Thesis (MSc Food Sc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Food-borne disease outbreaks caused by Escherichia coli have been linked to the use of faecally-polluted irrigation waters. Thus the overall aim of this research was to evaluate irrigation water and agricultural effluents as sources of antibiotic resistant E. coli in the Western Cape. The aim of the first study was to enumerate and characterise E. coli present in irrigation water and in potential contamination sources. Maximum total coliform and E. coli counts for irrigation sites was log 7.862 and log 5.364 MPN.100 mL-1, respectively. Five out of seven irrigation sites had E. coli counts exceeding national and international guidelines for ‘safe’ irrigation water (<1 000 counts.100 mL-1), making it unsafe for the irrigation of fresh produce. In this study, 46.6% of the E. coli strains were characterised in phylogenetic group B1. It has been shown that E. coli in group B1 have the ability to survive and persist in the external environment. Group B1 was also the most common group among isolates from irrigation sites (79.4%), while isolates from environmental sites grouped mainly in group A0 (54.1%). It was concluded that the wide variation of E. coli types present in irrigation water is a concern that should be further investigated. This raises human health implications since the increased exposure to faecal organisms increases the risk of food-borne outbreaks. The E. coli isolates (n = 120) and the marker (n = 37) and reference strains (n = 6), were evaluated for antibiotic resistance to seven medically-important antibiotics from different classes using the Kirby-Bauer disc diffusion method. Thirty-five strains (35/163 = 21.5%) exhibited resistance to one or more antibiotics. Piggery effluent was found to harbour the most antibiotic resistant E. coli isolates (9/35 = 25.7%). Among the resistant E. coli strains, the highest occurrence of antibiotic resistance was to trimethoprim (2.5 μg) (68.6%), tetracycline (30 μg) (57.1%), ampicillin (10 μg) (45.7%) and chloramphenicol (30 μg) (34.3%). Seventy-four percent (26/35) exhibited multiple antibiotic resistances to two or more antibiotics. The antibiotic resistant E. coli strains were evaluated for the presence of pathotypes using Polymerase Chain Reaction analysis to detect Intestinal Pathogenic E. coli (InPEC) and Extra-intestinal Pathogenic E. coli (ExPEC). Five InPEC strains were characterised as four Entero-Pathogenic E. coli (EPEC) strains resistant to three or four antibiotics and one Entero-Aggregative E. coli (EAEC) strain resistant to trimethoprim. The antibiotic resistant EAEC strain also possessed the ExPEC-related gene iutA. Two E. coli isolated from the Mosselbank River were both resistant to chloramphenicol and trimethoprim and also possessed the ExPEC-related gene iutA. It was concluded that the diverse antibiotic resistances of E. coli pathotypes present in irrigation water is a concern that should be further investigated. / AFRIKAANSE OPSOMMING: Voedselverwante siekte uitbrake wat deur Escherichia coli veroorsaak word, is gekoppel aan die gebruik van fekale besoedelde besproeiingswater. Dus was die hoof doel van die navorsing om besproeiingswater en landbou-afvalwater te evalueer as bronne van antibiotika-weerstandbiedende E. coli in die Wes-kaap. Die doel van die eerste studie was om die getalle en eienskappe van E. coli te bepaal wat in besproeiingswater en in ander potensiële besmettingsbronne teenwoordig is. Maksimum totale koliforme en E. coli-tellings vir besproeiingspunte was onderskeidelik log 7.862 en log 5.364 MPN.100 mL-1. Vyf uit sewe besproeiingspunte het E. coli-tellings gehad wat hoër is as die nasionale en internasionale riglyne vir ‘veilige’ besproeiingswater (<1 000 tellings.100 mL-1). Dit maak dit onveilig vir die besproeiing van vars produkte. In hierdie studie was 46.6% van die E. coli-stamme in filogenetiese groep B1 gegroepeer. Dit is reeds bewys dat E. coli in groep B1 oor die vermoë beskik om in die eksterne