South Africa is burdened with tuberculosis (TB) which is aggravated by the concurrent epidemic of HIV as well as the emergence of drug resistance. In most developed countries molecular techniques have been used to look at the dynamics of the TB epidemic however, despite the prevalence that is high in sub-Saharan Africa, there is little data on strain types that are available in Port Elizabeth. This study aims to find the major clades of M. tuberculosis that are circulating in Port Elizabeth. Two hundred MDR-TB DNA samples were obtained from the National Health Laboratory Services TB laboratory in Port Elizabeth. Spoligotyping and MIRU-VNTR were used to genotype the strains. Two hundred strains were sent to the University of Stellenbosch for spoligotyping and 179 of those were typed. Spoligotype defined families were further typed by MIRU-VNTR typing, so as to further differentiate and assess clonal diversity within the spoligotype families. The Beijing family was the dominant family and the MANU family being the least dominant, with percentages of 71 percent and 0.5 percent respectively. A comparison of spoligotyping results with the international spoligotyping database (SITVIT2) showed a total of 15 shared international types. Forty four percent (44 percent) of the isolates that were typed by MIRU-VNTR showed similarities, suggesting epidemiological relatedness. Thirty eight percent of isolates from spoligotyping were from the same family, the Beijing family, with the same shared international type STI1, but when typed by 12 MIRU-VNTR they showed no epidemiological relatedness and 18 percent of the isolates showed no relatedness when typed by 12 MIRU-VNTR but spoligotyping showed that they were from the LAM family. Results from our study illustrate the effectiveness of MIRU-VNTR typing together with spoligotyping in epidemiological studies in the region of Port Elizabeth.

Made available in DSpace on 2016-12-23T13:56:05Z (GMT). No. of bitstreams: 1 Maria Carolina Campana - texto completo.pdf: 1739324 bytes, checksum: 2c77a0541ea8a062d1765a13e6b4e9e5 (MD5) Previous issue date: 2010-09-02 / Immunity against tuberculosis depends on the establishment of an effective Th1 response, with IFN- production, a pivotal cytokine to the activation of macrophages and bacilli destruction. It is known that several factors are related to the host s resistance to tuberculosis. In the present work, the influence of T regulatory cells (Tregs) in the immune response against Mtb was evaluated in whole blood and PBMC cultures from tuberculin skin test positive (TST+) and tuberculin skin test negative (TST-) individuals. Paralelly, we also evaluated if different levels of resistance to anti-mycobacterial drugs displayed by different Mtb clinical isolates could interfere with the cytokine profile, Tregs frequency, and with its proliferative capacity on both PBMC and whole blood cultures from TST+ and TST- subjects. Twenty-two healthy volunteers were invited to participate in our study, 11 TST+ and 11 TST-. Twelve Mtb clinical isolates were used, 6 drug-resistant and 6 drug-susceptible. Our data shows that differences in drug susceptibility did not affected any of the evaluated parameters. In the other hand, TST status correlated with differences in Treg frequencies, which was significantly elevated among TST+ when compared to TSTsubjects. The increase in Tregs was also associated with a decrease in the microbicidal effect observed in whole blood cultures from TST+, when compared to TST-. This association was confirmed through the depletion of Tregs, which restored the microbicidal activity and an increase in IFN-, IL-2, IL10, and TNF- when compared to undepleted cultures. Data presented here supports the hypothesis that pre-exposure to MTB, represented by a positive TST result, may increase the number of Treg cells, which in turn could affect negatively the establishment of a protective immunity against TB in TST+ individuals / A resposta imune contra a tuberculose é mediada por uma resposta do tipo Th1, com a produção de IFN-γ, citocina fundamental para a ativação dos macrófagos e, consequentemente para a eliminação do bacilo. Vários fatores podem estar relacionados com a resistência a tuberculose. Neste trabalho, foi avaliado o impacto das células T reguladoras na resposta imune contra o Mtb em infecções de amostras de sangue total e CMSP de indivíduos positivos para o teste com PPD (PPD+) e negativos ao teste com PPD (PPD-). Além disso, avaliamos também se o perfil de resistência dos isolados de Mtb tem relação com o perfil de citocinas secretadas, com a frequência de células T reguladoras e com sua capacidade proliferativa em culturas de sangue total e CMSP dos indivíduos estudados. Para isso, foram arrolados 22 voluntários saudáveis. Desses indivíduos, 11 eram PPD+ e 11 