Purpose. The use of antiplatelet agents in patients hospitalized with ischemic stroke was studied. Methods. Patients with a primary or secondary diagnosis of noncardiogenic, thrombotic ischemic stroke from January 2002 through December 2004 were included in the analysis. Patients were then subdivided into four treatment groups and one no-treatment group based on whether they were charged for any of four antiplatelet regimens (low-dose aspirin [≤325 mg daily], extended-release dipyridamole 200 mg with aspirin 25 mg, clopidogrel 75 mg, and clopidogrel 75 mg [as the bisulfate] plus low-dose aspirin) at any time during hospitalization. Patients who did not receive any of these medications during hospitalization were defined as the no-treatment group. A patient's illness severity was measured and compared with other patients in the data set. Results. A total of 44,108 patients were assigned to the treatment group, and 14,255 patients were assigned to the no-treatment group. In general, longer lengths of stay and higher institutional costs were associated with the no-treatment group. Patients in the no-treatment group consistently displayed more comorbid conditions than did patients in the treatment group. The no-treatment group exhibited higher usage rates of both fibrinolytic agents and vitamin K. More patients in the treatment group were discharged to home or rehabilitation, while more patients in the no-treatment group were either discharged to another nursing facility or died before discharge. Conclusion. A retrospective analysis of a large national hospital database revealed that one quarter of patients who suffered an acute stroke did not receive antiplatelet drugs during their patient stay. Outcomes for such patients were poorer than for patients who had received antiplatelet therapy.

Background: Dual antiplatelet therapy (DAPT) is a mainstay treatment for hospital survivors of an acute myocardial infarction (AMI). However, there are extremely limited data on the prescribing patterns of DAPT among patients hospitalized with AMI. Objective: To examine decade-long trends (2001-2011) in the use of DAPT versus antiplatelet monotherapy and patient characteristics associated with DAPT use. Methods: The study population consisted of 2,389 adults hospitalized with an initial AMI at all 11 central Massachusetts medical centers on a biennial basis between 2001 and 2011. DAPT was defined as the discharge use of aspirin plus either clopidogrel or prasugrel. Logistic regression analysis was used to identify patient characteristics associated with DAPT use. Results: The average age of the study population was 65 years, and 69% of them were discharged on DAPT. The use of DAPT at the time of hospital discharge increased from 49% in 2001 to 74% in 2011; this increasing trend was seen across all age groups, both sexes, types of AMI, and in those who underwent a PCI. After multivariable adjustment, older age was the only factor associated with lower odds of receiving DAPT, while being male, receiving additional evidence-based cardioprotective therapy and undergoing cardiac stenting were associated with higher odds of receiving DAPT. Conclusions: Between 2001 and 2011, the use of DAPT increased markedly among patients hospitalized with AMI. However, a significant proportion of patients were not discharged on this therapy. Greater awareness is needed to incorporate DAPT into the management of patients with AMI.

Le CD40 ligand (CD40L) est une molécule thrombo-inflammatoire qui prédit des événements cardiovasculaires. Les plaquettes constituent la principale source du CD40L soluble (sCD40L) dans la circulation, avec la capacité d’influencer la fonction des plaquettes par l'intermédiaire de sa liaison à ses récepteurs plaquettaires : CD40, αIIbβ3 et α5β1. Nous avons précédemment démontré que la stimulation des plaquettes humaines avec le sCD40L induit une activation du nuclear factor kappa B (NF-κB), qui pourra jouer un rôle non génomique en amorçant les plaquettes. En effet, l’amorçage des plaquettes par le sCD40L augmente fortement l’activation et l’agrégation plaquettaire en réponse à des doses sous-optimales d’agonistes. Cependant, l’implication des différents récepteurs dans l’amorçage des plaquettes suite à