Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by 3H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.

Azithromycin, a macrolide antibiotic, specifically binds to the 50S ribosomal subunit of bacterial ribosomes and inhibits translation. Azithromycin also prevents 50S ribosomal subunit assembly by binding to a 50S ribosomal subunit precursor particle. When exposed to azithromycin, several ribonucleases in wild-type Escherichia coli cells degrade antibiotic-bound 50S precursor particles. Presumably, cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound precursor less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonucleaseûdeficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability, growth rates, and protein synthesis rates were measured. Degradation of 23S rRNA was examined by hybridization with a 23S specific probe. Ribonuclease II and polynucleotide phosphorylase mutants demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation, suggesting that these two ribonucleases are important for 23S rRNA turnover in azithromycin-treated Escherichia coli.

Bacteremia initiated by Staphylococcus aureus infections can be a serious medical problem. Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. Combination therapy for certain infections has been used to reduce the emergence of resistance when a single agent has become ineffective. We hypothesize that the use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects resulting from indirect effects on ribosomal subunit synthesis. To determine this we measured the effects of single and multiple antibiotics on cell growth rates, cell viability, and synthesis rates for DNA, RNA, and protein. We then measured synthesis rates of ribosomal subunits and the amounts of gyrase and RNAP. Effects of the antibiotic combinations on 70S ribosomes was assayed and the amounts of RNA and degradation was measured. We lastly studied the effects of these antibiotic combinations on mutation frequency in Staphylococcus aureus. Our data have shown support not only for the use of antibiotic combination therapy but have provided strong evidence of an increase in the inhibition of bacterial ribosome assembly in Staphylococcus aureus. The reduction of 50S ribosomal subunit synthesis and 23S ribosomal RNA in cells grown in the presence of azithromycin, already known for it’s inhibitory effects on the 50S subunit synthesis, in combination with rifampicin or in combination with rifampicin and ciprofloxacin was observed. This also resulted in a reduction or elimination in the frequency of resistant cells when grown in the presence of these combinations. These studies have shed light on the mechanism of action involved and synergistic effects occurring in combination antibiotic treatments and how ribosomal subunit assembly is affected. The insights gained through this research provide necessary information needed for the design of more potent antibiotic combinations. This will create a better understanding and new methods for eliminating the spread of harmful pathogens such as Staphylococcus aureus.

