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In this project, we synthesized a new homologous series of five long-chain, two-headed amphiphiles [2CAm13, 2CAm15, 2CAm17, 2CAm19, 2CAm21; CH3(CH2)n-1CONHC(CH3)(CH2CH2COOH)2, n = 13, 15, 17, 19, 21]. The synthesis of the 2CAmn series was accomplished in four steps. The first step involves a reaction of nitroethane and two equivalents of tert-butyl acrylate to create the nitrodiester synthon [O2NC(CH3)(CH2CH2COOtBu)2] by successive Michael additions. The second step in the synthesis consists of a reduction of nitrodiester with H2 and Raney nickel to give the diesteramine [H2NC(CH3)(CH2CH2COOtBu)2]. The third step is the condensation of an acid chloride with diesteramine to give an alkanamido diester [2EAmn; CH3(CH2)n-1CONHC(CH3)(CH2CH2COOtBu)2, n = 13, 15, 17, 19, 21]. The final step is the removal of the tert-butyl protecting groups to give 2CAmn. Critical micelle concentration measurements were collected by the pendant drop method for measuring surface tension for a series of triethanolamine/2CAmn solutions to establish the concentration required for detergency. The CMCs for the 2CAmn series were found to decrease in value from 3.0 Ã 10â 2 M (2CAm13) to 1.7 Ã 10â 4 M (2CAm21) in a linear fashion [log CMC = (â 0.28 ± 0.01)n + (2.2 ± 0.1)]. The CMCs for the 2CAmn series falls in between the CMCs for three series of homologues three-headed amphiphiles (3CAmn, 3CCbn, 3CUrn) and the CMCs for fatty acids, with fatty acids having the lowest CMCs. Antibacterial activity (minimal inhibitory concentrations, MICs) for a series of homologous dendritic two-headed amphiphiles and three series of homologous, three-headed amphiphiles against Staphylococcus aureus and methicillin-resistent S. aureus (MRSA) were measured by broth microdilution to compare the effect of chain length and, hence, hydrophobicity. Inoculum density affected antibacterial activity of the 2CAmn series against both S. aureus and MRSA. MIC measurements at different cell densities showed that activity decreased with higher cell densities. For all four series, the MICs were relatively flat at low inoculum densities. This flat region defines the intrinsic activity, MIC0. The MIC0 results revealed that inoculum density, chain-length, and hydrophobicity all influenced antibacterial activity and that activity correlates strongly with clogp, an established measure of hydrophobicity. The most hydrophobic members from each homologous series exhibited antibacterial activity. The most active homologue of the 2CAmn series was 2CAm21 with MIC0 of 2.0 ± 1.0 and 3.2 ± 1.0 μM against S. aureus and MRSA, respectively. The CMCs and MIC0s of the two- and three-headed amphiphiles were compared for both S. aureus and MRSA to gauge the effect that micelles may have on activity. Amphiphile 2CAm19 has the largest ratio between CMC and MIC0 (CMC/MIC0 = 205) against S. aureus and 3CUr20 has the largest ratio (CMC/MIC0 = 339) against MRSA. These ratios suggest that micelle formation is not a mechanism of action for anti-Staphylococcal activity. / Master of Science

This project is about the synthesis of homologous series of two-headed, long-chain amphiphiles (the 2CCbn series, where n = 16, 18, 20, 22, 30, 5α-cholestan-3Ã -ol). The 2CCbn series was synthesized in five steps. The first step involves a reaction of nitroethane and two equivalents of tert-butyl acrylate to form nitrodiester by successive Michael addition reaction. The second step is the reduction of nitrodiester with Raney nickel to form aminodiester. The third step involves a reaction of aminodiester with di-tert-butyl dicarbonate [(Boc)2O] to form isocyanatediester. The fourth step is addition of iscocyanatediester with aliphatic alcohol to give alkyl carbamate diester (2ECbn) series. The fifth step is the removal of the tert-butyl protecting group to give the 2CCbn series. The critical micelle concentrations (CMC) were measured by the pyrene-based fluorescent probe method. The pyrene excited at 345 nm and fluoresces with maxima at 374 nm (I1) and 385 nm (I3). The stock solution and the dilution series for each amphiphiles were made in 0.9% triethanolamine solution. The CMCs were measured at two pH ~9.2 and 7.4. The CMCs were determined by plotting I1/I3 vs. concentrations. The CMCs were decreasing with increasing chain length. The CMCs for the 2CCbn series are lower than the 3CCbn series but higher than the fatty acids. The minimal inhibitory concentrations were measured against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. These strains were grown on BHIB+S with 5% triethanolamine. The MICs of the 2CCbn series amphiphiles were measured by using microtiter plate reader and by looking turbidity. The cutoff effect was found for the 2CCbn series. The MIC decreased up to C20 chain length and started rising for C22. The 2CCb18 (MICâ 2.2 µg/mL) of the 2CCbn series was the most effective amphiphile against S. aureus and MRSA. The CMC/MIC ratio was used to determine the safety of an amphiphile as a drug use. The amphiphile 2CCb18 has given the largest safety ratio (CMC/MIC = 273) against S. aureus and MRSA. It suggests that micelle formation is not a mechanism of action for anti-Staphylococcal activity. / Master of Science

