Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Molecular Investigation of P. Aeruginosa in the Presence of 4-Hba(01. Izmir Institute of Technology, 2023) Ekenel, Nil Hazal; Soyer Dönmez, FerdaThe escalating threat to human health posed by bacterial pathogens is increasingly attributed to the growing prevalence and distribution of antibiotic-resistant bacteria. In response to antibiotics, microorganisms have developed resistance mechanisms to elude and survive the impacts of these drugs. Phenolic acids have emerged as potent candidates in the battle against bacterial infections due to their unique property of not inducing resistance. This study focuses explicitly on 4-hydroxybenzoic acid (4-HBA), a type of phenolic acid, as an effective antimicrobial agent. Proteomics research has become an indispensable tool in the fight against antimicrobial resistance. Pseudomonas aeruginosa, a bacterium capable of existing in both planktonic and biofilm states and known to cause numerous human diseases, is of particular significance in this context. Furthermore, the study explores the molecular aspects of P. aeruginosa when exposed to 4-HBA through proteomic analysis, revealing a significant impact on protein biosynthesis as a predominantly affected function. Additionally, P. aeruginosa, the study investigates the effects of phenolic acid and antibiotic-loaded bone cements on Methicillin-sensitive Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, and P. aeruginosa. Remarkably, 20 percent inhibition rate is achieved after 48 hours of treatment. Moreover, the study examines biofilm produced by P. aeruginosa in the presence of 4-HBA, utilizing both the Crystal Violet assay and Scanning Electron Microscopy. Significantly, the biofilm formation is observed to be disrupted by these methodologies. Overall, this study underscores the effectiveness of 4-HBA as antimicrobial compound against diverse range of pathogens, as demonstrated through both phenotypic and proteomic approaches.Master Thesis Investigation of Antimicrobial Properties of 4-Hydroxybenzoic Acid and Its Application in Bone Cement(01. Izmir Institute of Technology, 2022) Dokumacı, Ecem; Soyer Dönmez, FerdaThe bone cement is composed of polymethylmethacrylate powder and liquid methylmethacrylate which is commonly used in clinical applications. Nosocomial pathogens utilize the bone cement surface to induce infections, so that bone cements are loaded with various antibiotics to inhibit the infection. Nevertheless, the bacteria gain resistance against antibiotics since the antibiotics are released at slow rates. Phenolic acids are secondary metabolites of plants to prevent infections; thus, are promising antimicrobial chemicals against pathogens. The main aim of this study is the investigation of the antimicrobial effect of liquid and powder 4-hydroxybenzoic acid-loaded bone cement on Escherichia coli, methicillin-resistant and susceptible Staphylococcus aureus. Previously, liquid and powder vancomycin-loaded bone cement had been studied against MRSA and Candida albicans, caffeic acid-loaded bone cement is also investigated on the S. aureus. This study will be the first investigation that the antimicrobial effect of liquid and powder phenolic acid-loaded bone cement against pathogens. 4-HBA was added directly into polymethylmethacrylate powder and mixed with liquid methylmethacrylate. Cement discs were prepared by metal mold and the antimicrobial assay was carried out to determine the bacterial growth inhibition. The structural features of bone cements such as pores and surface were characterized SEM. The antimicrobial agent release of bone cement was also examined by spectrophotometer and HPLC was used for confirmation. Resistance development against 4-HBA was tested on MRSA and Acinetobacter haemolyticus by exposing them to the sub-inhibitory concentrations. The possible genes for the resistance were checked by ddPCR. The significant inhibition of bacterial growth by 4-HBA-loaded bone cement is expected and liquid 4-HBA is expected to be released at a higher rate. Therefore, the phenolic acids may be used as antimicrobial agents in bone cement to inhibit pathogenic bacterial growth.Master Thesis Investigation of Protein Profiles of Listeria Monocytogenes in the Existence of Phenolic Acids Using Mass Spectrometry(Izmir Institute of Technology, 2020) Şen, Sener; Yalçın, Talat; Soyer Dönmez, FerdaListeria monocytogenes is one of the foodborne pathogens (FBP), which are a threat to the consumers' health, able to cause listeriosis. L. monoytgenes cells, which can easily adapt and survive stresses, can develop resistance to antibiotics used in standard therapy. Phenolic acids that are a natural defense mechanism against