omgewing te oorleef en voort te bestaan. Groep B1 was ook die mees algemene groep onder die isolate van besproeiingspunte (79.4%), terwyl isolate van omgewingspunte meestal in groep A0 (54.1%) gegroepeer is. Die breë variasie E. coli tipes in die besproeiingswater is bekommerniswaardig en sal gevolglik verder ondersoek moet word. Dit bring gesondsheidsimplikasies mee vir mense aangesien die verhoogde blootstelling aan fekale organismes die risiko van voedselverwante uitbrake verhoog. Die E. coli isolate (n = 120) en die merker (n = 37) en verwysingsstamme (n = 6), is teen sewe medies belangrike antibiotikas uit verskillende klasse getoets vir antibiotika-weerstandbiedendheid. Die Kirby-Bauer skyfie diffusie metode is gebruik. Vyf-en-dertig stamme (35/163 = 21.5%) het weerstand teen een of meer antibiotika getoon. Dit is gevind dat vark-afvalwater die meeste antibiotika-weerstandbiedende E. coli-isolate (9/35 = 25.7%) bevat. Die weerstandbiedende E. coli-stamme het die hoogste antibiotika-weerstandheid getoon teen "trimethoprim" (2.5 μg) (68.6%), tetrasiklien (30 μg) (57.1%), ampisillien (10 μg) (45.7%) en chloramfenikol (30 μg) (34.3%). Vier-en-sewentig persent (26/35) het meervoudige weerstandbiedheid teen twee of meer antibiotikas getoon. Die antibiotika-weerstandbiedende E. Coli stamme is getoets vir die teenwoordigheid van patogene deur van Polimerase Ketting Reaksie analise gebruik te maak om ‘Intestinal Pathogenic’ E. coli (InPEC) en ‘Extra-intestinal Pathogenic’ E. coli (ExPEC) op te spoor. Vyf InPEC-stamme is geklassifiseer as vier ‘Entero-Pathogenic’ E. coli (EPEC)-stamme wat weerstandbiedend teen drie of vier antibiotika getoon het en een ‘Entero-Aggregative’ E. coli (EAEC)-stam wat weerstandbiedendheid getoon het teen "trimethoprim". Die antibiotika-weerstandbiedende EAEC-stam het ook die ExPEC-verwante geen, iutA, besit. Twee E. coli isolate van die Mosselbankrivier het weerstand teen beide chloramfenikol en "trimethoprim" getoon en het ook die ExPEC-verwante geen, iutA, besit. Daar is tot die gevolgtrekking gekom dat die diverse antibiotika-weerstandbiedenheid van E. coli patogene teenwoordig in besproeiingswaters bekommerniswaardig is en verder ondersoek behoort te word.

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-22Bitstream added on 2014-06-13T19:03:31Z : No. of bitstreams: 1 silva_rml_dr_jabo.pdf: 986300 bytes, checksum: 4a3600af533a0403550481d53c48eaae (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dentre os agentes bacterianos amplamente distribuídos no ecossistema aquático, destacam-se as famílias Aeromonadaceae e Enterobactereaceae. Os peixes são importantes veículos de infecções humanas causadas por essas bactérias. Com este enfoque, o estudo foi realizado com o objetivo de verificar a ocorrência de Aeromonas sp., coliformes termotolerantes e bactérias heterotróficas mesófilas em pisciculturas da Região da Baixada Ocidental Maranhense. Para tal, foram selecionadas no período de outubro de 2008 a março de 2009, doze propriedades nos municípios de Pinheiro, Palmeirândia e Perimirim e colhidas amostras de água dos viveiros e peixes de cada piscicultura, totalizando 96 amostras. Em 100% das amostras analisadas foi confirmada a presença de Aeromonas sp., classificadas em quatro espécies, A. hydrophila (87,03%), A. caviae (8,02%), A. veronii sobria (3,70%), A. schubertii (1,23%). Essas ainda apresentaram elevados percentuais de resistência e multiresistência a 12 antimicrobianos testados. As populações de bactérias heterotróficas mesófilas nas pisciculturas variaram de 102 UFC/mL a 104 UFC/mL de água. Das pisciculturas avaliadas, sete apresentaram pelo menos uma amostra em desacordo com o padrão para coliformes termotolerantes. As amostras analisadas revelaram - se como possíveis vias de transmissão de aeromonas potencialmente patogênicas para peixes e ser humano, representando risco para a saúde da população consumidora dos organismos cultivados nessas propriedades / Among widely distributed agents in the aquatic ecosystem can