Infections caused by antibiotic resistant pathogenic bacteria is an increasing issue inhealthcare, and development of new antimicrobial substances could contribute to combat the continued spread. Plants have historically been used in traditional medicine, have intrinsic defense mechanisms against microbes, and could therefore be a source for new antimicrobial agents. At the Department of Chemistry and Biomedicine at Linnaeus University, Kalmar, a total of 18 extracts made from 9 different combinations of Swedish-growing plants were available.The purpose of the current thesis was to investigate possible antimicrobial effects of the plant extracts in vitro, on a selection of Gram-positive (n=3) and Gram-negative (n=4) bacterial strains. Initial screening of all 18 extracts was performed with agar-based methods including agar well diffusion and direct application on inoculated agar. Detection of concentration-dependent antimicrobial effects was performed with four extracts on Staphylococcus aureus and Enterococcus faecalis. At inhibitory concentrations, viability was estimated as colony forming units/ml (CFU/ml).Screening showed that 11 of 18 extracts affected the growth of at least one of the strains tested. Gram-positive species were affected to a greater extent than Gram-negatives. Estimation of concentration dependency showed inhibitory effects at 50 mg/l in the most potent extracts. Viability estimation revealed an average reduction for both S. aureus and E. faecalis, as compared to the positive control. In conclusion, the study showed possible antimicrobial effects of several extract-bacteria combinations, disclosing potential substances for further investigations.

Purpose Anastomotic leakage (AL) and surgical site infection (SSI) account for most postoperative complications in colorectal surgery. The aim of this retrospective trial was to investigate whether perioperative selective decontamination of the digestive tract (SDD) reduces these complications and to provide a cost-effectiveness model for elective colorectal surgery. Methods All patients operated between November 2016 and March 2020 were included in our analysis. Patients in the primary cohort (PC) received SDD and those in the historical control cohort (CC) did not receive SDD. In the case of rectal/sigmoid resection, SDD was also applied via a transanally placed Foley catheter (TAFC) for 48 h postoperatively. A propensity score-matched analysis was performed to identify risk factors for AL and SSI. Costs were calculated based on German diagnosis-related group (DRG) fees per case. Results A total of 308 patients (154 per cohort) with a median age of 62.6 years (IQR 52.5–70.8) were analyzed. AL was observed in ten patients (6.5%) in the PC and 23 patients (14.9%) in the CC (OR 0.380, 95% CI 0.174–0.833; P = 0.016). SSI occurred in 14 patients (9.1%) in the PC and 30 patients in the CC (19.5%), representing a significant reduction in our SSI rate (P = 0.009). The cost-effectiveness analysis showed that SDD is highly effective in saving costs with a number needed to treat of 12 for AL and 10 for SSI. Conclusion SDD significantly reduces the incidence of AL and SSI and saves costs for the general healthcare system.

Antibiotikaresistens är en av de största globala utmaningarna i sjukvården och om den mängdantibiotika som används idag fortsätter att användas i framtiden riskerar det att kosta 10 miljonermänniskor om livet varje år från år 2050. Antibiotikarester i akvatiska miljöer ökar risken förspridning av antibiotikaresistenta bakterier. Vissa antibiotikum är även toxiska för vattenlevandeorganismer. Dagens vattenreningsverk är inte utformade att rena vatten från antibiotikaresteroch därför följer resterna med vattnet ut i recipienten. För att lösa det här problemet behövervattenreningsverk utvecklas med nya reningsmetoder för att kunna rena antibiotika och vårdenmåste minska användandet i sjukvården. De kopplade globala målen kopplade till detta problemär mål 3 “God hälsa och välbefinnande”, 6 “rent vatten och sanitet”, 11 “hållbara städer ochsamhällen”, 14 “hav och marina resurser” (Globala målen, 2021a; Globala målen, 2021b;Globala målen, 2021c; Globala målen, 2021d). Arbetet undersöker potentiella lösningar förutveckling av vattenreningsverk och alternativa behandlingsmetoder för att minska användandetav antibiotika inom sjukvården. För att göra detta har intervjuer med respondenter på Gävlesjukhus och Duvbackens reningsverk genomförts för att hitta brister i dagens teknik.Förbättringsförslagen är baserade på vetenskapliga artiklar och myndigheters hemsidor. I EUfinns det inga krav gällande att producenterna av läkemedel ska granska den verkligamiljöpåverkan läkemedlet har efter att det har godkänts för försäljning. Antibiotikaläkemedel ärbiologiskt aktiva föreningar vilka, även i små mängder, kan påverka det akvatiska ekosystemetnegativt. Penicillin är det vanligaste använda antibiotikan i Gävles sluten- och primärvård.Slutenvården utgör den största gruppen av antibiotikaanvändare men beaktas bör att det är okänthuruvida dessa användare tidigare kommer från primärvården. Det är också okänt hur stor andelav inköpta läkemedel som faktiskt använts. De reningstekniker bäst lämpade att använda påvattenreningsverk för att rena antibiotikarester varierar och beror bland annat på klimat,resurser och vilken typ av antibiotika som finns i avloppsvattnet. Ozonering är positivt eftersomdet är effektivt mot antibiotikasubstanser med brett spektrum. Det ger heller inte någonpåverkan på slam från vattenreningsverket eftersom ozonet löses upp och blir till syre efterreningen. Ozonering är ofta mest effektiv i kombination med andra reningstekniker vilket kallasför hybridprocesser. Adsorption är effektivt för rening av antibiotikarester och den vanligametoden kolbaserad adsorption är effektiv vid rening av tetracyklinrester. Metoden är effektiveftersom det tar upp mycket av resterna samt att energikostnaden är låg. Sandfilter är en brametod för att rena bort antibiotikaresistenta bakterier och dessutom renar filtrets adsorptionockså rester av tetracyklin. Genom att införa en reningsteknik kombinerad med aktivt kol ochozonering för att rena antibiotikarester från avloppsvattnet samt att använda vakuumassisteradsårbehandling kan användandet av antibiotika minska. / Antibiotic resistance is one of the biggest global challenges in modern day healthcare and ifcontinued use of antibiotics in the same amount as today, it will kill 10 million people every yeararound 2050. Antibiotic