Background: Resistance against antibiotics is a global problem that can lead to serious complications. Many bacteria have developed resistance against several antibiotics which causes infections that are hard to treat. Resistant bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococcus (VRE) are mainly spread within healthcare. To combat these infections, it is important to have reliable detection methods. Purpose: To qualitatively compare the Copan Transsystem 114c with theESwab-system for the detection of MRSA and VRE. Methods: For the study, Polymerase chain reaction (PCR) methods, Matrix-assisted laser desorption/ionization Time of Flight (Maldi-Tof), and microbiological cultivation methods was used on simulated and clinical samples. Results: For the VRE-samples no difference in results were identified. The MRSA samples showed significant differences in PCR-results where the Transsystem 114c detected 16/60 positive samples whereas the ESwab-system detected 12/60. But the cultivation results showed opposite results where the Transsystem 114c detected three positive samples and the ESwab-system detected four. Conclusion: The results show that both the Transsystem 114c and ESwab show similar results on simulated samples but differ on clinical samples. Further comparative tests need to be carried out to validate the methods before they can be used in routine operation thus no real conclusion can be drawn from this study.

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Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-28T19:21:24Z No. of bitstreams: 1 rodrigo-muller.pdf: 1238864 bytes, checksum: 6c267fda3ccb992508b5424e77147783 (MD5) / Made available in DSpace on 2012-11-28T19:21:24Z (GMT). No. of bitstreams: 1 rodrigo-muller.pdf: 1238864 bytes, checksum: 6c267fda3ccb992508b5424e77147783 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / As infecções causadas por Staphylococcus aureusresistentes à meticilina (MRSA) são temidas em virtude da dificuldade de seu tratamentoe da elevada morbidade associada. A PBP2a (penicillin binding protein 2a), enzima responsável pela síntese da parede celular em MRSA, apresenta baixíssima afinidade porbeta-lactâmicos. Pelo fato de a PBP2a estar localizada na superfície externa, a mesma seria acessível ao sistema imune. Durante as infecções causadas por MRSA, pouco se sabe se o hospedeiro infectado ou colonizado desenvolve anticorpos anti-PBP2a e se osmesmos seriam protetores ou não. O presente projeto tem por finalidade avaliar a presença e níveis de anticorpos anti-PBP2a em um grupo de 56 pacientes colonizados por MRSA e investigar se estes anticorpos seriam protetores ou não. Os resultados gerados permitiram observar que 71% das amostras analisadas apresentaram anticorposanti-PBP2a pelo teste ELISA. Estas amostras foram submetidas à Western blot paraconfirmação, demonstrando que 46% das amostras possuíram anticorpos anti-PBP2a. Após estes resultados, as amostras positivas foram submetidas à purificação para avaliar a cinética de crescimento de MRSA. Foi observado que houve ligeira redução do crescimento bacteriano entre amostras de soro com anticorpos MRSA comparadas comas de soro com anticorpos anti-MSSA (PBP2a negativos). Por conseguinte avaliamos a curva de crescimento de MRSA em presença de imunoglobulinas purificadas de soro de pacientes colonizados por MRSA, (quantificação bacteriana). Observamos que houve uma redução drástica do crescimento bacteriano em presença destas imunoglobulinas. Os resultados obtidos indicaram que: (i) pacientes colonizados por MRSA podem produzir anticorpos anti-PBP2a e (ii) estes anticorpos podem conferir proteção contra MRSA. Estes dados parecem indicar que uma potencial vacina anti-PBP2apoderia ser efetiva para prevenção de infecções causadas por MRSA, como também para o emprego de imunoterapia passiva para o tratamento de pacientesinfectados por este patógeno. / Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are feared because of the difficulty of their treatment and the high associated morbidity. The PBP2a (penicillin binding protein 2a), the enzyme responsible for cell wall synthesis in MRSA, presents low affinity for beta-lactams. Because of the PBP2a is located on the external surface, it would be accessible to the immune system. During the MRSA infections, little is known whether the infected orcolonized host can produce antibodies anti-PBP2a and whether these antibodies would be protective or not. This project aims to assess the presence and levels of anti-PBP2a in a group of 56 patients colonized by MRSA and investigate whether these antibodies wouldbe protective or not. The results showed that 71% of the samples presented anti-PBP2aantibodies in ELISA. These samples which were subjected to Western blot for confirmation, it was demonstrated that 46% of the samples presented antibodies anti-PBP2a. After these results, the positive samples were subjected to purification to assess the MRSA growth kinetics. It was observed that there was a slight reduction in bacterial growth between serum samples with antibodies and MRSA compared to those of the serum with anti-MSSA (PBP2a negative). Therefore, the curve of growth ofMRSA was evaluated in the presence of purified immunoglobulins from the serumof patients colonized by MRSA (bacterial quantification). We observed that there was a drastic reduction in bacterial growth in the presence of immunoglobulins, thus demonstrating that these antibodies could have a protective action. These results indicated that: (i) MRSA colonized patients can produce antibodies against PBP2a and (ii) these antibodies can be protective against MRSA. These data suggest that a potential vaccine anti-PBP2a could be effective for prevention of infections caused byMRSA and for the use of passive immunotherapy in the treatment of patients infectedby this pathogen.

