Ce travail de thèse a dans un premier temps permis de mettre en évidence les différentes pratiques de traitement de toiture existantes en France. Après avoir sélectionné la pratique de traitement majoritaire (à savoir le traitement biocide), le protocole d'analyse de la molécule la plus répandue dans les différents produits : le benzalkonium (ou alkyldiméthylbenzylammonium) a été développé. Ce protocole a ensuite été appliqué pour évaluer les niveaux de concentration en biocide dans l'eau de ruissellement de toiture après un traitement. Une double approche a été mise en œuvre : "Suivi sur une durée d'un an des flux de benzalkonium dans les eaux de ruissellement de 12 bancs d'essais exposés en conditions naturelles", évaluation en condition de pluie simulée au laboratoire de différents paramètres (nature du matériau de couverture, dosage du produit et intensité de la pluie) sur les processus d'émission du benzalkonium. Enfin, l'ensemble des données acquises ont permis d'évaluer l'incidence sur la contamination des eaux de ruissellement de toiture après un traitement biocide, à une échelle locale et à l'échelle d'un petit bassin versant résidentiel. Le suivi de la contamination des eaux de ruissellement par bancs d'essais a permis de mettre en évidence une très forte contamination en benzalkonium dans les premiers millimètres de pluie (5 à 27 mg/L), très supérieures aux EC50 disponibles pour les organismes aquatiques (5,9 µg/L) et dépendante de la nature du matériau de toiture utilisé, les tuiles en terre cuite ayant émis moins de biocide que les tuiles en béton. La contamination diminue avec la succession des pluies, mais reste significative plusieurs mois après le traitement. Par ailleurs, l'étude au laboratoire a montré que l'état de surface des tuiles influence beaucoup le lessivage. Ainsi, les tuiles très imperméables en surface ont émis une quantité de benzalkonium proportionnelle à la masse épandue lors du traitement, alors que les tuiles sans traitement de surface ne sont pas sensibles au dosage du produit. A l'échelle locale, des précautions doivent être prise lors d'un traitement de toiture vis-à-vis de la collecte / utilisation des eaux de ruissellement. En effet, les très fortes concentrations en benzalkonium dans les premières pluies suivant le traitement peuvent avoir des impacts sur les végétaux arrosés, sur l'équilibre microbien de la cuve de récupération, voir des effets irritants sur l'homme. Une déconnexion minimale de 3 à 5 mois de la cuve permettrait de limiter les risques. Enfin, sur la base des résultats des bancs d'essais et d'un travail d'enquête sur l'étendue des pratiques de traitement, les concentrations et flux de benzalkonium susceptibles d'être émis dans les eaux pluviales d'un bassin versant résidentiel ont été modélisés. Les résultats mettent en évidence une contamination significative des eaux pluviales, liée essentiellement à la phase particulaire. La masse annuelle de benzalkonium exportée dans les eaux pluviales pourrait être de l'ordre de 0,85 kg / ha imperméabilisé / an / Firstly, this thesis listed the different roof treatment practices in France. Once the major treatment practice selected (biocidal treament), the analytical protocol of the most widely used molecule (benzalkonium or alkyldimethylbenzylammonium) was developed. This protocol has been followed to evaluate the biocide concentration levels in roof runoff after a treatment. A double approach was considered : - Follow up of the benzalkonium flux for one year in runoff using 12 bench tests exposed to natural conditions. - Evaluation of the influence of different parameters (material, product dosage and rain intensity) on benzalkonium emmission processes with simulated rains. Finally, all the data acquired allowed the evaluation of the incidence of biocidal treatments on roof runoff contamination at different scales ( local and residential watershed). The results of the bench tests showed a very high benzalkonium contamination during the millimetres of rain (5 to 27 mg/L), higher than the different EC50s of aquatic organisms ( 5.9 ùg/L). Furthermore, the results showed an influence of the tile material on the release of benzalkonium : clay tiles were less washed off than concrete tiles. The contamination decreased with successive rains, but remained significantly high several months after treatment. Moreover, laboratory studies showed a proportional release of benzalkonium from waterproofed tiles with respect to the biocidal mass spread. On the contrary, tiles without any surface treatment were not sensitive to the product's dosage. On a local scale, precautions have to be taken during treatment with respect to the harvest / reuse of the runoff. Indeed, high benzalkonium concentrations during the first rains may have a potential impact on watered plants, microbial equilibrium in the collection tank and irritating effects on human. A deconnection of the tank for at least three to five months will reduce the risks. Finally, based upon the bench tests results and a survey on roof treatment practices, the benzalkonium concentrations and fluxes were injected in a mathematical model on a watershed scale. The results showed a significant contamination linked to the particles. The annual mass of benzalkonium transferred to the rainwaters could reach 0.85kg / waterproofed ha / year