eram PPD-. Foram também utilizados 12 isolados de Mtb, sendo 6 sensíveis e 6 resistentes a drogas antituberculose. Não foram encontradas diferenças entre os perfis de resistência dos isolados de Mtb e os parâmetros avaliados. O número de células T reguladoras encontrado nos indivíduos PPD+ foi maior em relação aos PPD- e a presença dessas células afetou negativamente o efeito microbicida nas culturas de sangue total de indivíduos PPD+, nas quais os isolados de M. tuberculosis proliferaram mais. Quando foram feitas infecções com isolados de Mtb em culturas de CMSP nas quais as células T reguladoras foram depletadas uma diminuição na proliferação do Mtb foi encontrada tanto nas amostras de indivíduos PPD+ quanto PPD-. Essa alteração na proliferação do Mtb foi acompanhada de maiores níveis de INF-γ, IL-2, IL-10 e TNF-α em relação às culturas de CMSP na presença das células T reguladoras. Os resultados desse trabalho sugerem que a pré-exposiçã o ao Mtb, evidenciada pelo teste positivo com o PPD, pode aumentar o número de células T reguladoras que, por sua vez, podem prejudicar a resposta imune protetora contra a tuberculose nos indivíduos PPD+

No description available.

Arranjos supramoleculares combinando o lípide catiônico brometo de dioctadecildimetilamônio (DOD) com polímeros, como carboximetilcelulose (CMC) e cloreto de poli(dialildimetilamônio) (PDDA), foram preparados na forma de nanopartículas (NPs), na ausência ou presença de antimicrobiano tradicional, como a claritromicina (CLA). NPs preparadas por atração eletrostática entre os fragmentos de bicamada (BF) de DOD, CMC e PDDA foram avaliadas, in vitro, quanto à atividade contra isolados clínicos de micro-organismos multirresistentes (MR) a antimicrobianos, como Pseudomonas aeruginosa MR, Klebsiella pneumoniae produtora da enzima carbapenemase do tipo KPC, Staphylococcus aureus resistente à meticilina/oxacilina (MRSA) e Candida albicans resistente ao fluconazol, através do método de plaqueamento e contagem de viáveis. As NPs de DOD BF/CMC/PDDA apresentam alta atividade de amplo espectro contra micro-organismos MR, em que o PDDA é o componente responsável pela excelente atividade biocida das NPs. O mecanismo de ação antimicrobiana indica a dissociação dessas NPs na presença dos micro-organismos, com a remoção de biopolímeros da parede celular microbiana pelo PDDA, conforme visualizado por microscopia eletrônica de varredura, ocorrendo lise da membrana microbiana e liberação de compostos fosforilados para o meio extracelular. Também foram desenvolvidas neste trabalho NPs carreadoras de CLA à base de DOD e polímeros. Solução etanólica contendo CLA/DOD foi injetada em solução aquosa de CMC, formando arranjos coloidalmente estáveis e aniônicos, que posteriormente foram adicionados de solução de PDDA, para a obtenção de arranjos estáveis e catiônicos. CLA/DOD/CMC e CLA/DOD/CMC/PDDA NPs incorporaram CLA em quantidade suficiente para inibir o crescimento de M. abscessus no interior de macrófagos bem como evitar a formação de biofilmes, sendo que altas doses de CLA foram tóxicas aos macrófagos, enquanto doses menores apresentaram baixa toxicidade e boa atividade antimicrobiana. NPs catiônicas carreando CLA foram tóxicas aos macrófagos nas concentrações de PDDA testadas. A natureza particulada das CLA NPs possivelmente aumenta a retenção intracelular de CLA em comparação com CLA livre, podendo prolongar atividade da CLA contra patógenos intracelulares. Desta maneira, arranjos supramoleculares combinando lípide e polímeros, com ou sem antimicrobianos tradicionais poderão encontrar diversas aplicações nas áreas farmacêutica, médica, alimentícia e biotecnológica. / Supramolecular assemblies combining cationic lipid dioctadecyldimethylammonium bromide (DOD) and polymers, such as sodium carboxymethylcellulose (CMC) and poly(diallyldimethylammonium chloride) (PDDA), were prepared as nanoparticles (NPs), in the absence or presence of traditional antibiotic, such as clarithromycin (CLA). NPs prepared by electrostatic attraction between DOD bilayer fragments (BF), CMC and PDDA were evaluated against clinical strains of multidrug resistant (MDR) microorganisms, such as Pseudomonas aeruginosa MDR, Klebsiella pneumoniae producer of KPC carbapenemase enzyme, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans fluconazole resistant, by plating and colony forming unities counting. DOD BF/CMC/PDDA NPs display high and broad-spectrum activity against MDR microrganisms, and PDDA is the excellent biocidal component in the NPs. The mechanism of antimicrobial action shows that NPs disassembly in the presence of microrganisms, with biopolymers withdrawn from the cell wall, as observed by scanning electron microscopy, consecutively lysing bacterial membrane as determined from the leakage of inner phosphorylated compounds. In