residues in aquatic environments increase the risk of spreading ofspreading antibiotic resistant bacteria. Some antibiotics are also toxic to aquatic organisms.Because the wastewater plants are not designed to purify the water from antibiotics the residuescontinue with the water to the recipient. To solve the problem the wastewater plants needs toupgrade with new technology and the healthcare industry reduces the amount prescribedantibiotics. The UN global goals connected to this issue is goal 3 “Good health and well-being”,6 “clean water and sanitary”, 11 “sustainable cities and communities” and 14 “life below water”(Global goals). This study explores potential developments in the wastewater plants andalternatives for antibiotics in healthcare by interviewing respondents at the hospital in Gävle andthe wastewater plant Duvbacken to find the deficiencies of today. Included suggestions are basedon scientific articles and Swedish agencies. There is no requirement for the medicalmanufacturers within the EU to examine the environmental impact of medicines after they havebeen approved for sale. Antibiotic drugs are biologically active compounds which, even in smallamounts, can adversely affect the aquatic ecosystem. Penicillin is the most widely used antibioticin Gävle's inpatient and outpatient care. Outpatient care constitutes the largest group ofantibiotic users, but it should be taken into account that people from this group may havepreviously belonged to inpatient care. It is also unknown how much of each purchased drug thatis used. The treatment techniques that are best suited to use at water treatment plants to purifyantibiotic residues vary and depend on the climate, resources and the type of antibiotics found inthe wastewater. Ozonation is positive as it is effective for broad spectrum antibiotics. It also hasno effect on sludge from the water treatment plant because the ozone dissolves and becomesoxygen after the treatment. Ozonation is often most effective when combined with otherpurification techniques which is called hybrid processes. Adsorption is effective for thepurification of antibiotic residues and the usual method of carbon-based adsorption is effective inthe purification of tetracycline residues. The method is effective because it absorbs much of theresidues at a low energy cost. Sand filter is an effective method to clean away antibiotic-resistantbacteria and in addition, the adsorption of the filter also cleans residues of tetracycline. Byintroducing a purification technique combined with activated carbon and ozonation to purify thewastewater in wastewater plants and to use vacuum assisted wound treatment, the use ofantibiotics can be reduced.

The most frequent intervention performed by physicians is the writing of a prescription. Modern medicine has been remarkably effective in managing diseases. Medicines play a fundamental role in the effectiveness, efficiency and responsiveness of health care systems. However, health care expenditure is a great cause for concern and many nations around the world struggle to contain rising health care costs. Pharmaceutical benefit management programmes such as pharmacoeconomics, drug utilisation review (DUR) and disease management have emerged as control tools to ensure cost effective selection and use of medicine. These managed care instruments are often used to determine whether new strategies or interventions, such as the implementation of a managed medicine reference price list, are appropriate and have "value". The general objective of this study was to investigate the influences of the implementation of a managed medicine reference price list on the usage and cost of antibiotic medicine in the private health care sector of South Africa. The research design used in this study was retrospective, non-experimental and quantitative. The data used for the analysis were obtained over a two-year study period (1 May 2001 to 31 April 2003) from the central medicine claims database of Medschem&. Data was analysed according to prevalence, cost and original (innovator) or generic medicine items. For the purpose of this study antibiotics referred to beta-lactams (penicillins, cephalosporins and "others"), erythromycin and other macrolides, tetracyclines, sulphonamides and combinations, quinolones, chloramphenicol and aminoglycosides. The results of the empirical investigation showed the total number of medicine items claimed during the study period amounted to 49098736 medicine items having a total expenditure of R7150344897.00. There was a decrease in the prevalence of original (innovator) products during the two-year period. The prevalence of generic products increased from 25.87% to 32.47%. A total of 4092495 antibiotic medicine items were claimed with a total cost of R526309279.43 representing 7.36% (n = R7150344897.00) of all pharmaceutical products purchased during the two-year period. Original antibiotics had a prevalence of 42.32%, while generic antibiotics constituted 57.68% of all antibiotic products claimed (n = 4092495). However, original (innovator) products contributed 62.32% and generic products 37.68% to the total cost of all antibiotics claimed. It was concluded that the beta-lactam antibiotics represented 56.99% of all antibiotics claimed (n = 4092495) and contributed 52.51% to the total antibiotic expenditure (n = R526309279.43) for the two-year period. The average cost of beta-lactam items ranged between R112.88 * 69.95 and R122.18 + 81.42. The Medschema Price List (MPL) was implemented in May 2001. The aim of this reference pricing system was to allocate a ceiling price to a group of drugs, which are similar in terms of composition, clinical efficacy, safety and quality, with the ultimate goal to reduce medicine expenditure. During the year of implementation of the MPL 62.24% of beta-lactam antibiotics claimed (n = 1303464) were MPL listed. These products contributed 43.25% to the total cost of all beta-lactam antibiotics (n = R157142778.38). Medical aid companies reimbursed R61649211.86 for penicillins claimed and MPL listed. If all penicillin products were claimed at the ceiling price set by the MPL, a cost saving of 2.79% could have been achieved. Cost analysis indicated that it is possible to reduce health care costs by implementing strategies with the aim to reduce medicine cost. Further research, however, is necessary and in this regard recommendations for further research were formulated. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2005.