Einleitung: Die Hände des medizinischen Personals gelten als wichtigste Übertragungsquelle von Krankheitserregern. Methicillin- und multiresistente Erreger stellen die Tiermedizin vor besondere Herausforderungen und limitieren therapeutische Optionen. Obwohl die chirurgische Händedesinfektion einen wichtigen und alltäglichen Bestandteil der Infektionsprävention darstellt, scheinen die Grundkenntnisse hierüber selbst bei chirurgischen Fachtierärzten gering zu sein. Studien haben gezeigt, dass 66 % der Chirurgen sich nicht an etablierte Standardprotokolle halten. Neben der Händedesinfektion stellen sterile OP-Handschuhe eine zusätzliche Barriere für die Übertragung von Bakterien dar. Allerdings sind perforierte Handschuhe mit einem höheren Risiko für postoperative Infektionen (SSI) verbunden, wobei die SSI-Rate in der Pferdechirurgie bis über 60 % reicht. Ziele der Untersuchungen: Die Hauptziele dieser Arbeit lagen in der Erhebung der individuellen Gewohnheiten bei der Durchführung der chirurgischen Händedesinfektion in einer Pferdeklinik (Phase 1) und dem Vergleich der Keimreduktion mit einem Standardprotokoll (Phase 2). Ferner wurden die Proben auf Bakterienspezies gescreent, die SSI induzieren können. Darüber hinaus wurde die Rate von Handschuhperforationen bestimmt. Material und Methoden: Die Observation der individuellen Gewohnheiten (Phase 1) umfasste die Dauer der Händewaschung und -desinfektion, die genutzte Desinfektionsmittelmenge und Zusammenfassung 62 die Verwendung von Bürsten. Das Standardprotokoll in Phase 2 beinhaltete eine 1-minütige Händewaschung mit flüssiger, pH-neutraler Seife ohne Bürsten und die Händedesinfektion über 3 Minuten. Alle Teilnehmer (2 Chirurgen, 8 Klinikmitarbeiter, 32 Studenten) verwendeten Sterillium® für die Händedesinfektion. Die Gesamtkeimzahlen wurden jeweils vor und nach dem Händewaschen, nach der Desinfektion und nach der Operation bestimmt. Zur Probennahme wurden die Hände für 1 Minute in 100 ml sterile phosphatgepufferte Lösung getaucht und die Bakterienkulturen auf Columbia-Schafblutagar angezüchtet. Die Bakterienkolonien wurden manuell ausgezählt und die Bakterienspezies mittels MALDI-TOF identifiziert. Die Handschuhe wurden postoperativ mit einem modifizierten Wasser-Leck-Test auf Perforationen untersucht. Ergebnisse: In Phase 1 und Phase 2 wurden 46 bzw. 41 Händedesinfektionen durchgeführt. Die individuellen Gewohnheiten unterschieden sich deutlich zwischen den Teilnehmern hinsichtlich der Dauer des Händewaschens (bis zu 8 min) und der Desinfektion, sowie der Menge des verwendeten Desinfektionsmittels (bis zu 48 ml). Die Dauer des Händewaschens in Phase 1 und 2 zeigte keinen statistisch signifikanten Effekt auf die Bakterienreduktion. Bei Verwendung des Standardprotokolls war die Reduktion der Keimzahlen nach der Desinfektion im Vergleich zur täglichen Routine signifikant höher (p < 0.001). Die mittlere Reduktion in Phase 1 betrug 90,72 % (LR = 3,23; rechte Hand) und 89,97 % (LR = 3,28; linke Hand) im Vergleich zu 98,85 % (LR = 3,29; rechte Hand) und 98,92 % (LR = 3,47; linke Hand) in Phase 2. Bei acht Teilnehmern (19 %) wurde MRSA (spa Typ t011, CC398) nachgewiesen. Die MRSA-Isolate konnten ferner einer Subpopulation zugeordnet werden, die besonders mit Pferdekliniken assoziiert wurde (hauptsächlich t011, ST398, Gentamicin-resistent). Handschuhperforationen traten bei 54 % (Chirurgen) bzw. 17 % (Assistenten) der Handschuhe auf, wobei eine höhere Prävalenz bei invasiven Eingriffen und Operationen mit einer Dauer von > 60 Minuten vorlag. Die Mehrheit (85 %) der Perforationen blieben vom Operationsteam unbemerkt, wobei Zeigefinger und Daumen die am häufigsten punktierten Stellen waren. Insgesamt nahmen die Bakterienzahlen an den Händen im Laufe der Zeit erneut zu, insbesondere wenn eine Handschuhperforation auftrat. Schlussfolgerung: Die Einhaltung eines Standardprotokolls nach neuestem Stand der Wissenschaft trägt zu einer quantitativ höheren und gleichmäßigeren Keimreduktion beider Hände bei. Die Implementierung eines standardisierten Händedesinfektionsplans sichert die Qualität der aseptischen Maßnahme und ist besonders für die Ausbildung und Schulung von Studenten mit geringer chirurgischer Erfahrung unerlässlich.:1 Einleitung ............................................................................................................................ 1 2 Literaturübersicht ............................................................................................................... 