Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Listeria monocytogenes is an important foodborne pathogen that can adapt to stress conditions to persist in food processing environments. Our findings show that there was a development of low-level tolerance to quaternary ammonium compound (QAC) and antibiotics ciprofloxacin and trimethoprim in L. monocytogenes after sublethal adaptation to QAC. Using eight L. monocytogenes strains, we determined the changes in MIC, growth rate, and surviving CFU for homologous and heterologous stress-response after sublethal exposure to daily cycles of fixed or gradually increasing concentration of QAC. Three main findings were observed: (1) Short-range MIC of QAC, ciprofloxacin, and trimethoprim increased by 1.6 to 2.3, 1.5 to 2.9, and 1.7 to 2.5 fold against QAC-adapted phenotypes of L. monocytogenes as compared to the non-adapted cells; (2) QAC-adapted phenotypes of L. monocytogenes exhibited a significant increase in growth rate by 2.5 to 7.1, 2.1 to 6.8, or 1.4 to 4.8 fold in the broth model containing QAC, ciprofloxacin, or trimethoprim respectively, as compared to non-adapted cells; and (3) QAC-adapted phenotypes of L. monocytogenes exhibited a significant increase in survival by 1.5 to 4, 2.2 to 4.3, or 1.3 to 3.2 log CFU/ml in the agar model containing QAC, ciprofloxacin, or trimethoprim respectively, as compared to non-adapted cells (P < 0.05). There were strain differences in QAC-adapted phenotypes of L. monocytogenes for both homologous and heterologous stress-response with some strains exhibiting a significant increase in short-range MIC, growth rate, and survival while others exhibiting no changes as compared to non-adapted cells. These findings suggest the potential formation of low-level QAC-tolerant and antibiotic-tolerant phenotypes in some L. monocytogenes strains under residual QAC concentrations (where QAC may be used widely) and such cells if not inactivated may survive longer to increase food safety risk.

In this master thesis project, the removal of micro-pollutants (MPs) from water and wastewater were investigated by ozonation and the peroxone process O3/H2O2. Themain aim of the study was to compare the degradation efficiency of the two processes for a selection of organic biocides and active pharmaceutical ingredients (APIs) in wastewater and synthetic water. Synthetic water and wastewater effluent were spiked with fifteen compounds including twelve biocides and three APIs and samples were taken at different time points to determine degradation kinetics in treatment by the two processes, respectively. Most of the biocides tested in this study showed moderately reactivity, or were non-reactive, with O3 (e.g., Carbendazim, 1H-Benzotriazole, 1, 2-Benzisothiazol-3(2H)-one, 2, 4-Dinitrophenol, 4-nitrophenol) and some of the biocides and APIs were readily reactive with ozone (O3) and were removed from the water by direct O3 oxidationduring both processes. Trimethoprim was found to be an ozone reactive micropollutants and showed highest reactivity with O3. In the abatement mechanism, these micro-pollutants are removed based on the ozone-based processes and with hydroxyl radical •OH in peroxone process. Comparing the two processes, the peroxone process showed highest percentage removal of compounds (90-100%) within moderate time periods (30 s - 2 min) in synthetic water and (30 s to 10 min) in wastewater. The results revealed that the peroxone process showed highest removal efficiency and with higher reproducibility. Further, generally

L'implication des tours aéro-réfrigérantes dites « ouvertes », dans les cas de légionellose, demeure à l'heure actuel un problème de santé public malgré la multitude de composés biocides disponibles qui, parfois, sont écotoxiques et appliqués à de forte concentrations. L'efficacité de la majorité de ces biocides est étudiée vis-à-vis des formes planctoniques des microorganismes. Cette étude s'intéresse à la mise au point de deux procédés de traitement du risque Legionella pneumophila : H2O2/UV et ClO2, avec la prise en compte particulière des communautés microbiennes fixées et notamment des protozoaires qui constituent l'hôte naturel de cette bactérie. La démarche expérimentale adoptée pour l'étude de l'efficacité de ces traitements, s'est appuyée sur des biofilms environnementaux naturellement contaminés par des légionelles et issus d'une