Dissertations / Theses on the topic 'Antimicrobial effect'

Tesis por compendio
[ES] La concienciación social con respecto al bienestar animal, la seguridad alimentaria, las resistencias antimicrobianas y la salud medioambiental ha incrementado en los últimos años, promoviendo la implementación de sistemas de producción que incluyan el concepto 'One Health' en su diseño. Por ello, en el sector avícola se han propuesto alternativas a nivel de campo centradas en la mejora de la bioseguridad, el uso de estirpes rústicas y de crecimiento lento y la implementación de ganadería de precisión. En este contexto, la microbiota intestinal tiene un papel importante, tanto en la salud de los animales, como en la diseminación de resistencias antimicrobianas y la transmisión de patógenos zoonósicos a lo largo de la cadena alimentaria. El bienestar animal promueve la presencia de microbiota beneficiosa y la integridad del epitelio intestinal, reduciendo las interacciones con las bacterias ambientales. Por ello, sería posible conseguir una reducción en la administración de antibióticos y reducir la presencia de patógenos en la cadena alimentaria. Salmonella spp. es la principal causa de brotes alimentarios en la Unión Europea, y la principal fuente de infección son los productos avícolas. Entre los principales serotipos se encuentra S. Infantis, actualmente muy prevalente en pollos de engorde. Por todo ello, el objetivo general de esta tesis doctoral fue evaluar el efecto de sistemas alternativos de producción avícola sobre el desarrollo y la composición de la microbiota, la evolución de las resistencias antimicrobianas y la epidemiología de Salmonella. En el primer experimento, se estudió el efecto de la estirpe genética, comparando una estirpe comercial de crecimiento rápido frente a una estirpe alternativa de crecimiento lento. Los objetivos fueron caracterizar la microbiota cecal e investigar la dinámica de las resistencias y multirresistencias antimicrobianas a lo largo del ciclo. Respecto a la composición de la microbiota, los resultados mostraron que Firmicutes fue el filo dominante y los géneros predominantes fueron Oscillospira spp., Ruminococcus spp., Coprococcus spp., Lactobacillus spp. y Bacteroides spp. Por otro lado, los resultados obtenidos en el estudio de resistencias antimicrobianas mostraron que, al inicio del ciclo, los animales de la estirpe de crecimiento rápido presentaron un mayor porcentaje de resistencia, pero al final del periodo no se encontraron diferencias. En el segundo experimento, se evaluó el efecto de las condiciones de manejo, comparando las condiciones comerciales de densidad y ventilación, frente a condiciones mejoradas. Los objetivos fueron caracterizar la microbiota cecal, evaluar la evolución de las resistencias y multirresistencias antimicrobianas, e investigar el desarrollo de S. Infantis y sus resistencias antimicrobianas a lo largo del periodo de engorde. Los resultados obtenidos en la evolución de la microbiota, mostraron un mayor nivel de diversidad en el grupo producido bajo condiciones mejoradas. Además, Firmicutes fue el filo dominante durante todo el ciclo y los géneros predominantes fueron Oscillospira spp., Ruminococcus spp., Bacteroides spp. y Coprococcus spp. Los resultados obtenidos en el estudio de resistencias antimicrobianas mostraron altas tasas de resistencia a lo largo de todo el ciclo, sin diferencias entre grupos. Por último, los recuentos más altos de excreción de Salmonella se observaron el día de sacrificio en ambos grupos. Además, el 100% de las cepas aisladas fueron multirresistentes tras la primera semana post-infección. En conclusión, los principales resultados obtenidos en esta tesis doctoral incluyen que la diversidad y la composición de la microbiota están en constante desarrollo a lo largo del periodo de engorde, viéndose afectadas por los factores de manejo estudiados. Además, las resistencias antimicrobianas están presentes en las bacterias comensales desde el día de llegada, y aumenta hasta el final del ciclo, destacando la necesidad de controlar la administración de antibióticos en todas las etapas de la producción avícola. En cuanto a la epidemiología de S. Infantis, la continua excreción durante todo el periodo de engorde y su capacidad de adquirir resistencias, independientemente de las condiciones de manejo en granja, sugieren la necesidad de realizar más estudios para poder establecer mejores programas de control de la bacteria a lo largo de la cadena alimentaria.
[CA] La conscienciació social amb respecte del benestar animal, la seguretat alimentaria, les resistències antimicrobianes i la salut mediambiental han incrementat en els últims anys, promovent la implementació de sistemes de producció alternatius que incloguen el concepte 'One Health'. Per aixó, en el sector avícola s'han proposat diverses alternatives en granja, centrades en la millora dels protocols de bioseguretat, l'ús d'estirps més rústiques i de creixement lent, així com la implementació de la ramaderia de precisió. En aquest context, la microbiota intestinal té un paper clau en la salut dels animals, la diseminació de resistències antimicrobianes i la transmissió de patògens zoonòsics al llarg de la cadena alimentària. El benestar animal promou la presència de microbiota intestinal beneficiosa i la integritat de l'epiteli intestinal, reduïnt les interaccions amb els bacteris ambientals. D'aquesta manera, es pot aconseguir una reducció de l'administració d'antibiòtics i la presència de patògens en la cadena alimentària. Salmonella spp. és la principal causa de brots alimentaris en la Unió Europea, i la principal font d'infecció són els products avícoles. Entre els principals serotips relacionats amb aquestos brots es troba S. Infantis, actualment molt prevalent en pollastres. Per tot açò, l'objectiu general d'aquesta tesi doctoral va ser avaluar l'efecte de sistemes alternatius de producció avícola sobre el desenvolupament i la composició de la microbiota, l'evolució de les resistències antimicrobianes i l'epidemiologia de Salmonella. En el primer experiment, es va estudiar l'efecte de l'estirp genètica, comparant una estirp comercial de creixement ràpid front a una estirp alternativa de creixement lent. Els objectius van ser caracteritzar la microbiota fecal i investigar la dinàmica de les resistències i multirresistències antimicrobianes al llarg del cicle productiu. Els resultats de la composició de la microbiota mostraren que Firmicutes va representar el fil dominant i els gèneres predominants van ser Oscillospira spp., Ruminococcus spp., Coprococcus spp., Lactobacillus spp. i Bacteroides spp. D'altra banda, els resultats obtinguts en el estudi de resistències antimicrobianes van mostrar que, a l'inici del cicle, els animals de l'estirp de creixement ràpid van presentar un major percentatge de resistència, però al final del període no es van encontrar diferències. En el segon experiment, es va avaluar l'efecte de les condicions de maneig de la granja, comparant les condicions comercials de densitat i ventilació, front a condicions millorades. Els objetius van ser caracteritzar la microbiota fecal, avaluar l'evolució de les resistències i multirresistències antimicrobianes, i investigar el desenvolupament de S. Infantis i les seues resistències antimicrobianes al llarg del cicle productiu. Els resultats obtinguts en la evolució de la microbiota van mostrar un major nivell de diversitat en el grup produït davall condicions de maneig òptimes. A més, Firmicutes va ser el fil dominant i els gèneres predominants van ser Oscillospira spp., Ruminococcus spp., Bacteroides spp. i Coprococcus spp. Els resultats obtinguts en el estudi de resistències antimicrobianes, van mostrar altes tases de resistència al llarg del període, sense diferències entre grups. Per últim, els recomptes més alts d'excreció de Salmonella es van observar en la en el dia de sacrifici en ambdós grups. A més, el 100% dels ceps aïllats van ser multirresistents després de la primera setmana post-infecció. En conclusió, els principals resultats obtinguts en aquesta tesi doctoral inclouen que la diversitat i la composició de la microbiota es troben en constant desenvolupament al llarg del període d’engreixament, veient-se afectades per els factors de maneig estudiats. A més a més, les resistències antimicrobianes es troben presents en els bacteris comensals des del dia d’arrivada, i augmenta fins al final del cicle, destacant la necessitat de controlar l’administració d’antibiòtics en totes les etapes de la producció avícola. Quant a l’epidemiologia de S. Infantis, la contínua excreció durant tot el període d’engreixament i la seua capacitat d’adquirir resistències, independentment de les condicions de maneig en granja, sugereixen la necessitat de realitzar més estudis per poder establir millors programes de control del bacteri al llargo de la cadena alimentària.
[EN] Social awareness regarding animal welfare, food safety, antimicrobial resistance and environmental health has increased, promoting the implementation of alternative sustainable production systems that include the 'One Health' concept. For this reason, in the poultry sector different alternatives at field level have been proposed, centred on the improvement of biosecurity protocols, the use of rustic slow-growing breeds and the implementation of precision livestock farming. In this context, intestinal microbiota play an important role in poultry health, in the spread of antimicrobial resistance and in the transmission of zoonotic pathogens throughout the poultry production chain. Animal welfare promotes the presence of beneficial microbiota and the integrity of the intestinal epithelium, reducing the interactions between environmental and intestinal bacteria. This way, it could be possible to achieve a reduction in antibiotic administration at field level, and also the presence of zoonotic pathogens in the food chain. Salmonella spp. is the main cause of human foodborne outbreaks in the European Union, and the main sources of infection are poultry products. Between the main serovars related to these outbreaks is S. Infantis, which is currently the most prevalent serovar isolated in broiler chickens. Therefore, the general objective of this doctoral thesis was to evaluate the effect of alternative production systems of poultry production on the microbiota composition development, antimicrobial resistance dynamics and Salmonella epidemiology. In the first experiment, the effect of the genetic breed was studied by comparing a commercial fast-growing breed vs. an alternative slow-growing breed. The objectives were to characterise the caecal microbiota and to investigate antimicrobial resistance and multidrug-resistance dynamics throughout the growing period. Regarding microbiota composition, results showed that Firmicutes represented the dominant phylum for both systems, and the most predominant genera were Oscillospira spp., Ruminococcus spp., Coprococcus spp., Lactobacillus spp. and Bacteroides spp. On the other hand, results obtained in the study of antimicrobial resistance showed that at the onset of the cycle, fast-growing day-old-chicks showed higher antimicrobial resistance rates. However, at the end of the period no significant differences were found. In the second experiment, the effect of the farm management conditions was evaluated by comparing commercial density and ventilation conditions vs. improved conditions. The objectives were to characterise the caecal microbiota, to evaluate antimicrobial resistance and multidrug-resistance dynamics, and to investigate the development of S. Infantis and its antimicrobial resistance throughout the growing period. Results obtained in microbiota development showed a higher level of microbiota complexity in the group reared under optimal farm conditions at the end of rearing. Moreover, Firmicutes was the dominant phylum during all the growing period, and the predominant genera were Oscillospira spp., Ruminococcus spp., Bacteroides spp. and Coprococcus spp. Results obtained in the study of antimicrobial resistance showed high antimicrobial resistance rates throughout rearing, and no statistical differences were observed between groups. Finally, Salmonella shedding showed that the highest counts were observed at slaughter day for both groups. Moreover, 100% of the isolates were multi-resistant after the first week post-infection. In conclusion, the main results obtained include that microbiota diversity and composition are in constant development throughout the growing period, and antimicrobial resistance is present as of the arrival day and increases until the end of rearing. Regarding S. Infantis epidemiology, it has been demonstrated the continuous shedding throughout the growing period and its ability to gain antimicrobial resistance.
Montoro Dasí, L. (2021). Effect of Farm Management on Antimicrobial Resistance and Intestinal Microbiota in Poultry Production [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172610
TESIS
Compendio

