Exposure to antibiotics is an important factor influencing the development of bacterial resistance.  In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on  in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with examples advocating regimens that (i) promote bacterial killing and reduce risk for toxicity in preterm and term newborn infants receiving gentamicin, (ii) achieve a fast initial bacterial killing and reduced resistance development of colistin in critically ill patients by application of a loading dose, and (iii) overcome existing meropenem resistance by combining colistin and meropenem

Current excessive use and abuse of antibiotics has resulted in increasing bacterial resistance to common treatment options which is threatening to deprive us of a pillar of modern medicine. In this work methods to optimize the use of existing antibiotics and to help development of new antibiotics were developed and applied. Semi-mechanistic pharmacokinetic-pharmacodynamic (PKPD) models were developed to describe the time course of the dynamic effect and interaction of combinations of antibiotics. The models were applied to illustrate that colistin combined with a high dose of meropenem may overcome meropenem-resistant P. aeruginosa infections. The results from an in vivo dose finding study of meropenem was successfully predicted by the meropenem PKPD model in combination with a murine PK model, which supports model based dosage selection. However, the traditional PK/PD index based dose selection was predicted to have poor extrapolation properties from pre-clinical to clinical settings, and across patient populations. The precision of the model parameters, and hence the model predictions, is dependent on the experimental design. A limited study design is dictated by cost and, for in vivo studies, ethical reasons. In this work optimal design (OD) was demonstrated to be able to reduce the experimental effort in time-kill curve experiments and was utilized to suggest the experimental design for identification and estimation of an interaction between antibiotics. OD methods to handle inter occasion variability (IOV) in optimization of individual PK parameter estimates were proposed. The strategy was applied in the design of a sparse sampling schedule that aim to estimate individual exposures of colistin in a multi-centre clinical study. Plasma concentration samples from the first 100 patients have been analysed and indicate that the performance of the design is close to the predicted. The methods described in this thesis holds promise to facilitate the development of new antibiotics and to improve the use of existing antibiotics.

A infecção de corrente sanguínea (ICS) causada por bactérias multirresistentes tem alta mortalidade em pacientes receptores de transplante de células-tronco hematopoiéticas (TCTH). A Pseudomonas aeruginosa é um dos agentes mais frequentes e de difícil tratamento nessa população de pacientes. Objetivos: Avaliar características clínicas, microbiológicas e moleculares de 30 isolados de P. aeruginosa resistente à carbapenêmicos (PARC) em ICS de pacientes submetidos a TCTH e a presença de sinergismo antimicrobiano in vitro. Métodos: Os dados clínicos foram obtidos retrospectivamente de prontuários médicos e registrados em banco de dados. Análises bivariadas e multivariadas foram realizadas para avaliar determinantes de desfechos clínicos e uma curva de sobrevida foi construída. Determinou-se a concentração inibitória mínima (CIM) dos antimicrobianos por meio de microdiluição, foram realizados ensaios de sinergismo por método de checkerboard e time-kill, avaliação da clonalidade por eletroforese em campo pulsado e detecção de genes codificadores de mecanismos de resistência e virulência por reação em cadeia de polimerase. O sequenciamento do genoma completo (WGS) dos principais clones foi realizado por Nextera XT, utilizando a tecnologia Illumina MiSeq. Resultados: A maioria dos pacientes era do gênero feminino, com mediana de idade de 48 anos. Neutropenia foi presente em 93% dos pacientes e colonização prévia por PARC em 32%. A mortalidade em 14 dias foi 68%; a maioria dos pacientes que morreram foram transplantados alogênicos (79% vs. 17% entre receptores de transplante autólogo; p=0,012). Pacientes tratados com duas ou três drogas não apresentaram diferença estatisticamente significante na mortalidade até 14 dias após a ICS. Foram avaliados 30 isolados bacterianos. Todos apresentaram alto nível de resistência ao meropenem (MERO): CIM90 > 512 ug/mL; dois terços eram resistentes à amicacina (AMK) (CIM 2-512 ug/mL) e todos mantinham sensibilidade à colistina (COL). Muitos isolados (17/30) alcançaram efeito sinérgico in vitro pelo método time-kill com a combinação MERO mais COL, mas não com AMK. Nenhum antagonismo foi observado. Houve menor mortalidade em pacientes cujo isolado apresentou sinergismo entre COL e MERO quando comparados a pacientes portadores de isolados sem sinergismo, sem significância estatística. O gene de carbapenamase mais identificado foi blaSPM e 6 isolados apresentaram blaSPM e blaKPC. Os isolados apresentaram genes relacionados com