Τα ριβοσώματα, οι μακρομοριακές μεταφραστικές μηχανές που είναι υπεύθυνες για την πρωτεϊνική σύνθεση, αποτελούν έναν από τους κυριότερους κυτταρικούς στόχους των αντιβιοτικών, που χορηγούνται για αντιμικροβιακή θεραπεία. Μελέτες για περισσότερο από 40 χρόνια δείχνουν ότι το κατάλληλο ιοντικό περιβάλλον (μονοσθενή, δισθενή κατιόντα και πολυαμίνες) είναι απαραίτητο για τη σωστή ριβοσωματική λειτουργία, ενώ παράλληλα επηρεάζει τις αλληλεπιδράσεις του με διάφορους προσδέτες. Παρόλα αυτά η μοριακή βάση της επίδρασης του ιοντικού περιβάλλοντος στο μηχανισμό δράσης των αντιβιοτικών δεν έχει ενδελεχώς μελετηθεί. Στόχος της παρούσας διατριβής είναι η διερεύνηση του μηχανισμού δράσης αντιβιοτικών που αναστέλλουν την πρωτεϊνική σύνθεση σε συνθήκες που προσομοιάζουν με τις φυσιολογικές του κυττάρου και η μελέτη της επίδρασης του ιοντικού περιβάλλοντος στη δράση αυτών. Τα αντιβιοτικά που μελετήθηκαν ήταν: α) η βλαστισιδίνη, ως κλασικός αναστολέας της πεπτιδυλοτρανσφεράσης (ΡΤάσης), β) το μακρολίδιο τυλοσίνη που αναστέλλει την ΡΤάση, αλλά παράλληλα προσδένεται στην αρχή του τούνελ εξόδου και παρεμποδίζει την πολυπεπτιδική αλυσίδα να εξέλθει από το ριβόσωμα, και γ) τα μακρολίδια ερυθρομυκίνη (πρώτης γενεάς), αζιθρομυκίνη (δεύτερης γενεάς) και τελιθρομυκίνη (μακρολίδιο τρίτης γενεάς ή κετολίδιο), η δράση των οποίων έγκειται στην παρεμπόδιση του τούνελ εξόδου. Εξίσου σημαντική φαίνεται να είναι η επίδραση των μακρολιδίων στη συγκρότηση της 50S ριβοσωματικής υπομονάδας. Ο μηχανισμός δράσης των αντιβιοτικών και η επίδραση του ιοντικού περιβάλλοντος στη δράση τους έγινε αρχικά με κινητικές μελέτες. Το πειραματικό σύστημα που χρησιμοποιήθηκε ήταν η αντίδραση πουρομυκίνης, η οποία μας έδωσε τη δυνατότητα τιτλοδότησης των ενεργών ριβοσωμάτων. Βάσει αυτού μελετήθηκαν τα αντιβιοτικά βλαστισιδίνη και τυλοσίνη που αναστέλλουν άμεσα την ΡΤάση, ενώ για τη μελέτη των υπολοίπων μακρολιδίων διεξήχθησαν πειράματα συναγωνιστικής αναστολής. Ως γνωστό, τα μακρολίδια ερυθρομυκίνη, αζιθρομυκίνη και τελιθρομυκίνη μοιράζονται κοινές θέσεις πρόσδεσης στο ριβόσωμα με την τυλοσίνη. Έτσι, για την εύρεση των σταθερών πρόσδεσης αυτών στο ριβόσωμα έγινε συναγωνισμός με τυλοσίνη. Τα πειράματα συναγωνισμού πραγματοποιήθηκαν, επωάζοντας το ριβόσωμα με μείγμα μακρολιδίου και τυλοσίνης, και τιτλοδοτώντας την απομένουσα δραστικότητα του ριβοσώματος με την αντίδραση πουρομυκίνης απομακρύνοντας την περίσσεια αντιβιοτικών. Σε παράλληλα πειράματα, το ριβόσωμα προεπωάστηκε αρχικά με το μακρολίδιο και στη συνέχεια προστέθηκε τυλοσίνη, η οποία ανιχνεύει το εναπομείναν ριβοσωματικό σύμπλοκο. Επειδή η σταθερά συγγένειας στη δεύτερη περίπτωση βρέθηκε μικρότερη (ισχυρότερη πρόσδεση αντιβιοτικού) συμπεράναμε, ότι ο μηχανισμός πρόσδεσης του αντιβιοτικού είναι βραδύς και ακολουθεί δυο στάδια. Βασιζόμενοι στις τιμές των σταθερών συγγένειας σε πειράματα αναγέννησης του ριβοσωματικού συμπλόκου από την απενεργοποιημένη μορφή του, προσδιορίστηκαν ξεχωριστά όλες οι κινητικές παράμετροι που χαρακτηρίζουν την πρόσδεση του αντιβιοτικού στο ριβόσωμα. Συγκρίναμε τις παραμέτρους αυτές και γενικότερα την ισχύ πρόσδεσης των αντιβιοτικών στο ριβόσωμα σε πέντε ιοντικές συνθήκες: (α) 4,5 mM Mg2+, 150 mM NH4+, (β) 4,5 mM Mg2+, 150 mM NH4+, 100 μΜ σπερμίνη, (γ) 4,5 mM Mg2+, 150 mM NH4+, 50 μΜ σπερμίνη και 2 mM σπερμιδίνη, (δ) 4,5 mM Mg2+, 150 mM NH4+ ριβοσωματικό σύμπλοκο φωτοσημασμένο με 100 μΜ ΑΒΑ-σπερμίνη, και (ε) 10 mM Mg2+, 100 mM NH4+. Τα αποτελέσματα έδειξαν ότι οι πολυαμίνες βελτιώνουν την πρόσδεση της βλαστισιδίνης, αλλά μειώνουν την πρόσδεση των μακρολιδίων. Η επίδραση των ιόντων Mg2+ προσομοιάζει εκείνης των πολυαμινών, αλλά είναι λιγότερο αποτελεσματική, αφού 100 μΜ σπερμίνης επιφέρουν μεγαλύτερη αναστολή πρόσδεσης, από ότι 10 mM Mg2+. Για να ερμηνευτεί σε μοριακό επίπεδο η επίδραση της σπερμίνης στη συγγένεια των αντιβιοτικών έναντι του ριβοσώματος, οι θέσεις πρόσδεσης των πολυαμινών στο ριβόσωμα προσδιορίστηκαν με φωτοσήμανση συγγένειας, χρησιμοποιώντας ως μοριακό ανιχνευτή ένα φωτοδραστικό ανάλογο της σπερμίνης, την ΑΒΑ-σπερμίνη. Οι θέσεις αυτές αποκάλυψαν ότι οι πολυαμίνες προσδένονται σε γειτονικές θέσεις προς τα