The most frequent intervention performed by physicians is the writing of a prescription. Modern medicine has been remarkably effective in managing diseases. Medicines play a fundamental role in the effectiveness, efficiency and responsiveness of health care systems. However, health care expenditure is a great cause for concern and many nations around the world struggle to contain rising health care costs. Pharmaceutical benefit management programmes such as pharmacoeconomics, drug utilisation review (DUR) and disease management have emerged as control tools to ensure cost effective selection and use of medicine. These managed care instruments are often used to determine whether new strategies or interventions, such as the implementation of a managed medicine reference price list, are appropriate and have "value". The general objective of this study was to investigate the influences of the implementation of a managed medicine reference price list on the usage and cost of antibiotic medicine in the private health care sector of South Africa. The research design used in this study was retrospective, non-experimental and quantitative. The data used for the analysis were obtained over a two-year study period (1 May 2001 to 31 April 2003) from the central medicine claims database of Medschem&. Data was analysed according to prevalence, cost and original (innovator) or generic medicine items. For the purpose of this study antibiotics referred to beta-lactams (penicillins, cephalosporins and "others"), erythromycin and other macrolides, tetracyclines, sulphonamides and combinations, quinolones, chloramphenicol and aminoglycosides. The results of the empirical investigation showed the total number of medicine items claimed during the study period amounted to 49098736 medicine items having a total expenditure of R7150344897.00. There was a decrease in the prevalence of original (innovator) products during the two-year period. The prevalence of generic products increased from 25.87% to 32.47%. A total of 4092495 antibiotic medicine items were claimed with a total cost of R526309279.43 representing 7.36% (n = R7150344897.00) of all pharmaceutical products purchased during the two-year period. Original antibiotics had a prevalence of 42.32%, while generic antibiotics constituted 57.68% of all antibiotic products claimed (n = 4092495). However, original (innovator) products contributed 62.32% and generic products 37.68% to the total cost of all antibiotics claimed. It was concluded that the beta-lactam antibiotics represented 56.99% of all antibiotics claimed (n = 4092495) and contributed 52.51% to the total antibiotic expenditure (n = R526309279.43) for the two-year period. The average cost of beta-lactam items ranged between R112.88 * 69.95 and R122.18 + 81.42. The Medschema Price List (MPL) was implemented in May 2001. The aim of this reference pricing system was to allocate a ceiling price to a group of drugs, which are similar in terms of composition, clinical efficacy, safety and quality, with the ultimate goal to reduce medicine expenditure. During the year of implementation of the MPL 62.24% of beta-lactam antibiotics claimed (n = 1303464) were MPL listed. These products contributed 43.25% to the total cost of all beta-lactam antibiotics (n = R157142778.38). Medical aid companies reimbursed R61649211.86 for penicillins claimed and MPL listed. If all penicillin products were claimed at the ceiling price set by the MPL, a cost saving of 2.79% could have been achieved. Cost analysis indicated that it is possible to reduce health care costs by implementing strategies with the aim to reduce medicine cost. Further research, however, is necessary and in this regard recommendations for further research were formulated. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2005.

Antibiotika sind für die Behandlung von bakteriellen Infektionskrankheiten in der Human- und Veterinärmedizin von immenser Bedeutung. Angesichts der Korrelation zwischen Antibiotika-Einsatzmengen und der Häufigkeit resistenter Organismen ist eine unsachgemäße bzw. übermäßige Verwendung dieser antibakteriellen Wirkstoffe sowie deren Eintrag über die Kläranlagen in die Umwelt äußerst problematisch. Neben Vermeidungs- und Verminderungsstrategien besteht ein Ansatz zur Problemlösung in der Entwicklung innovativer Technologien zur Entfernung von Antibiotikarückständen aus Wässern, da konventionelle Kläranlagen dieser Anforderung nicht vollständig genügen. Das im Rahmen dieser Arbeit entwickelte und charakterisierte biologische Verfahren basiert auf genetisch modifizierten Saccharomyces cerevisiae-Zellen, welche spezielle Enzyme sezernieren, die zur Umsetzung von Antibiotika herangezogen werden können. Als Modellsystem diente die enzymatische Hydrolyse des β-Lactam-Antibiotikums Ampicillin mit der β-Lactamase TEM-1. Unter Verwendung von enzymhaltigen Hefe-Kulturüberständen gelang es, die grundsätzliche Eignung des Systems zur Entfernung dieses Antibiotikums nachzuweisen. Untersuchungen mit weiteren β-Lactam-Antibiotika zeigten in Übereinstimmung mit der Literatur, dass TEM-1 Penicilline und Cephalosporine der 1. Generation hydrolysieren kann. Am Beispiel der TEM-8 wurde die Übertragbarkeit des