virulência, tais como toxA, exoS e lasB; pacientes com ICS causada por P. aeruginosa que abrigava o gene lasB apresentaram maior risco de evoluir para o óbito. O WGS mostrou que os clones abrigavam SPM-1, Tn4371, mutações em porinas, em partes das bombas de efluxo, nas proteínas ligadores de penicilina (PBP) e pertenciam a ST277. Conclusão: As ICS por PARC cursaram com alta mortalidade em pacientes submetidos à TCTH. Houve uma grande proporção de resultados positivos para sinergismo entre os antimicrobianos in vitro, mas não foi possível demonstrar benefício estatisticamente significante no uso da terapia combinada com três drogas. Os clones carreavam SPM-1, Tn4371 e pertenciam a ST277 / Bloodstream infection (BSI) has high mortality in hematopoietic stem cell transplant (HSCT) recipients and Pseudomonas aeruginosa is an important and challenging organism. Objectives: To evaluate clinical, microbiological and molecular features of carbapenem-resistant P. aeruginosa (CRPA) isolates from BSI identified among HSCT patients and address in vitro synergy of antibiotic combination. Methods: Patient medical records were retrospectively reviewed and registered in a database. We used bivariate and multivariate analyzes to investigate determinants of clinical outcomes, and demonstrated overall mortality using a survival curve. We determined minimal inhibitory concentrations (MIC) for antimicrobials and in vitro synergies using checkerboard and time-kill assays, pulsed-field electrophoresis (PFGE) for clonality assessment and polymerase chain reaction (PCR) to detect carbapenamases and virulence genes were performed for all isolates. Whole genome sequence (WGS) of main clones was performed by Nextera XT, using Illumina MiSeq technology. Results: Most patients were female, median age was 48 years old. Main baseline disease was acute leukemia and 68% received allogeneic HSCT. 93% of patients had neutropenia and 32% had prior CRPA gut colonization.14-day mortality was 68%; mortality was higher among allogeneic HSCT recipients compared to autologous HSCT recipients (79% vs. 17% p = 0,012). Patients treated with two or three drugs did not present a statistically significant difference in 14-day mortality after BSI. In total, 30 bacterial isolates were analyzed; all presented a high resistance level to meropenem (MERO): MIC90 > 512ug/mL; two thirds were also resistant to amikacin (AMK) (MIC 2-512 ug/mL) and all were susceptible to colistin (COL). Many (17/30) isolates achieved in vitro synergistic effect in time-kill assay with the association of MERO and COL, but synergistic effect was not observed with AMK, by time-kill. No antagonistic effect was observed. There was a tendency towards better survival in patients whose CRPA isolate had in vitro synergy between COL and MERO without statistical significance. The most frequent carbapenamase gene identified was blaSPM, and six co-harboured both blaKPC and blaSPM. Isolates presented genes related to virulence factors such as toxA, exoS and more patients with BSI caused by P. aeruginosa harbouring gene lasB evolved to death. WGS analysis showed that clones harboured SPM-1, Tn4371 and belonged to ST277. They also presented mutations in genes related with porins and efflux pumps, as well in penicillin binding proteins (PBPs). Conclusion: CRPA BSI as associated with high mortality in HSCT recipients. A large proportion of isolates had in vitro synergy; however, we could not demonstrate statistically significant benefit in the use of combination therapy. Clones carried SPM-1, Tn4371 and belonged to ST277

The treatment options for multidrug resistant bacteria are dwindling and it is an important issue of research in medicine to solve. One of the more problematic bacterial species is Klebsiella pneumoniae, it can cause infections with high morbidity that are difficult to treat. Common antibiotics for treatment of these infections are carbapenems but K. pneumoniae can produce enzymes called carbapenemases that can hydrolyze carbapenems and most other beta-lactam antibiotics. In this study carbapenemase genes were introduced chromosomally to a previously susceptible K. pneumoniae strain using λ-Red recombineering. Further constructs were made with non-functional porins to examine how they affect combination treatment with carbapenems. Antibiotic combination therapy was evaluated against constructed carbapenemase- (KPC-2, NDM-1 and OXA-48) producing K. pneumoniae strains. Screening was done using time-lapse microscopy (oCelloScope), and combinations with better effect than treatment with a single antibiotic were chosen for time-kill assays. The results shows that a triple combination of colistin, meropenem and the beta-lactamase inhibitor avibactam gives an improved effect, up to twice the effect compared to monotherapy and up to 1.8 times increased effect compared to double combination. The synergistic effect was greater when adding colistin to treat the strains with non-functional porins, indicating that colistin can increase the permeability for other antibiotics into the cell. This is an interesting finding that need to be researched further.