this work there have also been developed NPs, based on lipid and polymers, as carriers for CLA. Ethanolic solution co-solubilizing CLA/DOD was injected in CMC aqueous solution, yielding colloidaly stable and anionic NPs, that were further added of PDDA solution, yielding stable and cationic NPs. CLA/DOD/CMC NPs and CLA/DOD/CMC/PDDA NPs incorporated CLA at doses high enough to inhibit M. abscessus growth inside macrophages or in biofilms. Larger CLA doses were toxic to macrophages while lower CLA doses reduced toxicity to macrophages despite their high antimicrobial activity. Cationic CLA NPs exhibited substantial toxicity against macrophages at the PDDA concentrations tested. The particulate nature of these CLA NPs possibly increases intracellular CLA retention in comparison to free CLA, probably extending CLA activity against intracellular pathogens. In conclusion, supramolecular assemblies combining cationic lipid and polymers, with or without traditional antibiotics, may find multiple possibilities of applications at pharmaceutical, medical, food and biotecnological fields.

As infecções relacionadas à assistência à saúde (IRAS) são um grave problema de saúde pública cujo prognóstico tem sido desfavorecido pela emergência e endemicidade de bactérias multirresistentes (MRs). Neste cenário, seguindo uma tendência mundial, no Brasil, infecções por cepas de Acinetobacter baumannii MRs produtoras de carbapenemases do tipo OXA são atualmente consideradas uma emergência clínica e epidemiológica. Na falta de alternativas terapêuticas efetivas para infecções relacionadas, este trabalho objetivou avaliar efeitos sinérgicos (utilizando checkerboard e time-kill) decorrentes da combinação de 10 antimicrobianos de diferentes classes, contra 8 cepas MRs de A. baumannii, clonalmente não relacionadas, produtoras de carbapenemases do tipo OXA-23, OXA-72, OXA-58 e OXA-143, representativas de diferentes centros hospitalares do Brasil. Como resultado, a combinação amicacina/tigeciclina apresentou atividade sinérgica (S= ΣCIF ≤ 0,5) e parcialmente sinérgica (PS= &#931CIF ;0,5-0,75) contra 4 (50%) cepas produtoras de OXA-143 ou OXA-72, e 2 cepas (25%) produtoras de OXA-23, respectivamente. Por outro lado, a combinação polimixina B/imipenem apresentou atividade S e PS contra 3 (37,5%) isolados OXA-143, OXA-23 ou OXA-72 positivos, e 1 (12,5%) isolado produtor de OXA-58, respectivamente. Já, a combinação amicacina/ampicilina-sulbactam foi S contra 2 (25%) A. baumannii OXA-143 ou OXA-23 positivos, sendo PS contra dois (25%) A. baumannii OXA-58 ou OXA-143/23 positivos. De interesse, foi o efeito S da combinação polimixina B/vancomicina, contra 2 cepas (25%) produtoras de OXA-72 ou OXA-23. Por outro lado, a combinação ampicilina-sulbactam/rifampicina apresentou atividade PS contra 6 (75%) cepas produtoras das variantes OXA-23, OXA-143, OXA-72 ou OXA-58. Da mesma forma, rifampicina combinada com polimixina B foi sinérgica para uma cepa OXA-23 (12,5%) e PS para 5/8 (62,5%) cepas produtoras de OXA-72, OXA-58, OXA-23/-OXA143 ou OXA-143. O efeito sinérgico da combinação polimixina B/imipenem foi confirmado, in vivo, no modelo murino de infecção, tanto por avaliação histopatológica como por redução das UFC/g pulmão ou baço (p ≤ 0,05). Finalmente, foi avaliada a atividade, in vitro, do lípide catiônico brometo de dioctadecildimetilamônio (DDA), individualmente e em combinação com tigeciclina. DDA possui efeito bactericida, e potencializou sinergicamente a tigeciclina contra 2 (25%) cepas OXA-143 ou OXA-23 positivas. A atividade do DDA, assim como a atividade da sua combinação com tigeciclina foram efetivas já na segunda hora de interação, como avaliado pelas curvas de morte. Em resumo, o efeito sinérgico decorrente do uso combinado de amicacina, tigeciclina, polimixina B, imipenem, rifampicina ou ampicilina/sulbactam, pode constituir uma alternativa terapêutica para o tratamento de infecções produzidas por cepas de A. baumannii MRs produtoras de oxacilinases, sendo que nanofragmentos catiônicos de bicamada do lipídeo sintético de DDA tem potencial para consolidar um produto de aplicação clínica. / Healthcare-associated infections (HAIs) are a serious public health issue, which have been related with an unfavorable prognosis due to the emergence and endemicity of multidrug-resistant (MDR) bacteria. In this scenario, following a worldwide trend, in Brazil, infections produced by MDR OXA-type carbapenemase-producing Acinetobacter baumannii are currently considered a clinical and epidemiological urgency. In the absence of effective therapeutic alternatives for related infections, this study aimed to evaluate synergistic effects (by using time-kill and checkerboard assays) achieved by the combination of 10 different classes of antimicrobial against 8 strains of MDR, clonally