This thesis describes the development of new pathways towards the synthesis of novel antimicrobial (and anticancer) agents. Two synthetic protocols based on free radical chemistry are studied, which are used to access polycyclic heterocyclic compounds of potential biological importance. Both these procedures involve the generation of radicals using Bu3SnH and AIBN initiators, and the subsequent intramolecular radical cyclisation onto the imidazole ring. Radical cyclisations onto benzimidazoles and pyrroles are also described.

The microbial contamination of biomedical devices is a leading cause of hospital- acquired infection. A number of strategies aimed at developing device coatings that are refractory to microbial adhesion, colonisation and biofilm formation have been developed, but the problem remains. The incorporation of biocides into biomedical device surface coatings has shown promising results in preventing the establishment of infection. Current controversy over the possibility that extensive use of biocides could potentially lead to antimicrobial resistance has fuelled the search for new actives with good antimicrobial activity and low cytotoxicity, that maintain marked efficacy after prolonged use. This doctoral thesis aims to evaluate the antimicrobial potential of a novel peptide based on human apolipoprotein E receptor binding region (apoEdpL-W). The spectrum of antimicrobial activity and anti-biofilm efficacy of apoEdpL-W was compared to that of common biocides polyhexamethylene biguanide, triclosan, cetrimide and chlorhexidine. The potential to induce bacterial insusceptibility towards these agents after long-term sub-lethal level exposure was assessed. Initial examination against 18 test microorganisms, commonly associated with device infection, showed that apoEdpL-W displayed broad-range antimicrobial and anti-biofilm efficacy. ApoEdpL-W also maintained marked antibacterial activity after incorporation onto various biomaterial polymers, often used in device surface coatings. Alterations in bacterial susceptibility after prolonged exposure to apoEdpL-W, as well as to the other biocides, were often temporary and partially reverted once the bacteria had been grown in the absence of the antimicrobial agent. The adaption of Staphylococcus aureus to the presence of triclosan resulted in the formation of small colony variants (SVCs) with reduced triclosan susceptibility. Analysis of the physiological characteristics of the triclosan induced SCVs revealed the loss of virulence determinants and potentially reduced pathogenic capability, when compared to the parent strain. The biocompatibility index values of the test actives were determined by the parallel assessment of their antibacterial activity and in vitro cytotoxicity. ApoEdpL-W showed good antibacterial efficacy whilst remaining relatively less toxic to mammalian cells than triclosan or chlorhexidine. We studied the interactions of the test antimicrobials with a preformed phospholipid bilayer using the quartz crystal microbalance device and dual polarisation interferometry, to better understand potential mode of action. Analysis revealed that ApoEdpL-W and PHMB induced the highest level of bilayer disruption, of all the antimicrobials tested. These data suggest that apoEdpL-W demonstrates antibacterial activity; biocompatibility and long-term efficacy on a level that compares favourably to that of currently used biocides. The peptide demonstrates good antimicrobial efficacy when incorporated into a range of biomaterial polymers and shows the potential to be developed as an effective coating for the reduction of device associated infections.