be outstanding the families Aeromonadaceae and Enterobacteriaceae. The fishs are very important vehicles of human infections caused for these bacteria. With the approach the study intended to verify the occurrence of Aeromonas sp., thermotolerant coliforms and heterotrophic mesophilic bacteria in fish farms located in Occidental Baixada Maranhense Region. Twelve properties in the Pinheiro, Palmeirândia and Perimirim’ s cities were selected in the period from October of 2008 to March of 2009, and harvested water pond and fish samples of each fish farm, with the total of 96 samples. Aeromonas sp. was confirmed in 100% of samples, classified in four species, A. hydrophila (87,03%), A. caviae (8,02%), A. veronii sobria (3,70%), A.schubertii (1,23%). These bacteria showed high resistance and multiple resistance to the 12 antibiotics tested. The populations of heterotrophic mesophilic bacteria varied between 1,4 x 102 UFC/mL to 7,2 x 103 UFC/mL/. Seven fish farms showed at least one sample in disagreement with the standard to termotholerant coliforms. The samples of water and fish revealed the possible sources of potentially pathogenic contamination of aeromonas for fish and human being representing risk for health of the population healthy that consume the organisms cultivated in these properties

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-10-03Bitstream added on 2014-06-13T19:22:44Z : No. of bitstreams: 1 rodolpho_d_dr_jabo.pdf: 299270 bytes, checksum: 8a1668550cacade414ec08c3350e51f4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Escherichia coli Shigatoxigênica (STEC) tem sido implicada como agente causador de severas doenças humanas. Amostras de carne moída, moedor de carne e mãos de manipuladores de 23 estabelecimentos comerciais foram testadas para o isolamento de E. coli usando métodos microbiológicos padronizados. Um total de 287 cepas de E. coli isoladas destes diferentes locais foram submetidas ao PCR para detecção de genes stx 1, stx 2 e eae. As cepas positivas para o gene stx foram analisadas verificando se pertenciam ao sorogrupo 0157. Quatro cepas de STEC foram isoladas, sendo 2 de carne moída e 2 de moedor de carne, todas possuíam o gene stx 2, sendo negativas para a presença do gene eae e o sorogrupo 0157. Todas as E. coli isoladas, incluindo as 4 STEC, foram pesquisadas para sua resistência a 12 antibióticos. Altos níveis de resistência frente aos diferentes agentes antimicrobianos foram detectados; as resistências maiores foram observadas para a tetraciclina (76,6%), amoxicilina (64,1 %) e cefalotina (58,8%). Os altos níveis de resistência antimicrobiana salientam a necessidade para a utilização racional destes agentes em bovinos. Foram observadas índices elevados de sensibilidade frente a associação amoxicilina + ácido clavulânico (96,6%), ceftriaxona (92,7%) e gentamicina (90,3%). / Shiga toxigenic Escherichía colí (STEC) has been implicated as the causative agent of several human diseases. Samples from 23 retail meat stores (ground beef, grinding-machine and human hand) were assayed for E. calí isolation using microbiological standard methods. A total of 287 E. colí isolates from these different origins were submitted to polymerase chain reaction for the detection of stx 1, stx 2 and eae genes. The isolates positives for stx gene were serotyped for 0157. Four STEC isolates were recovered, 2 from ground beef and 2 from grinding-machine; ali harbored the stx 2 gene and were negative for the presence of the eae gene and the serogroup 0157. Ali E. colí isolates including the four STEC were screened for antibiotic resistance. High levels of resistance against different antimicrobial agents were detected; those most commonly observed were to tetracycline (76.6%), amoxicillin (64.1 %) and cephalothin (58.8%). Such high levels of antimicrobial agents' resistance highlight the need for a more rational use of these agents in cattle. Susceptibility was high for amoxicillin + clavulanic acid (96,6%), ceftriaxone (92,7%) and gentamicin (90,3%).