αντιβιοτικά, επηρεάζοντας την τοπική διαμόρφωση και το φορτίο. Επιβεβαίωση του μηχανισμού δράσης και της επίδρασης των πολυαμινών έγινε με ανάλυση αποτυπώματος. Σύμφωνα με την τεχνική αυτή, τα μακρολίδια προσδενόμενα στο ριβόσωμα προστατεύουν ορισμένα νουκλεοτίδια από την επίδραση χημικών τροποποιητών. Τα αποτελέσματα έδειξαν ότι τα αντιβιοτικά διέρχονται μια ενδιάμεση κατάσταση πρόσδεσης στο ριβόσωμα δεσμευόμενα αρχικά στην είσοδο του τούνελ εξόδου και στη συνέχεια εισχωρώντας βαθύτερα σε αυτό. Η φύση της ενδιάμεσης κατάστασης εξαρτάται από τα ιδιαίτερα χαρακτηριστικά του κάθε μακρολιδίου. Η επίδραση των πολυαμινών στο μηχανισμό πρόσδεσης ελέγχθηκε επαναλαμβάνοντας τα πειράματα χημικής προστασίας παρουσία αυτών. Τα αποτελέσματα έδειξαν ότι η μείωση της πρόσδεσης των μακρολιδίων στο ριβόσωμα επιτελείται κυρίως μέσω της επίδρασης των πολυαμινών στη δέσμευση του υδρόφοβου λακτονικού δακτυλίου. Τα ιδιαίτερα χαρακτηριστικά του κάθε αντιβιοτικού επηρεάζουν ποικιλοτρόπως το μέγεθος της επίδρασης αυτής. Στο τελευταίο κομμάτι της διατριβής μελετήθηκε η ισχύς των μακρολιδίων, υπολογίζοντας την αναστολή που προκαλούν στο συζευγμένο σύστημα μεταγραφής-μετάφρασης του γονιδίου της GFP πρωτεΐνης, και τα αποτελέσματα επιβεβαίωσαν τα κινητικά δεδομένα πρόσδεσης των μακρολιδίων στο ριβόσωμα-στόχο. Σε υψηλή συγκέντρωση ιόντων Mg2+ η τυλοσίνη έχει μεγαλύτερη ισχύ, ενώ σε χαμηλή συγκέντρωση ιόντων απουσία ή παρουσία πολυαμινών η αζιθρομυκίνη. Η τελιθρομυκίνη παρουσίασε τη χαμηλότερη ισχύ πρόσδεσης στο ριβόσωμα και αναστολής της πρωτεϊνικής σύνθεσης. Επιπρόσθετα, ελέγχθηκε πιθανή επίδραση των μακρολιδίων στην πρόσδεση των υποστρωμάτων (tRNAs) στην Α-, Ρ- και Ε- θέση του ριβοσώματος, στη μετατόπιση αυτών από την Α- στην Ρ- θέση και στη μεταφραστική πιστότητα του ριβοσώματος. Βρήκαμε ότι τα μακρολίδια δεν μπορούν να επηρεάσουν αυτά τα στάδια της ριβοσωματικής λειτουργίας. / Ribosomes, the macromolecular translating machines responsible for protein biosynthesis, are the most common targets for many antibacterial agents. Experiments for more than 40 years have demonstrated that a distinct ionic environment (monovalent, divalent cations and polyamines) is essential for ribosomal functions and their interactions with the ligands. Nevertheless, the molecular basis of the ionic environment’s influence on antibiotic mechanism of action has never been precisely elucidated. The aim of this thesis was first to investigate the mechanism of action of several antibiotics –inhibitors of protein synthesis, under ionic conditions close to the cell environment and second, to clarify the role of the ionic environment on their mechanism of action. The antibiotics studied were: a) blasticidin-S, a classic inhibitor of peptidyl tranferase (PTase) activity, b) tylosin which inhibits PTase, but in parallel binds at the entrance of exit tunnel and blocks the passage of the nascent polypeptide chain, and c) erythromycin (a first generation macrolide), azithromycin (a second generation macrolide), and telithromycin (a third generation macrolide, ketolide), that blocks the exit tunnel. The mechanism of action of antibiotics and the influence of ionic environment on antibiotic potency was studied primarily with kinetic methods. The experimental procedure was based on the puromycin reaction, performed under conditions allowing the estimation of the catalytic rate constant. Using this experimental approach we studied the mechanism of action of blasticidin and tylosin which directly inhibit PTase. For studying the other macrolides, experiments employing competitive kinetics were