The goal of this thesis work was to investigate the antimicrobial effect of chitosan from zygomyzetes cell wall material in nonwoven textiles and compare it to commercially available chitosan. This was done using two methods, a somewhat modified version of AATCC Test Method 100-2004 (a standard method for quantitative testing of antimicrobial effect in textiles developed by the AATCC Committee) focusing on CFU, and TTC, a tetrazolium salt that changes from colourless to red in the presence of living microorganisms under the right conditions. The CFU method was also used to detect if it is possible to add chitosan earlier in the production stages, by scanning for any antimicrobial effect in test samples produced that way. Commercial chitosan added to the test samples in 2 % citric acid showed the strongest antimicrobial effect, even reaching the detection limit of approximately 99.5 % inhibition for both E. coli and K. pneumoniae without any incubation. Medium molecular weight commercial chitosan added in 2 % citric acid solution was also the only tested compound that could eliminate C. ablicans after 24 h incubation. Both commercial chitosan added as a powder and cell wall extract showed a mediocre inhibition without incubation, but were able to reach >99 % inhibition after 24 h incubation. Generally speaking, the chitosan investigated is comparable to the chitosan available commercially today, even if it required somewhat longer to reach the same levels of inhibition. The other method for addition of chitosan, however, did not work properly.
Uppsatsnivå: D

Autologous platelet concentrates have been widely used in various medical fields in order to promote tissue regeneration. The significance behind their use lies in the presence of growth factors in platelets α-granules that enhance wound healing by promoting cell chemotaxis, proliferation and differentiation. In addition, anti-inflammatory and antibacterial properties of platelet concentrates have been recently pointed out. In this study, the antimicrobial effect of pure platelet-rich plasma (P-PRP) was evaluated against microorganisms isolated from oral cavity, such as Candida albicans, Enterococcus faecalis, Pseudomonas aeruginosa, Streptococcus agalactiae and Streptococcus oralis. Blood samples were obtained from healthy donors. The antibacterial activity of P-PRP, was evaluated as the minimum inhibitory concentration (MIC), determined through the microdilution two-fold serial method, and the minimum microbicidal concentration (MMC), determined by subculturing microbial samples from MIC tests to solid media. MIC results showed that P-PRP was able to inhibit the growth of E. faecalis, C. albicans, S. agalactiae and S. oralis, but not of P. aeruginosa strains. Moreover, MMB tests showed that C. albicans was less suceptible to P-PRP than other microorganisms. In conclusion, this study shows that, in adjunct to its well established regenerative properties, P-PRP possess an antimicrobial activity and so might represent a valuable product in the fight against infections

This study looked at effects of organic solvents dimethylsulfoxide, dimethylformamide and acetone at 0.01%, 0.05% and 0.1% concentration on germination and seedling development wetland plants. Even at 0.01% level, all solvents affected some aspect of seed germination or seedling growth. Acetone at 0.01% was least toxic. Root morphological characteristics were most sensitive compared to shoot morphological characteristics. This study also looked at bioconcentration patterns of antimicrobial compounds triclosan, triclocarban and methyl-triclosan in wetland plants exposed to Denton Municipal Waste Water Treatment Plant effluent. Bioconcentration patterns of antimicrobial compounds varied among species within groups as well as within organs of species. The highest triclocarban, triclosan and methyltriclosan concentration were in shoot of N. guadalupensis, root of N. lutea and in shoots of P. nodous respectively.

Group B Streptococcus (GBS) is a Gram-positive bacteria known for causing a wide range of illnesses in neonates, pregnant woman, elderly, and immunocompromised people. Intrapartum antibiotic prophylaxis is the current prevention measure for pregnant women used worldwide and has been highly successful thus far. However, it is a dangerous long-term approach with the rising antimicrobial resistance crisis. Vaccine development has been ongoing for several decades and remains a key area of study for GBS researchers worldwide. Finding an effective vaccine target candidate that can be packaged in a safe and inexpensive manner is a continuous challenge. The use of metal ions, such as zinc, as an antibacterial has dated back centuries. Its potential to be harnessed against GBS shows promise as an alternative treatment and prevention option, leading to recent characterisation of GBS zinc efflux machinery. It has been established that GBS employs robust strategies to overcome zinc stress and mediate survival. While bacteria require essential nutrient metals for survival, it is presented with nutritional challenges imposed by the vertebrate host during infection. One such strategy is intoxication of invading bacterial pathogens with transition metals. This work assessed 16 strains of GBS from diverse capsular serotypes, sequence types and isolation sources for susceptibility or resistance toward zinc intoxication. The findings show that strains of a variety of clinical backgrounds, capsular types and sequence-types differ in their resistance phenotypes toward zinc intoxication. For example, cpsV and ST-19 were found to be highly resistant to zinc stress, whereas cpsIII and ST-7 and ST-17 were most sensitive to metal intoxication. It is also first reported here that IS1381, a previously identified transposon, has been found in the main zinc export gene, czcD, of strain 834. Investigation of its possible involvement in strain 834’s increased resistance phenotype was inconclusive.
Thesis (Masters)
Master of Medical Research (MMedRes)
School of Pharmacy & Med Sci
Griffith Health
Full Text

Honey has been used to heal wounds since ancient times. There are many references in ancient literature that cite honey for its medicinal uses. It is used as an alternative agent to cure infections of wounds, burns, ulcers etc. Researchers have shown some of the antimicrobial properties of honey when used as an ointment. When applied to an affected area, it helps to promote the growth of healthy tissue. One of the factors on which the quality of the honey depends, is its geographical origin. Based on the location, honey types can vary as much as 100-fold from each other in color, aroma, viscosity, and antimicrobial properties. The important components in honey that play an essential part in healing wounds and contributing to the antimicrobial properties are enzymes. Their presence allows honey to kill various types of pathogenic bacteria, viruses, fungi etc. A higher antimicrobial effect is seen in monofloral honey (when a single plant species is the source of nectar), which is often more potent than other types of honey in terms of antibacterial activity. Resistance of pathogens to these antimicrobial actions has never been shown, which makes honey a more promising source of antimicrobial research. Presently, infections of burns and wounds are very challenging to treat, especially when they are caused by antibiotic-resistant bacteria. The purpose of this study was to examine the antimicrobial properties of honey from Utah and other locales, and to identify promising antimicrobial activities that could be useful in treating infections caused by resistant bacteria.

A utilização de substitutos ósseos para recuperação da função perdida é uma constante busca dentro da área médica. Por isso os biomateriais têm recebido uma atenção muito grande por parte da comunidade científica, dentre eles os materiais a base de fosfato de cálcio. A hidroxiapatita, Ca10 (PO4)6 (OH) 2, tem sido muito estudada, pois além de representar a constituição da massa dos ossos naturais e dentes em 30 a 70%, possui propriedades de bioatividade e osteocondutividade, favorecendo e auxiliando o crescimento do tecido ósseo. Em contrapartida, infecções bacterianas podem surgir após o implante ocasionando a perda da funcionalidade a curto e médio prazo. Várias alternativas estão sendo testadas, geralmente associadas ao uso de antibióticos convencionais incorporados aos biomateriais. Uma alternativa a tais antibióticos seria a utilização de metais que possuem propriedades antibacterianas. A prata (Ag) é conhecida como um metal bactericida e por isso ganhou lugar de destaque dentre os estudos como um aliado importante no controle das infecções pós-cirúrgicas. Este trabalho teve como objetivo sintetizar, caracterizar e avaliar o efeito antimicrobiano da adição de íons de prata em hidroxiapatita. Foram obtidos pós de hidroxiapatita contendo prata (HAAg), nas concentrações de 0,1M; 0,01M e 0,001M pelo método de precipitação em temperatura ambiente e por imersão do pó de hidroxiapatita em soluções aquosas. As fases cristalinas e os grupamentos iônicos foram analisados para cada condição por técnicas de difração de raios X (DRX) e espectroscopia no infravermelho (IV) respectivamente. As informações sobre a morfologia e identificação de elementos químicos foi realizado pela técnica de microscopia eletrônica de varredura com espectroscopia de energia dispersiva (MEV EDS). As avaliações antimicrobianas foram realizadas por ensaios qualitativos e quantitativos, o ensaio qualitativo utilizou o teste de halo de difusão em disco para Staphylococcus aureus e Escherichia coli e o ensaio quantitativo utilizou contagem de bactérias para as cepas de Staphylococcus aureus. Os resultados de DRX e IV indicaram que independentemente do método de obtenção da HAAg foi possível observar a presença de prata metálica caracterizada pelos picos em 2θ=38,1º e 44,3º nas amostras HAAg0,1Im, HAAg0,1Pr e HAAg0,01Pr. Observou-se também a presença de AgO, correspondente ao pico em 2θ=37,5º nas amostras de HAAg0,01Pr e HAAg0,001Pr. Nos espectros de IV estão presentes as bandas que caracterizam a fase HA, referentes aos grupamentos PO43-, OH- e CO32-. Analisados em conjunto os ensaios qualitativos e quantitativos, as amostras HAAg0,01Im e HAAg0,001Im sintetizadas por imersão indicaram os melhores resultados para o ensaio de disco difusão, por apresentarem formação de halo inibição do crescimento bacteriano para a bactéria S. aureus. Para os ensaios quantitativos as amostras obtidas por precipitação com concentrações 0,1M e 0,01M de prata apresentaram melhor resultado por inibirem o crescimento bacteriano para as cepas S. aureus.
The use of bone substitutes for recovery of lost function is a constant search within the medical field. So biomaterials have received a very large attention from the scientific community, including the materials the basis of calcium phosphate. Hydroxyapatite, Ca10(PO4)6(OH)2, has been studied as apart from representing the natural constitution of the mass of bones and teeth in 30 to 70 % , has properties of bioactivity and osteoconductivity, encouraging and assisting the growth of bone tissue. In contrast, bacterial infections can arise after implantation causing the loss of functionality in the short and medium term. Several alternatives are being tested, usually associated with the use of conventional antibiotics incorporated into biomaterials. An alternative would be to use antibiotics such metals that possess antibacterial properties. Silver (Ag) is known as a bactericidal metal and so gained a prominent place among the studies as an important ally in the control of post- surgical infections. This study aimed to synthesize, characterize and evaluate the antimicrobial effect of adding silver ions into hydroxyapatite. Were obtained hydroxyapatite powders containing silver (Haag) at concentrations 0,1M; 0,01M and 0,001M by the precipitation method at room temperature and by immersion the hydroxyapatite powder in aqueous solutions of AgNO3. The crystalline phases and the ionic groups were analyzed for each condition by techniques of X-ray diffraction (XRD) and infrared spectroscopy (IR) respectively. The information on the morphology and identification of chemical elements was performed by the technique of scanning electron microscopy with energy dispersive spectroscopy (SEM EDS). The antimicrobial evaluations were carried out by qualitative and quantitative assays, the assay used a qualitative diffusion halo disk test for Staphylococcus aureus, Escherichia coli and quantitative assay employed bacteria count for Staphylococcus aureus strains. The results of XRD and IR indicated that regardless of the method of obtaining Haag was possible to observe the presence of metallic silver characterized by peaks in 2θ=38,1º and 44,3º in HAAg0,1Im samples, HAAg0,1Pr and HAAg0,01Pr. It also observed the presence of AgO, corresponding to the peak in 2θ=37,5º in samples HAAg0,01Pr and HAAg0,001Pr. In the IR spectra are present bands that characterize the HA phase, referring to groups PO43-, OH- and CO32-. Taken together qualitative and quantitative assays, and HAAg0,01Im HAAg0,001Im synthesized by soaking samples showed the best results for the disk diffusion test by presenting halo formation bacterial growth inhibition for S. aureus. For quantitative assays, the samples obtained by precipitation with concentrations 0,1M and 0,01M silver showed better results by inhibiting bacterial growth for Staphylococcus aureus strains.