unrelated, A. baumannii strains producing OXA-23, OXA-72, OXA-58 and OXA-143 carbapenemases, being representatives of different medical centers in Brazil. As a result, the combination of amikacin / tigecycline showed synergistic (S = ΣFIC ≤ 0.5) and partially synergistic (PS = 0.5 to 0.75 ΣFIC) activity against 4 (50%) OXA-72 or OXA-143 producing A. baumannii strains, and two strains (25%) producing OXA-23, respectively. Moreover, the combination of polymyxin B / imipenem showed S and PS activity against 3 (37.5%) OXA-143, OXA-23 and OXA-72 positive isolates, and 1 (12.5%) OXA-58 producer, respectively. On the other hand, the combination amikacin / ampicillin-sulbactam was S against 2 (25%) OXA-143 and OXA-23 positive strains, being PS against two (25%) OXA-58- and OXA-143/23-producing A. baumannii. Of interest was the synergistic effect achieved by polymyxin B plus vancomycin against two strains (25%) producing OXA-72 and OXA-23, respectively. Furthermore, the ampicilina-sulbactam / rifampicin combination displayed a PS activity against six (75%) strains producing OXA-23, OXA-143, OXA-72 or OXA-58-type enzymes. Likewise, rifampicin combined with polymyxin B was S against 1 (25%) OXA-23-positive A. baumannii being PS to 5/8 (62.5%) strains producing OXA-72, OXA-58, OXA-23/-OXA143 or OXA-143. The synergistic effect of the combination polymyxin B / imipenem was confirmed, in vivo, in the murine model of infection, by using both histopathological studies and bacterial clearance from the lungs and spleen (CFU/g, p≤ 0.05). Finally, we evaluated the in vitro activity of the cationic lipid dioctadecyldimethylammonium bromide (DDA), alone and in combination with tigecycline. DDA display a bactericidal effect, enhancing synergistically the activity of tigecycline against 2 (25%) OXA-143 and OXA-23 positive strains, respectively. DDA activity alone and in combination with tigecycline was effective on the second hour of interaction, as evaluated by time-kill assays. In summary, the synergistic effect resulting from the combined use of amikacin, tigecycline, polymyxin B, imipenem, rifampicin or ampicillin / sulbactam, could be an alternative therapy for the treatment of infections caused by MDR A. baumannii strains producing oxacilinases. On the other hand, cationic bilayer nanofragments of DDA has potential for consolidating a product for medical application.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / House fly (Musca domestica L.) larvae develop in decaying organic substrates such as animal manure and adult flies likely play an important role in the ecology of fecal bacteria, including potentially virulent strains. House fly larval development strictly depends on an active bacterial community in the habitat. Although the principle of this symbiosis is not well understood, this association plays a fundamental role in transmission of microbes by this insect. In this study, enterococci were chosen as a model organism to assess the role of house flies in dissemination of multi-drug resistant bacteria in the agricultural environment. House flies (FF) and cattle manure (FM) from a cattle feedlot (frequent use of antibiotics) and house flies (BF) and manure of the American bison (BM) from the Konza Prairie Nature Preserve (no antibiotic use) were collected and analyzed. Results showed a significantly higher prevalence of enterococci resistant to tetracycline and erythromycin in FM and FF compared to that of BF and BM. Enterococcal diversity did not indicate the house fly development in manure in the corresponding habitats but the antibiotic resistance data showed very similar profiles among isolates from flies and corresponding locations. Resistance genes (tetM, tetS, tetO, ermB) and the conjugative transposon Tn916 were the most commonly detected determinants from resistant isolates from both environments. The house fly digestive tract was evaluated for the potential for horizontal transfer of antibiotic resistance genes among Enterococcus faecalis. Horizontal transfer of the pCF10 plasmid with the tetracycline resistance gene (tetM) occurred in the fly digestive tract with a transfer rate up to 101 T/D. In addition, eight enterococcal species were selected to evaluate their role and survival during house fly development. Overall, the survival rate (egg to adult) was significantly higher with E. hirae, E. durans and E. avium compared to other strains. These results indicate: a) house flies play an important role in the ecology of antibiotic resistant enterococci; b) the house fly digestive tract provides conditions for horizontal gene transfer among enterococci, and c) enterococci support the house fly development and can colonize the gut of newly emerging adult flies.