Expressionssystems auf andere Lactamase-Varianten erfolgreich demonstriert. Das erweiterte Wirkspektrum dieses Enzyms, welches neben Penicillinen auch Monobactame und Cephalosporine bis zur 4. Generation umfasst, konnte bestätigt werden. Eine mittels Histidin-tag gereinigte TEM-1-His wurde eingesetzt, um systematisch den Einfluss verschiedener Faktoren, wie Temperatur, Substratkonzentration oder pH-Wert, unbeeinflusst von der Matrix der Hefe-Kulturüberstände untersuchen zu können. In diesem Zusammenhang wurde auch die Übertragbarkeit der Ergebnisse von Modell- auf Realwässer, wie Kläranlagenzu- und -ablauf, untersucht, mit dem Ergebnis, dass zumindest die TEM-1 temporär in allen getesteten Matrices aktiv ist. Mit dem Ziel, auch weitere Antibiotikaklassen transformieren zu können, wurden Esterase Ere-A-produzierende Zellen zur Umsetzung von Makrolid-Antibiotika, wie Erythromycin, herangezogen. Die Analyse der gebildeten Transformationsprodukte ergab, dass die antibakterielle Wirkung jeweils durch hydrolytische Spaltung des β-Lactam- bzw. des Makrolid-Ringes irreversibel verloren geht. Somit kann dieses biologische Verfahren prinzipiell zur gezielten Inaktivierung von Antibiotika eingesetzt werden, wobei der größte Vorteil in der erheblichen Beschleunigung der natürlicherweise ablaufenden Umsetzungsprozesse besteht. Diese Methode kann als ergänzende Technologie bei der Aufbereitung von Konzentraten und Wässern aus Spezialanwendungen angewendet werden.:Glossar Abkürzungsverzeichnis Symbolverzeichnis Kurzfassung Abstract 1 Einleitung 1.1 Motivation 1.2 Zielstellung 2 Theoretische Grundlagen 2.1 Antibiotika und Antibiotikaresistenzen 2.1.1 Antibiotika: Definition, Bedeutung, Einsatzmengen, Klassifikation 2.1.2 Wirkmechanismen: Antibiotika versus Antibiotikaresistenzen 2.1.3 Antibiotika und antibiotikaresistente Organismen in der Umwelt 2.1.4 β-Lactam-Antibiotika 2.1.5 Resistenzen gegenüber β-Lactam-Antibiotika 2.1.6 Makrolid-Antibiotika 2.1.7 Resistenzen gegenüber Makrolid-Antibiotika 2.2 Gentechnische Methoden zur gezielten Proteinbiosynthese 2.3 Der eukaryotische Modellorganismus Saccharomyces cerevisiae 2.4 Enzymkinetik 2.5 Spurenstoffanalytik mittels LC-MS/MS-Technik 2.5.1 Einleitung, Entwicklung und Bedeutung 2.5.2 Elektrospray-Ionisation 2.5.3 Der Quadrupol als Massenanalysator 2.5.4 Analysenmodi bei der Tandem-Massenspektrometrie 3 Material und Methoden 3.1 Verwendete Geräte und Chemikalien 3.2 Arbeiten mit gentechnisch veränderte S. cerevisiae-Zellen 3.2.1 Eingesetzte S. cerevisiae-Stämme 3.2.2 Nährmedien und Kultivierung 3.3 Gewinnung von rekombinanten, in S. cerevisiae exprimierten Enzymen 3.3.1 Gewinnung von β-Lactamase-haltigen Kulturüberständen und gereinigter MFα-TEM-1-His 3.3.2 Zellaufschluss zur Gewinnung der intrazellulären Enzyme 3.4 Einsatz der rekombinanten Enzyme zur Umsetzung von β-Lactam- und Makrolid-Antibiotika 3.4.1 Herstellung und Lagerung von Antibiotika-Stammlösungen, internen Standards und Pufferlösungen 3.4.1.1 Antibiotika-Stammlösungen 3.4.1.2 Interne Standards 3.4.1.3 Herstellung von Kaliumphosphatpuffer 3.4.2 Einsatz von enzymhaltigen Kulturüberstand 3.4.2.1 Nitrocefin-Assay 3.4.2.2 Allgemeine Vorgehensweise und Standardversuchsbedingungen 3.4.2.3 Variation der Antibiotika Konzentration 3.4.2.4 Untersuchungen mit TEM-8-haltigen Kulturüberständen 3.4.3 Einsatz von gereinigter TEM-1 β-Lactamase 3.4.3.1 Proteinbestimmung 3.4.3.2 Allgemeine Vorgehensweise und Standardversuchsbedingungen 3.4.3.3 Variation der Enzymkonzentration 3.4.3.4 Einfluss der Art des Puffers 3.4.3.5 Pufferkonzentration und Leitfähigkeit 3.4.3.6 Variation des pH Wertes 3.4.3.7 Einfluss der Temperatur 3.4.3.8 Variation des eingesetzten β-Lactam-Antibiotikums (Substrat) 3.4.3.9 Variation der AMP-Konzentration 3.4.3.10 Bestimmung der Michaelis-Menten-Konstante Km bei der AMP-Umsetzung mittels TEM-1-His 3.4.3.11 Aktivität und Stabilität der TEM-1-His in Realwässern 3.4.3.12 Bestimmung der spezifischen Enzymaktivität 3.4.4 Einsatz von zellfreien Rohextrakten 3.4.4.1 Allgemeine Versuchsbedingungen 3.4.4.2 Untersuchungen zur Esterase Ere-A 3.5 LC-MS/MS-Analytik 3.5.1 Probenvorbereitung und Herstellung von Kalibrierstandards 3.5.2 HPLC-Parameter 3.5.2.1 Zusammensetzung der Eluenten 3.5.2.2 HPLC-Methoden 3.5.3 Massenspektrometrische Parameter 3.5.4 Auswertung mittels Analyst 3.5.5 Leistungsgrenzen für die qualitative und quantitative AMP Bestimmung 3.5.6 Charakterisierung von Transformationsprodukten 4 Ergebnisse und Diskussion 4.1 Einsatz von TEM-1-haltigem Kulturüberstand zur Transformation von β-Lactam-Antibiotika 4.1.1 Entwicklung einer Versuchsvorschrift zum Nachweis der Enzymaktivität gegenüber β-Lactam-Antibiotika im Kulturüberstand 4.1.1.1 Nachweis der enzymatischen Aktivität mittels Nitrocefin-Assay 4.1.1.2 Versuche mit β-Lactam-Antibiotika 4.1.1.3 Probenvorbereitung 4.1.2 Optimierung einer HPLC-MS/MS-Methode zur Quantifizierung von β-Lactam-Antibiotika unter Berücksichtigung der Probenmatrix 4.1.3 Nachweis der Antibiotika Umsetzung mit TEM-1-haltigen Kulturüberständen 4.1.4 Wirksamkeit der TEM-1-haltigen Kulturüberstände in Abhängigkeit von ausgewählten Randbedingungen 4.1.4.1 Einfluss der Enzymkonzentration 4.1.4.2 Einfluss der Leadersequenz 4.1.4.3 