Twenty southern African plants were selected based on traditional use. The ethanol extracts were tested for their antimicrobial activity against P. acnes (ATCC 11827) [Propionibacterium acnes (Gilchrist) Douglas and Gunter deposited as Corynebacterium acnes (Gilchrist) Eberson]. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of the extracts were also determined. Cytotoxicity on human macrophage cells (U937) was performed using the 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) reduction assay. The cytotoxicity was performed to ensure that the extracts are not toxic to human macrophage cells (U937) and to obtain a non-lethal range of concentrations to be tested in the anti-inflammatory assay. The anti-inflammatory potential was tested using IL-8 as a marker cytokine. This is a pro-inflammatory cytokine secreted when cells are stimulated with heat-killed P. acnes cultures. During the investigation of the antimicrobial activity, four plant extracts were found to have significant inhibitory activity against P. acnes. Helichrysum odoratissimum showed the best activity with a minimum inhibitory concentration (MIC) at 7.81μg/ml. Clausena anisata, Rapanea melanophloeos and Helichrysum kraussii were also active with MICs at 31.25μg/ml, 15.63μg/ml and 125μg/ml, respectively. All MICs were selected based on PrestoBlue as the growth indicator. Helichrysum odoratissimum showed also showed the best antioxidant activity with an IC50 of 3.86 ± 0.24μg/ml. It was also reported to have the best selectivity index (SI) of 2.76 on U937 cells. Clausena anisata exhibited good antimicrobial activity and low toxicity on U937 cells with an IC50 at 74.46μg/ml and an SI of 2.38. During the investigation of the synergistic activity of the extracts, the combination of 3.13μg/ml of Helichrysum odoratissimum and Helichrysum kraussii at 0.78μg/ml showed better antimicrobial activity of either of the plant extracts acting alone against P. acnes with a fractional inhibitory index (ΣFIC) of 0.42. Clausena anisata was selected as the extract for the Interleukin-8 (IL-8) inhibition assay as it was shown to be traditionally used for treatment of many inflammatory disorders or symptoms. The inhibition of IL-8 by C. anisata in vitro when plant extract was added to stimulated U937 cells was low but there was some inhibition. The IL-8 protein concentration produced by U937 cells treated with 100μg/ml of heat-killed P. acnes was 840.52pg/ml. Low levels of IL-8 inhibition were observed when cells stimulated with P. acnes were treated with non-lethal concentrations of C. anisata extract. Treatment with 50μg/ml, 25μg/ml, 12.5μg/ml and 6.25μg/ml showed a decrease in IL-8 to 322.48 ± 0.07pg/ml, 365.98 ± 0.24pg/ml, 383.62 ± 0.08pg/ml and 409.52 ± 0.13pg/ml, respectively. The untreated cell control however, seemed to show spontaneous production of IL-8 with quantified as 488.76 ± 0.06pg/ml, making it difficult to analyse effects of other cell stimulants. This spontaneous release was also inhibited with the addition of C. anisata extract at 50μg/ml, 25μg/ml, 12.5μg/ml and 6.25μg/ml which showed IL-8 levels at 299.24 ± 0.13pg/ml, 357.82 ± 0.07pg/ml, 387.14 ± 0.12pg/ml and 388.74 ± 0.19pg/ml, respectively The use of many polyherbal formulations is becoming popular practice. Due to the variety of symptoms observed with acne vulgaris it would be beneficial to investigate mixtures of plants showing good antimicrobial, antioxidant, anti-inflammatory and anti-pathogenic activity as potential treatments for acne vulgaris. This is the first report of the synergistic activity of Helichrysum odoratissimum and Helichrysum kraussii crude ethanol extracts used in a synergistic combination. Also the production of hyaluronidase by the tester strain P. acnes (ATCC 11827). The combination and some other active plants shown in the study should be further investigated as possible novel medicinal agents against acne vulgaris.
Dissertation (MSc)--University of Pretoria, 2014.
National Research Foundation (NRF)
Plant Science
MSc
Unrestricted

With increasing problem of resistance development in bacteria against conventional antibiotics, as well as problems associated with diseases either triggered or enhanced by infection, there is an urgent need to identify new types of effective therapeutics for the treatment of infectious diseases and its consequences. Antimicrobial and anti-inflammatory peptides have attracted considerable interest as potential new antibiotics in this context. While antimicrobial function of such peptides is being increasingly understood demonstrated to be due to bacterial membrane disruption, the mechanisms of their anti-inflammatory function are poorly understood. Since bacterial membrane component lipopolysaccharide triggers inflammation, this thesis aims at clarifying importance of lipopolysaccharide (LPS)-peptide interactions while investigating possible modes of action of peptides exhibiting anti-inflammatory effect. Furthermore, effect of poly(ethylene)glycol (PEG)-conjugation was investigated to increase performance of such peptides. Results presented in this thesis demonstrate that peptide-induced LPS- and lipid A binding/scavenging is necessary but not sufficient criterium for anti-inflammatory effects of peptides. Furthermore, preferential binding to LPS over lipid membrane, as well as higher binding affinity to the lipid A moiety within LPS, are seen for these peptides. In addition, results demonstrate that apart from direct LPS scavenging, membrane-localized peptide-induced LPS scavenging seem to contribute partially to anti-inflammatory effect. Furthermore, fragmentation and densification of LPS aggregates, in turn dependent on the peptide secondary structure on LPS binding, as well as aromatic packing interactions, correlate to the anti-inflammatory effect, thus promoting peptide-induced packing transition in LPS aggregates as key for anti-inflammatory functionality. Thus, peptide-induced LPS aggregate disruption together with reduction of the negative charge of LPS suggests the importance of phagocytosis as an alternative to the inflammatory pathway, which needs to be further investigated. Furthermore, PEG conjugation of peptide results in strongly reduced toxicity at a cost of reduced antimicrobial activity but markedly retained anti-inflammatory effect. Taken together, the results obtained in this work have demonstrated several key issues which need to be taken into consideration in the development of effective and selective anti-inflammatory peptide therapeutics for the treatment of severe Gram-negative bacterial infections.

A bstract Few in vitro screening studies on the biological activities of plant extracts that are intended for oral administration consider the effect of the gastrointestinal system. This study investigated this aspect on extracts of Camellia sinensis (green tea) and Salvia officinalis (sage) using antimicrobial activity as amodel for demonstration. Both the crude extracts and their products after exposure to simulated gastric fluid (SGF) as well as simulated intestinal fluid (SIF) were screened for antimicrobial activity. The chromatographic profiles of the crude plant extracts and their SGF as well as SIF products were recorded and compared qualitatively by means of high performance liquid chromatography coupled to mass spectrometry. The effect of epithelial transport on the crude plant extracts was determined by applying them to an in vitro intestinal epithelial model (Caco-2). The crude extracts for both plants exhibited reduced antimicrobial activity after exposure to SGF, while no antimicrobial activity was detected after exposure to SIF. These results suggested chemical modification or degradation of the antimicrobial compounds when exposed to gastrointestinal conditions. This was confirmed by a reduction of the peak areas on the LC–UV–MS chromatograms. From the chromatographic profiles obtained during the transport study, it is evident that some compounds in the crude plant extracts were either not transported across the cell monolayer or they were metabolised during passage through the cells. It can be deduced that the gastrointestinal environment and epithelial transport process can dramatically affect the chromatographic profiles and biological activity of orally ingested natural products.

Preliminary studies have shown that manuka honey affects the cell cycle of MRSA by impeding cell division, but mode of action was unknown. Cell division depends on the formation of septa and cleavage of peptidoglycan at cytokinesis. This study investigated how manuka honey might alter the cell cycle of EMRSA-15. Physiological and chemical changes in the bacteria exposed to manuka honey were determined using time to kill studies, confocal and electron microscopy. Data indicated that honey had a bactericidal effect on MRSA, inhibiting the cell cycle cytokinesis. Increased septum formation was noted in honey treated cells by transmission electron microscopy. Cell division components including FtsZ and Endo-B-N-Acetylglucosaminidase were investigated using cell wall turbidity assays, zymography, immunofluorescence and immuno gold labelling. Manuka honey treated MRSA cells showed a marked reduction in hydrolase activity after 12 hours compared to untreated cells. The immunofluorescence indicated an initial increase in FtsZ production followed by a significant decrease by 24 hours. PCR of FtsZ showed a 10% increase in production after 1 and 4 hours. Localization by gold labelling gave inconclusive results. Immunofluorescence of Endo-B-N-Acetylglucosaminidase showed a decrease in the amount of enzyme over 24 hours and localization by gold labelling indicated altered distribution of this enzyme. PCR showed no significant difference in expression. 2-D electrophoresis showed a differing proteomic profile between control cells and those treated with honey, with a potential target protein being identified. Methylglyoxal (an antibacterial component of manuka honey) was investigated after a report named this as potentially the active component of manuka honey. Results showed it has an effect but is not wholly responsible for the effects induced by manuka honey. It was concluded that increased numbers of cells with septa were formed and alteration in production of proteins and enzymes resulted in MRSA cells exposed to bactericidal concentrations of manuka honey. The work was also carried out with artificial honey controls, indicating that effects seen were not due to sugar content within honey or methylglyoxal content.