Einführung: Enterobacter (E.) cloacae subsp. cloacae sind wichtige Humanpathogene, insbesondere bei stationär untergebrachten Patienten. Sie sind in der Lage, Medizinprodukte zu kontaminieren, und es wurde über nosokomiale Krankheitsausbrüche in Verbindung mit der Kolonisation von chirurgischen Utensilien berichtet. Es ist daher wichtig, die Wirksamkeit und Effizienz von Desinfektionsmitteln gegenüber dieser Bakterienart zu bestimmen. Ziele: Die aktuelle Studie wurde durchgeführt, um nachzuweisen, ob Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit, welche weit verbreitet in kommerziellen Desinfektionsmitteln enthalten sind, eine ausreichende Wirksamkeit gegen multiresistente, von Patienten im Krankenhaus isolierte, E. cloacae aufweisen. Material und Methoden: Sechs multiresistente E. cloacae Isolate, die von Patienten in einem klinischen Umfeld gewonnen wurden, wurden getestet und mit dem E. cloacae Typstamm verglichen. Die Studien wurden in vitro mit Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit nach den Richtlinien der Desinfektionsmittel-Kommission des Verbundes für Angewandte Hygiene e.V. durchgeführt. Die Tests umfassten qualitative und quantitative Suspensionstests zur Bestimmung der bakteriziden Wirkung, den sogenannten Keimträgertest und die Bestimmung der minimalen Hemmkonzentrationen. Ergebnisse: Die Studienergebnisse zeigten, dass multiresistente E. cloacae Stämme genauso empfindlich gegenüber Desinfektionsmitteln waren wie der Typstamm. Organische Belastung interagierte stark mit Natriumhypochlorit und minderte dadurch seine Wirksamkeit, während Peressigsäure und Ethanol nicht durch organische Verunreinigung beeinflusst wurden. Die Kontaktzeit hatte nur einen geringen Einfluss auf die bakterizide Wirkung. Im Gegensatz dazu spielten bei Benzalkoniumchlorid organische Verunreinigung und die Kontaktzeit eine wichtige Rolle. Insgesamt waren die minimalen Hemmkonzentrationen und die bakterizid wirksamen Konzentrationen niedriger als die für kommerzielle Produkte gebräuchlichen Konzentrationen. In den Keimträgertests hatte das Trocknen auf einer glatten Oberfläche einen Einfluss auf das Überleben eines Stammes von E. cloacae. Die Ergebnisse zeigten auch, dass sich die Wirksamkeit der Desinfektionsmittel in den verschiedenen verwendeten Tests deutlich unterscheiden kann. Die Ergebnisse waren schwer mit anderen Studien zu vergleichen, da eine internationale Durchführungsrichtlinie für die Prüfung der Wirksamkeit von Desinfektionsmitteln gegen multiresistente Bakterien fehlt. Fazit: Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit eignen sich zur Desinfektion von multiresistenten E. cloacae. Die Wirksamkeit von Natriumhypochlorit und Benzalkoniumchlorid wird jedoch stark durch organische Stoffe beeinflusst. Dies unterstreicht die Bedeutung geeigneter Reinigungsmaßnahmen vor der Desinfektion. Wenn dies erfolgt ist, erweisen sich die getesteten Desinfektionsmittel gegen multiresistente E. cloacae genauso effektiv wie gegen den Typstamm.:1. Introduction ............................................................ 1 2. Literature Review ....................................................... 3 2.1 Enterobacteriaceae ..................................................... 4 2.1.1 General properties ................................................... 4 2.1.2 Enterobacter cloacae complex ......................................... 4 2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6 2.3 Disinfectant testing ................................................... 7 2.4 Active substances investigated in this study ........................... 8 2.4.1 Peracetic acid (PAA) ................................................. 8 2.4.2 Ethanol (ETH) ........................................................ 9 2.4.3 Benzalkonium chloride (BKC) .......................................... 9 2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10 3. Materials and Methods ................................................... 11 3.1 Materials .............................................................. 11 3.2 Methods ................................................................ 14 3.2.1 Culture and storage of bacteria ...................................... 14 3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14 3.2.3 Minimum inhibitory concentration (MIC) ............................... 15 3.2.4 Qualitative suspension test .......................................... 15 3.2.5 Quantitative suspension test.......................................... 16 3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16 3.2.7 Statistical analysis ................................................. 17 4. Results ................................................................. 18 4.1 Minimum inhibitory concentrations ...................................... 18 4.2 Qualitative suspension tests ........................................... 