performed. Erythromycin, azithromycin and telithromycin share common binding sites on ribosomes with tylosin. Thus, to estimate the kinetic constants of their interactions with ribosomes, competitive kinetic experiments were carried out in the presence of tylosin. Namely, a posttranslocation ribosomal complex formed from Escherichia coli 70S ribosomes bearing tRNAPhe at the E-site and AcPhe-tRNA at the P-site (complex-C) was incubated with a mixture of each macrolide and tylosin for the desired time intervals. The rest of ribosomal activity was titrated by the puromycin reaction. In parallel experiments, complex-C was pre-incubated with each one of the macrolides and then reacted with tylosin. The rest of complex-C activity was again titrated with the puromycin reaction. Since the affinity constant obtained by the second series of experiments was less than that obtained by the first series of experiments, we concluded that the mechanism of action of antibiotics follows a slow onset inhibition process, which includes two steps. Based on secondary plots and on kinetic plots derived from regeneration of complex-C, we measured the kinetic parameters participating in the kinetic model. Thus, the potency of each antibiotic was determined under five different ionic conditions: (a) 4,5 mM Mg2+, 150 mM NH4+, (b) 4,5 mM Mg2+, 150 mM NH4+, 100 μΜ spermine, (c) 4,5 mM Mg2+, 150 mM NH4+, 50 μΜ spermine and 2 mM spermidine, (d) 4,5 mM Mg2+, 150 mM NH4+, and ribosomal complex photolabelled with 100μΜ ΑΒΑ-spermine, and (e) 10 mM Mg2+, 100 mM NH4+. Processing of the data led us to the conclusion that polyamines and Mg2+ ions increase the potency of blasticidin, but decrease the potency of macrolides. To explain the diverse action of polyamines and of the ionic environment in general on antibiotic potency, the binding sites of spermine in ribosomes were localized by photoaffinity labeling, using a photoactive analogue of spermine, ABA-spermine. These experiments revealed that polyamines bind at the vicinity of antibiotics, influencing the ionic charge and the local conformation of rRNA. Confirmation of the macrolide mechanism of action and verification of the influence of polyamines on their potency was achieved by footprinting analysis. According to this technique, macrolides bind to ribosomes and protect specific nucleotides from modification by chemical reagents like DMS, CMCT and kethoxal. The results demonstrated that the antibiotics (I) form an encounter complex with complex-C (CI), in which the antibiotics occupy the entrance of the exit tunnel. This intermediate complex is then isomerized slowly to a tighter complex (C*I) with which antibiotics move deeply in the exit tunnel. The exact interactions stabilizing the intermediate complex depend on the characteristic groups of each macrolide. The influence of polyamines was checked by repeating the experiment in the presence of polyamines. The results showed that polyamines reduce the macrolide binding to ribosomes, by affecting mainly the interactions of the hydrophobic lactone ring with the ribosome. The special characteristic groups of each macrolide affect the polyamine action. The potency of macrolides action was also estimated using a coupled transcription-translation system for GFP expression. The results obtained were consistent with those produced by kinetic analysis. In addition, we check for possible macrolide effects on tRNA binding at the A-, P- and E- sites of the ribosome, on translocation, and on translational fidelity. No strong effects were identified excluding the macrolide from these ribosomal functions.