The work described in this thesis focused on the use of nuclear magnetic resonance spectroscopy (NMR) to study two classes of metallo enzymes - the Fe(II)- and 2oxoglutarate (2OG)-dependent dioxygenases and the metallo β-lactamases (MBLs). These enzymes are involved in clinically important biological processes, i.e. the hypoxic response and antimicrobial resistance, respectively. Both protein systems are interesting from an NMR perspective because they have dynamic regions involved in catalysis and ligand interactions. The work included mechanistic studies, protein-ligand interaction studies, and method development for inhibitor discovery. NMR was applied to study the human prolyl hydroxylase domain-containing protein 2 (PHD2), which is crucially involved in the chronic hypoxic response. The results reveal that binding of the C- and the N-terminus of the oxygen dependent degradation domains CODD and NODD, respectively, induce different interactions with PHD2. The substitution of a single amino acid, as occurs with PHD2 variants linked to erythrocytosis and breast cancer, can alter the selectivity of PHD2 towards its ODD substrates. Studies with the Trichoplax adhaerens PHD provide insights into the evolutionary substrate preference of the PHDs. Using ¹³C-labelled peptidyl-substrates; NMR was applied to investigate proposed 'alternative' PHD2 substrates/interaction partners. The product release mechanism of PHD2 was investigated using NMR; the results reveal that the presence of 2OG strongly discriminates between the binding of CODD and hydroxylated CODD to PHD2. NMR was also applied to monitor PHD2 kinetics and inhibition. Competition and displacement assays were designed and applied to investigate PHD inhibitor binding modes. Comparative studies on the activities and selectivities of PHD inhibitors in clinical trials should aid in the work on the therapeutic manipulation of the natural hypoxic response. Protein-observe ¹9F-NMR was used to study the São Paolo MBL (SPM-1). The results provide new structural insights into SPM-1 catalysis and the requirements for inhibitor development. They also reveal that the hydrolysed β-amino acid products of MBL catalysis can bind to SPM-1. They illustrate the utility of ¹⁹F-NMR for detecting metal chelation, which is not always readily tractable in studies on metallo enzyme inhibition, new binding modes, and stereoisomer binding/epimerisation in solution. The interaction of a cyclobutanone analogue, a broad-spectrum MBL inhibitor, with SPM-1 was investigated. A combination of ¹H, ¹⁹F, ¹³C-NMR and crystallographic analyses reveal that cyclobutanone binding may mimic formation of the oxyanion tetrahedral intermediate in β-lactam hydrolysis. The susceptibility of avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, to MBL-catalysed hydrolysis was studied. The results reveal that avibactam is not an MBL inhibitor and a poor substrate of most members of all three clinically relevant subclasses of MBLs. In some cases, avibactam undergoes slow hydrolysis in a process different from that observed with serine β-lactamases. Overall, the results illustrate the utility of NMR for studying dynamic aspects of enzyme catalysis and inhibitor binding.

This thesis describes the synthesis and biological evaluation of a library of compounds containing the tetramic acid core in search of novel antibacterial drug candidates. Chapter 1 discusses the need for new antibiotics due to the emergence of virulent bacterial strains resistant to clinically available drugs and the hiatus in the discovery of new replacement antibitoics that has become a global threat to human health. Different platforms for antibacterial drug discovery and the re-emergence of natural products-based approach that has gained importance in the quest for novel antibiotics are discussed. In this regard, the intrinsic antibacterial activity of natural products containing a tetramate core structure and the strategies developed to synthesise the core scaffold are described. Chapter 2 discusses the use of ʟ-serine and ʟ-cysteine in tetramic acid synthesis and the application of ʟ-cysteine-derived thiazolidine templates suitable for stereoselective ring closing reactions to obtain the tetramic acid core with scope for further functionalization. Chapters 3 and 4 describe a range of synthetic routes for appropriate substitutions of the tetramate core for compound library generation. Elaboration of the tetramate core via carboxamide tetramate synthesis, Suzuki-Miyaura cross-coupling reactions, glycosylations and their aglycone analogue synthesis, etherification, tetramate-pyroglutamate systems, Buchwald aminations/amidations, cycloadditions and β-lactam hybrids as possible chemical modifications of the tetramate core structure are discussed. Chapter 5 describes the antibacetiral activity and physicochemical properties of the library of compounds synthesised. A preliminary evaluation of their antibiotic activity was conducted against S. aureus and E. coli using the hole-plate method. MICs of the tetramates synthesised were determined against several Gram-negative strains; Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Gram-positive strains; MRSA, Enterococcus faecalis and Streptococcus pneumoniae, in whole-cell bioassays. Physicochemical properties of the compound library were analysed to map the chemical space occupied by tetramates with potent antibacterial activity. Enzyme inhibition studies were conducted to identify possible modes of action that contribute to whole-cell antibiotic activity and in this regard, the inhibition of enzymes S. aureus topoisomerase IV, S. aureus RNA polymerase, E. coli RNA polymerase, E. coli gyrase and M. tuberculosis gyrase are discussed. Since plasma protein binding of compounds is an important factor that determines the bioavailability of antibiotics and their clinical outcome, a study of the binding affinity of these drug candidates to Human Serum Albumin (HSA) by both whole-cell bioassay and NMR spectroscopy-based protein binding experiments are discussed. Finally, a brief note on the potential of tetramic acids to function as proteasome inhibitors in anticancer chemotherapy is included at the end of this chapter.