Einfluss des pH-Wertes 4.1.4.4 Einflüsse auf die Enzymkonzentration im Kulturüberstand 4.1.4.5 Enzymatische Stabilität bei Lagerung 4.1.4.6 Variation der Substratkonzentration 4.1.4.7 Substratspezifität 4.1.5 Zwischenfazit 4.2 Einsatz von TEM-8-haltigem-Kulturüberstand zur Transformation von β-Lactam-Antibiotika 4.2.1 Nachweis der enzymatischen Aktivität von TEM-8 4.2.1.1 Auswahl der Modellsubstanzen AMP und CEF 4.2.1.2 Versuche zum Nachweis der TEM-8-Aktivität in nicht-gepuffertem Kulturüberstand 4.2.1.3 Nachweis der TEM-8-Aktivität unter Verwendung von MES-gepuffertem Kulturmedium 4.2.2 Wirksamkeit der TEM-8-haltigen Kulturüberständen in Abhängigkeit von ausgewählten Randbedingungen 4.2.2.1 Einfluss der Leadersequenz 4.2.2.2 Einfluss des Polyhistidin-tags 4.2.2.3 Substratspezifität 4.2.3 Vergleich TEM-1 und TEM-8 4.2.3.1 Prinzipielle Unterschiede in der Kultivierung zur Gewinnung von TEM-8 4.2.3.2 Versuche zur AMP-Umsetzung 4.2.3.3 Abnahme der AMP-Konzentration in den Kontrollproben 4.2.3.4 Versuche zur CEF-Umsetzung 4.2.4 Zwischenfazit 4.3 Einsatz von isolierter TEM-1-His zur Transformation von β-Lactam-Antibiotika 4.3.1 Nachweis der Enzymaktivität 4.3.2 Wirksamkeit des Enzyms TEM-1-His in Abhängigkeit von ausgewählten Randbedingungen 4.3.2.1 Variation der Enzymkonzentration 4.3.2.2 Variation der Pufferkonzentration und Pufferart 4.3.2.3 Variation des pH-Wertes 4.3.2.4 Variation der Temperatur 4.3.2.5 Variation des Substrates 4.3.2.6 Variation der Substratkonzentration 4.3.2.7 Kinetik der enzymatischen Reaktion mit TEM-1-His 4.3.2.8 Untersuchungen zur Umsetzung in Realwässern 4.3.2.9 Langzeitstabilität der isolierten TEM-1-His 4.3.3 Zwischenfazit: spezifische enzymatische Aktivität der TEM-1-His in Abhängigkeit von verschiedenen Versuchsparametern 4.4 Einsatz von Esterase Ere-A-haltigen Rohextrakten 4.4.1 Nachweis der Enzymaktivität im Rohextrakt 4.4.2 Wirksamkeit der Esterase Ere-A in Abhängigkeit von ausgewählten Randbedingungen 4.4.2.1 Einfluss verschiedener Puffer 4.4.2.2 Einfluss von C- und N-terminal angefügten Sequenzen 4.4.2.3 Substratspezifität 4.4.3 Zwischenfazit 4.5 Charakterisierung von Transformationsprodukten 4.5.1 Vorbemerkungen 4.5.2 Transformationsprodukte bei der Umsetzung von β-Lactam-Antibiotika 4.5.3 Einsatz der Esterase Ere A zur Transformation von Makrolid-Antibiotika 4.5.3.1 Transformationsprodukte von Erythromycin 4.5.3.2 Transformationsprodukte von Clarithromycin 4.5.3.3 Transformationsprodukte von Roxithromycin 4.5.4 Zwischenfazit 5 Zusammenfassung 6 Ausblick Literaturverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Anhang A-1 Weitere Analysenmodi bei der Tandem-Massenspektrometrie A-2 Verwendete Geräte und Chemikalien A-3 HPLC-Methoden A-4 Massenspektrometrische Parameter A-5 Erster Versuch zum Nachweis der enzymatischen Aktivität von TEM-1-haltigen Kulturüberstand A-6 Nachweis der Inhibitorwirkung von Sulbactam in Kombination mit TEM-1-His A-7 TEM-1: Variation der Substratkonzentration A-8 TEM-8: Substratspezifität A-9 Vergleich der AMP-Umsetzung mit TEM-1- und TEM-8-haltigen Kulturüberständen sowie den Rohextrakten A-10 TEM-1-His: Variation des pH-Wertes A-11 TEM-1-His: Substratspezifität A-12 Ere-A: Variation der Puffer A-13 Ere-A: Substratspezifität Selbstständigkeitserklärung / Antibiotics play an important role in human and veterinary medicine for the treatment of bacterial infectious diseases. However, regarding the known correlation between the quantities of antibiotics applied and the frequency of resistant organisms, the improper and excessive use of these antibacterial agents leads to serious problems. Their presence in the environment is largely caused by sewage systems due to their incomplete removal in conventional wastewater treatment plants. Therefore, besides avoidance and reduction strategies, one approach to address this issue is to develop innovative technologies for the removal of antibiotic residues from water. The biological method developed and characterized within this work is based on genetically modified Saccharomyces cerevisiae cells, which secrete special enzymes that can be used for the transformation of antibiotics. The enzymatic hydrolysis of the β-lactam-antibiotic ampicillin by the β-lactamase TEM-1 was employed as a model system. By using enzyme-containing yeast culture supernatants, it was possible to prove the suitability of the developed system for the removal of the mentioned antibiotic. The obtained results with other β-lactam-antibiotics showed in accordance with the literature, that TEM-1 was able to hydrolyse penicillins and the cephalosporins of the first-generation. Taking TEM-8 as an example, the transferability of this expression system to alternative lactamase was successfully demonstrated. The activity of this enzyme toward an extended substrate spectrum, which includes not only penicillins but also monobactams and cephalosporins up to the fourth-generation, could be confirmed. The histidine-tagged purified TEM-1-His was used to systematically investigate the influence of various factors, such as temperature, substrate concentration or pH value, independently from the matrix of the yeast culture supernatants. Furthermore, the transferability of the results from model