O advento do uso de cateteres venosos centrais na prática médica trouxe muitos benefícios aos pacientes, porém está relacionado a um aumento na incidência de infecções por microrganismos multirresistentes. Além disso, freqüentemente ocorre colonização por bactérias produtoras de biofilme. Estes microrganismos se aderem ao material abiótico desses dispositivos intravenosos, ficando protegidos sob a matriz exopolissacarídica do biofilme. Isso faz com que sistema imunológico e antimicrobianos sejam incapazes de ter sua ação plena e, muitas vezes, não atingem os microrganismos mais internos. O motivo deste insucesso é porque muitos desses agentes biológicos e farmacológicos apresentam propriedades físico-químicas incompatíveis com a penetração nesta matriz. Com o objetivo de determinar quais antimicrobianos são mais adequados para uso quando o microrganismo é produtor de biofilme e quais as propriedades físico-químicas que estão diretamente relacionadas à penetração do antimicrobiano na matriz polissacarídica, utilizou-se método colorimétrico com cristal violeta em microplacas modificado para obtenção de concentração inibitória mínima em biofilme (MBIC) e método já padronizado para concentração inibitória mínima (MIC). Para isso foram testados 10 antimicrobianos em Staphylococcus spp.: rifampicina, azitromicina, claritromicina, eritromicina, levofloxacino, gentamicina, doxiciclina, cloranfenicol, clindamicina e vancomicina. Para Pseudomonas aeruginosa foram testados os mesmos, exceto rifampicina e vancomicina. A discrepância entre MIC e MBIC foi muito grande para vários fármacos, mostrando a necessidade de se avaliar estes parâmetros antes do início da farmacoterapia para uma escolha correta, especialmente em hospitais. Os fármacos que apresentaram melhores resultados foram a rifampicina e os macrolídeos, enquanto que os menos efetivos foram vancomicina e clindamicina. Isso foi atribuído ao perfil lipofílico, porém com alguma solubilidade em água das melhores moléculas. Em contra ponto, a elevada área polar, complexidade e massa molar foram características negativas para a penetração em biofilme, resultando numa ineficácia para essas moléculas. Além disso, também foi avaliado o tratamento de polímeros plásticos com EDTA, obtendo-se redução significativa da produção de biofilme nas placas tratadas com o agente químico. / The use of central venous catheters in medicine has brought benefits to the patients and represents a great advance in clinical practice, while on the other hand this device is related to an increase in the incidence of infections caused by multiresistant pathogens. Furthermore, frequently, the catheters get colonized by biofilm producing bacteria. These microorganisms adhere to the abiotic material of the catheters keeping themselves protected underneath the exopolysaccharide matrix of biofilm, this way the immune system and antimicrobials are incapable to fulfill their action and, many times, are unable to reach internal bacteria. This fact is explained by the fact that many of the biological and pharmacological agents have physical-chemical properties incompatible with the penetration into the matrix. Aiming to determine which antimicrobials are suitable for using when dealing with a biofilm producing microorganism and which physical-chemical properties are directly related to the agent penetration into the polysaccharide matrix, we used colorimetric method with crystal violet to obtain biofilm minimum inhibitory concentration (MBIC) and the already standardized method for minimum inhibitory concentration (MIC). To accomplish these 10 antimicrobials were tested in Staphylococcus spp.: rifampin, azithromycin, clarithromycin, erythromycin, levofloxacin, gentamicin, doxycycline, chloramphenicol, clindamycin and vancomycin. For Pseudomonas aeruginosa all antimicrobials except for rifampin and vancomycin were included. There was a great difference between MIC and MBIC for many drugs, showing the need to evaluate these parameters before beginning treatment. The drugs with better results were rifampin and macrolides, while the worse were vancomycin and clindamycin, which can be attributed to the lipophilic profile with some water solubility present in the molecules with better results. The characteristics associated with poor penetration into biofilm were high polar surface area, complexity e molecular weight. Furthermore, the previous treatment of the plastic polymers with EDTA was accessed resulting in statistically significant reduction of biofilm production.