Cancer immunotherapy is an encompassing term referring to therapeutic strategies that aim to boost the immune system to fight cancer. These strategies include administering immune cells that have been altered to have greater anti-tumor activity or using biologics and small molecules that target immune components to also promote tumor clearance. Natural Killer (NK) cells are cells of the innate immune system that recognize and kill abnormal cells such as cancer cells and play an important role in the anti-tumor response. Because of their crucial role in tumor immunity, NK cells are prime targets for immunotherapies. Repurposing small molecule drugs is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we screened 1,200 approved drugs from the Prestwick Chemical Library to identify drugs that increase NK cell cytotoxicity. We used a high-throughput luciferase-release cytotoxicity assay to measure the killing of the myeloid leukemia cell line, K562 cells expressing nano luciferase (NL) by NK92 cells, a human NK cell line. From the drug candidates identified from the screening assay, the antibiotic colistin sulfate increased cytotoxicity of the NK92 cell line and unstimulated human NK cells towards K562-NL cells. This increase in NK cytotoxicity was short-lived as pre-treating NK92 cells with colistin for 1 hour or 24 hours did not increase cytotoxicity. Also, we show pre-treating K562-NL target cells with colistin does not sensitize them to NK-mediated killing. Further studies are needed to uncover the mechanism of action of colistin, thus contributing to knowledge of fundamental NK cell biology regarding NK cell cytotoxicity which will aid in identifying additional small molecule drugs that enhance NK cell activity.

Les infections dues aux bactéries gram-négatif multi résistantes en particulier la résistance aux carbapénèmes, représentent un problème majeur de santé publique. La hausse des taux de résistance à ces antibiotiques a conduit à la réutilisation de la colistine, comme alternative thérapeutique de dernier recours. Notre travail de thèse s'est concentré sur l'étude épidémiologique de la résistance aux antibiotiques d’isolats cliniques au Liban. Nos travaux se sont scindés en 4 chapitres, avec trois objectifs principaux; (i) l'étude des bactéries résistantes aux carbapénèmes, (ii) l'élucidation des mécanismes moléculaires de la résistance à la colistine (iii) l'émergence de bactéries à Gram-positif résistantes à la vancomycine. Initialement, une revue de la littérature sur l'épidémiologie et les facteurs de risque associés à l'infection bactérienne au cours de conflits armés et catastrophes naturelles en Asie et au Moyen Orient a été rédigée. Dans le deuxième chapitre nous avons cherché à voir l'effet du changement de traitement de la combinaison colistine-carbapénème à la colistine en monothérapie sur la résistance d’A. baumannii, en plus de la détection du blaVIM-2 codé par plasmide. Dans le troisième chapitre, nous avons détecté la propagation de bactéries gram-négatif résistantes à la colistine en raison de la mutation des (pmrA /pmrB, phoP/phoQ), ou mgrB. Enfin, nous détectons l'émergence du gène vanA d'E. faecium. Il serait nécessaire de mettre en place des enquêtes de surveillance de l’usage des antibiotiques pour éviter la propagation de souches résistantes à ces antibiotiques au Liban. / Infections due to multidrug-resistant gram-negative bacteria especially the resistance to carbapenems, have become a major public health problem. This increase in resistance to antibiotics has led to the resuscitation of colistin, as a last-resort treatment option. Our PhD work focused on the epidemiological study of the antibiotic resistance of clinical isolates in Lebanon. This thesis is divided into 5 chapters with three main objectives; (1) the investigation of carbapenem-resistant bacteria in Lebanese hospitals. (2) the Elucidation of the molecular mechanisms of colistin-resistant bacteria in Lebanese patients, and (3) the emergence of vancomycin-resistant gram-positive bacteria in Lebanon. At the start of this thesis, we have prepared a literature review on the epidemiology and the risk factors associated with bacterial infection in conflict wounded and natural disaster in Asia and the Middle East. The second chapter aimed to see the effect of the shift of treatment from colistin-carbapenem combination to colistin monotherapy on the prevalence and resistance of A. baumannii, in addition to the detection of the plasmid-encoded blaVIM-2 gene. In the third chapter, we have detected the spread of colistin-resistant gram-negative bacteria due to mutation of the two-component systems (pmrA /pmrB, phoP/phoQ), or mgrB. We detect the emergence of vanA of Enterococcus faecium resistant to vancomycin. This observation confirms that colistin resistance in Gram-negative bacteria is indeed increasing. In conclusion, it appears necessary and urgent to set up surveys to monitor the use of antibiotics to prevent the spread of resistant strains in Lebanon.