3 2.1 Residente und transiente Hautflora ............................................................................. 3 2.2 Asepsis und Antisepsis ............................................................................................... 4 2.3 Grundlagen und allgemeine Voraussetzungen für eine effektive Händehygiene ......... 4 2.4 Händewaschung .......................................................................................................... 5 2.4.1 Limitationen der Händewaschung ........................................................................ 5 2.5 Händedesinfektion ....................................................................................................... 6 2.5.1 Historie der Händedesinfektion ............................................................................ 6 2.5.2 Hygienische Händedesinfektion ........................................................................... 8 2.5.3 Chirurgische Händedesinfektion .......................................................................... 8 2.5.4 Aliphatische Alkohole ......................................................................................... 10 2.5.5 Dauer und Wirksamkeit der chirurgischen Händedesinfektion .......................... 12 2.5.6 Compliance ........................................................................................................ 13 2.5.7 Zulassung und Prüfung von Händedesinfektionsmitteln .................................... 15 2.6 Nosokomiale Infektionen, Surgical Site Infections (SSI) ........................................... 16 2.6.1 Staphylococcus aureus ...................................................................................... 18 2.6.1.1 Methicillin-resistente Staphylococcus aureus (MRSA) ................................... 18 2.7 Handschuhe .............................................................................................................. 21 2.7.1 Nutzen und Limitationen von Handschuhen ...................................................... 21 3 Veröffentlichung ............................................................................................................... 24 3.1 Eigenanteil zur Veröffentlichung ................................................................................ 24 3.1.1 Publikation ......................................................................................................... 26 4 Diskussion ........................................................................................................................ 50 5 Zusammenfassung ........................................................................................................... 61 6 Summary .......................................................................................................................... 63 7 Literaturverzeichnis .......................................................................................................... 65 Danksagung .............................................................................................................................. 79 / Introduction: Hands of medical personnel are considered the most important source of pathogen transmission. Methicillin- and multiresistant strains of pathogens provide particular challenges to veterinary medicine and limit therapeutic options. Surgical hand disinfection is a major aspect of infection prevention, but basic knowledge seems to be low, even among specialized veterinary surgeons. Studies revealed that 66 % of surgeons do not adhere to established standard protocols. Besides hand disinfection, sterile surgical gloves provide an additional barrier to the transmission of bacteria. However, perforated gloves are associated with a higher risk of surgical site infections (SSI), with an SSI rate in equine surgery exceeding 60 %. Objective: The major objectives were to assess current habits for presurgical hand preparation (phase 1) among personnel in a veterinary equine hospital and to compare the effectiveness in reducing bacteria from hands with a standardized protocol (phase 2). Moreover, samples were screened for bacteria known to cause surgical site infection. The rate of glove perforation was determined, additionally. Material and methods: Individual habits were recorded with regards to the time taken for washing and disinfecting hands, the amount of disinfectant used, as well as the usage of brushes (Phase 1). In contrary to the personal habits, the applied standardized protocol (Phase 2) defined washing hands for 1 minute with liquid neutral soap without brushing and disinfection for Summary 64 3 minutes. All participants (2 surgeons, 8 clinic members, 32 students) used Sterillium® for disinfection. Total bacterial counts were determined before and after hand washing, after disinfection and after surgery. In brief, hands were immersed in 100 ml sterile phosphate-buffered saline for 1 minute and cultures were inoculated onto Columbia sheep blood agar using the spread-plate method. Bacterial colonies were manually counted. Surgical gloves were investigated for perforations after surgery using a modified water leak