SYSTEMIC AND LOCAL ANTIMICROBIAL ACTIVITY AGAINST PLANKTONIC AND BIOFILM STATE OF MICROORGANISMS IN VITRO AND IN VIVO

Implant-associated infections caused by Candida spp. are difficult to treat due to reduced antimicrobial susceptibility in biofilm. Antifungal susceptibility testing is important due to the increasing number of prosthetic infections caused by Candida spp. The aim of this thesis was to investigate innovative methods using calorimetry for microbial detection and antimicrobial susceptibility testing, as well as treatment activity in validated C. albicans foreign-body infection animal model (part 1). In addition, new materials with intrinsic antimicrobial activity (bioactive glass) were tested (part 2). The first part of the thesis describes a novel real-time method for evaluation of antifungals against yeast, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against yeast have several limitations, especially when combinations of antifungals are investigated. We therefore evaluated the activity of fluconazole, amphotericin B and two echinocandins (caspofungin and anidulafungin) against Candida spp. by microcalorimetry. The minimal heat inhibition concentration (MHIC) was defined as the lowest concentration inhibiting ≥50% (≥90% for amphotericin B) of the heat produced at 24 and 48h for planktonic and biofilm yeast, respectively. Agreement within two-fold dilutions between MHIC and MIC was 50% for fluconazole and 100% for caspofungin, anidulafungin and amphotericin B. As determined by microcalorimetry, echinocandins (especially anidulafungin) were the most active agents against planktonic Candida. Subsequently, antimicrobial treatment strategies for infections caused by Candida albicans in-vivo were investigated. Since C. albicans had not previously been tested in this animal model, we first established an infection profile and investigated the pharmacokinetics of antifungals. In untreated animals, planktonic Candida progressively decreased in cage fluid and was cleared in 8% to 24% of cage fluids, however, Candida biofilm persisted on all cages, i.e. no spontaneous cure of cage- associated infections was observed. In accordance with in vitro experiments, echinocandins showed the highest activity against planktonic C. albicans. Against C. albicans biofilm, caspofungin showed the highest cure rate (25%), whereas cure rates of other antifungals ranged between 8% - 17%, demonstrating the difficulty of eradicating Candida biofilms on implants. The second part of the thesis investigates the activity of bioactive glass (BAG) S53P4 (volumes 1 g and 2 g and sizes 0.5-0.8 mm and <45 µm) against Staphylococcus aureus, S. epidermidis, Escherichia coli, Enterococcus faecalis et Candida albicans by microcalorimetry. BAG is a surface-reactive glass-ceramic biomaterial which is used as implant material to repair and replace diseased or damaged bones. Besides binding chemically to the bone and being osteoconductive, this material has the property to inhibit the biofilm formation. BAG showed good activity against tested mircoorganisms, except for E. faecalis with granula 0.5-0.8 mm. -- Les infections associees aux implants causees par Candida spp. sont difficiles a traiter a cause de la faible susceptibilite du biofilm aux antifongiques. Le test de susceptibilite aux antifongiques est important vu l’augmentation des infections de protheses dues a Candida spp. Le but de cette these est d’etudier des nouvelles methodes de detection des microorganismes par calorimetrie et de tester la susceptibilite ainsi que l’activite des antifongiques dans un modele animal (partie 1). De plus, des nouveaux materiaux avec une activite antimicrobienne intrinseque (bioactive glass) ont ete testes (partie 2). La premiere partie de la these decrit une nouvelle methode d’evaluation de l’effet des antifongiques en temps reel en se basant sur la chaleur produite lors de la croissance mesuree par microcalorimetre isothermique. Les methodes actuelles visant a evaluer l’effet des antifongiques sont limitees, surtout lorsqu’il s’agit d’evaluer l’effet des combinaisons d’antifongiques. Nous avons evalue l’activite du fluconazole, de l’amphotericin B et des echinocandines (caspofungine et anidulafungine), sur differentes souches de Candida spp. La concentration minimale d’inhibition de chaleur (CMIC) a ete definie comme etant la plus petite concentration inhibant ≥50% (≥90% pour l’amphotericine B) de la chaleur produite a 24h et 48h pour la croissance planctonique et du biofilm. La concordance entre CMIC et la concentration minimale d’inhibition (CMI), avec 2 dilutions de marge, etait de 50% pour fluconazole et 100% pour les echinocandines et l’amphotericin B. Comme determine par microcalorimetrie, les echinocandines (surtout l’anidulafungine) ont montre une meilleure activite contre la croissance planctonique de Candida. Nous avons ensuite etudie les traitements antimicrobiens contre C. albicans in vivo. Etant donne l’absence d’etudes avec ce modele animal avec C. albicans, nous avons d’abord etabli un profil d’infection et etudie la pharmacocinetique des antifongiques. Chez les animaux non traites, Candida planctonique a montre une decroissance progressive dans le fluide des cages, tout en restant present sous forme de biofilm. Pas de cure spontanee des cages infectees a ete observee. En accord avec les experiences in vitro, les echinocandines ont montre une meilleure activite contre C. albicans planctonique. Contre le biofilm la caspofongine montre le plus haut taux de guerison (25%), contrairement aux autres antifongiques ou le taux de guerison allait de 8% a 17%, demontrant ainsi la difficulte rencontree dans l’eradication du biofilm a C. albicans. La deuxieme partie etudie l’activite du bioactive glass (BAG) S53P4 (volumes 1 and 2 g et diametres de 0.5-0.8 mm et <45 µm) contre Staphylococcus aureus, S. epidermidis, Escherichia coli, Enterococcus faecalis et C. albicans. BAG est un biomateriel en vitre-ceramique a surface reactive utilise en tant qu’implant pour reparer et remplacer les os endommages ou fractures. En plus d’avoir la capacite de se lier chimiquement a l’os et etre osteoconductif, ce materiel a la caracteristique d’inhiber la formation du biofilm. BAG a montre une bonne activite contre les microorganismes testes a l’exception de E. faecalis avec les granules de 0.5-0.8 mm de diametre.