18 4.3 Quantitative suspension tests .......................................... 21 4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24 5. Discussion .............................................................. 28 6. Summary ................................................................. 31 7. Zusammenfassung ......................................................... 33 8. References .............................................................. 35 9. Appendix ................................................................ 49 Acknowledgments ............................................................ 50 / Introduction: Enterobacter (E.) cloacae subsp. cloacae are important human pathogens, particularly in hospitalized patients. They tend to contaminate various medical devices and nosocomial outbreaks have been reported to be associated with the colonization of surgical equipment. Therefore, it is critical to determine the efficacy and effectiveness of disinfectants against this bacterial species. Objectives: The current study was undertaken to prove whether single active ingredients (i.e. peracetic acid, ethanol, benzalkonium chloride, and sodium hypochlorite) of widely used commercial disinfectants provide proper efficacy against multidrug-resistant human isolates of E. cloacae. Material and Methods: Six multidrug-resistant E. cloacae isolates obtained from patients in a clinical setting were tested and compared to the E. cloacae type strain. The studies were performed in vitro using peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite following the guidelines specified by the Disinfectants Commission within the Association of Applied Hygiene. Tests included determination of minimum inhibitory concentrations, bactericidal values by qualitative and quantitative suspension tests, and so-called germ carrier tests. The influence of exposure time and organic load on bacteriostatic and bactericidal concentrations was evaluated for each disinfectant using the two-tailed Mann-Whitney U-test. Results: Study results showed that multidrug-resistant E. cloacae strains were equally susceptible to disinfectants as the type strain. Organic matter highly interfered with sodium hypochlorite thereby decreasing its efficacy whereas peracetic acid and ethanol were not influenced by organic soiling. Contact time had only a minor effect on bactericidal values. This was in contrast to benzalkonium chloride where organic soiling and contact time played an important role. On the whole, minimum inhibitory concentrations and bactericidal concentrations were lower than in-use concentrations of commercial products. Drying on smooth surfaces in the carrier tests had an effect on the survival of one E. cloacae strain. Results also showed that efficacious values determined by the different tests used may differ distinctly. Results were difficult to compare with other studies because an international practical standard for testing disinfectant efficacy against multidrug-resistant bacteria is missing. Conclusion: Peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite are suitable to disinfect multidrug-resistant E. cloacae but the effectiveness of sodium hypochlorite and benzalkonium chloride is strongly influenced by organic matter. This underlines the importance of proper cleaning measures before disinfection. When this is done, the tested disinfectants proved to be as efficient against multidrug-resistant E. cloacae as against the type strain.:1. Introduction ............................................................ 1 2. Literature Review ....................................................... 3 2.1 Enterobacteriaceae ..................................................... 4 2.1.1 General properties ................................................... 4 2.1.2 Enterobacter cloacae complex ......................................... 4 2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6 2.3 Disinfectant testing ................................................... 7 2.4 Active substances investigated in this study ........................... 8 2.4.1 Peracetic acid (PAA) ................................................. 8 2.4.2 Ethanol (ETH) ........................................................ 9 2.4.3 Benzalkonium chloride (BKC) .......................................... 9 2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10 3. Materials and Methods ................................................... 11 3.1 Materials .............................................................. 11 3.2 Methods ................................................................ 14 3.2.1 Culture and storage of bacteria ...................................... 14 3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14 3.2.3 Minimum inhibitory concentration (MIC) ............................... 