stress conditions in plants, might be used as an antibacterial-candidate in foodborne diseases, so there is a need for a better understanding of the stress-induced responses and mechanisms of bacteria against these substances. Proteomic approaches are an invaluable method for identifying the stress response in pathogenic bacteria. For this purpose, in this study firstly, the antibacterial effects of two phenolic acids (3-HPAA and 4-HBA) on bacteria were investigated by determining the minimum inhibitory concentrations (MIC). Subsequently, target changes in the protein profile due to antimicrobial effects of phenolic acids were evaluated using a soft ionization technology and mass spectrometry-based comparative gel-free proteomic approach (Shotgun proteomics). According to the results, Listeria monocytogenes could not develop resistance to both phenolic acids. This study emphasizes the importance of using of phenolic acids as a novel and natural therapy methods to overcome antibiotic-resistant pathogenic bacteria.Master Thesis Assesment of Genes That Play Role in Phenolic Compound Degradation in Olive Orchard Microbiome(Izmir Institute of Technology, 2019) Acar, Ertuğrul; Soyer Dönmez, FerdaThe olive tree (Olea Europea L.) is one of the most important fruit trees in Mediterranean countries. Its products, olive oil and table olives, are important components of the Mediterranean diet and widely consumed all around the World. Olives and virgin oil provide a rich source for phenolic compounds. The plant phenolics are secondary metabolites, and possesses several common biological and chemical properties. In this study, microorganisms were collected from soil, olive leaves, fruits, and Olive fruit fly larva and Olive mill wastewater (OMWW). They were characterized by 16S rRNA analysis. The microorganisms that were tolerant to phenolic compounds were selected in order to seek which genes were associated with the phenolic compound degradation. The genes related to the degradation of the selected organisms were identified by Sanger Sequencing and the level of phenol-degrading gene expression were aimed to be compared by using qPCR and Droplet Digital PCR (ddPCR). Microorganisms which degrade phenolic compounds can be harnessed for the purpose of bioremediation. However, the number of defined phenolic compound degrading microorganisms is still low in the literature. For this reason, many different microorganisms were used at the same time for bioremediation. Investigation of olive orchard microorganisms and phenolic-degrading genes might benefit bioremediation in the future. In this study, 8 different bacterial strains were identified and characterized from olive orchards. After that, their phenol hydroxylase and catechol 1,2 dioxygenase genes tried to be sequenced with primers designed by using of reference strains in NCBI database.Master Thesis Efficacy of Natural Antimicrobials on Food-Borne Pathogens and Their Applications(Izmir Institute of Technology, 2008) Eroğlu, Erdal; Soyer Dönmez, FerdaMicrobial contamination of food products increases the risk of food-borne infections and intoxications. In recent years, more studies have been performed for development of natural alternatives to control the growth of pathogenic microorganisms in food rather than chemical origin. In the present study, natural antimicrobial agents from different sources, activated lactoferrin-(ALF), rosemary extract-(RE), jenseniin-G (JG) and natamycin-(NA) were used.The antimicrobial activity of agents (ALF-RE-JG) and their combinations against L. monocytogenes, E. coli O157:H7 and S. Enteritidis, and NA against A. niger and P. roquefortii was tested. These antimicrobials, found effective in vitro, applied also on meat and cheese samples. The minimum inhibitory concentrations (MIC) of ALF, RE and JG against pathogens were determined via disc diffusion and microtiter plate growth assay. Dipping method was applied on meat samples to test the efficacy of antimicrobials. MIC of NA was determined via disc diffusion assay. NA was applied on cheese samples to test the efficacy against both molds by dipping method.The data from microtiter well plate assay showed that ALF is effective on three pathogens. Rosemary extract inhibited only growth of L. monocytogenes and JG did not show any significant activity on the growth of same pathogens. RE enhanced the activity of ALF on E. coli O157:H7. Neither individual activity nor synergistic activity was determined by disc diffusion assay. The data from disc diffusion assay revealed that the effective NA concentration on both molds was 750 ppm. Some of dipping applications on both meat and cheese displayed promising results.Master Thesis Molecular Characterization of