Orientadores: Pedro Luiz Rosalen, Severino M. Alencar / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-10T01:20:04Z (GMT). No. of bitstreams: 1 Bueno-Silva_Bruno_M.pdf: 1697425 bytes, checksum: 055f2dd91e407acf734bcc51d0a438bd (MD5) Previous issue date: 2008 / Resumo: Entre as própolis brasileiras, urna nova própolis ainda não classificada de acordo com Park et al., 2002 e denominada de própolis vermelha originária do estado de Alagoas (Nordeste do Brasil), tem mostrado resultados interessantes em relação a sua composição química e atividade biológica em estudos preliminares. Assim, o objetivo principal deste trabalho é identificar a origem botânica da própolis vermelha, a composição química e atividade biológica do extrato etanólico da própolis (EEP) e do extrato etanólico da resina da planta (EER), avaliar o efeito sazonal sobre a composição química e atividade biológica do EEP e do EER. Esses objetivos foram atingidos através das seguintes metodologias: 1- observação do comportamento de visita das abelhas à . vegetação próxima à colméia; 2- comparação dos perfis químicos dos vegeÜlis visitados pelas abelhas coletoras de resina e da própolis vermelha, obtidos por cromatografia líquida de alta eficiência em fase reversa e análises complementares, estabelecendo-se assim, as características entre ambos os materiais, visando à identificação do marcador biológico botânico e 3- avaliação da influência do efeito sazonal anual sobre a própolis vermelha e sua vegetação fonte por meio da atividade antimicrobiana e perfil químico, com coletas bimensais das amostras durante o período de 1 ano. A atividade antimicrobiana foi avaliada por meio da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM) e os microrganismos usados foram: O Streptococcus mutans UA159, Streptococcus sobrinus 6715, Staphylococcus aureus ATCC25923 e Actinomyces naeslundii ATCC 12104. Os resultados demonstraram o mesmo perfil quimico entre o EEP e o EER da planta Dalbergia ecastophyllum (L.) Taub., cuja característica foi a alta concentração relativa das isoflavonas 3-hidroxi-8,9-dimetoxipterocarpin e medicarpina. Os perfis químicos do EEP e do EER, obtidos ao longo do ano, através do testes químicos, apresentaram-se distintos dos perfis dos demais 12 tipos de própolis brasileiras já classificadas e variaram quantitativamente de acordo com a sazonalidade. A CIM variou entre 15,6-125 'mu'g/rnL e a CBM de 31,2 - 500 'mu'g/mL considerando os 4 microrganismos àvaliados. Conclui-se que esta própolis, cuja origem botânica é a Dalbergia ecastophyllum, pode ser classificada corno o 13° tipo de própolis, de acordo Park et al., 2002, e tanto o EEP quanto o EER apresentaram alta atividade antimicrobiana, os quais poderão ser utilizados para pesquisas futuras de novas moléculas no controle da cárie dental / Abstract: Among the Brazilian propolis, new propolis not yet classified, according to Park et al., 2002 and called of red propolis, originary from Alagoas state (Northeast of Brazil), have showed interesting results in preliminary studies in our laboratories. Thus, the main objective of this work is to identify the botanical marker of red propolis, the chemical composition and biological activity of extract ethanolic of propolis (EEP) and the extract ethanolic of resin of plant (EER), verifying the effect of seasonal period on chemical composition and antimicrobial activity of EEP and EER. This objective was reached through the following methodologies: 1- observation of bee behavior in visiting vegetation next beehive; 2- comparison of chemical profile of vegetables visited by bees for collecting resin with red propolis, gotten for liquid chromatography of high efficiency in phase reverse and complementary analyses, for establishing the common characteristics between both materiaIs, aiming to identification of botanical biological marker; 3- evaluation of influence seasonal annual effect on the red propolis and its vegetation source by means of the antimicrobial activity and chemical profile, with bymonthly collections of the samples during the period of 1 year. The antimicrobial activity was evaluated by Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) and microorganism used were Streptococcus mutans UA159, Streptococcus sobrinus 6715, Staphylococcus aureus ATCC25923 e Actinomyces naeslundii ATCC 12104. The results had the same demonstrated to chemical profile between the EEP and the EER of the plant Dalbergia ecastophyllum (L.) Taub. Whose characteristic was the high relative concentration of isoflavonas 3-hidroxi-8,9 dimetoxipterocarpin and medicarpina. The chemical profiles of EEP and EER varied quantitatively according to seasonal effect The CIM varied between 15.6-125 'mu'g/rnL and the CBM of 31.2 - 500 'mu'g/rnL. One concludes that these propolis, whose botanical origin is the Dalbergia ecastophyllum, can be classified as 13° type of propolis, according to Park et al., 2002 and the EEP and EER showed high antibacterial activity which can be used for future molecule research in the control of caries dental / Mestrado / Farmacologia, Anestesiologia e Terapeutica / Mestre em Odontologia

