein Vga(A) gives staphylococci resistance to streptogramins A. The recently discovered protein Vga(A)LC differs from Vga(A) only by 7 amino acid residues, but this difference is sufficient for shift of its substrate specificity towards lincosamides. The group of four amino acids in the central part of protein (LGAG in Vga(A) and SVTS in Vga(A)LC) was detected to be crucial for the substrate specificity. In this diploma thesis 5 alternativesets of vga(A)LC gene point mutations were prepared in order to determine the impact of individual amino acids of the aforementioned group on the resistance phenotype. Mutations were prepared in vector pGEM® -T and cloned into shuttle vector pRB374. The prepared constructs were transformed by electroporation into the sensitive strain of Staphylococcus aureus RN4220 and values of minimum inhibitory concentration (MIC) were measured for lincomycin, clindamycin and pristinamycin IIA by the agar dilution method. The transformation was not successful in one of the mutations. Results of setting MIC for the remaining four mutations do not make it possible to specify uniquely the ratio of individual amino acids for determining substrate specificity. Two of the amino acids were found to be important. We anticipate preparation of more mutations.