Heteropyxis natalensis (Heteropyxidaceae) is traditionally used to treat respiratory disorders, and as a decongestant and antimicrobial agent. The seasonal variation of the hydrodistilled essential oil was investigated. Three trees in the Johannesburg Botanical Garden (Gauteng) indicated similar chemical profiles with fluctuation in the levels of the two major constituents (1,8-cineole and limonene). Little variation between the antimicrobial activity of seasonally collected samples was documented, with standard deviations of ±0.3 to ±3.3 depending on the pathogen studied. Moderate antimicrobial activity (3.0–16.0 mg/ml) was noted for most pathogens tested with Cryptococcus neoformans exhibiting the highest sensitivity (2.0–3.0 mg/ml). The chemogeographical variation of the oil composition from five of the seven distinct localities studied all contains 1,8-cineole and limonene as major constituents. The antimicrobial study of these samples indicated little variability between localities (standard deviation of ±0.5 to ±3.8). As observed in the seasonal variation study, C. neoformans displayed the highest sensitivity (0.5–2.0 mg/ml). One oil sample (Lagalametse), was distinctly different both chemically and microbiologically.

Citrus essential oils (EO) are potential antimicrobials, first described as such in 1949 by Piacentini. Due to their acceptability in terms of fragrance and flavour characteristics, they lend themselves to use both in food and in clinical practice. Lemon (Citrus limon) or sweet orange (Citrus sinensis) or bergamot (Citrus bergamia) essential oils and their components (limonene, linalool, citral, hersipidin and neoericitrin) and vapours at different temperatures and pHs were tested for their anti-microbial activity against vancomycin-sensitive Enterococcus faecium and Enterococcus faecalis. Lemon essential oil alone or blended and orange EO alone were not as effective as citral or linalool alone or bergamot blends with inhibition diameters of > 9cm. The ranges established for differences in inhibition of growth were; 5-15°C, 20-25°C, 30-45°C and 50°C (p ≤ 0.005) and pHs 4.5-6.5, pH 7.5, and pHs 8.5 —10.5 (p ≤ 0.001). A blend of 1:1 (v/v) orange/bergamot EO was the most effective with MICs at 25°C and pH 5.5 of 0.25% - 0.5% (v/v) and an MID of 50mg/i at 50°C at pH 7.5, under these conditions viable counts were reduced by 5.5 - 10 log10 cfu/ml (p ≤ 0.001). The mechanisms by which a blend of orange/bergamot EO and its vapour bring about their antimicrobial effect were assessed. Transmission electron microscope (TEM) images established morphological changes as well as suggesting EO blend uptake into the cell. The permeability of the cell increased by x2 after being subjected to the EO and by x40 after exposure to the vapour. There were decreases of 1.5 in intracellular pH, 20 a.u. in membrane potential and 18 pmol/mg protein of intracellular ATP. The application of the EO blend impregnated into wipes reduced surface microbial load of vancomycin resistant and vancomycin susceptible E. faecium and E. faecalis by up to 4 log10 compared with control wipes (p ≤ 0.00 1). The use of the EO blend vapour on surfaces resulted in a 2.5 log10 reduction over 24 hours, whilst the vapours (15mg/L air) showed a microbial reduction of up to 4 log10 on cucumber skin and lettuce leaf after 45 seconds exposure via a heat diffuser at 25°C (p ≤ 0.001). Sensory tests demonstrated that there were no changes to the taste of the foodstuff after treatment with the EO blend vapour compared with untreated controls. Investigations into the applications of the EO blend showed no significant difference in effect of EO blend between vancomycin resistant and vancomycin susceptible strains. The results of this study suggest that the use of a citrus essential blend both in oil and vapour form could be a potential alternative to chemical based antimicrobial

Abstract Few in vitro screening assays for biological activities of plant extracts consider the potential effect of the gastrointestinal system on orally consumed plant extracts. Crude water and methanol extracts of Tarchonanthus camphoratus (wild camphor) and Agathosma betulina (‘buchu’) were prepared and exposed to simulated gastric fluid and simulated intestinal fluid during dissolution studies to address this aspect. The crude extracts and resulting simulated gastric fluid and simulated intestinal fluid products were screened for antimicrobial activity against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Proteus vulgaris (ATCC 33420). The T. camphoratus crude extract exhibited antimicrobial activity which was reduced after exposure to simulated gastric fluid. After exposure to simulated intestinal fluid no antimicrobial activity was detected, which suggests chemical alteration or degradation of the active compounds. For A. betulina, the crude water extract and simulated gastric fluid product exhibited no antimicrobial activity, while the simulated intestinal fluid product exhibited antimicrobial activity. This suggests activation of antimicrobial constituents during exposure to simulated intestinal fluid. The chemical composition profiles of the crude extracts and products were determined by means of liquid chromatography coupled to an ultraviolet detector (LC-UV) and a mass spectrometer (LC-MS) to qualitatively assess the effect of exposure to simulated gastrointestinal conditions on the chemical composition of the extracts. In many cases, the peak area of compounds decreased after exposure to simulated gastric fluid and simulated intestinal fluid, while the peak area of other compounds increased. Thus, it can be deduced that the antimicrobial activity and chemical composition was altered after exposure to intestinal conditions during dissolution studies.

Foodborne illness is a growing concern world-wide, and Campylobacter in particular has been reported to cause approximately 145,000 foodborne illness cases every year in Canada. A recent annual report from the Canadian Integrated Program for Antimicrobial Resistance Surveillance reveals an increasing trend of antibiotic-resistant Campylobacter isolated from poultry sources across Canada. The large number of foodborne illnesses and emergence of the resistant strains of Campylobacter pose a serious threat in the agri-food industry. Hence, there is increasing urgency to find alternatives to conventional antimicrobials to reduce the prevalence of Campylobacter in the food supply chain while reducing the likelihood of resistance. Combining antimicrobials is a potential intervention strategy to reduce the growth of pathogens by expanding the spectrum of antimicrobial activity. In this study, carvacrol and zinc oxide nanoparticles (ZnO NPs) were investigated in regards to their synergistic antimicrobial effect against C. jejuni. The combination of these two agents for treatment is based upon current evidence of their individual antimicrobial activity. The objectives of this thesis project were to (1) determine the synergistic antimicrobial effect of carvacrol and ZnO NPs against C. jejuni, (2) investigate the macromolecular fingerprints and gene expression profile of C. jejuni after the combinational treatment, and (3) explain the potential mechanism of the synergistic antimicrobial effect. In this work, a macrobroth dilution method was used to test the antimicrobial effect of the compounds against C. jejuni. The macromolecular fingerprints of C. jejuni cells treated with carvacrol and ZnO NPs were investigated using confocal micro-Raman spectroscopy, whereas Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was employed to study the variation in bacterial gene expression after the antimicrobial treatment. The synergistic antimicrobial effect of carvacrol and ZnO NPs against C. jejuni was clearly demonstrated using the time-kill curve. The macromolecular fingerprints and gene expression profile revealed the role of carvacrol in the synergistic antimicrobial effect against C. jejuni. The results of this study provide fundamental knowledge about bacterial stress in response to the synergistic antimicrobial effects. This antimicrobial combination may be utilized as an intervention strategy to reduce the prevalence of C. jejuni in agri-foods.
Land and Food Systems, Faculty of
Graduate

The purpose of this study was to compare the antimicrobial effect of MTAD, two of its components, doxycycline and citric acid, and NaOCl on E. faecalis in two in vitro models: the bovine tooth model and the agar diffusion model. In the tooth model, the lumens of thirty bovine dentin discs were standardized, autoclaved and infected with E. faecalis for 14 days. The infected dentin discs were treated with either MTAD, doxycycline 100mg/ml, 10% citric acid, 5.25% NaOCl or saline for 10 minutes. Dentinal shavings were collected using sterile 037 and 040 round burs. The number of bacteria in the shavings was enumerated by overnight CO2 culturing on TSA agar plates. For the agar diffusion model, E. faecalis was plated before placing paper discs wetted with three dilutions of each irrigant. The zone of inhibition was recorded after overnight culture. The data were analyzed with a two-way ANOVA and Tukey-Kramer post-hoc test. In the bovine tooth model, NaOCl and doxycycline were more effective than control in killing E. faecalis at the shallow bur depth but at the deeper bur depth, only NaOCl was significantly more effective (psE. faecalis.

The study objective was to compare the antimicrobial activity of STERIPLEX™ HC with 5.25% sodium hypochlorite (NaOCl) at different dilutions (50%, 25%, 10%, 1%, 0.1%) and different time intervals (1, 3, 5 minutes) on Enterococcus faecalis. All data was analyzed using an ANOVA. The 50%, 25%, and 10% dilutions of both disinfectants reduced the colony forming unit (CFU) count to below the limit of detection (50 CFU/ml) after one minute. The 1% dilutions at each of the time intervals show NaOCl was significantly more effective than STERIPLEX™ HC (all Ps < .0001) in reducing the CFU/ml count. The 0.1% dilutions of NaOCl and STERIPLEX™ HC at 1 minute, were not different (P = 0.7808), while at 3 minutes and 5 minutes NaOCl was significantly more effective (P = 0.0098 and P < .0001, respectively).

Silver was used for its antimicrobial effect by the ancient Greeks, long before the existence of microorganisms were first suspected. Nowadays a wide range of antimicrobial dressings containing silver, either incorporated within or applied on the dressings, are available for clinical use. This type of dressings is designed to provide the antimicrobial activity of silver in a more convenient application. The aim with this master thesis was to evaluate if silver release and antimicrobial effect of nine silver containing dressings are dependent on the test medium and if there is any relation between silver release and antimicrobial effect. Release of silver and antimicrobial effect was evaluated by using a 6-well co-culture system, with inoculated test medium in the wells and dressing pieces in the culture well inserts. Three different test media with increased complexity and nutrient value were inoculated with either Results show that release of silver depends on the test fluid used; for phosphate buffered saline (PBS), the silver concentration was as most 1.2 ppm, but for a complex media containing calf serum (SWF), it varied from 9 ppm to 134 ppm. The viable counts in PBS were reduced by at least 3 log units for all dressings and bacteria, whereas in SWF there were no reduction and instead growth was observed. In general, a high release resulted in less bacterial growth. Results also indicated that kinetics of silver release affect the antimicrobial effect. It is likely to assume that it is important for a dressing to release silver quickly. It has previously not been possible to correlate silver release of wound care dressings and antimicrobial effect, since the two factors have been measured in different test systems and in different media. Since both factors depend on test medium and method used, it is shown in the present study that it is important to use relevant test medium for in-vitro evaluation. When measuring silver release and antimicrobial effect in the same test system, a relation is found.