Biofilm Properties of Listeria Monocytogenes in the Presence of O-Coumaric Acid and 4-Hydroxybenzoic Acid(Izmir Institute of Technology, 2013) Ekin, Umut; Soyer Dönmez, FerdaBiofilms in medical and food industries are difficult to deal with. The formation of biofilms on every type of surfaces, ability to withstand in biofilm structures against thousand folds of lethal antibiotic doses and reasoning for different kinds of chronic diseases prove dangerousness of biofilms. Food borne pathogens that survive in biofilms can contaminate foods and feeding tubes which results food poisonings, outbreaks and infections. Listeria monocytogenes EGDe is one of the dangerous food borne pathogens with ability to form biofilms and cause listeriosis among elderly people, immunocompromised patients or pregnant women, which results lethal consequences. In this study, 4-hydroxybenzoic acid and o-coumaric acid that have potential antimicrobial and antibiofilm effect on Listeria monocytogenes EGDe were examined. Furthermore, protein profiles were investigated in the presence of phenolic acids. The antimicrobial assays of selected phenolic acids revealed the MICs against Listeria monocytogenes EGDe which are 18 mM for 4-hydroxybenzoic acid and 10 mM for ocoumaric acid. In the presence of MICs, while o-coumaric acid reduced initial attachment at 58%, 4-hydroxybenzoic acid reduced 48%. 4-hydroxybenzoic acid and ocoumaric acid also showed inhibitory effects on preformed biofilms as 34% and 56% respectively. The inhibitory effects of selected phenolic acids were ensured by visualization with SEM and Flourescence Microscopy. Afterwards, Listeria monocytogenes EGDe was treated with MIC of phenolic acids for total protein isolation. Isolated proteins were subjected to in-gel digestion and analysed in nanoLC-ESIMS/ MS system to interpret the mode of actions of antimicrobial and antibiofilming propertiesMaster Thesis A Proteomic Analysis of the Response of Listeria Monocytogenes To the Stress Induced by O-Coumaric Acid and Ferulic Acid(Izmir Institute of Technology, 2013) Cerit, Ender Efe; Soyer Dönmez, FerdaPhenolic acids are known for their potential antimicrobial action, however their mechanisms of action are still unraveled. In this study, effect of phenolic acid stress on Listeria monocytogenes, a dangerous foodborne pathogen, was studied with a proteomic approach. Two phenolic acids found in olive oil, namely o-coumaric acid and ferulic acid, were applied on this bacterium via microtiter plate assay. The concentrations of these phenolic acids that inhibit the bacterial growth by 50% were determined as 7.5 mM for o-coumaric acid, and 8 mM for ferulic acid. Besides, minimum bactericidal concentration of o-coumaric acid was also found as 12 mM. Cellular proteomes of phenolic acid-treated and untreated bacteria were analysed with nanoLC-ESI-MS/MS system, and identified with MaxQuant/Andromeda bioinformatic toolbox. According to the results, both phenolic acids induced oxidative stress in L. monocytogenes cells. In addition to this, DNA replication and repair enzymes, nutrient uptake proteins and several proteins directly related with the virulence of L. monocytogenes were among the targets of phenolic acid stress. Last but not least, proteins associated with biosynthetic pathways were found to be predominantly downregulated. Moreover, the morphology of phenolic acid-treated and untreated cells was visualized with scanning electron microscope to support these findings. The images showed that lengths of treated cells were either abnormally elongated or shortened when compared to untreated cells. A possible reason for these abnormalities is the unequal fission promoted by two factors; excessive production of cell envelope biosynthesis and fission regulatory proteins, and repression of aforementioned DNA replication and repair enzymes.Master Thesis Characterization of Outer Membrane Proteins of Salmonella Enteritidis in Response To Phenolic Acids Stress(Izmir Institute of Technology, 2012) Özdemir, Özgün Öykü; Soyer, Ferda; Soyer Dönmez, FerdaSalmonella enterica serovar Enteritidis (Salmonella Enteritidis) is one of the most reported foodborne pathogen bacterium throughout the world that causes large outbreaks and may result in deaths. Phenolic acids, synthesized in almost all plants, are important for plant metabolism and protection of plants. They can be thought as promising antimicrobial agents against Salmonella Enteritidis. Outer membrane proteins are unique structures to Gram-negative bacteria including Salmonella genus and they are the first targets to environmental changes. This study showed the antimicrobial effect of 3-HPAA, cinnamic acid and o-coumaric acid on Salmonella Enteritidis in vitro. Scanning electron microscopy was used for showing the changes in bacterial shapes. Their effects on the outer membrane protein profile were investigated via proteomic approach. According to results of 96-well microtiter plate assay used for antimicrobial effect determination, among these phenolic acids, 3-HPAA showed 100% growth inhibition and other phenolics retarded the bacterial growth. 