Polymyxins are potent antibiotics that bind to the outer membrane of Gram-negative bacteria, entering the cell and disrupting the inner membrane, resulting in cell death. They were traditionally used as antibiotics of last resort, but the recent surge of multidrug resistant pathogens has renewed interest in these antibiotics. The emergence of polymyxin resistance determinants such as the recently discovered plasmid-mediated phosphoethanolamine transferase MCR-1 may put a strain on the future effectiveness of this antibiotic. One method to combat the rise in antibiotic resistant bacteria is through the identification and development of antibiotic adjuvants. These are small molecules that are able to inhibit the resistance mechanism, allowing previously ineffective antibiotics to once again become effective at treating bacterial infections. In this work, a high throughput cell-based screen was conducted using an in-house library of Actinomycete-derived crude cell extracts in order to search for a natural product inhibitor of an E. coli strain expressing mcr-1. In addition, the development of a new enzyme assay was attempted using purified MCR-1 C-terminal catalytic domain and a chromogenic substrate to test enzymatic activity in vitro, in hopes of establishing a simple means of studying inhibition of MCR-1. The structure-function relationship of MCR-1 was also explored by generating amino acid substitutions and studying their effect on the ability of the enzyme to confer resistance to colistin, as well as the generation of MCR-1 transmembrane truncation mutants to determine if it was possible to generate a shorter variant of MCR-1 that retained its enzymatic activity. This work furthers our understanding of the biochemistry and enzymology of MCR-1, and outlines attempts to identify inhibitors of MCR-1 in order to re-sensitize resistant bacteria to polymyxins. / Thesis / Master of Science (MSc) / Polymyxins are potent antibiotics that are threatened by the spread of multi-drug resistant bacteria. Resistance to these antibiotics is relatively rare, although the recent discovery of a mobile polymyxin resistance enzyme, MCR-1, threatens the future use of this antibiotic for treating infections, as it can readily transfer to other bacteria. The goal of this work was to search for a natural product inhibitor of MCR-1 in order to reverse its ability to confer resistance to polymyxins. A color-changing assay was conducted with MCR-1 in hopes of establishing a method to study the inhibition of MCR-1 in vitro. Additionally, amino acid substitutions were generated in MCR-1 to better understand how key amino acids affect enzyme function, as well as transmembrane domain truncations to determine if it was possible to create a shorter functioning variant of MCR-1.

In view of the increasing global challenge of bacterial resistance, there exists an urgent need for the rationale development of antibacterial compounds with either novel or multiple mechanisms of action. Two chalcone-derivatives, F1 and F23, demonstrated MICs within the range of 16 to >512 μg/ml against two plant pathogens (P. caratovoram and C. michiganensis subsp. michiganensis) as well as important clinical bacterial species. Both compounds displayed an MIC of 32 μg/ml against quinolone-resistant S. aureus. Whilst possessing weak activities individually, each semi-synthetic agent displayed notable synergistic action with polymyxin B against S. aureus, C. violaceum, E. coli and Ps. aeruginosa, thereby recording FICs within the range of <0.093 to 2 that indicated the existence of synergism in some instance. These chalcone compounds applied with polymyxin B displayed a notable FICindex of <0.093 against the Neisseriaceae C. violaceum, and a potential noteworthy capacity to extend the spectrum of activity of the latter antibiotic to include Gram-positive S. aureus species. F1 inhibited staphylococcal replication in broth and the combination of either of both chalcone-derivatives with polymyxin B instituted a metabolic blockage in S. aureus and other bacterial species as determined through a modified MTT reduction assay. The combined agents inflicted major disruptions to the S. aureus cytoplasmic membrane bilayer as evidenced by the release of intracellular potassium as well as the influx of Sytox Green fluorescent stain. Notable levels of cell membrane potential dissipation, leakage of intracellular potassium ions and blockage of reducing enzymes activities occurred within the first 30 minutes, well in advance of significant loss in cell viability that was recorded usually after 4 – 8 hours, suggesting these activities were prerequisites to cell death. In erythrocyte lysis assay, the synergistic combinations of 128 μg/ml of either of both chalcone derivatives with 128 μg/ml polymyxin B displayed the lowest degree of haemolysis, followed by that occurring with 32 μg/ml of the chalcone-derivatives combined with 256 μg/ml of the polypeptide antibiotic. In conclusion, further structure activity modifications aimed at improving the aqueous solubility of these chalcone-derivatives as well as the antibacterial activity recorded for certain combination concentrations of polymyxin B with either of these semi-synthetic agents may be required before considerations are made for the possibility for potential external formulations. Such preparations may include antiseptic creams, lotions, ointments, as well as aerosols that can be applied with nebulizers in targeted delivery for such cases like cystic fibrosis.