Master of Science
Department of Diagnostic Medicine/Pathobiology
Sanjeev K. Narayanan
Antibiotics are substances produced by bacteria or fungi that are inhibitory to other bacteria and fungi. Antimicrobial compounds include substances that are naturally produced, chemically modified or completely synthetic (chemically designed or synthesized). The chemical modification of naturally produced antibiotic generally results in increase stability, solubility, increased spectrum of activity, or efficacy. Antimicrobial compounds are used in animals to treat and control infectious diseases, and also for growth promotion. Bacteria may gain resistance to antibacterial agents via a variety of mechanisms. There is growing evidence that antimicrobial resistance has significant public health consequences. Rationale use of antimicrobial drugs using appropriate medication at the proper dosage and for duration is one of the important means to reduce selective pressure that helps reduce life of resistant organism. It is also vital to reduce the spread of multi drug resistant organisms in the environment especially in health care facilities. Bacteria evolve rapidly not only by mutation, but also by horizontal gene transfer through the transformation, transduction, and conjugation. Conjugation involves a close contact between two bacteria and transfer of the plasmid that carry many genetic elements. The pathogenic bacteria have the ability to sense as well as respond to the stress in the recipient. The epinephrine and norepinephrine play a key role in stress situations in animals. A previous study showed that norepinephrine (NE), a catecholamine at physiological concentrations promoted the conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella typhimurium to an E. coli recipient in vitro. The objective of this study was to determine the effect of norepinephrine on conjugation of two E. coli strains. Both filter mating and liquid mating assays were used. The results revealed that there was no significance difference between the presence and the absence of norepinephrine on conjugative transfer of RP4 plasmid between E. coli strains (FS1290 and C600N) either in filter mating or liquid mating. Further studies are needed to determine whether higher concentration of (more than 20 mM) has any effects on conjugation in E. coli.

Os ramnolipídeos (RL) são biossurfatantes que apresentam características de grande interesse para a indústria de alimentos, tais como alta biodegradabilidade, baixa toxicidade e propriedades emulsionantes. Este trabalho teve como objetivo avaliar a atividade antimicrobiana do ramnolipídeo comercial (RL-Com - Jeneil Co.) e do ramnolipídeo produzido e purificado no Laboratório de Biotecnologia Microbiana (RL-LBM) sobre culturas de Listeria monocytogenes, através da determinação da concentração inibitória mínima (CIM) utilizando o método de microdiluição em caldo. Os valores de CIM dos RLs variaram entre 78,1 μg mL-1 e 2500 μg mL-1 sendo que a CIM predominante foi de 2500 μg mL-1. Dentre as 32 L. monocytogenes testadas 68,7% foram sensíveis ao RL-LBM e 90,6% sensíveis ao RL-Com. O efeito do ramnolipídeo sobre L. monocytogenes mostrou-se predominantemente bacteriostático. O RL aumentou a permeabilidade celular de L. monocytogenes, contudo este efeito não foi relacionado à sua atividade antimicrobiana. A interação entre os antimicrobianos RL-Com e nisina foi avaliada sobre dois isolados de L. monocytogenes com sensibilidades diferentes ao RL, o L17 menos sensível, com CIM de 2500 μg mL-1, e o L12 mais sensível, com CIM de 156,2 μg mL-1. O índice CIF obtido para os isolados foi de 0,078 e 0,18 , para L17 e L12 respectivamente, caracterizando o efeito sinérgico da combinação entre RL e nisina. As curvas de sobrevivência dos isolados L12 e L17 mostraram que a interação entre a nisina e o RL foi bactericida em concentrações menores que as obtidas individualmente para cada antimicrobiano. Para a L12 a combinação de 78,1 μg mL-1 de RL e 160 UI mL-1 de nisina reduziu completamente a população em 30 min de tratamento, e para a L17 a combinação de 156,2 μg mL-1 de RL e 320 UI mL-1 de nisina foi bactericida após 2 h. Os resultados obtidos neste trabalho demonstraram que o ramnolipídeo possui potencial como agente de controle de L. monocytogenes assim como efeito sinérgico quando combinado à nisina.
The rhamnolipids (RL) are biodegradable biosurfactants which have low toxicity and surface activity properties that can be useful for food processing industries. The objective of this study was to evaluate the susceptibility of Listeria monocytogenes with two rhamnolipids products: the rhamnolipid from P. aeruginosa LBI that was produced and purified in the Microbial Biotechnology Laboratory (RLMBL) and a commercial product (RL-Com - Jeneil Co.). Susceptibility tests were performed by the minimal inhibitory concentration (MIC) using the micro-broth dilution technique. The MIC values varied from 78.1 μg mL-1 to 2500 μg mL-1 and the 2500 μg mL-1 concentration was the predominant value . Among the 32 tested cultures, 68.7% were susceptible to RL-MBL and 90.6% to RL-Com. Results showed that the rhamnolipid activity was primarily bacteriostatic. The RL increases the membrane permeability of L. monocytogenes, however this effect was not directly related to its antimicrobial activity. The combined effect of nisin and RL-Com was evaluated against two wild-type isolates of L. monocytogenes, L12 more sensitive (MIC 156.2 μg mL-1) and L17 less sensitive (2500 μg mL-1). The FIC index for the isolates were 0.18 and 0.078 for L12 and L17 respectively, indicating a particular synergistic effect. The survival curve of isolates L12 and L17 showed that the combination between nisin and RL was bactericidal at lower concentration than for the individual antimicrobials. For L12 isolate 78.1 μg mL-1 of RL and 160 UI mL-1 of nisin eliminated the population after 30 min of incubation. The combination of 156.2 μg mL-1 of RL and 320 UI mL-1 of nisin reduced completely L17 population after 2 h of incubation. This work demonstrated the potential antimicrobial activity of rhamnolipids against L. monocytogenes, as well as the synergistic effect of this biosurfactant with nisin.

Esse trabalho teve como objetivo o estudo da toxicidade aguda e crônica da ação isolada e de misturas binárias de três medicamentos veterinários (Monensina, Sulfametazina e Enrofloxacina) para o organismo teste Daphnia magna. A toxicidade aguda da enrofloxacina determinada foi de CE50 - 54.36 mgL-1, da monensina CE50 - 15.11mgL-1 e da sulfametazina CE50 - 183.80 mgL-1. Para os ensaios de toxicidade crônica foram determinados 3 \"endpoints\" (sobrevivência, reprodução e tamanho do adulto) e foi determinado o CEO para a enrofloxacina de 0,33 mgL-1, da monensina 0,09 mgL-1 e da sulfametazina de 6,8 mgL-1. Para fazer uma comparação entre os testes das substâncias isoladas e das misturas binárias foi utilizado o conceito de unidade tóxica (UT), essa comparação foi feita através da soma das UT dos ensaios individuais e comparando com os resultados dos ensaios de misturas para determinar se houve ação sinérgica, aditiva ou antagônica. O ensaio agudo de mistura monensina/enrofloxacina apresentou ação sinérgica já os ensaios monensina/sulfametazina e sulfametazina/enrofloxacina apresentaram ação antagônica. Os ensaios crônicos de mistura monensina/enrofloxacina e monensina/sulfametazina apresentaram ação sinérgica, porém não foram dosedependente e o ensaio sulfametazina/enrofloxacina apresentou ação antagônica. Com base nesse estudo é possível concluir que a mistura desses medicamentos interfere na sua toxicidade, podendo causar efeitos sinérgicos ou antagônicos
This work aims to study the acute and chronic toxicity of the isolated and binary mixtures action of three veterinary drugs (Monensin, Sulfamethazine and Enrofloxacin) for the test organism Daphnia magna. The determined acute toxicity of enrofloxacin was EC50 - 54.36 mgL-1, monensin EC50 - 15.11 mgL-1 and sulfamethazine EC50 - 183.80 mgL-1. In the chronic toxicity tests were determined 3 endpoints (survival, reproduction and adult size) and the LOEC determined for enrofloxacina was 0.33 mgL-1, monensin 0.09 mgL-1 and sulfamethazine 6.8 mgL-1. To make a comparison between the tests of isolated substances and binary mixtures it was used the concept of toxic unit (TU), this comparison was made by adding the UT of individual studies and comparing the test results of mixtures to determine whether there was a synergistic action, additive or antagonistic. The acute mixture assay monensin/enrofloxacin showed synergistic action yet the assays monensin/sulfamethazine and sulfamethazine/enrofloxacin showed antagonistic action. The chronic mixing assays monensin / enrofloxacin and monensin / sulfamethazine showed synergistic action, but were not dose-dependent and testing sulfamethazine / enrofloxacin present antagonistic action. Based on this study it can be concluded that the mixing of these drugs interferes with its toxicity, and may cause synergistic or antagonistic effect

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed September 24, 2009). Thesis advisor: Dr. Andrew D. Clarke. Includes bibliographical references.

The efficacy of ozone and ultraviolet light, used in combination, to inactivate Listeria monocytogenes in fresh (9% NaCl, 91.86% transmittance at 254 nm) and spent chill brines (20.5% NaCl, 0.01% transmittance at 254 nm) was determined. Preliminary studies were conducted to optimize parameters for the ozonation of â freshâ and â spentâ brines. These include diffuser design, comparison of kit to standard methods to measure residual ozone, studying the effect of ozone on uridine absorbance and determining presence of residual listericidal activity post ozonation. An ozone diffuser was designed using 3/16 inch PVC tubing for the ozonation of brines. The sparger was designed to facilitate better diffusion and its efficiency was tested. The modified sparger diffused 1.44 ppm of ozone after 30 minutes of ozonation and the solution had an excess of 1 ppm in 10 minutes of ozonating fresh brine solution (200ml). Population levels of L. monocytogenes were determined at various time intervals post-ozonation (0, 10, 20, 60 min) to determine the presence of residual listericidal activity. The population post ozonation (0 minutes) was 5.31 Log CFU/ml and was 5.08 Log CFU/ml after a 60 minute interval. Therefore, residual antimicrobial effect was weak. Accuracy of the Vacu-vial Ozone analysis kit was evaluated by comparing the performance of the kit to the standard indigo colorimetric method for measuring residual ozone. The kit was inaccurate in determining residual ozone levels of spent brines and 1% peptone water. Uridine was evaluated as a UV actinometric tool for brine solutions that were ozonated before UV treatment. The absorbance of uridine (A262) decreased after ozonation from 0.1329 to 0.0512 for standard 10 minutes UV exposure duration. Absorbance of uridine was influenced by ozone indicating that the presence of ozone may hamper UV fluence determination accuracy in ozone-treated solutions. Upon completion of diffuser design and ozone/UV analysis studies, the effect of ozone-UV combination on L. monocytogenes in fresh and spent brines was evaluated. Ozonation, when applied for 5 minutes, caused a 5.29 mean Log reduction while 5 minutes of UV exposure resulted in a 1.09 mean Log reduction of L. monocytogenes cells in fresh brines. Ten minutes of ozonation led to a 7.44 mean Log reduction and 10 minutes of UV radiation caused a 1.95 mean Log reduction of Listeria in fresh brine. Spent brines required 60 minutes of ozonation for a 4.97 mean Log reduction in L. monocytogenes counts, while 45 minutes resulted in a 4.04 mean Log reduction. Ten minutes of UV exposure of the spent brines resulted in 0.30 mean Log reduction in Listeria cells. A combination of 60 minutes ozonation and 10 minute UV exposure resulted in an excess of 5 log reduction in cell counts. Ozonation did not cause a sufficient increase in the transmittance of the spent brine to aid UV penetration but resulted in apparent color change as indicated by change in L*a*b* values. Ozonation for sufficient time had considerable listericidal activity in fresh brines and spent brines and when combined with UV treatment, is effective reducing L. monocytogenes to undetectable levels in fresh brines.
Master of Science in Life Sciences