source thermale. La caractérisation des légionelles isolées des biofilms de cette source montre le maintien du sérogroupe 1 de L. pneumophila et l'apparition de deux autres sérogroupes non reportés dans des études précédentes (sg10 et sg12), avec toutefois une prédominance du sérogroupe 12. Quel que soit le sérogroupe, ces souches environnementales se sont avérées plus virulentes vis-à-vis de l'amibe Acantamoeba castellanii, que les souches cliniques de Lp1 répertoriées. A l'état planctonique elles se sont également avérées très sensibles aux traitements H2O2/UV et UV seul. A l'échelle expérimentale, les deux traitements, H2O2/UV et ClO2, montrent une performance élevée vis-à-vis des biofilms. L'étude a particulièrement mis en évidence le rôle du facteur trophique et de l'adaptation des bactéries au stress oxydatif dans la performance des traitements mais aussi dans l'apparition de la reviviscence. L'application des traitements à des installations en vraie grandeur a permis de conforter les résultats expérimentaux et de mettre l'accent, dans le contexte des sites étudiés, sur les limites de leur efficacité et sur la nécessité d'ajustements des doses appliquées (concentrations des biocides, flux d'irradiation UV, mode d'application) aux particularités des contextes industriels. / Cooling towers are considered as one of the major sources of Legionella pneumophila, the causal agent of Legionnaires' disease. Despite the high number of commercial disinfectants dedicated to it, the Legionella threat is still rising especially in connection with cooling towers. The efficacy of most disinfectants is demonstrated on planktonic bacteria while very few studies have been devoted to their effects on biofilms. The main objective of the study was to optimize two treatments dedicated to the control of biofilms in cooling towers, H2O2/UV and ClO2, so as to reduce the risk associated to Legionella pneumophila while minimizing environmental damages. Their efficacy was studied against environmental biofilms developed in a hot sulphur spring where presence of L. pneumophila is permanent. Their analysis revealed the occurrence of Lp serogroups 1 (Lp1) and of two serogroups not reported in previous studies that were oriented toward water samples, though (Lp10 and Lp12). The environmental strains we isolated display a higher cytotoxicity and virulence towards the amoeba Acantamoeba castellanii than those of known Lp1 epidemic strains and a higher sensitivity to UV and H2O2/UV. In the experimental part of the study, ClO2 and H2O2/UV display a high efficacy against biofilms. Furthermore the study showed the role of trophic parameters and of bacterial adaptation to oxidative stress on the performance of the treatments but also on biofilms regrowth. The experiments performed at the industrial scale corroborate the results gained at the pilot scale and focus on the relationships between the dose and the effectiveness of each treatment. Our results suggest the possibility to apply the process to an industrial scale with necessary adjustments about doses and injection modalities to the context of the considered sites.

Le travail de thèse décrit dans ce manuscrit porte sur l’étude de la présence de biocides désinfectants dans les produits laitiers et sur l’impact des procédés de transformation du lait sur l’apparition de produits néoformés. Les biocides désinfectants sont des composés chimiques utilisés quotidiennement en industrie laitière lors des procédures de nettoyage-désinfection (ND) des surfaces en contact avec les aliments. Les ammoniums quaternaires de type chlorure de Benzalkonium (BACs) et chlorure de dialkyldimethylammonium (DDACs), et l’Aminopropyldodécylpropane diamine sont les désinfectants les plus largement utilisés en industrie laitière. Ces biocides peuvent entrainer des résidus sur les surfaces alimentaires, ce qui présente un risque pour la santé du consommateur. Dans le but de mesurer l’exposition des consommateurs, deux méthodes analytiques fiables ont été développées pour l'analyse de ces substances dans les produits laitiers impliquant la chromatographie liquide couplée à la spectrométrie de masse en tandem. Le lait cru de vache, la poudre de lait entier, les fromages à pâte pressée cuite et les fromages fondus ont été sélectionnés pour représenter la diversité des produits laitiers. L'évaluation des performances de chacune des