l’activation du NF-κB par le sCD40L demeure inconnue. De plus, le transforming growth factor-B (TGF-B)-activated Kinase (TAK1), un régulateur majeur de l’inflammation, est activé par le sCD40L dans les cellules nucléées, et pourra ainsi être impliqué dans la signalisation sCD40L/NF-κB plaquettaire. D’autre part, le sCD40L se trouve en quantité élevée dans la circulation sanguine des patients coronariens. De plus, le traitement antiplaquettaire à l’Aspirine (ASA) est inefficace chez certains patients et son efficacité est réduite chez les patients à hauts risques d’évènements coronariens, ce qui augmente leur risque de sur-activation plaquettaire et de développer une thrombose. Des taux sanguins élevés de médiateurs thrombo-inflammatoires, tels que le sCD40L, peuvent expliquer de telles variabilités. Ainsi, ce projet a été entrepris dans le but d’élucider l’impact de l’axe sCD40L/NF-κB sur la fonction plaquettaire et sa modulation par l’ASA. Dans le premier volet de mon projet, nous avons identifié les récepteurs de sCD40L impliqués dans l'activation du NF-κB plaquettaire, leur signalisation en aval et leur implication dans l'agrégation plaquettaire. Nous avons montré que les plaquettes exprimaient les récepteurs du CD40L : CD40, αIIbβ3 et α5β1 et libéraient du sCD40L. Le sCD40L seul a induit une activation de NF-κB plaquettaire. Cet effet était absent des plaquettes de souris déficientes en CD40 (CD40-/-) et inhibé par le blocage de CD40, mais n'était pas affecté par le blocage de αIIbβ3 ou α5β1. L’axe sCD40L/CD40 a aussi activé TAK1 en amont de NF-κB. Dans les études fonctionnelles, le sCD40L seul n’a pas eu d’effet sur l’agrégation plaquettaire, mais a potentialisé l’agrégation en présence de doses sous-optimales de thrombine; cet effet a été aboli par des inhibiteurs de CD40, TAK1 et NF-κB. Cette première étude nous a permis de conclure que le sCD40L amorce les plaquettes via des voies de signalisation impliquant CD40/TAK1/NF-κB, ce qui prédispose les plaquettes à une activation et agrégation accrues en réponse à des stimuli thrombotiques. Dans le deuxième volet de mon projet, nous avons émis l’hypothèse qu’en présence de taux élevés de sCD40L, l’efficacité de l’ASA peut varier et avons visé à déterminer les effets de l’ASA sur la signalisation et l’agrégation des plaquettes en présence de sCD40L. Les effets de l'ASA sur les plaquettes humaines traitées au sCD40L, en réponse à des concentrations sous-optimales de collagène ou de thrombine, ont été évalués sur l'agrégation, la sécrétion de thromboxane A2 (TxA2) et la phosphorylation de p38 MAPK, NF-κB, TAK1 et la chaîne légère de la myosine (MLC). Le sCD40L a significativement augmenté la sécrétion de TxA2 par les plaquettes, en réponse à des doses sous-optimales de collagène et de thrombine; cet effet a été inversé par l'ASA. L'ASA n'a pas inhibé la phosphorylation de p38 MAPK, NF-κB, TAK1, que ce soit avec une stimulation plaquettaire par le sCD40L seul ou en présence des agonistes. Le sCD40L a potentialisé l'agrégation plaquettaire, un effet complètement inversé et partiellement réduit par l'ASA en réponse à une dose sous-optimale de collagène et de thrombine, respectivement. Les effets de l'ASA sur les plaquettes traitées au sCD40L avec du collagène étaient liés à l'inhibition du changement de forme des plaquettes et à la phosphorylation de la MLC. En résumé, l'ASA n'affecte pas la signalisation plaquettaire du sCD40L, mais empêche son effet sur la sécrétion de TxA2 et l'agrégation plaquettaire en réponse au collagène, via un mécanisme impliquant l'inhibition de la MLC. En conclusion, ce projet nous a permis de déterminer que l’amorçage des plaquettes par le CD40L via l’activation de NF-κB dépend du récepteur CD40 et la signalisation via TAK1. Ainsi, l’axe sCD40L/CD40/TAK1/NF-κB potentialise l’activation et l’agrégation en réponse à des stimuli thrombotiques, ce qui peut favoriser l’occurrence d’événements athéro-thrombotiques chez les patients coronariens. De plus, étant donné que l’ASA n’a pas d’effet sur la signalisation via l’axe sCD40L, le ciblage de cet axe dans les plaquettes peut avoir un potentiel thérapeutique chez les patients coronariens présentant des taux élevés de sCD40L et ne réagissant pas ou moins à