to real water (e. g. influent and effluent from a sewage treatment plant) was investigated with the result that TEM-1 was at least temporarily active in all tested matrices. In order to be able to transform other classes of antibiotics, esterase Ere-A-producing cells were employed to transform macrolide antibiotics, such as erythromycin. The analysis of the formed transformation products revealed that the antibacterial activity is irreversibly lost by the hydrolytic cleavage of the β-lactam or macrolide ring. Therefore, the biological process can be generally used for the selective inactivation of antibiotics, affording a considerable acceleration of the naturally occurring transformation process as its greatest advantage. This method is considered to be a complementary technology for the treatment of concentrates and water from special applications.:Glossar Abkürzungsverzeichnis Symbolverzeichnis Kurzfassung Abstract 1 Einleitung 1.1 Motivation 1.2 Zielstellung 2 Theoretische Grundlagen 2.1 Antibiotika und Antibiotikaresistenzen 2.1.1 Antibiotika: Definition, Bedeutung, Einsatzmengen, Klassifikation 2.1.2 Wirkmechanismen: Antibiotika versus Antibiotikaresistenzen 2.1.3 Antibiotika und antibiotikaresistente Organismen in der Umwelt 2.1.4 β-Lactam-Antibiotika 2.1.5 Resistenzen gegenüber β-Lactam-Antibiotika 2.1.6 Makrolid-Antibiotika 2.1.7 Resistenzen gegenüber Makrolid-Antibiotika 2.2 Gentechnische Methoden zur gezielten Proteinbiosynthese 2.3 Der eukaryotische Modellorganismus Saccharomyces cerevisiae 2.4 Enzymkinetik 2.5 Spurenstoffanalytik mittels LC-MS/MS-Technik 2.5.1 Einleitung, Entwicklung und Bedeutung 2.5.2 Elektrospray-Ionisation 2.5.3 Der Quadrupol als Massenanalysator 2.5.4 Analysenmodi bei der Tandem-Massenspektrometrie 3 Material und Methoden 3.1 Verwendete Geräte und Chemikalien 3.2 Arbeiten mit gentechnisch veränderte S. cerevisiae-Zellen 3.2.1 Eingesetzte S. cerevisiae-Stämme 3.2.2 Nährmedien und Kultivierung 3.3 Gewinnung von rekombinanten, in S. cerevisiae exprimierten Enzymen 3.3.1 Gewinnung von β-Lactamase-haltigen Kulturüberständen und gereinigter MFα-TEM-1-His 3.3.2 Zellaufschluss zur Gewinnung der intrazellulären Enzyme 3.4 Einsatz der rekombinanten Enzyme zur Umsetzung von β-Lactam- und Makrolid-Antibiotika 3.4.1 Herstellung und Lagerung von Antibiotika-Stammlösungen, internen Standards und Pufferlösungen 3.4.1.1 Antibiotika-Stammlösungen 3.4.1.2 Interne Standards 3.4.1.3 Herstellung von Kaliumphosphatpuffer 3.4.2 Einsatz von enzymhaltigen Kulturüberstand 3.4.2.1 Nitrocefin-Assay 3.4.2.2 Allgemeine Vorgehensweise und Standardversuchsbedingungen 3.4.2.3 Variation der Antibiotika Konzentration 3.4.2.4 Untersuchungen mit TEM-8-haltigen Kulturüberständen 3.4.3 Einsatz von gereinigter TEM-1 β-Lactamase 3.4.3.1 Proteinbestimmung 3.4.3.2 Allgemeine Vorgehensweise und Standardversuchsbedingungen 3.4.3.3 Variation der Enzymkonzentration 3.4.3.4 Einfluss der Art des Puffers 3.4.3.5 Pufferkonzentration und Leitfähigkeit 3.4.3.6 Variation des pH Wertes 3.4.3.7 Einfluss der Temperatur 3.4.3.8 Variation des eingesetzten β-Lactam-Antibiotikums (Substrat) 3.4.3.9 Variation der AMP-Konzentration 3.4.3.10 Bestimmung der Michaelis-Menten-Konstante Km bei der AMP-Umsetzung mittels TEM-1-His 3.4.3.11 Aktivität und Stabilität der TEM-1-His in Realwässern 3.4.3.12 Bestimmung der spezifischen Enzymaktivität 3.4.4 Einsatz von zellfreien Rohextrakten 3.4.4.1 Allgemeine Versuchsbedingungen 3.4.4.2 Untersuchungen zur Esterase Ere-A 3.5 LC-MS/MS-Analytik 3.5.1 Probenvorbereitung und Herstellung von Kalibrierstandards 3.5.2 HPLC-Parameter 3.5.2.1 Zusammensetzung der Eluenten 3.5.2.2 HPLC-Methoden 3.5.3 Massenspektrometrische Parameter 3.5.4 Auswertung mittels Analyst 3.5.5 Leistungsgrenzen für die qualitative und quantitative AMP Bestimmung 3.5.6 Charakterisierung von Transformationsprodukten 4 Ergebnisse und Diskussion 4.1 Einsatz von TEM-1-haltigem Kulturüberstand zur Transformation von β-Lactam-Antibiotika 4.1.1 Entwicklung einer Versuchsvorschrift zum Nachweis der Enzymaktivität gegenüber β-Lactam-Antibiotika im Kulturüberstand 4.1.1.1 Nachweis der enzymatischen Aktivität mittels Nitrocefin-Assay 4.1.1.2 Versuche mit β-Lactam-Antibiotika 4.1.1.3 Probenvorbereitung 4.1.2 Optimierung einer HPLC-MS/MS-Methode zur Quantifizierung von β-Lactam-Antibiotika unter Berücksichtigung der Probenmatrix 4.1.3 Nachweis der Antibiotika Umsetzung mit TEM-1-haltigen Kulturüberständen 4.1.4 Wirksamkeit der TEM-1-haltigen Kulturüberstände in Abhängigkeit von ausgewählten Randbedingungen 4.1.4.1 Einfluss der Enzymkonzentration 4.1.4.2 Einfluss der Leadersequenz 4.1.4.3 Einfluss des pH-Wertes 4.1.4.4 Einflüsse auf die Enzymkonzentration im Kulturüberstand 4.1.4.5 Enzymatische Stabilität bei Lagerung 4.1.4.6 Variation der Substratkonzentration 4.1.4.7 Substratspezifität 4.1.5 Zwischenfazit 4.2 Einsatz von TEM-8-haltigem-Kulturüberstand zur Transformation von β-Lactam-Antibiotika 4.2.1 Nachweis der enzymatischen Aktivität von TEM-8 4.2.1.1 Auswahl der Modellsubstanzen AMP und CEF 4.2.1.2 Versuche zum Nachweis der TEM-8-Aktivität in nicht-gepuffertem Kulturüberstand 4.2.1.3 Nachweis der TEM-8-Aktivität unter Verwendung von MES-gepuffertem Kulturmedium 4.2.2 Wirksamkeit