Legionella pneumophila est une bactérie à Gram négatif, intracellulaire facultative, responsable de la légionellose (ou maladie des Légionnaires) chez l'Homme. Les fluoroquinolones et les macrolides sont utilisés en première intention dans le traitement antibiotique de cette maladie. Cependant, les échecs thérapeutiques sont fréquents, et le taux de mortalité demeure élevé (10-15% des cas, plus de 30% chez le patient immunodéprimé). Bien qu'aucune souche de L. pneumophila résistante à ces antibiotiques n'ait été isolée à ce jour, ces échecs peuvent faire évoquer la possibilité d'une sélection in vivo de mutants résistants. Le mécanisme génétique principal d'acquisition de la résistance aux fluoroquinolones correspond à l'accumulation de mutations au niveau des gènes codant pour l'ADN gyrase et la topoisomérase IV ; en particulier celles affectant les codons en positions 83 et 87 du QRDR (quinolone resistance determining region) du gène gyrA entrainent une résistance de haut niveau à ces antibiotiques. Le première aspect de notre projet était d'élaborer un test de PCR en temps réel permettant de détecter chez L. pneumophila des mutants gyrA résistants aux fluoroquinolones et de les différencier des souches sauvages par analyse des températures de fusion des amplifias obtenus. Après optimisation, ce test nommé qPCRgyrALp amplifie spécifiquement une portion du QRDR du gène gyrA de l'espèce L. pneumophila et permet de détecter et de différencier les mutations gyrA83 et gyrA87. Nous avons ensuite utilisé ce test pour la recherche de mutants gyrA directement dans divers prélèvements respiratoires provenant de 82 patients atteints de légionellose, certains en échec thérapeutique après traitement par une fluoroquinolone. Les résultats ont montré pour quatre patients un profil de courbe de fusion semblable à celui du mutant gyrA83. Le séquençage du QRDR de gyrA à partir de ces prélèvements respiratoires a confirmé cette mutation chez deux patients. L'utilisation de la technique de séquençage à haut débit a permis de quantifier ces mutants gyrA83 chez ces deux patients, permettant de montrer un remplacement progressif in vivo de la population de L. pneumophila sensible aux fluoroquinolones par une population résistante à ces antibiotiques. Le deuxième aspect de notre travail a été de développer des tests de PCR quantitative en temps réel (qPCR) permettant de quantifier la charge bactérienne à L. pneumophila dans les prélèvements cliniques des patients infectés, avant et au cours du traitement antibiotique, dans la but de prédire l'évolution clinique et le pronostic final de ces patients. Nous avons utilisé deux tests de qPCR, ciblant soit le gène codant pour l'ARNr16s (qPCR16S) soit le gène mip (qPCRmip) dans des prélèvements respiratoires de 116 patients atteints de légionellose. Chez certains patients, nous avons pu déterminer la cinétique de la charge bactérienne au cours du temps, alors que les patients recevaient une antibiothérapie adaptée. Les premières cinétiques recueillies montrent la possibilité de différencier les patients qui répondent rapidement au traitement antibiotique et évoluent favorablement au cours de la 1ère semaine d'hospitalisation, de ceux qui présentent une réponse modeste au traitement et nécessitent une hospitalisation prolongée, voire décèdent. La PCR en temps réel semble donc représenter un outil pronostique d'intérêt au cours de la légionellose. Le type de cinétique observé chez un patient donné semble pouvoir prédire l'évolution des patients et la nécessité d'ajuster le traitement antibiotique. / Legionella pneumophila is a Gram- negative, facultative intracellular bacterium responsible for legionellosis (or Legionnaires' disease ) in humans. The fluoroquinolones and the macrolides are used as first-line antibiotic treatment of this disease. However, treatment failures are common, and the mortality rates remain high (10-15 % of cases, more than 30% in immunocompromised patients). Although L. pneumophila strain resistant to these antibiotics have never been isolated, treatment failures may suggest the possibility of in vivo selection of resistant mutants. The main genetic mechanisms associated with acquired resistance to fluoroquinolones correspond to the accumulation of mutations in the genes encoding DNA gyrase and topoisomerase IV, especially those affecting codons 83 and 87 of the QRDR (quinolone resistance determining region) of the gyrA gene, which are associated with high level resistance to these antibiotics. The first aspect of our project was to develop a real-time PCR test to detect gyrA QRDR mutants and differentiate them from wild-type strains of L. pneumophila by analysis of melting temperatures of the amplified DNA. After optimization, the qPCRgyrALp test specifically amplified a portion of the gyrA QRDR of L. pneumophila and could detect and differentiate gyrA83 and gyrA87 mutations. Then, we checked the presence of gyrA mutants directly in respiratory samples collected in 82 legionellosis patients, including some after treatment failure with a