Healthcare workers (HCWs) uniforms are subject to increasing debate regarding the levels of bacterial contamination and the clinical significance of any pathogenic contaminants. Some information is available within the literature, however much of this is dated, and there stilI lacks a standard, reproducible sampling method for use on HCWs uniforms that could be easily implemented worldwide. During this study there was recovery of S. aureus, MRSA, and Enterococcus spp. in communal staff changing rooms, clearly demonstrating that such non-patient areas present sources for the transmission of pathogenic bacteria between wards within the hospital. It was established that current international standard methods of determining antimicrobial activity in fabrics may not provide accurate indications of the efficacy of such products under ward conditions, or dealing effectively with "wild type" agents of hospital acquired infections. These findings suggest that further work is necessary in the development and use of such fabrics. Furthermore, contact plates were optimised as a method for the sampling of used nurses scrub suits and assessing the 'in-use' effect of antimicrobial impregnated fabrics. The main finding of this project was that scrub suits frequently became (and remained) heavily contaminated with pathogenic bacteria during ward use. MRSA was recovered from 40 % of standard nurses scrub suits, and 38% of antimicrobial impregnated (Permagard) scrub suits. Thus, the incorporation of Permagard fabric had no observable effect on the incidence of contamination on scrub suits under ward conditions. MRSA was enumerated at 1.65 cfus per 25 cm2 suggesting counts of around 100-200 cfus per 25 cm''.

Photodynamic therapy is a promising new strategy for the treatment of superficial skin infections and periodontitis. A limitation of antibiotic treatment for these diseases is that even after successful killing of the infecting organism, secreted virulence factors may still be present and cause significant damage to host tissues. If light-activated antimicrobial agents can inactivate microbial virulence factors in addition to killing the pathogenic microorganisms, this would represent an advantage of photodynamic therapy over conventional treatment options. The light-activated antimicrobial agents methylene blue and tin chlorin e6 in combination with laser light of 665 and 633 nm respectively, were assessed for their antibacterial activity and ability to reduce the activity of selected virulence factors of Staphylococcus aureus and Porphyromonas gingivalis. In addition to successfully reducing the microbial burden, it was demonstrated that photosensitisation was able to cause significant reductions in the activity of a number of secreted and cell wall-associated virulence factors produced by these species when irradiated with laser light of the appropriate wavelength. Photosensitisation was also shown to reduce the biological activities of the proinflammatory cytokines tumour necrosis factor-alpha and interleukin-6, which are produced in response to infecting bacteria and are associated with damage to host tissues. The results of these studies indicate that light-activated antimicrobial agents may be useful in reducing the pathology associated with bacterial virulence factors and host-mediated inflammation when used as part of an antimicrobial treatment regimen.

Master of Science
Department of Biomedical Sciences
Sanjeev K. Narayanan
Cefovecin is an extended-spectrum long-acting third generation cephalosporin used to treat canine infections. The study objective was to determine the effect of cefovecin on the absolute number and antimicrobial susceptibility of fecal enteric bacteria in healthy dogs. Fourteen Beagles were randomly assigned to a treated (n = 7, 8 mg/kg cefovecin subcutaneously on day 1) or untreated (n = 7) group. LC/MS was used to determine plasma cefovecin concentration on day 14. E. coli, enterococci, and Salmonella were isolated and enumerated from fecal samples collected on days 0, 3, 7, 14, and 28. Antimicrobial resistance was determined using disc diffusion, MIC, and detected using PCR for the bla[subscript CMY-2] gene on select isolates. Mean plasma concentration of cefovecin on day 14 was 9.59 µg/mL in treated dogs; untreated dogs had no measurable plasma cefovecin. The absolute number of E. coli was lower in treated dogs on day 3 (P ≤ 0.0001), and the absolute number of cefovecin-resistant E. coli was higher in treated dogs on days 7 (P = 0.002), 14 (P = 0.004) and 28 (P ≤ 0.0001), compared to untreated dogs. Enterococci increased and were higher in the treatment group on day 7 (P = 0.0226). Isolation of Salmonella was rare. After cefovecin treatment, beta-lactam resistance was more common in fecal E. coli from treated dogs than untreated dogs, while resistance of enterococci was not altered. On day 28, treated dogs were 3.25 times more likely to carry the bla[subscript CMY-2] gene than untreated dogs (95% CI 1.27 – 8.35). The implications of these findings in clinically ill patients require further research.

M. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology
The present study includes the use of a green synthetic method to prepare copper and silver nanoparticles using chitosan, aqueous extracts of Camellia sinensis, Combretum molle and Melia azedarach linn leaves. This study aims to investigate the influence of capping and precursor concentration on the properties of silver nanoparticles with emphasis on the medicinal plants chosen. The effect of capping agent on the properties of copper nanoparticles is also investigated. The phytochemical properties of plant extracts and the antimicrobial activity of the synthesized particles were also studied; this was achieved by using microdilution bioassay. Decoction method was used to extract secondary metabolites from plant leaves. Preliminary phytochemical screening carried out on the aqueous extracts of the plant leaves showed the presence of tannins, proteins, flavonoids, phenols, and carbohydrates. The total phenolic and flavonoids content of the aqueous extract was determined using spectroscopic methods. The highest phenolic content was found in the aqueous extract of Combretum molle (135 mg/g), and the highest flavonoid content was found in the aqueous extract of Camellia sinensis (0.4 mg/g). Characterization was done by a combination of spectroscopic, microscopy and XRD techniques. Both the size and shape of the synthesized silver nanoparticles were dependent on the identity of the capping molecule, precursor and capping agent concentration as depicted from their TEM and XRD results. Silver nanoparticles were found to be predominantly spherical. The capping agent concentration was also found to influence the degree of agglomeration, with an increase in capping agent concentration giving lesser agglomeration. FTIR spectral analysis showed that silver nanoparticles interact with bioactive compounds found in the plants through the hydroxyl functional group. Other shapes including diamond were observed for the effect of precursor concentration. The XRD micrographs revealed a face-centered cubic geometry and the phase remained the same with an increase in precursor concentration. The synthesized silver nanoparticles were all blue shifted compared to the bulk material. The TEM results revealed that copper nanoparticles with different sizes and shapes were successfully synthesized. All the prepared copper and silver nanoparticles showed satisfactory antifungal and antibacterial activity against Candida albicans, Cryptococcus neoformans, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia and Pseudomonas aeruginosa. The capping molecules used in this study also showed some antibacterial and antifungal activity against the selected strains. However nanoparticles performed better than these capping molecules. Both silver and copper nanoparticles were found to be more active against gram-negative bacteria compared to gram-positive bacteria. Amongst all the prepared silver nanoparticles Combretum molle capped nanoparticles were found to be the most active nanoparticles. Also with copper nanoparticles, it was found that Combretum molle capped nanoparticles were the most active nanoparticles. Between the two metal nanoparticles, silver nanoparticles showed high antibacterial and antifungal activity compared to copper nanoparticles. The antioxidant activity of silver nanoparticles was assessed using 2.2-diphenyl-1-picrylhydrazyl. Silver nanoparticles were found to have some antioxidant activity. However, the capping molecules were found to be more active than the synthesized nanoparticles. This observation is attributed to the presence of some bioactive compounds in the plant extracts.

High strength wastewater (HSWW) generated in food processing industries is characterized by high organic carbon and nitrogen content, and thus high oxygen demand. Biological nitrogen removal (BNR) is a technology widely used for the treatment of HSWW. Food processing facilities practice sanitation to keep food contact surfaces clean and pathogen-free. Benzalkonium chlorides (BACs) are cationic quaternary ammonium antimicrobial compounds (QACs) common in industrial antimicrobial formulations. BAC-bearing wastewater generated during sanitation applications in food processing facilities is combined with other wastewater streams and typically treated in BNR systems. The poor selectivity and target specificity of the antimicrobial BACs negatively impact the performance of BNR systems due to the susceptibility of BNR microbial populations to BAC. Objectives of the research were: a) assessment and quantification of the inhibitory effect of QACs on the microbial groups, which mediate BNR in HSWW treatment systems while treating QAC-bearing HSWW; b) evaluation of the degree and extent of the contribution of QAC adsorption, inhibition, and biotransformation on the fate and effect of QACs in BNR systems. A laboratory-scale, multi-stage BNR system was continuously fed with real poultry processing wastewater amended with a mixture of three benzalkonium chlorides. The nitrogen removal efficiency initially deteriorated at a BAC feed concentration of 5 mg/L due to complete inhibition of nitrification. However, the system recovered after 27 days of operation achieving high nitrogen removal efficiency, even after the feed BAC concentration was stepwise increased up to120 mg/L. Batch assays performed using the mixed liquors of the BNR system reactors, before, during, and post BAC exposure, showed that the development of BAC biotransformation capacity and the acquisition of resistance to BAC contributed to the recovery of nitrification and nitrogen removal. Kinetic analysis based on sub-models representing BNR processes showed that BAC inhibition of denitrification and nitrification is correlated with BAC liquid-phase and solid-phase concentrations, respectively. Simulations using a comprehensive mathematical BNR model developed for this research showed that BAC degradation and the level of nitrification inhibition by BAC were dynamic brought about by acclimation and enrichment of the heterotrophic and nitrifying microbial populations, respectively. The fate and effect of BACs in the BNR system were accurately described when the interactions between adsorption, inhibition, and resistance/biotransformation were considered within the conditions prevailing in each reactor. This work is the first study on the fate and effect of antimicrobial QACs in a continuous-flow, multi-stage BNR system, and the first study to quantify and report parameter values related to BAC inhibition of nitrification and denitrification. Results of this study enable the rational design and operation of BNR systems for the efficient treatment of QAC-bearing wastewater. The outcome of this research provides information presently lacking, supporting the continuous use of QACs as antimicrobial agents in food processing facilities, when and where needed, while avoiding any negative impacts on biological treatment systems and the environment.