der TEM-8-haltigen Kulturüberständen in Abhängigkeit von ausgewählten Randbedingungen 4.2.2.1 Einfluss der Leadersequenz 4.2.2.2 Einfluss des Polyhistidin-tags 4.2.2.3 Substratspezifität 4.2.3 Vergleich TEM-1 und TEM-8 4.2.3.1 Prinzipielle Unterschiede in der Kultivierung zur Gewinnung von TEM-8 4.2.3.2 Versuche zur AMP-Umsetzung 4.2.3.3 Abnahme der AMP-Konzentration in den Kontrollproben 4.2.3.4 Versuche zur CEF-Umsetzung 4.2.4 Zwischenfazit 4.3 Einsatz von isolierter TEM-1-His zur Transformation von β-Lactam-Antibiotika 4.3.1 Nachweis der Enzymaktivität 4.3.2 Wirksamkeit des Enzyms TEM-1-His in Abhängigkeit von ausgewählten Randbedingungen 4.3.2.1 Variation der Enzymkonzentration 4.3.2.2 Variation der Pufferkonzentration und Pufferart 4.3.2.3 Variation des pH-Wertes 4.3.2.4 Variation der Temperatur 4.3.2.5 Variation des Substrates 4.3.2.6 Variation der Substratkonzentration 4.3.2.7 Kinetik der enzymatischen Reaktion mit TEM-1-His 4.3.2.8 Untersuchungen zur Umsetzung in Realwässern 4.3.2.9 Langzeitstabilität der isolierten TEM-1-His 4.3.3 Zwischenfazit: spezifische enzymatische Aktivität der TEM-1-His in Abhängigkeit von verschiedenen Versuchsparametern 4.4 Einsatz von Esterase Ere-A-haltigen Rohextrakten 4.4.1 Nachweis der Enzymaktivität im Rohextrakt 4.4.2 Wirksamkeit der Esterase Ere-A in Abhängigkeit von ausgewählten Randbedingungen 4.4.2.1 Einfluss verschiedener Puffer 4.4.2.2 Einfluss von C- und N-terminal angefügten Sequenzen 4.4.2.3 Substratspezifität 4.4.3 Zwischenfazit 4.5 Charakterisierung von Transformationsprodukten 4.5.1 Vorbemerkungen 4.5.2 Transformationsprodukte bei der Umsetzung von β-Lactam-Antibiotika 4.5.3 Einsatz der Esterase Ere A zur Transformation von Makrolid-Antibiotika 4.5.3.1 Transformationsprodukte von Erythromycin 4.5.3.2 Transformationsprodukte von Clarithromycin 4.5.3.3 Transformationsprodukte von Roxithromycin 4.5.4 Zwischenfazit 5 Zusammenfassung 6 Ausblick Literaturverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Anhang A-1 Weitere Analysenmodi bei der Tandem-Massenspektrometrie A-2 Verwendete Geräte und Chemikalien A-3 HPLC-Methoden A-4 Massenspektrometrische Parameter A-5 Erster Versuch zum Nachweis der enzymatischen Aktivität von TEM-1-haltigen Kulturüberstand A-6 Nachweis der Inhibitorwirkung von Sulbactam in Kombination mit TEM-1-His A-7 TEM-1: Variation der Substratkonzentration A-8 TEM-8: Substratspezifität A-9 Vergleich der AMP-Umsetzung mit TEM-1- und TEM-8-haltigen Kulturüberständen sowie den Rohextrakten A-10 TEM-1-His: Variation des pH-Wertes A-11 TEM-1-His: Substratspezifität A-12 Ere-A: Variation der Puffer A-13 Ere-A: Substratspezifität Selbstständigkeitserklärung

The main aim of the study was to determine the prescribing patterns of antibiotics with an emphasis on fluoroquinolones in the private health sector of South Africa. The empirical study followed a quantitative, descriptive, observational method using retrospective, longitudinal medicine claims data provided by a nationally representative Pharmaceutical Benefit Management company (PBM) from 1 January 2005 to 31 December 2012. Penicillins, cephalosporins, carbapenems, aminoglycosides, chloramphenicol, fluoroquinolones, macrolides, tetracyclines, sulphonamides and trimethoprim were considered in the study. A total of 5 155 262 (44.8%) patients received at least one antibiotic prescription out of the total number of registered beneficiaries included in the database. The average number of antibiotic prescriptions per patient per year ranged from 2.22 ± 1.89 (95% CI 2.22-2.22) in 2005 to 1.98 ± 1.62 (95% CI 1.98-1.99) in 2012. The number of antibiotics per prescription per year remained fairly constant at 1.05 ± 0.19 (95% CI 1.05-1.05) in 2005 to 1.06 ± 0.21 (95% CI 1.06-1.06) in 2012. The prevalence of patients receiving antibiotic prescriptions decreased from 46.1% (n = 789 247) in 2005 to 38.2% (n = 480 159) in 2012. Antibiotics were mostly prescribed for females (54.9%, n = 2 831 686) and in patients aged 0 to 18 years (26.5%, n = 1 366 824) and least in patients above 65 years (9.5%, n = 490 496). The prevalence of patients receiving antibiotic prescriptions was highest in Gauteng (41.9%, n = 2 159 360) and lowest in the Northern Cape (1.7%, n = 87 720). Antibiotics were mostly prescribed during the winter period. Penicillins were the most prescribed antibiotics (43%) and carbapenem the least (0.1%) out of the total number of antibiotics claimed. No practically significant association was found between antibiotic prescribing and gender, age, province and season. A total of 1 983 622 prescriptions for fluoroquinolones were claimed in patients older than 18 years. The average number of fluoroquinolone prescriptions per patient per year ranged from 1.45 ± 0.92 (95% CI 1.44-1.45) in 2005 to 1.31 ± 0.71 (95% CI 1.31-1.32) in 2012. The highest prevalence of fluoroquinolone prescribing was observed in females (64.1%, n = 850 253) and in patients between 45 and 65 years (38.6%, n = 511 542). The total fluoroquinolone use by the study population decreased from 2.85 DID in 2005 to 2.41 DID in 2012. Norfloxacin was the only first-generation fluoroquinolone prescribed. The second-generation fluoroquinolones accounted for more than 50% of the