fluoroquinolone. For four patients, results corresponded to a melting curve profile similar to that of the gyrA83 mutant. Amplification and sequencing of the gyrA QRDR directly from these respiratory samples confirmed this mutation in two patients. The use of high-throughput sequencing technology allowed us to quantify the gyrA83 mutants in these two patients, allowing demonstration of in vivo gradual replacement of the fluoroquinolones susceptible population of L. pneumophila by a resistant one. The second aspect of our work was to develop quantitative real-time PCR tests offering the possibility to quantify the L. pneumophila bacterial load in respiratory specimens before and during antibiotic treatment, in order to predict the clinical course and the final prognosis of these patients. We used two qPCR tests, either targeting the gene encoding 16S rRNA (qPCR16S ) or the mip gene (qPCRmip ) in respiratory samples from 116 patients with Legionnaires' disease. In some patients, we determined the kinetics of bacterial loads over time, while patients received appropriate antibiotic therapy. The kinetics we observed allowed differentiation of patients who respond quickly to antibiotic treatment and were released from hospital within the first week following admission, from those with a modest response to treatment and requiring prolonged hospitalization or finally died. Thus, our real-time PCR tests seem to be good prognostic tools for evaluation of legionellosis prognosis. The type of kinetics observed in a given patient may allow the clinician to predict the evolution of patients and the need to adjust the antibiotic treatment.

Health Care Associated Infections (HCAIs) are a concern especially in regards to antibiotic resistance and effective treatments. Staphylococcus aureus is often the main focus for eradication and prevention procedures, however, other bacterial species are also problematic. These include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis amongst others. Chronic infections caused by these bacteria are often biofilm related, and include dental caries, otitis media, osteomyelitis, burns & chronic wounds, and device related & prosthetic joint infections. Prosthetic joints and indwelling devices, such as catheters, are a prime environment on which biofilms can develop. This thesis aims to look at biofilms, investigating how they are established, the development of resistance against individual antibiotics and the antibiotic concentrations required to reduce biofilm load. A novel biofilm system – the alginate bead method will be used for these experiments, The alginate bead method was developed by a previous student in the Gallagher Laboratory, due to a need to have a reliable, robust and inexpensive technique to examine formation of biofilms and antibiotic resistance. There are devices and assays available, such as the Calgary Biofilm Device, which are extensively used for these purposes. However, the cost is prohibitive. This thesis found that the development of biofilms occurs much earlier than expected, with stable, fixed formation after just four hours of growth. Depending upon the antibiotic, resistance can develop within the first two hours of growth and thereafter steadily increases. By 24 hours the biofilms are fully resistant to all the tested antibiotics. In mixed species biofilms, the two species act synergistically protecting each other against the antibiotics, resulting in a much higher antibiotic concentration required. Common antibiotics used to treat staphylococcal infections are often combined to enhance their destructive effect and prevent the development of resistance. The effects of these antibiotics, when combined was explored. Biofilm resistance against gentamicin, one of the most common antibiotics used to treat staphylococcal infections develops quickly. However, when combined with other antibiotics gentamicin resistance is delayed. As antibiotic concentrations have to be extremely high in order to have any effect on established biofilms, alternative methods need to be investigated. Any alternative approaches would be employed in conjunction with conventional therapies preventing stable biofilm formation and disrupting established biofilms. Such methods may include sugar metabolites, enzymatic disruption, D-amino acids and activation of the quorum sensing system. The main conclusion which can be taken from this work are that firstly the alginate bead method of a viable, suitable alternative to the Calgary Biofilm Device and supports biofilm formation and testing. Secondly that biofilms form and are resistant to antibiotics much earlier than expected, and extreme concentrations of antibiotics are required to have an effect. Thus the inclusion of alternative methods which disrupt biofilms would be beneficial to clinical practice. However, the alternative methods investigated within this thesis (D-amino acids and sugar metabolites) failed to show any inhibition of biofilms. There are other possible choices which would need to be investigated.