l'ASA. / CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets are the main source of soluble CD40L (sCD40L) in the circulation, with the ability to influence platelet function through its binding to platelet receptors: CD40, αIIbβ3, and α5β1. We have previously demonstrated that stimulation of human platelets with sCD40L induces activation of nuclear factor kappa B (NF-κB), which may play a non-genomic role in priming platelets. Indeed, platelet priming by sCD40L strongly enhances platelet activation and aggregation in response to suboptimal doses of agonists. However, the involvement of the different receptors in platelet priming following activation of NF-κB by sCD40L remains unknown. In addition, the transforming growth factor-B (TGF-B) -activated Kinase (TAK1), a major regulator of inflammation, is activated by sCD40L in nucleated cells, and may be involved in sCD40L/NF-κB signaling. On the other hand, sCD40L is high in the bloodstream of coronary patients, who receive Aspirin (ASA) for secondary prevention. However, antiplatelet therapy with ASA is ineffective in some patients and its efficacy is reduced in patients at high risk for coronary events, increasing their risk of platelet over-activation and developing thrombosis. High blood levels of thrombo-inflammatory mediators, such as sCD40L, may explain such variability. Thus, we undertake this project to elucidate the impact of the sCD40L/NF-κB axis on platelet function and its modulation by ASA. In the first part of our project, we aimed to identify the sCD40L receptors involved in the activation of platelet NF-κB, their downstream signaling and their involvement in platelet aggregation. We have shown that platelets express CD40L receptors: CD40, αIIbβ3 and α5β1 and release sCD40L. sCD40L alone induced activation of platelet NF-κB. This effect was absent in CD40-/- mouse platelets and inhibited by CD40 blockade, but was not affected by αIIbβ3 or α5β1 blockade. The sCD40L/CD40 axis has also activated TAK1 upstream of NF-kB. In functional studies, sCD40L alone had no effect on platelet aggregation but potentiated aggregation in the presence of suboptimal thrombin doses. The inhibitors of CD40, TAK1 and NF-κB abolished this effect. This first study concluded that sCD40L primes platelets via CD40/TAK1/NF-κB signaling pathways, which predisposes platelets to increased activation and aggregation in response to thrombotic stimuli. In the second part of our project, we hypothesized that in the presence of high levels of sCD40L; the effectiveness of ASA may vary. Accordingly, we aimed at determining the effects of ASA on signaling and aggregation of platelets in the presence of sCD40L. The effects of ASA on human platelets treated with sCD40L, in response to suboptimal concentrations of collagen or thrombin, were evaluated on aggregation, thromboxane A2 (TxA2) secretion and phosphorylation p38 MAPK, NF-κB, TAK1 and myosin light chain (MLC). sCD40L significantly increased TxA2 secretion by platelets in response to suboptimal doses of collagen and thrombin; this effect has been reversed by the ASA. ASA did not inhibit the phosphorylation of p38 MAPK, NF-κB, TAK-1, with platelet stimulation by sCD40L either alone or in the presence of agonists. sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to a suboptimal dose of collagen and thrombin, respectively. In addition, the effects of ASA on sCD40L-treated platelets with collagen related to inhibition of platelet shape change and phosphorylation of MLC. In summary, ASA does not affect platelet signaling via sCD40L, but prevents its effect on TXA2 secretion and platelet aggregation in response to collagen, via a mechanism involving inhibition of MLC. In conclusion, this project allowed us to determine that platelet priming by sCD40L via activation of NF-κB depends on the CD40 receptor and signaling via TAK1. Thus, the sCD40L/CD40/TAK1/NF-κB axis potentiates activation and aggregation in response to thrombotic stimuli, which may promote occurrence of atherothrombotic events in coronary patients. In addition, since ASA has no effect on platelet signaling via the sCD40L axis, targeting of this axis in platelets may have therapeutic potential in coronary patients with high levels of sCD40L that are none or less responding to ASA.