Cathelicidins are antimicrobial peptides (AMPs) that were first discovered to have microbicidal properties but more recently to be multifunctional immunomodulators and thus important in influencing host defence against infectious disease. Whilst roles in various organs have been demonstrated, their expression patterns in health and disease in other organs are less clear and their key immunomodulatory functions remain undefined, particularly with regard to the balance of immunomodulatory properties and microbicidal activity in their ability to promote defence against infection. I therefore set out to describe LL-37 expression (human cathelicidin) in the female reproductive tract (across the menstrual cycle) and in the lung (during specific lung diseases), to define the effects on the function of airway epithelial cells during bacterial infection and to evaluate the key in vivo roles of endogenous cathelicidin (using a knockout mouse model) as well as the effect of therapeutic administration of LL-37 in a pulmonary Pseudomonas aeruginosa infection model. I demonstrated that cathelicidin protein and transcription shows a cyclical pattern of expression in female reproductive tissues which is maintained at high levels in decidua. LL- 37 protein was also detected in hTERT endometrial epithelial cells but despite the suggestion that cathelicidin may be regulated by steroid hormones there was no direct effect of progesterone on transcription. LL-37 is barely detected in healthy airways however is well known to increase during infection or inflammation. I observed that sputum from patients with bronchiectasis showed a correlation between the level of LL-37, TNF, MPO and chronic colonisation of Pseudomonas aeruginosa. Patients with lung cancer expressed much less LL- 37 than the bronchiectasis patients but there was a trend towards increased production postsurgery compared to pre-surgery. LL-37 was previously shown by our lab to selectively promote BAX and caspase-dependant death of infected epithelial cells. I went on to show that this appears to be a partially caspase- 1 dependent mechanism and that human bronchial epithelial (HBE) cells and A549 cell lines both express several of the components required to form inflammasomes, a caspase-1 dependant form of inflammatory cell death. Finally, I showed using murine models that cathelicidin enhances bacterial clearance during pulmonary infection in vivo, a response which is defective in mice lacking endogenous cathelicidin and that administration of exogenous, synthetic LL-37 at the time of infection can promote an early protective neutrophil influx in the absence of endogenous cathelicidin production.

Background: Antimicrobial Stewardship Programs (ASPs) have been promoted to optimize antimicrobial usage and patient outcomes, and to reduce the emergence of antimicrobial-resistant organisms. However, the best strategies for an ASP are not definitively established and are likely to vary based on local culture, policy, and routine clinical practice, and probably limited resources in middle-income countries. The aim of this study is to evaluate structures and resources of antimicrobial stewardship teams (ASTs) in surgical departments from different regions of the world. Methods: A cross-sectional web-based survey was conducted in 2016 on 173 physicians who participated in the AGORA (Antimicrobials: A Global Alliance for Optimizing their Rational Use in Intra-Abdominal Infections) project and on 658 international experts in the fields of ASPs, infection control, and infections in surgery. Results: The response rate was 19.4%. One hundred fifty-six (98.7%) participants stated their hospital had a multidisciplinary AST. The median number of physicians working inside the team was five [interquartile range 4-6]. An infectious disease specialist, a microbiologist and an infection control specialist were, respectively, present in 80.1, 76.3, and 67.9% of the ASTs. A surgeon was a component in 59.0% of cases and was significantly more likely to be present in university hospitals (89.5%, p < 0.05) compared to community teaching (83.3%) and community hospitals (66.7%). Protocols for pre-operative prophylaxis and for antimicrobial treatment of surgical infections were respectively implemented in 96.2 and 82.3% of the hospitals. The majority of the surgical departments implemented both persuasive and restrictive interventions (72.8%). The most common types of interventions in surgical departments were dissemination of educational materials (62.5%), expert approval (61.0%), audit and feedback (55.1%), educational outreach (53.7%), and compulsory order forms (51.5%). Conclusion: The survey showed a heterogeneous organization of ASPs worldwide, demonstrating the necessity of a multidisciplinary and collaborative approach in the battle against antimicrobial resistance in surgical infections, and the importance of educational efforts towards this goal.