Introdução: Sepse relacionada ao cateter é a principal causa de hospitalizações e a segunda causa de morte em pacientes com insuficiência renal crônica em hemodiálise. O uso de medicações que possam diminuir a incidência de bacteremia relacionada ao cateter parece uma alternativa para diminuir o número de eventos. Objetivo: Comparação do uso de heparina contra minociclina e edetato dissódico (MEDTA) como selo de cateter de longa permanência em pacientes com insuficiência renal crônica em hemodiálise, comparando desfecho de sobrevida de cateter em relação ao tempo livre de bacteremias, alterações de fluxo, necessidade de trombolítico e cultura positiva de material de descarte. Método: Ensaio clínico, randomizado, multicêntrico, aberto, controlado, comparativo entre heparina e MEDTA. Resultados: 28 pacientes foram incluídos no total do estudo (14 no grupo heparina e 14 no grupo MEDTA). Os grupos não apresentaram diferenças estatisticamente significativas em relação à idade, sexo, níveis de albumina e hemoglobina, tempo de permanência de cateter, alterações de fluxo do cateter (fluxo < 250mL/min) e necessidade de uso de trombolítico. O grupo MEDTA apresentou significativamente menos episódios de bacteremia e menor número de culturais de material de descarte positivos (P< 0,05). Conclusões: O uso de MEDTA como selo parece diminuir a incidência de bacteremias em pacientes com insuficiência renal crônica em hemdiálise emuso de acesso vascular por cateter, apresentando índices semelhantes de patência de cateter. / Introduction: Catheter-related sepsis is the leading cause of hospitalization and the second cause of death in patients with chronic renal failure undergoing hemodialysis. The use of medications that can reduce the incidence of catheter-related bacteremia seems as an alternative to reduce the number of events. Objective: Comparison of heparin against minocycline and disodium edetate (MEDTA) as a tunneled catheter lock in patients with chronic renal failure on hemodialysis, comparing outcome of catheter survival over time free survival time of bacteremia, flow alterations, need of thrombolytic therapy and positive culture of material from discard. Method: Clinical trial, randomized, multicenter, open, controlled comparative between heparin and MEDTA. Results: 28 patients were included in the overall study (14 in the heparin group and 14 in MEDTA group). The groups showed no statistically significant differences regarding age, sex, albumin and hemoglobin levels, length of catheter, catheter flow changes (flow <250mL/min) and required use of thrombolytic therapy. MEDTA group had significantly fewer episodes of bacteremia and fewer positive cultural of discarded material (P <0.05). Conclusions: The use of MEDTA as a catheter lock appears to decrease the incidence of bacteremia in patients with chronic renal failure in hemdiálise inuse vascular access catheter, showing similar rates of patency of the catheter.

Objetivo: avaliar o possível efeito da obesidade sobre o tratamento periodontal não-cirúrgico associado ou não a antibiótico em mulheres de peso normal e obesas após 6 meses de acompanhamento. Metodologia: a partir de um delineamento do tipo ensaio clínico randomizado, controlado por placebo, em paralelo, 32 mulheres (35-55 anos) com periodontite crônica foram incluídas e distribuídas em dois grupo experimentais (16 peso normal e 16 obesas). Mulheres fumantes, diabéticas e com sobrepeso não foram incluídas. As pacientes foram alocadas randomicamente para receber tratamento periodontal subgengival associado ao uso de placebo ou metronidazol (400mg, 3x/dia, durante 10 dias) concomitantemente. Os parâmetros clínicos periodontais foram avaliados por um examinador treinado e calibrado no baseline e 6 meses após o tratamento periodontal. Resultados: Todos os parâmetros clínicos apresentaram melhora durante o período de acompanhamento. O Índice de Placa Visível foi positivo em aproximadamente 25% dos sítios nos grupos experimentais após 6 meses do tratamento, enquanto que o Índice de Sangramento Gengival e o Sangramento à Sondagem foram observados em aproximadamente 10% e 25% dos sítios, respectivamente. Esses parâmetros não apresentaram diferenças significativas entre os grupos experimentais aos 6 meses. A redução na Profundidade de Sondagem variou entre 0.52 ± 0.3 e 0.81 ± 0.5mm e o ganho de Inserção Clínica variou entre 0.23 ± 0.3 e 0.45 ± 0.5mm. Não foram observadas