Nosocomial pathogens are usually organisms such as fungi and bacteria that are associated with infections caused in a hospital environment. Examples include Clostridium difficile, Pseudomonas aeruginosa, Vancomycin-resistant enterococcus and Methicillin-resistant Staphylococcus aureus (MRSA). MRSA, like most of the nosocomial pathogens, is resistant to antibiotics and is one of the most serious causes of infections. In this thesis we assess the effects of antibiotics and antiseptics on carriage and transmission of MRSA. We use highly detailed patient level data taken from two Intensive Care Unit (ICU) wards in St. Guys and Thomas’s hospital in London, where patients were receiving daily antimicrobial treatment and a decolonisation protocol was used. We work in discrete time and employ three different patient-level stochastic models in a Bayesian framework to explore the effectiveness of antimicrobial treatment on MRSA in discrete time. We also develop suitable methods of model assessment. The first two models assume that there is no transmission between patients in the ICU wards. Initially a Markov model is used, assuming perfect swab test specificity and sensitivity, to describe the colonisation status of an individual on a daily basis. Results are obtained using Gaussian random walk Metropolis- Hastings algorithms. We find some evidence that decolonisation treatment and Oxazolidinone have a positive effect in clearing MRSA carriage. The second model is a hidden Markov model and assumes perfect swab test specificity but imperfect sensitivity. We obtain the results using data- augmented Markov Chain Monte Carlo (MCMC) algorithms to make inference for the unobserved patient colonisation states. We find evidence that the Antiseptic treatment used during the decolonisation period is effective in the clearance of MRSA carriage. In the third case we assume that there is MRSA transmission between the patients in the ICUs. We use three different stochastic transmission models which overcome many of the unrealistic assumptions of other models. A data- augmented MCMC algorithm is employed in order to estimate the transmission rates of MRSA between the patients assuming imperfect swab test sensitivity. We found no or limited evidence that antibiotic use affects the transmission process, whereas antiseptic treatment was found to have an effect.

Thesis (M.S. in food science)--Washington State University, December 2009.
Title from PDF title page (viewed on Jan. 20, 2010). "School of Food Science." Includes bibliographical references (p. 55-59).

Orientadores: Pedro Luiz Rosalen, Cínthia Pereira Machado Tabchoury
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-26T11:28:01Z (GMT). No. of bitstreams: 1 Paula-Eduardo_LailaFacinde_M.pdf: 1396217 bytes, checksum: ee32ae3fccb5fc73fe047b8152a78b6e (MD5) Previous issue date: 2014
Resumo: Os produtos naturais, comprovadamente, têm sido uma fonte promissora para descoberta de novos compostos bioativos. Dentre eles, a própolis coletada por abelhas Apis mellifera possui atividades biológicas descritas na literatura como anticárie, antibacteriana, anti-inflamatória, entre outras. Entretanto, a maioria dos estudos sobre própolis se refere àquelas coletadas por A. mellifera e pouco se tem conhecimento de outras, como a geoprópolis, produzida por abelhas sem ferrão do gênero Melipona. Em estudos recentes, a geoprópolis apresentou promissoras atividades antimicrobiana e anti-inflamatória, porém estas pesquisas ainda não evidenciaram quais as substâncias responsáveis por tais ações biológicas, especialmente contra o biofilme oral cariogênico. Portanto, o objetivo desse trabalho foi isolar e identificar o composto ativo da geoprópolis de Melipona scutellaris com atividade contra biofilme formado por Streptococcus mutans. Este objetivo foi alcançado por meio das seguintes metodologias: 1- fracionamento bioguiado do extrato etanólico da geoprópolis (EEGP); 2- isolamento e identificação do composto ativo; 3- avaliação do potencial anticárie do composto ativo utilizando modelo in vitro de inibição de biofilme oral monoespécie. Como resultado do fracionamento bioguiado foi isolado e identificado o composto nemorosona (C33H42O4, MM= 502 g/mol), uma benzofenona prenilada. A concentração inibitória mínima da nemorosona foi de 6,25 ¿ 12,5 ?g/mL e na concentração de 100 ?g/mL foi capaz de inibir em 95% a aderência do S. mutans em biofilme formado em microplacas de fundo côncavo. Em biofilme formado em discos de hidroxiapatita, a nemorosona na concentração 250 ?g/mL (0,50 mM) reduziu 65 % do peso seco, mais de 70% dos polissacarídeos e 48% da quantidade proteica além de diminuir a viabilidade bacteriana, quando comparada com o controle negativo (veículo, p<0,05). Estes resultados não diferiram estatisticamente da clorexidina a 0,12% (1,33 mM) (p>0,05). Portanto, concluímos que a nemorosona é um composto ativo isolado e identificado da geoprópolis com atividade antibiofilme de S. mutans com capacidade de alterar a composição bioquímica da matriz do biofilme de S. mutans, o que torna este composto promissor agente químico para o controle do biofilme oral
Abstract: Natural products have been demonstrated a promising source to discover new bioactive compounds. Among then, the propolis collected by Apis mellifera bees has biological activity described in the literature as anticairies, antimicrobial, anti-inflammatory, besides other activities. However, most of the studies on propolis refer to those collected by A. mellifera and little is known about others as geopropolis, which is collected by stingless bees of the genus Melipona. In recent studies, geopropolis presented promising antimicrobial and anti-inflammatory activities, but these studies have not revealed which is (are) the substance(s) responsible(s) for such biological activities, especially against the cariogenic oral biofilms. Therefore, the objective of this study was to isolate and identify the active compound from Melipona scutellaris geopropolis, which has activity against the biofilm formation by Streptococcus mutans. This goal was achieved by the following methodologies: 1- bioassay-guided fractionation of the goeporpolis ethanolic extract (EEGP); 2- isolation and identification of the active compound; 3- anticarie potential assessment of the active compound using an in vitro model of inhibition of the oral mono-species biofilm. As result of the bioassay-guided fractionation, the poliprenil benzophenone compound named nemorosone (C33H42O4, MW=502 g/mol) was isolated and identified. The nemorosone¿s minimum inhibitory concentration (MIC) was 6.25 ¿ 12.5 ?g/mL and the concentration of 100 ?g/mL was capable to inhibit by 95% the adherence of S. mutans¿s biofilm formed in U-bottom microtiter plates. In biofilm formed in hydroxyapatite disks, the nemorosone concentration of 250 ?g/mL (0.5 mM) reduced 65% of the dry weight, more than 70% of the polysaccharides and 48% of the protein content. In addition, it reduced the bacterial viability when compared to negative control (vehicle, p<0.05). These results did not differ statistically from chlorhexidine 0.12% (1.33 mM) (p> 0.05). Therefore, the conclusion is that nemorosone is the active compound isolated and identified from geopropolis with antibiofilm activity that is able to alter the biochemical composition of the S. mutans biofilm matrix, it makes this chemical compound promising to oral biofilm control
Mestrado
Farmacologia, Anestesiologia e Terapeutica
Mestra em Odontologia

[Truncated abstract] Antimicrobial growth promoters are added to feed to prevent lactic acidosis in ruminant animals by selectively inhibiting rumen bacteria that produce lactic acid. However, recently imposed or impending bans on the use of antimicrobial growth promoters in animal production have lead to a critical need to find practical alternatives that are safe for the animal and consumer and that obtain similar production benefits. I investigated bioactive plants of Australia for their potential to prevent lactic acidosis in ruminants. The unifying hypothesis tested was that plants would be identified that selectively inhibit lactic acid-producing bacteria and consequently protect against lactic acidosis. This hypothesis was tested in a three phase process: phase 1, plant selection and collection; phase 2, a three stage protocol for screening plants and essential oils; phase 3, in vivo experiments and chemical fractionation of the most promising plant. I developed an in vitro bioassay that simulated acidosis by adding glucose to rumen fluid in Bellco tubes and incubating for 5 h (Chapter 4). The pH and gas production were used as indicators of acidosis and fermentation activity. I used this bioassay to screen ninety-five plants (dried and ground material from 79 species) and ten essential oils and included a negative control (oaten chaff) and a positive control (virginiamycin). One plant, Eremophila glabra, produced a similar pH (5.63) to the positive control (5.43) although it inhibited gas production to a moderate extent (P < 0.05). ... Seven serrulatane diterpenes were identified to be the major secondary metabolites in E. glabra. The metabolites were screened using a broth dilution and microtitre spectrophotometry method and were selective against S. bovis at between 320 and 1077 [mu]g/ mL. The serrulatanes from E. glabra were probably responsible for the activity against acidosis that I observed in vitro, because they selectively inhibited lactateproducing bacteria. It is also possible that a synergy between serrulatanes and possibly other metabolites are responsible for the activity observed in vitro. The results from my experiments support the role that bioactive plants may have to replace the antibiotics that are added to livestock feed. Australian plants were identified containing compounds that were active against the bacterial processes responsible for ruminant acidosis. To my knowledge this is the first work undertaken to identify bioactive plants of Australia for their potential to prevent acidosis. I developed in vitro screening bioassays that targeted key indicators of acidosis. These bioassays enabled me to identify 5 plants from the 104 screened that could potentially control acidosis. One of these plants in particular, E. glabra, showed a level of activity in vitro that was comparable to antibiotic protection against acidosis. The exciting in vitro results were not demonstrated in vivo but only one dose level of E. glabra was used, which was based on the in vitro work. In contrast to the in vitro system the rumen is a continuous flow system with greater complexity and it is possible that the concentration of E. glabra that I used in vivo was not optimum. This places importance on future dose response experiments to confirm the efficacy of E. glabra in vivo.

Some yeast strains under certain conditions secrete into the medium polypeptide toxins which are inhibitory to sensitive cells. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component &
#946
-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. We have previously characterized the K5 type killer protein. This protein is an exo &
#946
-1,3-glucanase which is stable at pH&rsquo
s and temperatures appropriate for its medical usage. &
#946
-1,3- glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antimycotic agent. Antifungal activity of the K5 type yeast killer protein was tested against 26 human pathogenic yeast and 9 dermathophyte strains and found to be affective on all of the tested strains. Toxin MIC50, MIC100 and MFC values were found to be between 0.25-4, 0.5-8, 1-8 µ
g/ml respectively except Candida krusei isolates. Cell killing analysis revealed that toxin activity starts within first 2 hours and complete cell death time differs due to the susceptibility of strains to the K5 type yeast killer protein. K5 type yeast killer protein would be used as a novel and selective agents with the results obtained from this study.

Philosophiae Doctor - PhD
Background: Oropharyngeal candidiasis, caused by the fungus Candida, is the most common opportunistic infection affecting the quality of life of immunocompromised patients. Fluconazole is widely used as the first line of treatment for fungal infections. However, the inappropriate and misguided use of the drug has led to the evolvement of fluconazole-resistant Candida organisms. This arising resistance resulted in the urgent need for the development of new antimicrobial drugs. The aim of the present study was to investigate the antifungal action of K21, a novel antimicrobial quarternary ammonium compound, on fluconazole-resistant Candida species.