O advento do uso de cateteres venosos centrais na prática médica trouxe muitos benefícios aos pacientes, porém está relacionado a um aumento na incidência de infecções por microrganismos multirresistentes. Além disso, freqüentemente ocorre colonização por bactérias produtoras de biofilme. Estes microrganismos se aderem ao material abiótico desses dispositivos intravenosos, ficando protegidos sob a matriz exopolissacarídica do biofilme. Isso faz com que sistema imunológico e antimicrobianos sejam incapazes de ter sua ação plena e, muitas vezes, não atingem os microrganismos mais internos. O motivo deste insucesso é porque muitos desses agentes biológicos e farmacológicos apresentam propriedades físico-químicas incompatíveis com a penetração nesta matriz. Com o objetivo de determinar quais antimicrobianos são mais adequados para uso quando o microrganismo é produtor de biofilme e quais as propriedades físico-químicas que estão diretamente relacionadas à penetração do antimicrobiano na matriz polissacarídica, utilizou-se método colorimétrico com cristal violeta em microplacas modificado para obtenção de concentração inibitória mínima em biofilme (MBIC) e método já padronizado para concentração inibitória mínima (MIC). Para isso foram testados 10 antimicrobianos em Staphylococcus spp.: rifampicina, azitromicina, claritromicina, eritromicina, levofloxacino, gentamicina, doxiciclina, cloranfenicol, clindamicina e vancomicina. Para Pseudomonas aeruginosa foram testados os mesmos, exceto rifampicina e vancomicina. A discrepância entre MIC e MBIC foi muito grande para vários fármacos, mostrando a necessidade de se avaliar estes parâmetros antes do início da farmacoterapia para uma escolha correta, especialmente em hospitais. Os fármacos que apresentaram melhores resultados foram a rifampicina e os macrolídeos, enquanto que os menos efetivos foram vancomicina e clindamicina. Isso foi atribuído ao perfil lipofílico, porém com alguma solubilidade em água das melhores moléculas. Em contra ponto, a elevada área polar, complexidade e massa molar foram características negativas para a penetração em biofilme, resultando numa ineficácia para essas moléculas. Além disso, também foi avaliado o tratamento de polímeros plásticos com EDTA, obtendo-se redução significativa da produção de biofilme nas placas tratadas com o agente químico. / The use of central venous catheters in medicine has brought benefits to the patients and represents a great advance in clinical practice, while on the other hand this device is related to an increase in the incidence of infections caused by multiresistant pathogens. Furthermore, frequently, the catheters get colonized by biofilm producing bacteria. These microorganisms adhere to the abiotic material of the catheters keeping themselves protected underneath the exopolysaccharide matrix of biofilm, this way the immune system and antimicrobials are incapable to fulfill their action and, many times, are unable to reach internal bacteria. This fact is explained by the fact that many of the biological and pharmacological agents have physical-chemical properties incompatible with the penetration into the matrix. Aiming to determine which antimicrobials are suitable for using when dealing with a biofilm producing microorganism and which physical-chemical properties are directly related to the agent penetration into the polysaccharide matrix, we used colorimetric method with crystal violet to obtain biofilm minimum inhibitory concentration (MBIC) and the already standardized method for minimum inhibitory concentration (MIC). To accomplish these 10 antimicrobials were tested in Staphylococcus spp.: rifampin, azithromycin, clarithromycin, erythromycin, levofloxacin, gentamicin, doxycycline, chloramphenicol, clindamycin and vancomycin. For Pseudomonas aeruginosa all antimicrobials except for rifampin and vancomycin were included. There was a great difference between MIC and MBIC for many drugs, showing the need to evaluate these parameters before beginning treatment. The drugs with better results were rifampin and macrolides, while the worse were vancomycin and clindamycin, which can be attributed to the lipophilic profile with some water solubility present in the molecules with better results. The characteristics associated with poor penetration into biofilm were high polar surface area, complexity e molecular weight. Furthermore, the previous treatment of the plastic polymers with EDTA was accessed resulting in statistically significant reduction of biofilm production.