diferenças estatisticamente significativas entre os grupos experimentais para estes parâmetros (P=0.59 e 0.67, respectivamente). Conclusão: Ponderando as limitações desta análise preliminar, obesidade parece não afetar negativamente os resultados da terapia periodontal não-cirúrgica. Além disso, o uso de Metronidazol não forneceu um benefício adicional a terapia periodontal. / Aim: to evaluate the possible effects of obesity on clinical parameters after systemic antibiotic adjunctive scaling and root planning (SRP) in women with 6 month follow-up. Methods: a randomized clinical trial was conducted. Thirty-two women (35 - 55 years old) with chronic periodontitis were allocated into obesity (n=16) and normal weight (n=16) groups. Smokers, diabetics and overweight were not included. Patients were randomized to SRP plus Metronidazole (400 mg, 3 time/day, 10 days) or SRP plus placebo. Periodontal parameters were evaluated by one calibrated examiner at baseline and 6 month follow-up. Results: all periodontal parameters showed improvement during follow-up. Visible Plaque Index was positive around 25% of sites in the experimental groups after 6 months of treatment, while the Gingival Bleeding Index and Bleeding on Probing were observed in approximately 10% and 25% of the sites, respectively. These parameters showed no significant differences between experimental groups at 6 months. Probing Pocket Depth was reduced ranged 0.52 ± 0.3 and 0.81 ± 0.5 mm and Periodontal attachment loss get ranged 0.23 ± 0.3 and 0.45 ± 0.5 mm. There were no statistically significant differences between experimental groups for these parameters (P=0.59 and 0.67, respectively). Conclusion: Considering the limitations of this preliminary analysis, obesity seems not affect the results of nonsurgical periodontal therapy. Furthermore, the use of Metronidazole was not provided an additional benefit to periodontal therapy.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A taxa de partos cesáreos no Brasil é muito alta, consequentemente ocorrendo uma maior incidência de infecção puerperal. A infecção puerperal é a principal complicação clínica que acomete muitas gestantes submetidas a uma cesariana, ocasionando maior morbimortalidade materna e ocupando um dos primeiros lugares dentre as doenças próprias do ciclo gravídico puerperal. Em função da importância dessa complicação e do risco obstétrico e neonatal que a mesma determina, fundamenta-se a importância da realização do presente estudo, cujo principal objetivo foi caracterizar o perfil epidemiológico das gestantes submetidas a cesariana com o uso da antibioticoprofilaxia como prevenção para infecção puerperal, afim de possibilitar a melhoria da assistência, no contexto da Saúde da Mulher. Tratou-se de um estudo transversal, observacional e prospectivo, realizado no período de agosto de 2009 a junho de 2010. Os dados foram obtidos através de formulário específico contendo 35 perguntas, as quais abordaram a situação socioeconômica, questões reprodutivas e questões ligadas à gestação atual. Os dados foram processados no programa Epi Info, versão 3.2.2. Foram entrevistadas 855 puérperas, cuja média de idade foi de 24,8 ± 6,4 anos. Observou-se no presente estudo que, no contexto socioeconômico, as únicas variáveis que apresentaram significância estatística para o desenvolvimento de infecção puerperal foram possuir a cor branca (efeito protetor) (OR=0,378; p=0,0227) e a cor negra (efeito promotor) (OR=2,487; p=0,0028) e a utilização do antibiótico profilático que confere proteção à puérpera (OR=0,403; p=0,0070). O estudo evidencia ainda que a realização de parto cesáreos, em maternidade pública, oferece maiores chances de infecção puerperal (OR=2,111; p=0,0086). Atualmente ainda não existe um procotolo para o uso da antibioticoprofilaxia. Cada unidade hospitalar respeita a conduta proposta pelo médico que presta assistência a gestante nesse procedimento. Considerando que o uso da antibioticoprofilaxia tem ajudado a diminuir os índices de infecção puerperal é preciso que sejam implantados protocolos, por meio de método prospectivo de vigilância com relação à infecção puerperal, ainda na admissão, de forma que possibilite a implementação de ações sistematizadas e direcionadas à população parturiente, bem como para estabelecer medidas de prevenção e controle das infecções, além de possibilitar o conhecimento do perfil microbiológico das infecções ora detectadas.