total DID, with ciprofloxacin being the most used active ingredient in this generation. Moxifloxacin was the most prescribed third-generation fluoroquinolone; its use ranging from 0.51 DID in 2005 to 0.44 DID in 2012. Between 2005 and 2012, a total of 57 325 prescriptions for fluoroquinolones were claimed by patients 18 years and younger. The prevalence of patients receiving fluoroquinolone prescriptions decreased from 3.6% (n = 8 329) in 2005 to 2.9% (n = 3 310) in 2012. Fluoroquinolones were mostly prescribed to females and in patients between 12 and 18 years. In all age groups, prescribing was mainly done by general medical practitioners. Ciprofloxacin was the most prescribed fluoroquinolone, followed by levofloxacin. In conclusion, this study established estimates on the prevalence of antibiotic prescribing covering an eight-year period. Secondly, baseline estimates for fluoroquinolone prescribing in adults using the ATC/DDD methodology were determined. Fluoroquinolone prescribing patterns in children and adolescents were determined, with specific reference to the comparison between the prescribed daily and recommended daily dosages in the different age groups and by prescribers’ specialties. / MPharm (Pharmacy Practice), North-West University, Potchefstroom Campus, 2015

The main aim of the study was to determine the prescribing patterns of antibiotics with an emphasis on fluoroquinolones in the private health sector of South Africa. The empirical study followed a quantitative, descriptive, observational method using retrospective, longitudinal medicine claims data provided by a nationally representative Pharmaceutical Benefit Management company (PBM) from 1 January 2005 to 31 December 2012. Penicillins, cephalosporins, carbapenems, aminoglycosides, chloramphenicol, fluoroquinolones, macrolides, tetracyclines, sulphonamides and trimethoprim were considered in the study. A total of 5 155 262 (44.8%) patients received at least one antibiotic prescription out of the total number of registered beneficiaries included in the database. The average number of antibiotic prescriptions per patient per year ranged from 2.22 ± 1.89 (95% CI 2.22-2.22) in 2005 to 1.98 ± 1.62 (95% CI 1.98-1.99) in 2012. The number of antibiotics per prescription per year remained fairly constant at 1.05 ± 0.19 (95% CI 1.05-1.05) in 2005 to 1.06 ± 0.21 (95% CI 1.06-1.06) in 2012. The prevalence of patients receiving antibiotic prescriptions decreased from 46.1% (n = 789 247) in 2005 to 38.2% (n = 480 159) in 2012. Antibiotics were mostly prescribed for females (54.9%, n = 2 831 686) and in patients aged 0 to 18 years (26.5%, n = 1 366 824) and least in patients above 65 years (9.5%, n = 490 496). The prevalence of patients receiving antibiotic prescriptions was highest in Gauteng (41.9%, n = 2 159 360) and lowest in the Northern Cape (1.7%, n = 87 720). Antibiotics were mostly prescribed during the winter period. Penicillins were the most prescribed antibiotics (43%) and carbapenem the least (0.1%) out of the total number of antibiotics claimed. No practically significant association was found between antibiotic prescribing and gender, age, province and season. A total of 1 983 622 prescriptions for fluoroquinolones were claimed in patients older than 18 years. The average number of fluoroquinolone prescriptions per patient per year ranged from 1.45 ± 0.92 (95% CI 1.44-1.45) in 2005 to 1.31 ± 0.71 (95% CI 1.31-1.32) in 2012. The highest prevalence of fluoroquinolone prescribing was observed in females (64.1%, n = 850 253) and in patients between 45 and 65 years (38.6%, n = 511 542). The total fluoroquinolone use by the study population decreased from 2.85 DID in 2005 to 2.41 DID in 2012. Norfloxacin was the only first-generation fluoroquinolone prescribed. The second-generation fluoroquinolones accounted for more than 50% of the total DID, with ciprofloxacin being the most used active ingredient in this generation. Moxifloxacin was the most prescribed third-generation fluoroquinolone; its use ranging from 0.51 DID in 2005 to 0.44 DID in 2012. Between 2005 and 2012, a total of 57 325 prescriptions for fluoroquinolones were claimed by patients 18 years and younger. The prevalence of patients receiving fluoroquinolone prescriptions decreased from 3.6% (n = 8 329) in 2005 to 2.9% (n = 3 310) in 2012. Fluoroquinolones were mostly prescribed to females and in patients between 12 and 18 years. In all age groups, prescribing was mainly done by general medical practitioners. Ciprofloxacin was the most prescribed fluoroquinolone, followed by levofloxacin. In conclusion, this study established estimates on the prevalence of antibiotic prescribing covering an eight-year period. Secondly, baseline estimates for fluoroquinolone prescribing in adults using the ATC/DDD methodology were determined. Fluoroquinolone prescribing patterns in children and adolescents were determined, with specific reference to the comparison between the prescribed daily and recommended daily dosages in the different age groups and by prescribers’ specialties. / MPharm (Pharmacy Practice), North-West University, Potchefstroom Campus, 2015

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

No description available.