La diarrhée colibacillaire post-sevrage (DCPS) est une infection intestinale endémique dans les fermes porcines à l’échelle mondiale. Cette maladie est causée principalement par la présence et la multiplication au niveau de l’intestin des porcelets d’un pathotype d’Escherchia coli, nommé E. coli entérotoxinogène (ETEC) et en particulier celui qui exprime l’adhésine F4 (K88) (ETEC: F4). Le sérogroupe ETEC: O149 a été le plus isolé à partir des cas de DCPS à travers le monde. Plusieurs études ont rapporté un taux de résistance important des souches O149: F4 contre les antibiotiques qui sont classiquement utilisés pour traiter cette infection et en particulier les aminoglycosides. Ainsi, pour remédier aux échecs thérapeutiques observés dans les fermes porcines au Canada, les vétérinaires ont commencé à utiliser, sous leurs responsabilités, un antibiotique, la colistine sulfate (CS), qui n’est pas homologué en production animale au Canada. Cette étude avait pour buts d’étudier la pharmacocinétique de la CS in vitro et in vivo, de développer une technique sensible pour une quantification plasmatique de la CS, de déterminer son efficacité thérapeutique in vivo dans un modèle d’infection expérimentale de DCPS et de caractériser la résistance d’E. coli consécutive à l’utilisation thérapeutique de la CS chez le porc. Une simulation du liquide gastrique (SLG) a été préparée, et après l’ajout de la CS et de la pepsine à cette solution, les concentrations de la CS ont été mesurées par chromatographie liquide à haute performance couplée à la spectrométrie de masse en tandem (HPLC-MS/MS). Une dégradation rapide de CS a été constatée dans la SLG et a été accompagnée par la formation de produits de dégradation qui ont démontré une activité microbienne plus importante par comparaison avec la molécule mère (CS). Dans un volet in vivo, l’infection expérimentale des porcelets sevrés par une souche ETEC: F4 n’a pas augmenté l’absorption digestive de la CS dans un modèle subclinique de DCPS chez le porc. L’administration orale de la CS à la dose thérapeutique de 50,000 UI/kg à raison de 2 fois par jour pendant 5 jours pour traiter la DCPS dans des conditions expérimentales a entraîné une réduction significative de l’excrétion fécale de la souche infectieuse (ETEC : F4), de la population totale d’E. coli et des scores de diarrhée, uniquement pendant la période du traitement. Cependant, ces résultats ont été accompagnés par une légère augmentation dans l’excrétion fécale des E. coli résistants à la colistine, et le traitement n'a pas empêché la perte de poids des porcs infectés. En revanche, l’infection expérimentale des porcelets par ETEC: F4 a augmenté l’absorption digestive de la CS dans un modèle clinique de diarrhée colibacillaire chez le porc. Cette étude a permis de générer pour la première fois des données scientifiques concernant l’efficacité thérapeutique, la pharmacocinétique et la résistance à la colistine dans un modèle de DCPS chez le porc. Elle a également remis en doute la pertinence économique d’augmenter la dose de CS pour accélérer le rétablissement clinique des porcs. Finalement, elle a indiqué que des conditions d’élevage optimales, sans autres facteurs prédisposants, étaient aussi efficaces que la CS dans l’amélioration des symptômes cliniques de la DCPS. / Post-weaning diarrhea (PWD) caused by Escherichia coli is an endemic intestinal infection in pig farms worldwide. This disease is mostly the consequence of the presence and the multiplication in piglet’s gut of an Escherchia pathotype, named enterotoxigenic E. coli (ETEC) and in particular those that express the F4 (K88) fimbrial adhesin (ETEC: F4). The predominant serogroup of E. coli isolated from piglets with PWD worldwide is O149. Several studies have reported a significant resistance rate of O149 ETEC strains against commonly used antibiotics for the treatment of PWD, particularly, aminoglycosides. Thereby, to address therapeutic failures observed in pig farms during PWD treatment, veterinarians in Canada started using, under their responsibilities, the colistin sulfate (CS), an antibiotic not approved for farm animals in Canada. The objectives of this thesis were: to study the pharmacokinetics of CS in vitro and in vivo, to develop a sensitive method for the quantification of CS plasma concentrations in pigs, to determine the therapeutic efficacy of CS in an