15 3.2.4 Qualitative suspension test .......................................... 15 3.2.5 Quantitative suspension test.......................................... 16 3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16 3.2.7 Statistical analysis ................................................. 17 4. Results ................................................................. 18 4.1 Minimum inhibitory concentrations ...................................... 18 4.2 Qualitative suspension tests ........................................... 18 4.3 Quantitative suspension tests .......................................... 21 4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24 5. Discussion .............................................................. 28 6. Summary ................................................................. 31 7. Zusammenfassung ......................................................... 33 8. References .............................................................. 35 9. Appendix ................................................................ 49 Acknowledgments ............................................................ 50

Cancer is responsible for upward of 13% of human deaths. Contemporary chemotherapy of disseminated cancer is often thwarted by dose limiting systemic toxicity and by multi-drug resistance (MDR). Riminophenazines are a novel class of potential anticancer agents that possess a potent multi-mechanistic antineoplastic action. Apart from their broad action against intrinsic, non-classical resistance, Riminophenazines inhibit the action of Pgp and hypothetically all ABC transporters demonstrating their great utility against classical MDR. Considering that combination chemotherapy is the norm, the vision directing R&D efforts was that Riminophenazines could be used with benefit within many standard chemotherapeutic regimes. The strategic intent of this project was to attain improved therapeutic benefit for patients through gains in both pharmaco dynamic and pharmacokinetic specificity for cancer cells over what is currently available. Tactically, this was driven through the use of synergistic Fixed-Ratio Drug Combinations (FRDC) encapsulated within tumour-targeting Nanoparticulate Drug Delivery Systems (NDDS). Long-term aims of this R&D project were to: 1) Screen FRDC of clofazimine (B663) and the lead derivative (B4125) with etoposide, paclitaxel and vinblastine for synergistic drug interactions in vitro. 2) Design, assemble and characterize a novel nanoparticulate, synergistic, anticancer co-formulation. 3) Evaluate the in vivo safety and efficacy of the developed product/s in accordance with international regulatory guidelines. Using the median effect and combination index equations, impressive in vitro synergistic drug interactions (CI<1) were shown for various FRDC of the three standard chemotherapeutics tested (etoposide, paclitaxel and vinblastine) in combination with either B663 or B4125 against MDR neoplastic cell cultures. Considering in vitro results and with the view to advance quickly to clinical studies, the already approved clofazimine (B663) was elected as the combination partner for paclitaxel (PTX). Considering the potency and wide action of PTX, a novel coformulation (designed to circumvent drug resistance) has the potential to greatly impact upon virtually all cancer types, particularly if selectively delivered through innovative delivery systems and loco-regional administration. A passively tumour targeting, micellular NDDS system called Riminocelles™ that encapsulates a synergistic FRDC of B663 and PTX has been designed, assembled using thin film hydration methods and characterized in terms of drug loading, particle size, zeta potential, CMC and drug retention under sink conditions. An acute toxicity and a GLP repeat dose toxicity study confirmed Riminocelles to be well tolerated and safe at clinically relevant dosages whilst Taxol® (QDx7) produced statistically significant (P<0.05) weight loss within 14 days. The same study demonstrated statistically significant (P<0.05) tumour growth delays superior to that of Taxol at an equivalent PTX dosage of 10 mg/kg. Importantly, all components (amphiphiles and drugs) used in assembly of Riminocelles are already individually approved for medicinal use - this promotes accelerated development towards advanced clinical trials and successful registration. Although these results are very promising (outperforming Taxol), this system was however found in a pharmacokinetic study to suffer from in vivo thermodynamic instability due to the high concentration (abundance) of albumin present in plasma. For this reason, in vivo longevity within circulation, permitting passive tumour accumulation was not fully realized. A second NDDS called the RiminoPLUS™ imaging system was additionally