test. Results: Fourty-six and 41 hand disinfection preparations were carried out during phase 1 and phase 2, respectively. Individual habits differed distinctly between participants regarding the duration of handwashing (up to 8 min) and disinfection as well as the amount of disinfectant used (up to 48 ml). The duration of hand washing in phase 1 and 2 revealed no statistically significant effect on reducing bacteria. In contrary, using the standardized protocol in phase 2, reduction in bacterial numbers after disinfection was significantly higher (p < 0.001) compared to current habits. The mean reduction in phase 1 was 90.72 % (LR = 3.23; right hand) and 89.97 % (LR = 3.28; left hand) compared to 98.85 % (LR = 3.29; right hand) and 98.92 % (LR = 3.47; left hand) in phase 2. Eight participants (19 %) carried MRSA (spa type t011, CC398) which is well established as a nosocomial pathogen in veterinary clinics. The isolates were further assigned to a subpopulation which is particularly associated with equine clinics (mainly t011, ST398, gentamicin-resistant). Glove perforation occurred in 54 % (surgeons) and 17 % (assistants) of gloves, respectively, with a higher number in invasive procedures and operations lasting > 60 minutes. The majority (85 %) of perforations was unnoticed by the surgical team, with index fingers and thumbs most frequently affected. Overall, bacterial numbers on hands mainly increased over time during surgery, especially when glove perforation occurred. Conclusion: Adherence to state-of-the-art standardized protocols contributes to a quantitatively higher and constant germ reduction on both hands. The implementation of a standardized hand disinfection protocol ensures a high quality of aseptic measures and is essential for the education and training of students with little surgical experience.:1 Einleitung ............................................................................................................................ 1 2 Literaturübersicht ............................................................................................................... 3 2.1 Residente und transiente Hautflora ............................................................................. 3 2.2 Asepsis und Antisepsis ............................................................................................... 4 2.3 Grundlagen und allgemeine Voraussetzungen für eine effektive Händehygiene ......... 4 2.4 Händewaschung .......................................................................................................... 5 2.4.1 Limitationen der Händewaschung ........................................................................ 5 2.5 Händedesinfektion ....................................................................................................... 6 2.5.1 Historie der Händedesinfektion ............................................................................ 6 2.5.2 Hygienische Händedesinfektion ........................................................................... 8 2.5.3 Chirurgische Händedesinfektion .......................................................................... 8 2.5.4 Aliphatische Alkohole ......................................................................................... 10 2.5.5 Dauer und Wirksamkeit der chirurgischen Händedesinfektion .......................... 12 2.5.6 Compliance ........................................................................................................ 13 2.5.7 Zulassung und Prüfung von Händedesinfektionsmitteln .................................... 15 2.6 Nosokomiale Infektionen, Surgical Site Infections (SSI) ........................................... 16 2.6.1 Staphylococcus aureus ...................................................................................... 18 2.6.1.1 Methicillin-resistente Staphylococcus aureus (MRSA) ................................... 18 2.7 Handschuhe .............................................................................................................. 21 2.7.1 Nutzen und Limitationen von Handschuhen ...................................................... 21 3 Veröffentlichung ............................................................................................................... 24 3.1 Eigenanteil zur Veröffentlichung ................................................................................ 24 3.1.1 Publikation ......................................................................................................... 26 4 Diskussion ........................................................................................................................ 50 5 Zusammenfassung ........................................................................................................... 61 6 Summary .......................................................................................................................... 63 7 Literaturverzeichnis .......................................................................................................... 65 Danksagung .............................................................................................................................. 79