méthodes a été réalisée par une approche globale basée sur le profil d'exactitude. Pour la plupart des composés et des matrices étudiés, les méthodes d'analyse ont été validées sur l’intervalle de dosage de 5 à 150 μg / kg. Pour répondre au questionnement de l'impact des procédés de transformation du lait sur les résidus de biocides désinfectants, l'évolution des teneurs en composés et leur devenir dans les différentes matrices issues du lait ont été étudiées. Pour cela, deux études de faisabilité mettant en oeuvre des comparaisons d'empreintes chimiques globales, acquises par spectrométrie de masse à haute résolution, de fromages fondus et de fromages à pâte dure (contaminés vs témoins) ont été réalisées. Ces études ont permis de détecter 4 ions discriminants liés à la présence de biocides dans le fromage fondu. Leur identification reste à réaliser. Tout ce travail a été réalisé à des fins de sécurité alimentaire. La première partie est à l'élaboration de méthodes analytiques ciblées pour les résidus de biocides dans le lait et les produits laitiers permettant ainsi de mesurer les résidus de biocides sur les denrées alimentaires. Ces mesures sont nécessaires pour effectuer une analyse du risque liée à ces résidus. La deuxième partie est en relation avec la question du comportement des résidus de biocides lors de la transformation du lait présentant la stratégie, les résultats que nous pourrions obtenir et la perspective de travaux futurs. / The thesis work focuses on the presence of disinfectants biocides in dairy products and on the impact of milk processing on the possible appearance of process-induced food contaminants related to the exposition of milk with biocides. Disinfectants biocides are chemicals daily used in the dairy industry in cleaning-disinfection (CD) processes of food contact surfaces. Quaternary ammoniums as benzalkonium chloride (BACs) and dialkyldimethylammonium chloride (DDACs), and the Aminopropyldodecylpropane diamine are the most widely used disinfectants in dairy industry. These biocides can lead residues on the surfaces of food contact materials therefore present a health risk for the consumer. With aim of measuring consumers exposure, two reliable analytical methods have been developed for the analysis of these substances in dairy products involving liquid chromatography hyphenated with tandem mass spectrometry. Raw cow's milk, whole milk powder, hard pressed and processed cheeses were selected as representing the diversity of dairy products. The evaluation of the performances of each of the methods was carried out by the global approach based on the accuracy profile. For most of compounds and matrices studied, analytical methods were validated within the range of 5 to 150 μg/kg. To answer to the questioning of the impact of milk processes on biocides disinfectants residues, the evolution of compounds contents and their fate in the various matrices resulting from the milk were studied. For this, two proof-of-concept studies implementing global chemical fingerprint comparisons, acquired by High Resolution Mass Spectrometry, of processed cheese and hard pressed cheese (contaminated vs control) samples were carried out. These studies allowed to detect 4 discriminant ions linked to the presence of biocides in processed cheese. Their identification remains to be done. Whole this work is related for food safety purposes. The first part was linked to elaborate targeted analytical methods for biocides residues in milk and milk products thus allowing the measurement of biocides residues on food. These measurements are necessary for the risk analysis linked to these residues. The second part is in relation with the question of the behavior of biocides residues within milk processing presenting the strategy, the results we could obtain and the perspective for future works.