The mutant prevention concentration (MPC) is defined as the lowest antimicrobial concentration required to inhibit the growth of the least susceptible bacterial cell based on an inoculum of ≥109 colony forming units (CFUs). The current protocol for MPC testing is technically demanding and time-consuming which limits its implementation into clinical microbiology laboratories. In an attempt to simplify the current MPC protocol we developed a modified MPC method, the microbroth dilution method, which requires two fewer days to complete than the current or traditional method. MPC values were consistent for all organisms and strains tested using both the traditional MPC method and the modified microbroth dilution MPC method.<p> Tigecycline is the first of a new class of compound glycylcyclines- with potent in vitro activity against Gram-positive organisms including penicillin-resistant and multi-drug resistant <i>Streptococcus pneumoniae</i> (SP) and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). We measured minimum inhibitory concentration (MIC) and MPC values for tigecycline against 47 clinical isolates of SP and found that the MPC90 values were >500 fold higher than the MIC90 values. To determine if MPC testing of tigecycline against SP is impacted by blood in the medium, we developed a new medium able to sustain the growth of SP without the need for blood; solidified Todd-Hewitt broth (sTHB). The MPC90 values of tigecycline against SP on sTHB were only 2 fold higher than the MIC90 values. When blood was added to the sTHB, the MPC90 values again became much greater than the MIC90 values (> 256 fold higher). MPC results for <i>Staphylococcus spp.</i> against tigecycline were not impacted by blood in the medium.<p> Benzalkonium chloride (BAK) is a cationic surface-acting agent that acts on bacterial cells by disrupting the intermolecular interaction of the lipid bilayer. To determine if the <i>fluoroquinolones gatifloxacin</i> (Gfx) and moxifloxacin (Mfx) are more active (lower MIC values) in the presence of BAK, we conducted MIC, MPC, and time-kill assays. MIC testing showed that in the presence of 3.125 to 50 µg/ml of BAK, the MIC of Gfx and Mfx decreased by 8- to 5000-fold against clinical isolates of methicillin-susceptible <i>Staphylococcus aureus</i> (MSSA), MRSA, Coagulase-negative <i>Staphylococci</i>(CNS), SP, <i>Escherichia coli</i> (EC), and <i>Pseudomonas aeruginosa</i> (PA). MPC testing showed that the presence of 7 to 10 µg/ml of BAK, the MPC of Gfx and Mfx decreased by 32- to 1000-fold against clinical isolates of MRSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) showed that the killing activity of Gfx against clinical MRSA isolates was enhanced in the presence of BAK with a log10-reduction (percent kill) of 1.6 (76.08%) for Gfx alone at 180 minutes compared to a log10-redecution (percent kill) of 5.4 (100%) for Gfx plus BAK at 180 minutes.<p> Alexidine (Alx) is a bisbiguanide that has been used as an effective disinfectant in the dental industry and is potentially being developed for use as an antimicrobial agent for ocular infections. We conducted susceptibility testing of Alx using MIC testing, MPC testing, and time-kill assays against Gram-positive and Gram-negative pathogens. MIC testing showed that Alx is more active against Gram-positive pathogens than Gram-negative pathogens and showed better activity than the fluoroquinolones Gfx, Mfx, and levofloxacin (Lfx) against MRSA. The MPC values measured for MRSA and MSSA against Alx were non-reproducible using the traditional MPC method. Using the microbroth dilution MPC method, MPC90 values were found to be 32 fold higher than the MIC90 values. If the experimentally determined MPC values are true MPC values, initial MPC testing indicates that Alx may have a high likelihood for selecting for resistance, however, if the MPC values are not accurate it may be necessary to modify the MPC protocol in order to complete MPC testing of Alx against MRSA and MSSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) measured bactericidal activity (> 3 log10-reduction) against MRSA, MSSA, SP, and PA.

