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...

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.

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.

The objective of this research was to study the impact of ripening inhibitor level and type on the formation, stability, and activity of antimicrobial thyme oil nanoemulsions formed by spontaneous emulsification. Oil-in-water antimicrobial nanoemulsions (10 wt%) were formed by titrating a mixture of essential oil, ripening inhibitor, and surfactant (Tween 80) into 5mM sodium citrate buffer (pH 3.5). Stable nanoemulsions containing small droplets (d < 70 nm) were formed. The antimicrobial activity of the nanoemulsions decreased with increasing ripening inhibitor concentration, which was attributed to a reduction in the amount of hydrophobic antimicrobial constituents transferred to the separated hydrophobic domain, mimicking bacterial cell membranes, by using dialysis and chromatography. The antimicrobial activity of the nanoemulsions also depended on the nature of the ripening inhibitor used: palm ≈ corn > canola > coconut which also depended on their ability to transfer hydrophobic antimicrobial constituents to the separated hydrophobic domain.

Background: Increasingly frequent reports of vancomycin treatment failures for serious methicillin-resistant Staphylococcus aureus (MRSA) infections provide impetus for comparative in vitro studies to assess the activity of newer antimicrobial agents against a range of MRSA isolates. Methods: A sample of 168 MRSA derived from a long-term MRSA collection was subjected to susceptibility testing to telavancin, daptomycin, linezolid, tigecycline and vancomycin by broth micro-dilution. Data were reviewed for sporadic occurrence of isolates with reduced susceptibility. Analyses were performed to test for temporal trends toward decreasing susceptibility and to compare susceptibility of isolates from different infection sites. Results: No MRSA isolate from any time period was resistant to test antibiotics. For daptomycin, linezolid and tigecycline, there were no susceptibility differences between the pre- and postclinical availability periods. All newer agents here active against MRSA isolates with minimum inhibitory concentrations (MICs) of vancomycin >1 mg/l, but there were significant correlations in susceptibility among several pairs of antibiotics. Conclusions: Telavancin and other newer antistaphylococcal agents were fully active against MRSA from various infection sites including isolates with vancomycin MIC >1 mg/l.

This study is a part of a project at Q-linea that aims to present a rapid diagnostic instrument to speed up the process of identification of pathogens and determination of MIC-values (Minimal Inhibitory Concentration) of antibiotic needed to treat patients with sepsis. Specifically, this report is aimed to describe the development and implementation of algorithms that examine susceptibility profiles ofsepsis related pathogens where the bacteria have been exposed to different antibiotics and by different lapse of concentrations. The developed algorithms are based on a clustering technique that identify inhibited growth and present the lowest concentration needed to slow down the growth of the pathogen. The implemented solution was tested on sepsis related pathogens and the determined MIC values were compared to MIC values generated with a method commonly used in healthcare today. Approximately 90% of instances were correctly classified based on data from six hours long tests which is significantly faster than the reference method which takes 16-24 hours to complete. Furthermore, each result comes with a set of quality measures for validation of the algorithm results. Although, further studies are necessary to increase the performance at the four-hour target time, and more data is needed to validate the developed quality measures.

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