A molecule can crystallise in more than one crystal structure, a common phenomenon in organic compounds known as polymorphism. Different polymorphic forms may have significantly different physical properties, and a reliable prediction would be beneficial to the pharmaceutical industry. However, crystal structure prediction (CSP) based on the knowledge of the chemical structure had long been considered impossible. Previous failures of some CSP attempts led to speculation that the thermodynamic calculations in CSP methodologies failed to predict the kinetically favoured structures. Similarly, regarding the stabilities of co-crystals relative to their pure components, the results from lattice energy calculations and full CSP studies were inconclusive. In this thesis, these problems are addressed using the state-of-the-art CSP methodology implemented in the GRACE software. Firstly, it is shown that the low-energy predicted structures of four organic molecules, which have previously been considered difficult for CSP, correspond to their experimental structures. The possible outcomes of crystallisation can be reliably predicted by sufficiently accurate thermodynamic calculations. Then, the polymorphism of 5- chloroaspirin is investigated theoretically. The order of polymorph stability is predicted correctly and the isostructural relationships between a number of predicted structures and the experimental structures of other aspirin derivatives are established. Regarding the stabilities of co-crystals, 99 out of 102 co-crystals and salts of nicotinamide, isonicotinamide and picolinamide reported in the Cambridge Structural Database (CSD) are found to be more stable than their corresponding co-formers. Finally, full CSP studies of two co-crystal systems are conducted to explain why the co-crystals are not easily obtained experimentally. / University of Bradford

The genotoxicological effects of five intra-peritoneal administered oestrogens (17β- oestradiol, daidzein, diethylstilboestrol, genistein, and equol), were examined. Male hooded- Lister rats were used to examine to what extent DNA damage occurred. The alkaline Comet assay was the chosen method used to assess double-strand DNA breakage by examining the Olive tail moment and %age tail DNA. Tissues from the testis, bone marrow, liver and blood were analysed after an 8-day duration of exposure. Statistically significant increases in DNA damage were observed in the testis with daidzein and in the blood with diethylstilboestrol. In addition, a further study was carried out to examine the effects of bulk and nanotised forms of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and ibuprofen, in the Comet and micronucleus assays, on whole blood taken from prostate cancer patients or volunteers. These were used because it is known that the sensitivity of DNA to genotoxins can be heightened in patients with cancer. Patients’ and volunteers’ blood was cultured with either the bulk or nano-forms for 44 hours at 37°C, 5% CO2. Data were obtained for the Comet assay as above and the number of binucleated cells scored for the micronucelus assay. The results show the nanotised forms of the NSAIDs decreased the levels of strand breakage and lowered the numbers of micronuclei generated compared with their bulk forms. There was no clear difference between the sensitivity of the healthy controls and the prostate cancer patients, with only one individual showing evidence of heightened sensitivity.

This project focuses on two important topics which may pose hazards to human health. Firstly, drinking water disinfection by-products (DBPs), which are generated by the chemical disinfection of water have been investigated. What has not been shown is the effect of DBPs in human germ cells as well as somatic cells and whether oxidative stress is involved in the mechanism of genotoxic action. Three different DBPs (halo acetic acids: HAAs), together with the antioxidants – catalase and butylated hydroxyanisole (BHA), were investigated in peripheral blood cells and sperm from healthy individuals using the Comet assay and lymphocytes only using the micronucleus assay. Secondly, nanoparticles of the non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and ibuprofen, have been investigated in patients with respiratory diseases, in the micronucleus assay and the Comet repair assay. NSAIDs inhibit cyclooxygenase enzyme activity, which plays part in tumour progression. In the Comet assay, BHA and catalase were able to reduce DNA damage in both cell types compared to HAAs alone. Similarly, in the micronucleus assay, micronuclei were reduced with the antioxidants, suggesting oxygen radical involvement in both assays. With the NSAIDs, reductions were seen for DNA damage in the micronucleus assay with aspirin and ibuprofen nanoparticles compared to their bulk forms. Using the Comet repair assay, aspirin and ibuprofen nanoparticles aided repair of DNA to a greater extent than their bulk counterparts, which in turn showed better repair compared to samples repaired without NSAIDs. These observations show the importance of DBPs and NSAIDs in genotoxic public health issues. / United Kingdom India Education and Research Initiative (UKIERI).