L’objectif de ce travail a été d’étudier la présence, le devenir et le traitement de biocides dans les rejets d’un établissement hospitalier. Pour cela, 3 substances biocides, représentatives d’une activité de soins (i.e. didécylmethylammonium (DDAC), digluconate de chlorhexidine (CHD) et bis(aminopropryl)laurylamine (BAPLA)) ont été ciblées et 5 campagnes de prélèvements réalisées sur l’ensemble du réseau de collecte des eaux usées du CHU de Poitiers ont été menées. Dans ces conditions, une émission continue de biocides sur 24 h, avec des quantités rejetées en journée plus importantes du fait de la plus forte activité de soins et de nettoyage durant cette période a été montrée. Des concentrations de l’ordre de quelques mg/L pour le DDAC et de quelques dizaines de µg/L pour le CHD et le BAPLA ont été observées. En termes de flux massiques, la comparaison des quantités retrouvées dans les rejets aux quantités utilisées par le CHU a permis de préciser la nature des sources d’émission ainsi que les usages et/ou les pratiques les plus propices à ces rejets.Parallèlement, une étude laboratoire de la stabilité et du devenir des biocides dans les eaux résiduaires a été menée. Une première estimation des quantités adsorbées sur les matières en suspension a été obtenue. De même, les produits de dégradation susceptibles d’être générés lors du rejet de biocides en présence d’un résiduel de chlore ont été identifiés et l’évolution de l’écotoxicité vis-à-vis de Vibrio fisheri a été évaluée.Enfin, la possibilité d’un traitement à la source utilisant l’ozone pour limiter les rejets de biocides a été envisagée et une étude pilote sur le terrain a été menée. / The objectives of this study was to study the presence, the fate and the treatment of some biocides in the wastewaters from a hospital. 3 biocidal substances specific to healthcare activities (i.e. didecylmethylammonium chloride (DDAC), chlorhexidine digluconate (CHD) and bis(aminopropryl)laurylamine (BAPLA)) were selected and 5 sampling campaigns were conducted throughout the entire Poitiers University Hospital sewage system. Under these conditions, a continuous biocide discharges over 24 h was shown, with higher quantities discharged during the day. This was pointing out the greater healthcare and facility cleaning activities taking place during this period. Concentrations of a few mg/L for DDAC and a few dozens of µg/L for CHD and BAPLA were detected. In terms of mass flows, the comparison of the quantities found in the discharges with the quantities used by the university hospital made it possible to specify the nature of the sources of emission as well as the uses and / or the practices most favorable to these discharges.In parallel, a laboratory study of the stability and fate of biocides in wastewater was conducted. A first estimate of the amounts adsorbed on suspended solids was obtained. Similarly, the degradation products likely to be generated during the release of biocides in the presence of a chlorine residual have been identified and the evolution of the ecotoxicity towards Vibrio fisheri has been evaluated.Finally, the proposal of an ozone based treatment at source was carried out to limit biocide discharges and a pilot study was conducted on the hospital wastewaters.

Cooling water systems are needed to dissipate heat. The mist from open system cooling towers has been implicated as a source of infections of Legionella pneumophila. As a result biocide is added to cooling water systems, but the addition of biocide worsens biofouling, scaling and corrosion. Increasing environmental pressure has resulted in a move away from biocides which are usually chlorine based chemicals, so that the use of ozone instead has recently been reported as a way of controlling microbiological growth. This study aims to compare the corrosion rates (using calculated and measured surface areas) of different metals exposed to chemically treated and ozone treated cooling water in an industrial cooling water system. The types of corrosion were also observed and recorded. The scales from different components of the cooling systems as well as scale from chemically and ozone treated D tower water were characterized qualitatively using X-ray diffraction (XRD), Thermo gravimetric Analysis (TGA) and Atomic Absorption Spectroscopy. The D tower is a cooling water circuit in which the cooling water is from the Vaal River. As expected, the stainless steel has the lowest corrosion rate of 0.000 milli inches/year followed by brass with 1.531 milli inches/year and lastly mild steel (2.098 milli inches/year). Water quality rather than the presence or absence of ozone determines the corrosion rate. This confirms the findings reported in the literature. Scale from chemically treated water contains many different compounds while scale from ozone treated contains only different polymorphs of CaCO3, which is present in the water source and magnesium calcite.