There is a constant need of new synthetic emulsifiers in the food industry. Sugar esters are widely used as food grade synthetic emulsifiers, amongst which sucrose esters are the most common. Although sucrose esters are used very frequently, little is known about the use of lactose esters in food. There is a need for characterization of lactose esters before they can be used in foods. The objective of this study was to characterize a lactose ester, lactose monolaurate (LML) as an antimicrobial agent on food pathogens, evaluate its effect on 20 % oil-in-water emulsions as an emulsifier, and to explore its effect on crystallization behavior of anhydrous milk fat. In the first study (Chapter 3), the effect of LML was evaluated on survival of some Gram-positive and Gram-negative bacteria. For Listeria monocytogenes, a concentration of 1 mg/ml showed some inhibition in growth media whereas the cells were completely killed at 5 mg/ml. For Mycobacteria, an LML concentration between 0.1-1mg/ml was lethal. Scanning electron microscopy was also conducted to examine any changes in the morphology of cells. Listeria exhibited a change in morphology and a wrinkling effect was shown in Mycobacteria. In the second study (Chapter 4), the effect of LML as an emulsifier was evaluated in 20 % oil-in-water emulsions. The use level of LML was comparable to commercially available emulsifier polysorbate 20, and produced comparable stabilization in the emulsions upon use. In this study, an attempt was also made to optimize the synthesis of LML with respect to the immobilized enzyme and solvent combination. It was concluded that for 20 % oil-in-water emulsions, LML is a promising emulsifier at 0.5%. In the third study (Chapter 5), the effect of LML was evaluated at two concentrations on the crystallization behavior of anhydrous milk fat at two temperatures with high and low supercooling. On application of high intensity ultrasound (HIU) to anhydrous milk fat (AMF) at 31°C and 0.05 % LML the effect on viscosity of sample and crystallization behavior was evaluated. It was concluded that the viscosity of AMF decreased with the addition of 0.05% LML. The lower viscosity of anhydrous milk fat on addition of LML could be restored with the application of HIU.

This dissertation describes the synthesis, antimicrobial properties, and catalytic activity of a variety of eta5-ligand rhodium and iridium complexes. Cp*RM(beta-diketonato)Cl (Cp*R = R-substituted tetramethylcyclopentadienyl ligand) complexes were found to have selective activity against Mycobacterium smegmatis, with activity highly dependent upon the substituents on the Cp*R ligand as well as on the beta-diketonato ligand. These complexes were synthesized in good yield from the reaction of the chloro bridged dimers ([Cp*RMCl2]2) with the desired beta-diketonato ligand under basic conditions. All complexes were fully characterized by 1H and 13C NMR. Twenty single crystal X-ray structures were solved. The success of these syntheses led to investigation of another beta-diketonato ligand: 1,1,1,5,5,5-hexafluoroacetylacetonate (hfac). Though many metal complexes of this ligand are known, reaction with [Cp*MCl2]2 did not yield the desired Cp*M(hfac)Cl complexes. Instead, a variety of products were obtained, three of which were characterized crystallographically. The most interesting structure featured a non-coordinating trifluoroacetate (TFA) anion and a [Cp*Ir]3Na1O4 cubane structure, which is an unprecedented and highly unusual arrangement for iridium. Attempts to synthesize this cluster rationally through reactions of [Cp*IrCl2]2 with TFA yielded instead a chloro bridged [Cp*IrCl(TFA)] dimer. Reaction of [Cp*MCl2]2 with 1,1,1-trifluoroacetylacetonate (tfac) yielded the expected Cp*M(tfac)Cl complex, indicating that the problem lies with using hfac as a ligand for Cp*M(III) complexes. Finally, the indenyl effect was investigated for the oxidative annulation of 2-phenylimidazole with 1-phenyl-1-propyne catalyzed by a series of methyl-substituted [(indenyl)RhCl2] dimers. [(Ind*)RhCl2]2 was found to have significantly greater activity than [Cp*RhCl2]2 (100% vs. 51%). Two plausible catalytic cycles were proposed, one of which invokes a ring slip transition state. Though it is unclear if the "indenyl effect" is responsible for this differing activity, it is certainly apparent that using an indenyl ligand has a notable effect in this catalytic reaction. Cyclometalation was also investigated stoichiometrically for 2-phenyl-1H-imidazole and 1-phenylpyrazole and found to proceed readily for [(Ind*)RhCl2]2. Additionally, the crystallographic structure of a Rh+ /Rh– ionic pair was solved. Ionic pairs such as this are rarely found in the literature.
Doctor of Philosophy

Brassica vegetables such as broccoli and red cabbage have been associated with positive health effects due to their phytochemical content. More specifically, isothiocyanates, the hydrolysis products of glucosinolates that are present in Brassica, have been shown to exert potential anti carcinogenic effects and antimicrobial activities against various bacterial and fungal pathogens. The overall aims of this work was to: (i) increase our understanding of how the cooking preparation methods affect the synthesis of the bioactive glucosinolate hydrolysis products in broccoli, super broccoli and a glucoraphanin-rich variety red cabbage, (ii) investigate the antimicrobial activity of broccoli extracts against potentially pathogenic Gram positive and Gram negative bacteria and study the mechanisms of action using a large library of E.coli K-12 single gene deletion mutants, and (iii) develop a sulforaphane-rich broccoli soup formulation with good organoleptic characteristics based on a consumer evaluation test and carry out a dietary intervention study with subjects ileostomy patients, i.e. patients who have their large intestine surgically removed and their wastes flow through the end of the small intestine into a special bag (stoma bag), with the aim to evaluate the absorption of sulforaphane by measuring the sulforaphane concentration in the collected ileal fluids. The results indicated that cooking broccoli, super broccoli and red cabbage in vacuum sealed bags (sous vide) prevented leaching of glucosinolates into boiling water. However, very little hydrolysis of the glucosinolates took place most likely due to the inactivation of myrosinase and the epithiospecifier protein (ESP). The addition of myrosinase sources, i.e. horseradish, mustard seed, rocket and watercress during the cooling stage of the cooked broccoli and red cabbage resulted in a significant increase by one to ten-fold for most isothiocyanates with very little amounts of their nitrile analogues being produced, indicating the absence of ESP in most cases in the myrosinase sources used. A wider range of hydrolysis products were detected in red cabbage including sulforaphane nitrile, iberin nitrile, erucin nitrile, iberverin nitrile, 1-cyano,2,3-epithiopropane and l-cyano-2-hydroxy-3-butene, formed by hydrolysis of various glucosinolates. The proposed cooking strategy can be used in home cooking as well as industrial cooking to increase the levels of the bioactive isothiocyanates. The antimicrobial assays demonstrated that broccoli extracts had a wide spectrum of strong inhibition against both Gram positive and Gram negative bacteria and that there was a significant positive correlation between antimicrobial activity and sulforaphane concentration. Several pathogens that were resistant to ampicillin, such as Salmonella Typhimurium DTI 04 TID, Escherichia coli 0157:H7 VT and E. coli K-12, and to tetracycline, such as Salmonella hadar, were sensitive to sulforaphane. The results from assessing the antimicrobial activity of pure sulforaphane against the E. coli K-12 mutant strains indicated that the deletion of genes such as acrR, ToIR, hfq, which are associated with antimicrobial resistance, increased the sensitivity to sulforaphane. Several genes were also identified that are associated with membrane integrity, such as BolA and Sip, whereas others such as malS, milE, to metabolic functions and cell wall recycling. Interestingly, some genes identified in this study, such as moeA, fdnH, lehB, and ycdB, are known to be important for microbial resistance to various metals, although sulforaphane has not been previously shown to be related to metals. Overall, the study suggests that sulforaphane, due to its strong antibacterial activity, could be used as a natural antibacterial agent for food and medical applications. The results also indicate the possibility to increase the antimicrobial effects of broccoli by reducing the temperature of the thermal treatment during cooking or through the addition of myrosinase sources. The developed soup formulations had good organoleptic properties and got satisfactory scores by the consumer panel for overall liking followed by liking of appearance, taste and texture. The best soup formulation was prepared in two ways, one was a standard formulation, and one had increased sulforaphane content through the addition of myrosinase containing mustard seeds during the cooling stage of the cooked soup. Analysis of the ileal fluids indicated that although the ileal fluids collected after feeding with sulforaphane-rich broccoli soup had a higher sulforaphane content, for both types of soups the concentration of sulforaphane was extremely low. Overall, the results demonstrated that more than 98% of sulforaphane was absorbed rapidly, i.e. within 4 hours after feeding. Sulforaphane was also extracted from the ileal fluid and re-constituted at a similar concertation to that found in the ileal fluid, and tested for its antimicrobial activity against E. coli. No inhibitory activity was observed from this test, which was expected taking into account the high minimum inhibitory concentration (MIC); however, the high initial sulforaphane concentration in the broccoli soups and the high microbial inhibitory activity obtained in the in vitro studies suggest that sulforaphane might be exerting its antimicrobial effects during its transit through the stomach and the small intestine, particularly against bacteria associated with small intestinal bacterial overgrowth (SmO) disorder and potentially Helicobacler pylori.

Microbial community in the gut of pigs provides a vast and complex microbial network of community diversity important for its health and development. Pathogenic Escherichia coli are responsible for acute profuse diarrhoea with resultant high morbidity and mortality. Antibiotics are used as growth promoters and for therapeutic purposes in pigs. Misuse, abuse and overuse of these antibiotics have led to development of resistant bacterial strains. This study reports the effect of antimicrobial usage on frequency in which growing pigs habour ETEC and VTEC virulence genes and compared phenotypic and genotypic antibiotic resistance pattern of E. coli and metagenomics analysis of fecal samples collected from; (i) pigs receiving normal farm treatment without antibiotics usage, over a 70 day period, and (ii) pigs allowed treatment with antibiotics and monitored over a 70 day period. Our hypothesis was that the use of antibiotics in commercial pig farms affect gut microbial population. A total of 241 E.coli strains were isolated and antibiotics resistance testing through disk diffusion and PCR was conducted. Sequencing was also done using the Miseq Illumina platform. Virulence genes were detected in [24.8% (Cl95%: 18.2-32.7)] of the antibiotic group isolates and [43.5% (Cl95%: 34.5-52.9)] of the non antibiotic group with a significant difference (P=0.002). Phenotypic resistance to oxytetracycline was most common and were significant (P = 0.03) in samples of days 10 (P = 0.02) and 21 (P = 0.01). Furthermore, [63.9% (Cl95%: 57.6, 69.7)] possesed one or more of the four tested tetracycline resistance genes. Significant statistical difference exists in bacterial structure and composition in the gut of growing pigs P<0.05. Firmicutes, Bacteriotedes and Proteobacteria were the three most abundant phyla and composition was statistically significant during the growing period. The study showed that antibiotics usage increases gut bacterial population in growing pigs. Disease causing virulence genes and antibiotics resistance genes may occur even without antibiotics usage in growing pigs and other factors may be involved.
Dissertation (MSc)--University of Pretoria, 2018.
Veterinary Tropical Diseases
MSc