Chronic inflammation is intimately linked with cancer development and progression and therefore reducing or eliminating inflammation represents a logical treatment and prevention strategy. Studies have shown that anti-inflammatory agents have anti-tumour effects in cancers, with reduced metastases and mortality. Current use of anti-inflammatory agents in the treatment and prevention of cancer is limited by their toxicity and side effects. The emerging field of nanotechnology allows the fundamental properties of a drug to be altered, creating a product with improved reactivity and bioavailability, leading to more targeted treatments and reduced dosage. In the present study, the genotoxic effects of three commonly used anti-inflammatory drugs; aspirin, ibuprofen and hydrocortisone, in their bulk and nano forms were evaluated on peripheral blood lymphocytes of healthy donors using the comet assay and the micronucleus assay. In order to determine any anti-cancer effects, these agents were also tested in peripheral blood lymphocytes in patients with haematological cancers. The glucocorticoid hydrocortisone was also evaluated for anti-oxidant capacity. Our results demonstrate that the nano versions of each drug produced a different response than the bulk counterpart, indicating that a reduction in particle size had an impact on the reactivity of the drug. Our results also indicate that the nano versions of each drug were less genotoxic than the bulk formulation, further emphasising the potential of nanoparticles as an improvement to current treatment options. We also found an anti-oxidant effect with hydrocortisone, with a more profound effect seen with the nano formulation.

Background: Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) via Ser108 of the AMPK β1 subunit, a mechanism that is distinct from metformin, which increases AMP:ATP. Many cancers have high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is unknown if targeting the AMPK-ACC-lipogenic pathway using salicylate and metformin may be effective for inhibiting cancer cell survival. Results: Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations of aspirin. These clinically achievable concentrations of salicylate activated AMPK per the increasing phosphorylation of ACC and suppressing the activity of mTOR effectors kinase p70-S6 kinase and S6; effects that were enhanced with the addition of metformin and blunted in mouse embryonic fibroblasts (MEFS) deficient in AMPK β1. MEF cells deficient in AMPK β1 were more resistant to salicylates inhibitory effect on proliferation. Supplementation of media with fatty acids and mevalonate reverses the suppressive effects on cell survival indicating the inhibition of de novo lipogenesis is likely important. Conclusions: Salicylate increases ACC phosphorylation, reduces phosphorylation of mTOR targets and inhibits de novo lipogenesis in prostate and lung cancer cells, with concentrations of salicylate achievable through the ingestion of Aspirin (0.25-1.0mM) these effects are blunted in AMPK β1 deficient cells. Effects on AMPK activity via ACC phosphorylation as well as reductions in mTOR signalling targets and de novo lipogenesis are enhanced when used in combination with metformin. Suppressive effects on prostate and lung cancer cell survival are ameliorated when media is supplemented with mevalonate and fatty acids. Pre-clinical studies evaluating the use of salicylates alone and with metformin to inhibit de novo lipogenesis and the growth of prostate and lung cancers are warranted. / Thesis / Master of Science (MSc)

Inhibition of cyclooxygenase (COX)-2 increases cardiovascular deaths. Identifying a biomarker of COX-2 is desirable but difficult, since COX-1 and COX-2 ordinarily catalyze formation of an identical product, prostaglandin H2. When acetylated by aspirin, however, COX-2 (but not COX-1) can form 15(R)-HETE, which is metabolized to aspirin-triggered lipoxin (ATL), 15-epi-lipoxin A4. Here we have used COX-1- and COX-2-knockout mice to establish whether plasma ATL could be used as a biomarker of vascular COX-2 in vivo. Vascular COX-2 was low but increased by LPS (10 mg/kg; i.p). Aspirin (10 mg/kg; i.v.) inhibited COX-1, measured as blood thromboxane and COX-2, measured as lung PGE2. Aspirin also increased the levels of ATL in the lungs of LPS-treated wild-type C57Bl6 mice (vehicle: 25.5±9.3 ng/ml; 100 mg/kg: 112.0±7.4 ng/ml; P<0.05). Despite this, ATL was unchanged in plasma after LPS and aspirin. This was true in wild-type as well as COX-1−/− and COX-2−/− mice. Thus, in mice in which COX-2 has been induced by LPS treatment, aspirin triggers detectable 15-epi-lipoxin A4 in lung tissue, but not in plasma. This important study is the first to demonstrate that while ATL can be measured in tissue, plasma ATL is not a biomarker of vascular COX-2 expression.—Kirkby, N. S., Chan, M. V., Lundberg, M. H., Massey, K. A., Edmands, W. M. B., MacKenzie, L. S., Holmes, E., Nicolaou, A., Warner, T. D., Mitchell, J. A. Aspirin-triggered 15-epi-lipoxin A4 predicts cyclooxygenase-2 in the lungs of LPS-treated mice but not in the circulation: implications for a clinical test.