Bacterial resistance to non-antibiotic agents is being increasingly studied. Plasmid-mediated resistance to cationic agents, which are important biocides, has been described in antibiotic-resistant Staphylococcus aureus. Multi-resistant Staphylococcus aureus (MRSA) are often found to express resistance to a range of cationic biocides including quaternary ammonium compounds (QACs), biguanides, diamidino compounds, cationic dyes and nuclear stains. Three resistance determinants, qacA, qacB and smr genes, have been identified that confer resistance to cationic biocides in staphylococci. These genes encode multi-drug efflux pumps that remove the cationic biocides from the cytoplasm using a membrane bound pumping mechanism dependent on the cell's proton-motive force (PMF). This prevents the build up of lethal concentrations of cationic compounds within the cytoplasm avoiding cell death.This research project has focused on the S. aureus strain WBG4364, a transcipient strain carrying the cationic biocide resistant plasmid pWBG1773. The plasmid encodes resistance to several QACs, including benzalkonium chloride and CTAB, and cationic dyes rhodamine 6G, crystal violet and safranin O but not to the dye ethidium bromide and therefore differing from other cationic biocide resistant plasmids previously identified in staphylococci (Emslie et al. 1986). This unique phenotype was further classified in this study alongside those strains carrying the qac gene families, qacA/B and smr.Plasmid pWBG1773 was cloned, sequenced and analysed to reveal a unique plasmid of 2,916 bp in length. Plasmid pWBG1773 was placed with the pC194 family of rolling-circle replicating plasmids. This family appear to be largely composed of interchangeable cassette structures.The plasmid was found to carry three ORFs, designated ORF1, ORF2 and ORF3. ORF1 was homologous to rep genes of small staphylococcal ++ / plasmids belonging to the pC194 rolling-circle replication family and has been redefined as repWBG1773. ORF2 was found to have no similarity to any proteins of known function in the GenBank database whereas ORF3 was found to have homology to the marR gene, a regulator of the multiple antibiotic resistance (mar) operon of Gram-negative organisms. MIC analysis of these ORFs found both ORF2 and ORF3 were essential for expression of resistance to cationic biocides. The exact ORF2 sequence required for resistance to be expressed was reduced to only 141 nt in size. This translated to a 47 aa sequence that contained a hydrophobic C-terminus indicating ORF2 to be a membrane-bound protein. The aa sequence of ORF3 contained a helix-turn-helix motif characteristic of the DNA binding domains of MarR-like proteins. Further analysis of pWBG1773 identified a putative 'marbox', a binding site for the homologous transcriptional activators of mar, within the ORF2 sequence. This indicated that ORF3 was binding to the 'marbox' sequence and activating transcription. Induction studies have not been able to ascertain any compounds capable of interacting with the ORF3 regulatory protein resulting in induction of cationic biocide resistance. Each ORF when analysed alone had no effect on the expression of cationic biocide resistance and it is thought that a efflux pump was not involved. This is further corroborated by the CCCP efflux experiments performed in an attempt to determine the mechanism of resistance. The unique ORFs of plasmid pWBG1773 appears to encode a novel cationic biocide resistance phenotype and mechanism.MRSA strains from all around the world were analysed to determine if they possessed sequences homologous to ORF2 and ORF3. Sequences sharing a high degree of homology to ORF2 and/or ORF3 were detected in several MRSA strains including strains sensitive to all cationic ++ / biocides tested. These findings suggest that the appearance of ORF2 and ORF3 sequences in MRSAs was not an isolated event and the fact that some MRSAs do not carry both ORF2 and ORF3 sequences simultaneously indicates that these genes have another role that does not involve expression of resistance to cationic biocides.Bacteria are noteworthy for their remarkable ability to adapt to changes in their environments and possess an impressive set of tools with which to adjust the blueprint of the cell to this change. The acquisition of a single system that may decrease a potential pathogenic organisms susceptibility to a wide range of cationic biocides, such as seen in pWBG1773, poses a clinical threat, one that needs to be thoroughly investigated.

Biocides are widely used as a preservative or as an antiseptic agent in consumer care products such as toothpaste, mouthwash, and soaps, as well as in household cleaners and even in textiles due to their high antimicrobial effectiveness. The usage of this compounds results in discharge to wastewater treatment plants and so into surface waters. Their existence in the environment is of importance due to their negative effects on aquatic environment microorganisms and human health in terms of occurrence in surface waters and their fate in wastewater treatment plants. In this scope, this study focuses on occurrence and fate of selected biocides, namely triclosan (TCS) and chlorhexidine (CHD), in wastewater treatment plants and in surface waters. It was aimed to determine the biocides levels in surface water and wastewater in Turkey. For the wastewater treatment plant (WWTP) studies, several WWTPs with different process configurations, namely, Tatlar WWTP, METU WWTP, Kayseri WWTP and Antalya WWTPs were selected. Composite wastewater samples were taken from various points along the WWTPs on a seasonally basis for one year period. For the surface water part, samples were taken monthly from three different sources with different pollution levels, namely, Kesikk&ouml / pr&uuml / Reservoir, &Ccedil / amlidere Reservoir and Eymir Lake for one year period. All water samples were analyzed for their biocide level using liquid chromatography, following solid phase cartridge extraction. As a result of analyses, TCS concentration in surface water samples was detected as in the range of 0.65-11.15 ng/L, 0.86-48.96 ng/L and 0.86-757.7 ng/L for clean, moderately polluted and polluted water sources respectively. The recovery of solid phase extraction analyses for TCS was achieved as %92. CHD concentration was determined as in the range of &lt / 1.33-5.31 ng/L for surface water samples and the recovery of extraction were calculated as %96 for CHD. The concentration of TCS in wastewater samples was measured as in the range of 1.77-94.47 ng/L and 1.40-15.09 ng/L for influent and effluent samples respectively. These ranges became 1.39-10.45 ng/L and &lt / 1.32-2.44 ng/L for CHD. The highest concentrations of biocides were observed in sludge samples with concentrations of 1117-3687 &mu / g/kg and 510-2742 &mu / g/kg for TCS and CHD. Biocide removal efficiency of primary and biological treatment together was reported as % 67.5&plusmn / 8.2 in January 2012 Tatlar WWTP analyses.