For centuries medicinal plants have been used all over the world for the treatment and prevention of various ailments, particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years the use of and search for drugs derived from plants have been accelerated. Ethnopharmacologists, botanists, microbiologists, and natural-product chemists are trying to discover phytochemicals and “leads” which could be developed for the treatment of infectious diseases. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties. The evaluation of these plants for biological activity is necessary, both to substantiate their use by communities, and also to discover possible new drug or herbal preparations. Twenty five plants selected through ethno-botanical surveys in Mozambique which are used to treat respiratory diseases, wounds, viruses, stomach ailments and etc., were collected and investigated for antimicrobial activity. Acetone extracts of selected plants were tested for antibacterial, antimycobacterial and anti-HIV-1 activity. Antibacterial activity was evaluated using the agar diffusion method. Five Gram positive (Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis) and five Gram negative (Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens) bacterial species were used in this study. The extracts of each plant were tested at concentrations ranging from 0.125 to 5.0 mg/ ml. Most of the plant extracts inhibited the growth of the Gram-positive microorganisms. The minimum inhibitory concentration of eight plants (Cassia abbreviata, Elephanthorrhiza elephantina, Hemizygia bracteosa, Hoslundia opposita, Momordica balsamina, Rhoicissus tomentosa and Salvadora australis) against Gram-positive bacteria was found to be 0.5 mg/ml. Gram-positive bacteria were found to be susceptible to extracts of Lippia javanica at concentration of 0.125 mg/ml. Among the 22 acetone extracts tested, two were found to have activity against Gram-negative bacteria at a concentration of 5.0 mg/ml (Adenia gummifera and Momordica balsamina). Rhoicissus revoilli inhibited E. cloacae, a Gram-negative strain, at a concentration of 2.5 mg/ml. To evaluate antimycobacterium activity ten plants species were tested against H37Rv, a drug-sensitive strain of Mycobacterium tuberculosis at concentrations ranging from 0.5 to 5.0 mg/ml using BACTEC radiometric method. Four of the plant species tested (Cassia abbreviata, Hemizigya bracteosa, Lippia javanica and Melia azedarach) were observed to be active against the H37Rv. (ATCC 27294) strain of TB at a concentration of 0.5 mg/ml which was the lowest concentration used in this study. Seventeen plant species, were screened for anti-HIV bioactivity in order to identify their ability to inhibit the enzymes glycohydrolase (á -glucosidase and â- glucuronidase) and eleven species were further tested against Reverse transcriptase. It was found that 8 plant species (Cassia abbreviata, Elephantorrhiza elephantina, Rhoicissus tomentosa, Pseudolachnostylis maprouneifolia, Lippia javanica, Litogyne gariepina, Maerua junceae and Momordica balsamina) showed inhibitory effects against á-glucosidase and â-glucuronidase at a concentration of 200 ìg/ml. The results of the tests revealed that the plant extracts of Melia azedarach and Rhoicissus tomentosa appeared to be active, showing 49 and 40% inhibition of the enzyme activity respectively. Lippia javanica was found to have the best activity exhibiting a minimum inhibitory concentration of 0.125 mg/ml. The extracts also showed positive activity against Mycobacterium tuberculosis at concentration of 0.5 mg/ml and HIV-enzyme glycohydrolase was (á-glucosidase and â-glucuronidase) inhibited by 62 % and 73 % respectively. Considering its medicinal use local for HIV and various infections, it was therefore, selected for identifying its bioactive constituents. In the initial screening of plants used in Mozambique Hoslundia oppositademonstrated good antitubercular activity. It was therefore, selected to identify its bioactive constituents. A Phytochemical investigation of L. javanica led to the isolation of eight compounds, 4-ethyl-nonacosane (1), (E)-2(3)-tagetenone epoxide (2), myrcenone (3), piperitenone (4), apigenin (5), cirsimaritin (6), 6-methoxyluteolin 4'-methyl ether (7), 6-methoxyluteolin and 3',4',7-trimethyl ether (8). Three known compounds, 5,7-dimethoxy-6-methylflavone (9), hoslunddiol (10) and euscaphic acid (11) were isolated from H. opposita. This is the first report of compounds (1), (2), (5-8) from L. javanica and of compound (9) from H. opposita. The compounds were tested against Mycobacterium tuberculosis and HIV-1 reverse transcriptase for bioactivity. It was found that compounds (2), (4) and (9) inhibited the HIV-1 Reverse transcriptase enzyme by 91%, 53% and 52% respectively at 100 ìg/ml. Of all the compounds tested against a drug-sensitive strain of Mycobacterium tuberculosis, euscaphic acid (11) was found to exhibit a minimum inhibitory concentration of 50 ìg/ml against this strain. The present study has validated scientifically the traditional use of L. javanica and H. opposita and a few other Mozambican medicinal plants to some extent. / Thesis (PhD)--University of Pretoria, 2010. / Plant Science / unrestricted

