Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Bc. Nela Drnková Supervisor: Mgr. Marcela Vejsová, Ph.D. Title of diploma thesis: Evaluation of activity of potencial antifungal substances trought the use of microdilution broth method Background: The aim of this thesis was evaluation of antifungal activity of 70 substances trought the use of microdilution broth method. Substances for testing were synthesized at the Faculty of Pharmacy and they were divided into 5 groups based on chemical structure. Methods: The microdilution broth method was used for testing. The antifungal activity of substances was tested on 8 strains of yeasts and filamentous fungi: Candida albicans, C. tropicalis, C. glabrata, C. krusei, Trichosporon asahii, Aspergillus fumigatus, Absidia corymbifera a Trichophyton mentagrophytes. Results: The most active substances were in group of salicylanilide N- monosubstituted and N,N-disubstituted carbamates and thiocarbamates. The most effective substances were SAL-1L, SAL-1M, SAL-1N. They were effective on Trichophyton mentagrophytes already at concentration of 1,95 µmol.l-1 . The widest spectrum had substance GP-137, it inhibited the growth of all tested strains, but only at high concentrations. Rhodamine...

Department of Biological and Medical Sciences Faculty of Pharmacy in Hradec Králové Charles University in Prague Author: Ivana Ťapuchová Title of diploma thesis: Evaluation of activity of potentional antifungal substances through the use of microdilution broth method Supervisor: Mgr. Marcela Vejsová, Ph.D. Background: The aim of this diploma thesis was to evaluate potential antifungal activity of 52 test substances which were develope at the Department of anorganic and organic chemistry Faculty of Pharmacy in Hradec Králové Charles University in Prague. Methods: For testing we used microdilution broth method on eight strains of yeasts and filamentous fungi in the laboratory of Department of Biological and Medical Sciences. Results: Overall 26 test substances with various substituents had antifungal activity. These substances was derivates of 4-aminobenzoic acid, 4-aminosalicylic acid, 5-aminosalicylic acid and large group derivates of sulfonamids. The most sensitive strain was dermatophyt Trichophyton mentagrophytes, the least sensitive strain was filamentous fungi Absidia corymbifera. Conclusions: Despite there was observed antifungal activity in low concentrations of derivates, it is necessary to perform next tests and clinical studies to prove efficiency and safety for use. Key words: yeasts,...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of thesis: Evalution of activity of potentional antimycotic substances through the use of microdilution broth method III Autor of thesis: Alena Zavřelová Supervisor: Mgr. Klára Konečná, Ph.D. Background: The aim of this thesis, due to the growing incidence of fungal diseases and the growing incidence of resistance, was to determine the activity of potentially antimycotic active substances. We tested newly synthesized compoused, structurally based on the substance of pyrazinamide which is significant antituberculotic. Methods: Testing of substances was carried out using a broth microdilution method on eight strains of pathogenic fungi consisting of five yeast strains: Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Trichosporon asahii and three strains of filamentous fungi: Aspergillus fumigatus, Absidia corymbifera, Trichophyton mentagrophytes. Results: From the range of tested concentrations and from the total number of the thirty tested substances, the most noticeable antifungal aktivity was revealed in the substance of 5-methyl-6-(2-phenylhydrazinyl)pyrazine-2,3- dicarbonitrile. All strains, except Trichophyton mentagrophytes strain, showed a relatively...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biological and medical sciences Study program: Pharmacy Autor: Klára Kopečná Supervisor: Mgr. Klára Konečná, Ph.D. Title of diploma thesis: Evaluation of activity of potencional antibiotic substances through the use of microdilution broth method II Background: To evaluate the antibacterial activity of the compounds synthesized by the Department of Pharmaceutical Chemistry and Drug control, Faculty of Pharmacy in Hradec Králové, Charles University in Prague under the leadership of prof. PharmDr. Martin Doležal, Ph.D. Methods: Using broth microdilution method was tested antibacterial activity at eight bacterial strains, namely Staphylococcus aureus, Staphylococcus aureus methicilin resistant, Staphylococcus epidermidis, Enterococcus sp., Escherichia coli, Klebsiella pneumoniae, Klebsiella pneumoniae ESBL positive and Pseudomonas aeruginosa. Results: Test substances were divided into 5 groups according to similarities in the chemical structure. The best antibacterial activity was observed in the group of derivatives of N-benzyl- 6-(alkylamino)pyrazine-2-carboxamide. Conclusion: Antibacterial activity was detected in 10 of 24 tested compounds. From active substances, all but one danced only Gram-positive bacteria. Among...