30 mM was found as minimum inhibitory concentration (MIC) of 3-HPAA at 24th hour of the experiment. SEM results demonstrated that 3-HPAA causes bacteria to have collapses on cell surface while cinnamic acid and o-coumaric acid cause cells to elongate abnormally. SDS PAGE of the OMPs of bacteria who were exposed to phenolic acids showed changes in amounts of proteins in some protein bands compared to control OMPs. 2-D PAGE results of OMPs of control bacteria via OMPs of 3-HPAA, cinnamic acid and o-coumaric acid treated bacteria, changes in sizes of spots were observed.Master Thesis Outer Membrane Protein Profiling of Escherichia Coli O157:h7 in Response To Phenolic Acid Stress(Izmir Institute of Technology, 2012) Keman, Deniz; Soyer Dönmez, FerdaEscherichia coli O157:H7 is a Gram-negative foodborne and waterborne pathogenic bacterium. Low doses of E. coli O157:H7 are adequate for infection that results in clinical cases, namely hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome. These diseases can be lethal for children, elderly and immunocompromised people. Phenolic acids are herbal secondary metabolites. They are important for the response to environmental stresses. They are significant for human diet due to their health beneficial properties. The objective of this study was the elucidate the antimicrobial mode of action of selected phenolic acids against E. coli O157:H7. The antimicrobial activities of caffeic, rosmarinic, and vanillic acids on E. coli O157:H7 were investigated via microtiter plate assay. Minimum inhibitory concentrations were found as 6.5 mM for caffeic acid, 15 mM for rosmarinic acid and 8 mM for vanillic acid by applying a range of concentrations for each phenolic acid against the bacteria. Effect of each phenolic acid on the cell surface of E. coli O157:H7 was visualized by scanning electron microscopy. Microscopic examination demonstrated that while the surfaces of control group E. coli O157:H7 remained intact, cells treated with phenolic acids displayed disrupted cell surfaces. The bacteria were treated with each phenolic acid at a concentration which resulted in 30-50% inhibition for evaluation of outer membrane protein (OMP) profiles in response to phenolic acids stress. Following OMP isolation, SDS-PAGE and 2D-PAGE analyses were performed. By examining the OMP profiles of phenolic acid treated-bacteria, differences in expression of some proteins were observed.Master Thesis Antimicrobial and Antioxidant Activities of Turkish Extra Virgin Olive Oil From Different Varieties(Izmir Institute of Technology, 2009) Karaosmanoğlu, Hande; Soyer Dönmez, FerdaOlive oil is the main source of fat in Mediterranean diet. The major active components of olive oil include oleic acid, phenolic compounds and squalene which have different benefits such as cancer prevention, antimicrobial and antioxidant activities, and lowering the incidence of skin cancer, respectively. In recent years, the number of studies about the biochemical properties of different varieties of olive oils and their phenolic contents has dramatically increased.In this study, Turkish EVOO from different varieties and their phenolic compounds were investigated in terms of their antimicrobial and antioxidant properties as well as refined olive, hazelnut and canola oils. Antimicrobial activities of EVOOs and ten phenolic compounds were tested against three foodborne pathogenic bacteria, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Enteritidis which are the foremost bacterial cause of the largest number of outbreaks, cases and deaths in the world. While all EVOOs showed bactericidal effect the phenolics demonstrated slight antimicrobial activity with percent inhibition between 0-25 of growth rate of bacteria. Moreover, refined olive, hazelnut and canola oils did not show any antimicrobial activity.Finally, antioxidant activities of EVOOs, refined olive, hazelnut and canola oils were determined by B-carotene-linoleic acid model system and ABTS radical scavenging method. According to B-carotene-linoleic acid method, it was observed that antioxidant activities of oil samples varied between 21.19% and 64.54%. On the other hand, it was found that free radical scavenging activities varied between 1.21% and 21.97% as a result of ABTS method. These results were correlated with TPC values of oils.