O advento do uso de cateteres venosos centrais na prática médica trouxe muitos benefícios aos pacientes, porém está relacionado a um aumento na incidência de infecções por microrganismos multirresistentes. Além disso, freqüentemente ocorre colonização por bactérias produtoras de biofilme. Estes microrganismos se aderem ao material abiótico desses dispositivos intravenosos, ficando protegidos sob a matriz exopolissacarídica do biofilme. Isso faz com que sistema imunológico e antimicrobianos sejam incapazes de ter sua ação plena e, muitas vezes, não atingem os microrganismos mais internos. O motivo deste insucesso é porque muitos desses agentes biológicos e farmacológicos apresentam propriedades físico-químicas incompatíveis com a penetração nesta matriz. Com o objetivo de determinar quais antimicrobianos são mais adequados para uso quando o microrganismo é produtor de biofilme e quais as propriedades físico-químicas que estão diretamente relacionadas à penetração do antimicrobiano na matriz polissacarídica, utilizou-se método colorimétrico com cristal violeta em microplacas modificado para obtenção de concentração inibitória mínima em biofilme (MBIC) e método já padronizado para concentração inibitória mínima (MIC). Para isso foram testados 10 antimicrobianos em Staphylococcus spp.: rifampicina, azitromicina, claritromicina, eritromicina, levofloxacino, gentamicina, doxiciclina, cloranfenicol, clindamicina e vancomicina. Para Pseudomonas aeruginosa foram testados os mesmos, exceto rifampicina e vancomicina. A discrepância entre MIC e MBIC foi muito grande para vários fármacos, mostrando a necessidade de se avaliar estes parâmetros antes do início da farmacoterapia para uma escolha correta, especialmente em hospitais. Os fármacos que apresentaram melhores resultados foram a rifampicina e os macrolídeos, enquanto que os menos efetivos foram vancomicina e clindamicina. Isso foi atribuído ao perfil lipofílico, porém com alguma solubilidade em água das melhores moléculas. Em contra ponto, a elevada área polar, complexidade e massa molar foram características negativas para a penetração em biofilme, resultando numa ineficácia para essas moléculas. Além disso, também foi avaliado o tratamento de polímeros plásticos com EDTA, obtendo-se redução significativa da produção de biofilme nas placas tratadas com o agente químico. / The use of central venous catheters in medicine has brought benefits to the patients and represents a great advance in clinical practice, while on the other hand this device is related to an increase in the incidence of infections caused by multiresistant pathogens. Furthermore, frequently, the catheters get colonized by biofilm producing bacteria. These microorganisms adhere to the abiotic material of the catheters keeping themselves protected underneath the exopolysaccharide matrix of biofilm, this way the immune system and antimicrobials are incapable to fulfill their action and, many times, are unable to reach internal bacteria. This fact is explained by the fact that many of the biological and pharmacological agents have physical-chemical properties incompatible with the penetration into the matrix. Aiming to determine which antimicrobials are suitable for using when dealing with a biofilm producing microorganism and which physical-chemical properties are directly related to the agent penetration into the polysaccharide matrix, we used colorimetric method with crystal violet to obtain biofilm minimum inhibitory concentration (MBIC) and the already standardized method for minimum inhibitory concentration (MIC). To accomplish these 10 antimicrobials were tested in Staphylococcus spp.: rifampin, azithromycin, clarithromycin, erythromycin, levofloxacin, gentamicin, doxycycline, chloramphenicol, clindamycin and vancomycin. For Pseudomonas aeruginosa all antimicrobials except for rifampin and vancomycin were included. There was a great difference between MIC and MBIC for many drugs, showing the need to evaluate these parameters before beginning treatment. The drugs with better results were rifampin and macrolides, while the worse were vancomycin and clindamycin, which can be attributed to the lipophilic profile with some water solubility present in the molecules with better results. The characteristics associated with poor penetration into biofilm were high polar surface area, complexity e molecular weight. Furthermore, the previous treatment of the plastic polymers with EDTA was accessed resulting in statistically significant reduction of biofilm production.