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / Antimicrobial resistance (AMR) has arisen as one of the most important public health concerns in the last 60 years. AMR results from pathogenic strains of bacteria adapting to antimicrobial-containing environments through mutations or through horizontal gene transfer (HGT) of genetic material containing resistance genes. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. This dissertation explores the factors, specifically catecholamines and antimicrobials that influence the conjugation frequencies of enteric bacteria including Salmonella, E. coli and Enterococcus. We found that the catecholamine norepinephrine (NE) at physiological concentrations enhanced conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an E. coli recipient in vitro. Additional experiments determined the influence of the antimicrobial concentrations above, equal to and below the minimum inhibitory concentration (MIC) under in vitro conditions on conjugation efficiencies using an Enterococcus to Enterococcus mating pair in addition to the Salmonella to E. coli mating pair. Conjugation occurred in all concentrations, but efficiencies of transfer were consistently low in 0 MIC and 1 MIC, with increased activity both above and below 1 MIC. These data were fit to a previously described mathematical model and the rate constant E that relates the rate of gene transfer to drug concentration was determined. The data showed highly similar patterns of conjugation efficiencies when compared to the rate constant E. A final study we measured conjugation frequencies when donor Salmonella Typhimurium and the E. coli recipient were exposed to both variable concentrations of oxytetracycline and NE. Conjugation was increased pre- and post- MIC, but conjugation frequencies were not enhanced further by the combination of the oxytetracycline and the NE. This dissertation defines the role of outside factors in conjugative gene transfer, and may provide future insight into better control of AMR.

The increasing infection of Cryptococcus species and Tricosporon asahii emerged in clinical patients who were immunocompromised. They usually induce lung, skin, brain and systemic infection. Morbidity and mortality of immunocompromised patients are higher than normal healthy people. Cryptococcus neoformans var. grubii ¡]serotype A¡^ infections were reported in clinical cases predominantly and they were isolated from birds¡¦ droppings in large amount. Cryptococcus neoformans var. gattii ¡]serotype B, C¡^ had a natural life in plants, especially Eucalypticus trees. Isolations from other trees were reported increasingly in the tropical and subtropical areas. Comparing to Cryptococcus species, Tricosporon asahii is the normal mycoses of soil. In this study, we performed an environmental investigation concerning Cryptococcus species and Tricosporon asahii in Southern Taiwan. 120 droppings of racing pigeons and 114 samples from Eucalypticus trees were obtained. The results revealed that 30 Cryptococcus neoformans were isolated from racing pigeons¡¦ droppings ¡]25%¡^, as well as 4 Cryptococcus laurentii ¡]3.3%¡^ and 2 Cryptococcus albidus ¡]1.7%¡^. In addition, 25 Tricosporon asahii ( 20.8% ) were isolated from droppings of racing pigeons. But, none of Cryptococcus species or Tricosporon asahii is isolated from Eucalypticus trees ¡]0%¡^. All of Cryptococcus neoformans isolated from pigeons¡¦ droppings were var. grubii ¡]serotype A¡^ and their drug susceptibility tests showed sensitive to Amphotericin B ¡]minimal inhibitory concentration ¡Ø0.25£gg/ml¡^ and Fluconazole ¡]minimal inhibitory concentration 2£gg/ml¡^ and Flucytosine ¡]minimal inhibitory concentration ¡Ø1£gg/ml¡^. To sum up, both Cryptococcus species and Tricosporon asahii were isolated from droppings of racing pigeons in our study, especially Tricosporon asahii in large amount. Opportunistic infection caused by these species should be given more attention to racing pigeons which have close contact with human . Intensive investigation and surveillance should be carried out in the future to provide an information for the control and prevention of diseases.

No description available.