Orientador: Márcia Justino Rossini Mutton / Banca: Maria das Graças Cardoso / Banca: Flávia Cecílio Ribeiro Bregagnoli / Resumo: Na produção de cachaça, a microbiota contaminante afeta diretamente o processo fermentativo, originando fermentações indesejáveis, contribuindo para menores rendimentos em álcool e um desbalanceamento na formação dos compostos secundários que caracterizam a bebida. Portanto, são necessárias práticas para minimizar e controlar essas contaminações, sendo a aplicação de antimicrobianos uma alternativa eficaz para tal controle. Dentro desse enfoque, o objetivo deste trabalho foi avaliar a eficiência de antimicrobianos naturais e sua interação com antibióticos convencionais no controle da contaminação bacteriana na produção cachaça. Para as análises tecnológicas e microbiológicas do vinho e pé-de-cuba, utilizou-se o delineamento inteiramente casualizado em parcelas sub-subdivididas, em esquema fatorial 5x2x10 com três repetições, sendo quatro biocidas (extrato de própolis marrom, extrato de própolis verde, ampicilina e interação própolis/ampicilina (P/A)) e controle (sem adição de antimicrobiano), combinando-se os tratamentos em início e final de safra por 10 ciclos fermentativos. Para as análises tecnológicas do mosto, utilizou-se o delineamento inteiramente casualizado em parcelas subdivididas, em esquema fatorial 2x10 com três repetições, sendo duas épocas da safra (inicio e final) por 10 ciclos fermentativos. Foi observado um comportamento distinto entre épocas, com maiores valores de contaminantes e acidez em mosto no final da safra. Dentre os tratamentos, o extrato de própolis marrom, própolis verde, ampicilina e interação (P/A) mantiveram os índices de viabilidade celulares a níveis elevados e superiores a 90%, com menores índices observados para o tratamento controle. As concentrações de bactérias no pé-de-cuba foram significativamente menores com o emprego dos biocidas. No tratamento controle a acidez total do vinho foi superior... (Resumo completo, clciar acesso eletrônico abaixo) / Abstract: During the cachaça production, the contaminant microbiota affects directly the fermentative process, originating undesirable fermentations, minimizing the alcohol production as well an unbalancing in the secondary compounds formation that characterizes the beverage. Therefore, practices that minimize those contaminations are necessary and the antimicrobial use is one of the efficient alternatives the control. This work aimed to evaluate the natural antimicrobials efficiency as well its interaction with conventional antibiotics to control the bacterial contamination in the cachaça production. A completely randomized design in sub-divided parcels in a factorial 5x2x10 scheme with 3 replications was used, being four biocides (brown propolis extract, green propolis extract, ampicillin and propolis/ampicillin interaction - P/A) and control, combining them in the beginning and final of harvest by 10 fermentative cycles. A different behavior between treatments was observed, being the higher contamination and acidity found in the end of the harvest. Among treatments, the brown propolis extract, green propolis extract, ampicillin and P/A kept cellular viability indexes higher than 90%, and the control treatment was lower. The bacterial concentration in the inoculums was significantly smaller when biocides were used. In the control treatment the residual reducing sugars. The propolis extract and its combination with synthetic antimicrobials were efficient to control contaminant bacteria during the fermentative process / Mestre