Self-medication impacts both negatively and positively the health of people, which has become evident during the COVID-19 pandemic. The study aimed to assess the prevalence of self-medicated drugs used for respiratory symptoms, as COVID-19 preventive, for its symptoms or once tested positive. To determine the perception of symptom relief and demographic variables that promote self-medication in Peru. We performed a cross-sectional, analytical, multicenter study in 3792 study respondents on the use, the reason for use, and perception of relief after the use of six drugs during the quarantine period. An online questionnaire was developed, pretested and submitted to the general public. Multivariable logistic regression was used to ascertain factors that influence an individual's desire to self-medicate, associations were considered significant at p < 0.05 and using region (coast, mountain and jungle) as cluster group. The majority of respondents self-medicated with acetaminophen for respiratory symptoms and mainly because they had a cold or flu. It was observed that all the surveyed drugs (acetaminophen, ibuprofen, azithromycin, penicillin, antiretrovirals and hydroxychloroquine) were consumed for various symptoms including: fever, fatigue, cough, sneezing, muscle pain, nasal congestion, sore throat, headache and breathing difficulty. Over 90% of respondents perceived relief of at least one symptom. Multivariable logistic regression showed that older people have a higher frequency of antiretroviral self-medication, respondents who currently have a job had a higher frequency of penicillin self-medication, and that respondents from the Andes consumed less acetaminophen, while the ones from the rainforest consumed it more. There were significant percentages of self-medication, including drugs without sufficient scientific evidence. Age, region where one lived and job status were variables associated with self-medication frequency. Continuous awareness and sensitization about the risks of self-medication are warranted. / Revisión por pares

Chlamydia trachomatis serovars D-K are the leading cause of bacterially-acquired sexually transmitted infections in the United States. As an obligate intracellular pathogen, C. trachomatis infects columnar epithelial cells of the genital mucosae and can cause deleterious sequelae such as pelvic inflammatory disease, infertility, and ectopic pregnancy. Several chlamydial antigens reach the host cell cytosol prior to the natural release of chlamydiae at the end of the developmental cycle. While some of these extra-inclusion antigens traffic to the host cell surface, others remain intracellular where they are proposed to influence vital host cell functions and antigen trafficking and presentation. The research herein examines the escape and trafficking of the immunodominant chlamydial antigens MOMP, LPS, and cHsp60 within C. trachomatis serovar E-infected polarized human endometrial epithelial cells. Studies using high-resolution transmission electron microscopy (TEM) and immuno-TEM report the novel escape mechanism of chlamydial antigens via vesicles everted/pinched off from the inclusion membrane, an occurrence observed both in the presence and absence of the antibiotic azithromycin. These extra-inclusion vesicles were differentiated from Golgi vesicles and were shown to deliver chlamydial heat shock protein 60 (cHsp60)-homologs 2 and 3, but not homolog 1, to the infected cell surface. Examination of the iron-responsiveness of the three cHsp60 homologs by immuno-TEM revealed a significant increase in cHsp60-2 following iron deprivation. Further investigation of the trafficking of chlamydial MOMP and LPS antigens enveloped within the protective everted inclusion membrane vesicles within host cells involved density gradient centrifugation for the separation of epithelial secretory pathway components followed by SDS-PAGE and Western blot to determine whether the chlamydial antigen-containing vesicles could fuse with and deliver the antigens to host cell organelles. Coupled with immuno-TEM, these data confirmed the presence of major chlamydial antigens within the endoplasmic reticulum of infected host cells. Additionally, chlamydial lipopolysaccharide (LPS) was co-localized with CD1d, a lipid antigen-presenting molecule. Collectively, these studies (i) establish a novel escape mechanism for chlamydial antigens, (ii) identify cHsp60-2 as a marker of iron stress response in C. trachomatis, and (iii) define for the first time the host cell ER as a destination for selected chlamydial antigens during infection.

The aim of the diploma thesis was to monitor seasonal concentration changes of 7 antibiotics norfloxacin, levofloxacin, ciprofloxacin, azithromycin, erythromycin, trimethoprim and sulfamethoxazole in wastewater influent and (cleaned) water effluent in the Sewage Treatment Plant (STP) České Budějovice. Time-proportional 24 hours pooled samples of wastewater were collected every month from March 2011 to February 2012 in the influent and effluent pof the STP. The concentrations of target compounds were determined by using in line SPE/LC-MS/MS analysis. The highest average concentration in the influent was detected in case of norfloxacin (0.563 microgram/l) and ciprofloxacin (0.406 microgram/l). The highest average concentration in the effluent was detected in the case of trimethoprim (0.255 microgram/l) and erythromycin (0.117 microgram/l). Higher concentration of antibiotics was measured in the colder periods of the year. It was connected with increased use of antibiotics and with less cleaning efficiency of the STP in this season. The highest removal efficiency was determined for norfloxacin and ciprofloxacin, the lowest for erythromycin. The influence of the season on the removal efficiency of antibiotics was found esp. for azithromycin,trimethoprim and sulfamethoxazole.