Raw and processed foods are vulnerable to contamination during their production, distribution and sale. Thus, a wide variety of chemical preservatives are used in the food industry to prevent the growth of food spoilage and pathogenic bacteria. However, health and economic concerns have led to an intensive search for natural alternatives, such as plant extracts, that can safely be used as substitutes for synthetic antimicrobials and preservatives to partially or completely inhibit the growth of bacteria. This study evaluated the antimicrobial effects of natural phenolic compounds extracted from vegetables, fruits, herbs and spices. The main objective was to determine the lowest concentration of phenolics to inhibit the visible growth of the pathogenic bacteria which is defined as the minimum inhibitory concentration (MIC). Some of the most common Gram-positive and Gram-negative foodborne pathogens were treated with several natural phenolic compounds. Concentrations of 5, 10, 15, and 20 ppm (pH 5-6) of each compound were evaluated by broth micro-dilution method and the MICs were determined by using official density (OD) assay. The results demonstrated that the phenolic compounds have varying antimicrobial activities against foodborne pathogens. Natural sources of phenolic compounds contain major antibacterial components and have great potential to be used as natural antimicrobials and food preservatives.

The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

Doctor of Philosophy / Department of Animal Sciences and Industry / Cassandra K. Jones / Porcine epidemic diarrhea virus (PEDV) is a heat-sensitive virus that devastated the United States swine industry. Because of its heat sensitivity, it was hypothesized that a pellet mill mimicking commercial thermal processing may mitigate PEDV infectivity. From the results, it was determined that a conditioning time of 30 sec or greater and temperatures above 54.4°C were effective point-in-time kill steps to inactive PEDV in a research setting. However, this does not prevent subsequent recontamination after pelleting as it is a point-in-time mitigation step. To further explore this, various mitigation additives were evaluated to prevent or mitigate PEDV post-pellet contamination in swine feed and ingredients. Various additives were examined across 3 experiments and included mitigation additives of medium chain fatty acids (MCFA), organic acids (OA), essential oils (OA), formaldehyde based products, and sodium bisulfate. From Exp. 1, formaldehyde, medium chain fatty acids (MCFA), essential oils (EO), and organic acid (OA) each decreased detectable PEDV RNA compared to the control (P<0.05). Additionally, PEDV stability over time was influenced by matrix as the meat and bone meal and spray-dried animal plasma resulted in a greater (P<0.05) quantity of detectable PEDV RNA over 42 days compared to that of the swine diet and blood meal. In Exp. 2, the 1% MCFA inclusion was equally effective at mitigating PEDV as a commercially available formaldehyde product in the complete swine diet. To further explore the effects of MCFA against PEDV, Exp. 3 was conducted to evaluate lower inclusion levels of MCFA and fat sources containing MCFA. It was noted that formaldehyde, 1% MCFA (1:1:1: of caproic, caprylic, and capric acids), 0.66% caproic, 0.66% caprylic, and 0.66% capric acids enhance the RNA degradation of PEDV in swine feed as determined by a bioassay. The MCFA were also evaluated against Salmonella Typhimurium, Generic Escherichia coli, Enterotoxigenic Escherichia coli, and Campylobacter coli. It was noted that the efficacy of the MCFA varied between each bacteria species with caproic and caprylic being the most effective. Commercial developmental products were also tested and determined that Product A and B provided the lowest MIC values across Salmonella Typhimurium, Generic Escherichia coli, and Enterotoxigenic Escherichia coli (P < 0.05). Product A and B were further tested in an animal disease trial utilizing a strain of enterotoxigenic Escherichia. coli O149:K91: K88. From d 7 to 14, chlortetracycline, 1:1:1 blend, and Product B, all improved G:F compared to the control (P<0.05). This also led to chlortetracycline and Product B having an improvement (P<0.05) over the control diet from d 0 to 14. A treatment × day interaction for the enterotoxigenic E. coli plate scores was observed (P < 0.05), which occurred because of the decrease (P<0.05) in plate scores for Product B from d 1 to d 14 and an increase (P<0.05) in chlortetracycline from d 7 to 14. A decrease (P<0.05) in plasma urea nitrogen and haptoglobin was observed as time increased from d -2 to 14. In summary MCFA have shown to be an effect interventional mitigation strategy against PEDV and various bacteria.