A platform for anti-biofilm assays combining viability, biomass and matrix quantifications in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Bacteria can exist as planktonic, the lifestyle in which single cells exist in suspension, and as biofilms, which are surface-attached bacterial communities embedded in a selfproduced matrix. Most of the antibiotics and the methods for antimicrobial work have been developed for planktonic bacteria. However, the majority of the bacteria in natural habitats live as biofilms. Biofilms develop dauntingly fast high resistance towards conventional antibacterial treatments and thus, there is a great need to meet the demands of effective anti-biofilm therapy. In this thesis project it was attempted to fill the void of anti-biofilm screening methods by developing a platform of assays that evaluate the effect that screened compounds have on the total biomass, viability and the extracellular polysaccharide (EPS) layer of the biofilms. Additionally, a new method for studying biofilms and their interactions with compounds in a continuous flow system was developed using capillary electrochromatography (CEC). The screening platform was utilized with a screening campaign using a small library of cinchona alkaloids. The assays were optimized to be statistically robust enough for screening. The first assay, based on crystal violet staining, measures total biofilm biomass, and it was automated using a liquid handling workstation to decrease the manual workload and signal variation. The second assay, based on resazurin staining, measures viability of the biofilm, and it was thoroughly optimized for the strain used, but was then a very simple and fast method to be used for primary screening. The fluorescent resazurin probe is not toxic to the biofilms. In fact, it was also shown in this project that staining the biofilms with resazurin prior to staining with crystal violet had no effect on the latter and they can be used in sequence on the same screening plate. This sequential addition step was indeed a major improvement on the use of reagents and consumables and also shortened the work time. As a third assay in the platform a wheat germ agglutinin based assay was added to evaluate the effect a compound has on the EPS layer. Using this assay it was found that even if compounds might have clear effect on both biomass and viability, the EPS layer can be left untouched or even be increased. This is a clear implication of the importance of using several assays to be able to find “true hits” in a screening setting. In the pilot study of screening for antimicrobial and anti-biofilm effects using a cinchona alkaloid library, one compound was found to have antimicrobial effect against planktonic bacteria and prevent biofilm formation at low micromolar concentration. To eradicate biofilms, a higher concentration was needed. It was also shown that the chemical space occupied by the active compound was slightly different than the rest of the cinchona alkaloids as well as the rest of the compounds used for validatory screening during the optimization processes of the separate assays.

Effects of feed processing on the apparent ileal digestibility of amino acids in pig diets containing wheat bran or wheat middlings

Selostus: Prosessoinnin vaikutus vehnän sivutuotteita sisältävien rehuseosten aminohappojen ohutsuolisulavuuteen sioilla

Apparent ileal digestibility of amino acids in wet wheat protein and soya bean meal for growing pigs

Selostus: Kuivaamattoman vehnäproteiinin ja soijarouheen aminohappojen ohutsuolisulavuus sioilla

Universally primed polymerase chain reaction analysis of Fusarium avenaceum isolated from wheat and barley in Finland

Selostus: Vehnästä ja ohrasta eristettyjen F. avenaceum -punahomekantojen analysointi UP-PCR-menetelmällä

First report of Diuraphis (Holcaphis) frequens as a pest of wheat in Finland

Selostus: Diuraphis frequens -kirva vehnässä

Effects of expanding on the nutritive value of wheat bran in pig diets

Selostus: Ekspanderkäsittelyn vaikutus vehnänleseen rehuarvoon lihasian ruokinnassa

Photosynthesis and Rubisco kinetics in spring wheat and meadow fescue under conditions of simulated climate change with elevated CO[sub 2] and increased temperatures

Selostus: Kevätvehnän ja nurminadan fotosynteesi ja Rubisco-kinetiikka simuloidun ilmastonmuutoksen eli kohotetun hiilidioksidipitoisuuden ja kohotetun lämpötilan oloissa

Preparation, properties and applications of wheat gluten edible films

Selostus: Syötävien gluteenikalvojen valmistus, ominaisuudt ja eräät käyttösovellukset

Simulation of spring wheat responses to elevated CO‚́‚ and temperature by using CERES-wheat crop model

Selostus: Kohotettujen CO‚́‚:n ja lämpötilan vaikutukset kevätvehnän fenologiseen kehitykseen ja sadontuottomahdollisuuksiin

Germ bilolja

kuv., 11 x 14 cm

Germ autoöljyjä

kuv., 11 x 14 cm