developed. This lipopolymeric nanoemulsion system has successfully entrapped Lipiodol® Ultra fluid (an oil based contrast agent) within the hydrophobic core of a monodisperse particle population with a size of roughly 100 nm and a stability of one week. This formulation is therefore thought capable of CT imaging of tumour tissue and drug targeting after either intravenous or loco-regional injection. In vivo proof of the imaging concept is warranted. The results of this study serve to highlight the great potential of in vitro optimized synergistic FRDC against drug resistant cancers. Lipopolymeric micelles are an effective way to formulate multiple hydrophobic drugs for intravenous administration and present a means by which cancer can be readily targeted; provided that the delivery system possess the prerequisite in vivo stability and surface attributes. Further experiments exploring synergistic drug and biological combinations as well as “intelligent” NDDS actively guided through specific molecular recognition are called for. / Thesis (PhD)--University of Pretoria, 2012. / Pharmacology / unrestricted

This quantitative, descriptive study investigated risk factors for MDR-TB in Addis Ababa, Ethiopia. A total of 439 medical records belonging to MDR-TB and non MDR-TB patients managed in public health centres from January 2008 to December 2011 were analysed. Data were transcribed from each TB patient‟s medical records using a specifically designed checklist. The findings revealed that male gender, previous history of TB treatment, poor treatment adherence, an outcome of failure after TB re-treatment, previous category of failure, pulmonary involvement of TB infection and HIV infection were associated with MDR-TB. The findings illustrate that efforts should be made to prioritise the development and implementation of effective MDR TB screening and treatment protocols for these high risk groups to improve treatment outcome and minimize the emergence of XDR TB. / Health Studies / M. Public Health

L'augmentation et la dissémination de la résistance aux antibiotiques chez les bactéries à gram-negatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique au niveau mondial. Les infections nosocomiales causées par les bactéries multi-résistantes (BMR) ont conduit non seulement à une augmentation de la mortalité, de la morbidité, et du coût de traitement, mais aussi continuent de mettre en danger la vie des patients surtout immunodéprimés en milieu hospitalier. Bien entendu, l'utilisation abusive et non contrôlée des antibiotiques a grandement contribué à la large diffusion des déterminants de la résistance; cependant, des études récentes ont démontré que ces déterminants de la résistance pouvaient émerger à partir de sources anciennes et/ou environnementales. Ainsi, face à cette préoccupation mondiale, plusieurs études ont été rapportées avec des recommandations importantes de conduire des études épidémiologiques, moléculaires, et génomiques afin de contrôler la diffusion et l'augmentation de la résistance aux antibiotiques. De plus, durant ces 10 dernières années, nous avons assisté à l'emergence et au développement de nouvelles technologies de séquençage à haut débit coïncidant avec une augmentation exponentielle du nombre de genomes bactériens séquencés. / The increase and spread of multidrug-resistant (MDR) gram-negative bacteria especially Enterobacteriaceae, Pseudomonas, and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led not only to an increase in mortality, morbidity, and cost of treatment, but also continue to endanger the life of patients, especially those immunocompromised. Although the frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination and resistance to antibiotics; recent studies have shown that these resistance determinants could emerge from ancient or environmental sources. Front of this worldwide concern, several studies have been reported with significant recommendations to conduct molecular epidemiology, and genomic studies, in order to control the increase and the dissemination of the antibiotic resistance. Moreover, during these last 10 years, we are witnessing the emergence and development of new technologies of high throughput sequencing and coinciding with an exponential increase of number of bacterial genomes sequenced today. Therefore, it is in this context that the project of this thesis was conducted with three essential objectives: (i) the genome sequencing of clinical MDR bacteria, the analysis and the identification of the mechanisms and the genetic determinants of antimicrobial resistance (ii) the achievement of molecular epidemiology studies from clinical MDR bacteria responsible of outbreak (iii) the development and implementation of molecular tools for monitoring and diagnosis of potential MDR bacteria.