Aunque la plata se usa como componente clave en el control microbiano en incontables aplicaciones, las tecnologías basadas en plata disponibles son escasas. Esto radica en la dificultad para evaluar su eficacia debido a problemas de estabilidad y de especiación. En la presente tesis, iones de plata fueron incorporados en matrices biopoliméricas para obtener materiales de prolongada capacidad antimicrobiana basados en su liberación sostenida. Se realizó un estudio profundo de las interacciones químicas entre las especies activas de plata, las bacterias, y posibles ligandos presentes en el medio de acción. En condiciones óptimas, la plata demostró ser eficaz en el rango de los nanomoles. Sin embargo, interacciones químicas con varios ligandos afectaron drásticamente tanto su eficacia como la evaluación de la viabilidad bacteriana. La incorporación de iones de plata en películas de EVOH no alteró las propiedades físico-químicas de los materiales que mostraron una rápida liberación del contenido de plata al entrar en contacto con la humedad. Esto se reflejó en la inactivación de las bacterias a concentraciones muy bajas (0.0001wt.%) en condiciones óptimas. Cuando se incorporaron iones de plata en PLA por casting o mezclado-fundido, la liberación y el rendimiento antimicrobiano se prolongaron de días a meses, dependiendo del contenido, el método de incorporación, la humedad o el pH del medio de liberación. Una etapa inicial de liberación mayor pudo ser atenuada gracias a la aplicación de una capa de cera de abejas, lo que permitió adaptar los perfiles de liberación a demanda y cumplir con la legislación vigente en diversas condiciones de liberación. Las películas demostraron un alto efecto antibacteriano y antiviral contra los patógenos transmitidos por los alimentos más comunes en medios sintéticos, en superficie y en alimentos líquidos y sólidos. Este estudio representa un avance en la comprensión de la eficacia antimicrobiana de la plata y destaca su posible idoneidad para la fabricación de materiales de envasado de alimentos, de contacto con alimentos u otras aplicaciones. / Martínez Abad, A. (2014). Development of silver based antimicrobial films for coating and food packaging applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36738 / TESIS / Premios Extraordinarios de tesis doctorales

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. Mutants with deletions in two PEA transferase genes were significantly more susceptible to β-defensins, and the triple mutant was significantly more susceptible to both α- and β-defensins, but not LL-37; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface, suggesting these three genes contribute to the addition of positively charged moieties on the cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdtA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin may be more important than defensin resistance to H. ducreyi pathogenesis.

Biofilm formation within burn wounds pose numerous health-related problems as they prolong recovery, inhibit antimicrobial treatments, and serve as a reservoir to spread new infections. In the United States alone there are half a million burn wounds each year. These burn wounds result in tens of thousands of patients to be admitted to hospitals and thousands of deaths. Burn wound infections alone account for over half of these deaths. Currently, standard models of burn wound biofilms, both in-vivo and in-vitro, have their benefits and limitations. These models include skin explants, animal models, and complex growth media. For the examination of microbial biofilms and rapid screening of potential antimicrobial topical treatments, a physiologically relevant media that more closely mimics what would be found in the host’s tissue would be advantageous. This pilot study was conducted to examine different formulations of a synthetic tissue-like media, the biofilm growth of common burn wound infectious microbes, and served as a high-throughput means of testing current and potentially new antimicrobials. Our laboratory has begun characterizing a new antimicrobial gel and its ability to eradicate microorganisms that commonly infect burn wounds, specifically focusing on the common wound microbe Staphylococcus aureus. Utilizing a constitutively expressed green fluorescent protein, both the growth on the textured media, as well as, biofilm inhibition by the antimicrobial gel showed significant reduction in S. aureus. On a molecular level, we examined biofilm gene expression, via reverse transcription polymerase chain reaction, of adhesion, quorum sensing, and drug resistance markers in our new model in conjunction with our antimicrobial gel. Our new synthetic wound-like media supported the growth of S. aureus and was successful in its ability to quickly screen different formulations of topical antimicrobial treatments. The antimicrobial gel produced significant reduction of S. aureus burden. The results of this study indicate that our formulated synthetic burn wound media model supports microbial growth, is efficient in the ability to rapidly screen antimicrobials, and could lead to a better understanding of the etiology of burn wound infections.

Salmonella and Campylobacter are two foodborne pathogens that continue to persist in broiler processing. Various studies have demonstrated that peracetic acid can effectively reduce the prevalence pathogens on broiler meat. However, there are a limited number of studies comparing the effects of peracetic acid on broiler meat from different processing plants. The objective of this study was to determine the prevalence of Salmonella, Campylobacter, and spoilage microbes on broiler meat at different stages of poultry processing in commercial plants that use peracetic acid as the primary antimicrobial. Results indicated that there was a high initial microbial presence on broiler meat at initial stages of processing in all three plants. Peracetic acid effectively reduced the prevalence and microbial load of all microbes analyzed in this study. All microbes were reduced to nondetectable levels in the finishing chiller. However, contamination of all microbes in mechanically deboned meat closely resembled initial carcass contamination. In conclusion, the intervention with the greatest effect on microbial prevalence was peracetic acid in carcass chilling tanks, and, given the level of contamination in mechanically deboned meat, an intervention at this step would be worth investigating.