experimental model of PWD, and to characterize the resistance of E. coli to colistin consecutive to its therapeutic use in pigs. Simulated gastric fluid (SGF) was prepared, and after the addition of CS and pepsin to this solution, the concentrations of CS were followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A rapid degradation of CS in the SGF was observed, and the degradation products showed a greater antimicrobial activity compared to the native CS. On the other hand, the experimental challenge of piglets with an ETEC: F4 strain has not increased the CS intestinal absorption in a subclinical model of PWD in pigs. The oral administration of a therapeutic dose of CS at 50,000 IU/kg twice a day for 5 successive days to treat an experimental PWD in pigs, resulted in a significant reduction of fecal ETEC: F4 and total E. coli shedding, and in diarrhea scores but only during the treatment period. However, CS treatment resulted in a slight increase in fecal shedding of CS resistant E. coli and did not prevent weight loss in challenged pigs. In addition, challenge with ETEC: F4 resulted in an increase of CS intestinal absorption in a clinical model of PWD. This study has generated, for the first time, scientific data regarding CS therapeutic efficacy, its pharmacokinetic and the selection of E. coli colistin resistant in an experimental model of PWD in pigs. It also challenged the economic relevance of increasing CS oral doses to accelerate the clinical recovery of pigs. Finally, it indicated that optimal housing conditions were without other predisposing factors, effective as CS in improving clinical symptoms of experimental PWD in pigs.

Les antibiotiques sont des polluants présents dans les écosystèmes aquatiques, réceptacles ultimes des substances anthropiques. L’étude de ces composés porte sur leur rémanence dans le milieu ou leurs effets sur des organismes naturels. De nombreux efforts ont été faits à l’échelle mondiale pour l’évaluation de la qualité environnementale des différentes ressources en eau pour la survie des espèces aquatiques mais aussi pour la consommation humaine et le risque sanitaire lié. Dans ce but, l’optimisation des techniques analytiques pour ces composés dans les systèmes aquatiques demeure une nécessité. Notre objectif est de développer des méthodes d’extraction et de détection pour 12 molécules appartenant à la famille des aminoglycosides et de la colistine dans les eaux des stations d’épuration et les eaux hospitalières. L’absence des méthodes d’analyse pour ces composés ainsi que le manque des études permettant leur détection dans l’eau sont les raisons de leur étude. L’Extraction sur Phase Solide (SPE) en mode classique (hors ligne) ou en ligne, suivie d’une analyse par la Chromatographie Liquide couplée à la Spectrométrie de Masse (LC/MS/MS) est la méthode la plus couramment employée pour ce type d’analyse. Les paramètres sont optimisés et validés afin d’assurer les meilleures conditions utilisées dans les analyses environnementales. Cette technique a été appliquée sur des échantillons réels des eaux des stations d’épuration à Bordeaux et au Liban. / Antibiotics are pollutants present in aquatic ecosystems ultimate receptacles of anthropogenic substances. These compounds are studied as their persistence in the environment or their effects on natural organisms. Numerous efforts have been made worldwide to assess the environmental quality of different water resources for the survival of aquatic species, but also for human consumption and health risk related. Towards goal, the optimization of analytical techniques for these compounds in aquatic systems remains a necessity. Our objective is to develop extraction and detection methods for 12 molecules of aminoglycosides and colistin in sewage treatment plants and hospitals waters. The lack of analytical methods for analysis of these compounds and the deficiency of studies for their detection in water is the reason for their study. Solid Phase Extraction (SPE) in classic mode (offline) or online followed by Liquid Chromatography analysis coupled with Mass Spectrometry (LC/MS/ MS) is the most method commonly used for this type of analysis. The parameters are optimized and validated to ensure the best conditions for the environmental analysis. This technique was applied to real samples of wastewater treatment plants in Bordeaux and Lebanon.