Le but de ce travail de mémoire était d'explorer des moyens pour augmenter la perméabilité des biofilms de Streptococcus mutans aux macromolécules en utilisant des agents potentiellement perturbateurs de la structure des biofilms. L’acide éthylènediamine tétraacétique (EDTA) ainsi que l’acide acétylsalicylique (aspirine) sont les agents perturbateurs choisis. Le changement de perméabilité des biofilms de S. mutans a été déterminé en mesurant les coefficients de diffusion globale du polyéthylène glycol (PEG) et de diffusion locale de dextrans. Les coefficients de diffusion globale ont été mesurés par spectroscopie infrarouge avec un échantillonnage par réflexion totale atténuée (ATR) alors que la spectroscopie par corrélation de fluorescence (SCF) a été utilisée pour la mesure des coefficients de diffusion locale. Les résultats ont démontré que l’incorporation de l’EDTA à une concentration de 7.5 (m/v) % dans la solution de diffusion permet d’améliorer les propriétés de transport du PEG dans les biofilms en augmentant sa pénétrabilité et son coefficient de diffusion globale. Par contre, aucune variation n’a été constatée dans la valeur du coefficient de diffusion locale de dextran fluorescent. Cette différence peut être expliquée, entre autres, par l'échelle des mesures et la nature différente des molécules diffusantes. L’aspirine n’a démontré aucun effet sur le transport du PEG à travers les biofilms de S. mutans. La pénétration accrue du PEG en présence de l’EDTA a été corrélée aux tests de viabilité des cellules bactériennes. En effet, la combinaison de la pénicilline G (PenG) avec l’EDTA 2 (m/v) % a eu comme effet l’augmentation du pouvoir biocide d’un facteur 3. De plus, les images de microscopie à épifluorescence et de microscopie confocale à balayage de laser ont démontré que les bactéries dans le cœur des microcolonies sont plus affectées par la PenG lorsque le milieu contient de l'EDTA. A la lumière des résultats obtenus, il s’avère que l’incorporation d'agents perturbateurs de la structure des biofilms est une option sérieuse à considérer dans l’éradication des biofilms microbiens. Plus d’études devront être effectuées afin d’investiguer l’effet d’autres molécules possédant les propriétés perturbatrices de la structure des biofilms sur la résistance de ces derniers aux agents antimicrobiens. / The aim of this study was to investigate the role of biofilm structure disrupting potential agents in the biofilm permeability to macromolecules. The effect of ethylenediamine tetraacetic (EDTA) and acetylsalicylic acid (aspirin), two potential disrupting agents, on the diffusion of poly (ethylene glycol) (PEG) and dextran in Streptococcus mutans biofilms was investigated. Attenuated total reflection (ATR) infrared spectroscopy was used to measure the global diffusion coefficients, while the local diffusion coefficients were measured using fluorescence correlation spectroscopy (FCS). On one hand, the results showed that EDTA, at a concentration of 7.5 (w/v) %, increased PEG penetrability and global diffusion coefficient in biofilms. No effect was noticed for the local diffusion coefficient of fluorescent dextran molecules. This difference was associated with, amongst others, the scale of the measurements and the different nature of the diffusing species. On the other hand, aspirin had no effect on the S. mutans biofilm permeability for PEG. The enhanced penetration of PEG in the presence of EDTA was correlated with bacterial cell viability. The bactericidal effect of penicillin G (PenG) alone and in combination with EDTA, was assessed for the biofilms. The results showed that antibiotic effect of PenG was improved by a factor of 3 when the antibiotic was used in combination with EDTA compared to the same antibiotic used alone. Moreover images obtained by epifluorescence microscopy and laser scanning confocal microscopy indicated that the bacteria in the core of the microcolonies were more affected by PenG when the milieu contained EDTA. These results revealed that the use of disrupting agents of biofilm structure is a promising approach to eradicate biofilms showing resistance to classical antimicrobial agent treatments. More studies should be done to investigate the effect of others potentials disrupting agents on the biofilm permeability to macromolecules.