The mutant prevention concentration (MPC) is defined as the lowest antimicrobial concentration required to inhibit the growth of the least susceptible bacterial cell based on an inoculum of ≥109 colony forming units (CFUs). The current protocol for MPC testing is technically demanding and time-consuming which limits its implementation into clinical microbiology laboratories. In an attempt to simplify the current MPC protocol we developed a modified MPC method, the microbroth dilution method, which requires two fewer days to complete than the current or traditional method. MPC values were consistent for all organisms and strains tested using both the traditional MPC method and the modified microbroth dilution MPC method.<p> Tigecycline is the first of a new class of compound glycylcyclines- with potent in vitro activity against Gram-positive organisms including penicillin-resistant and multi-drug resistant <i>Streptococcus pneumoniae</i> (SP) and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). We measured minimum inhibitory concentration (MIC) and MPC values for tigecycline against 47 clinical isolates of SP and found that the MPC90 values were >500 fold higher than the MIC90 values. To determine if MPC testing of tigecycline against SP is impacted by blood in the medium, we developed a new medium able to sustain the growth of SP without the need for blood; solidified Todd-Hewitt broth (sTHB). The MPC90 values of tigecycline against SP on sTHB were only 2 fold higher than the MIC90 values. When blood was added to the sTHB, the MPC90 values again became much greater than the MIC90 values (> 256 fold higher). MPC results for <i>Staphylococcus spp.</i> against tigecycline were not impacted by blood in the medium.<p> Benzalkonium chloride (BAK) is a cationic surface-acting agent that acts on bacterial cells by disrupting the intermolecular interaction of the lipid bilayer. To determine if the <i>fluoroquinolones gatifloxacin</i> (Gfx) and moxifloxacin (Mfx) are more active (lower MIC values) in the presence of BAK, we conducted MIC, MPC, and time-kill assays. MIC testing showed that in the presence of 3.125 to 50 µg/ml of BAK, the MIC of Gfx and Mfx decreased by 8- to 5000-fold against clinical isolates of methicillin-susceptible <i>Staphylococcus aureus</i> (MSSA), MRSA, Coagulase-negative <i>Staphylococci</i>(CNS), SP, <i>Escherichia coli</i> (EC), and <i>Pseudomonas aeruginosa</i> (PA). MPC testing showed that the presence of 7 to 10 µg/ml of BAK, the MPC of Gfx and Mfx decreased by 32- to 1000-fold against clinical isolates of MRSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) showed that the killing activity of Gfx against clinical MRSA isolates was enhanced in the presence of BAK with a log10-reduction (percent kill) of 1.6 (76.08%) for Gfx alone at 180 minutes compared to a log10-redecution (percent kill) of 5.4 (100%) for Gfx plus BAK at 180 minutes.<p> Alexidine (Alx) is a bisbiguanide that has been used as an effective disinfectant in the dental industry and is potentially being developed for use as an antimicrobial agent for ocular infections. We conducted susceptibility testing of Alx using MIC testing, MPC testing, and time-kill assays against Gram-positive and Gram-negative pathogens. MIC testing showed that Alx is more active against Gram-positive pathogens than Gram-negative pathogens and showed better activity than the fluoroquinolones Gfx, Mfx, and levofloxacin (Lfx) against MRSA. The MPC values measured for MRSA and MSSA against Alx were non-reproducible using the traditional MPC method. Using the microbroth dilution MPC method, MPC90 values were found to be 32 fold higher than the MIC90 values. If the experimentally determined MPC values are true MPC values, initial MPC testing indicates that Alx may have a high likelihood for selecting for resistance, however, if the MPC values are not accurate it may be necessary to modify the MPC protocol in order to complete MPC testing of Alx against MRSA and MSSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) measured bactericidal activity (> 3 log10-reduction) against MRSA, MSSA, SP, and PA.

Master of Science / Food Science Institute / Sara E. Gragg / Leafy greens have been recognized as vehicles for transmission of foodborne pathogens and an effective pre-harvest intervention to control them is currently lacking. After harvest, lettuce is often subjected to chlorinated water to reduce the microbial load in the water and on the lettuce tissue. While moderately effective, there is also a need for improved postharvest interventions. The purpose of Objective I was to 1) determine potassium bisulfate efficacy at reducing populations of Escherichia coli (E. coli) and Listeria innocua (L. innocua) when applied pre-harvest to lettuce, and 2) assess the impact on product quality at harvest. Potassium bisulfate reduced E. coli populations on inoculated lettuce by 1.32 log₁₀ CFU/g (P=0.0002) and L. innocua by 1.18 log₁₀ CFU/g (P=0.0017). No detectable differences were observed in color (P>0.05); however, brown spots were observed on various leaves sprayed with potassium bisulfate. The purpose of Objective II was to employ a blend of benzalkonium chloride, acetic acid, and methyl paraben (BAM) as a postharvest wash on romaine and iceberg lettuce and to 1) determine efficacy at reducing populations of Listeria monocytogenes (L. monocytogenes), E. coli O157:H7 and Salmonella, 2) measure changes in aerobic bacteria throughout the shelf life, and 3) quantify benzalkonium chloride and methyl paraben residues post-washing. To quantify efficacy of BAM reducing pathogenic bacterial populations, fresh-cut romaine and iceberg lettuce were inoculated with L. monocytogenes, E. coli O157:H7, or Salmonella and washed in BAM at concentrations of 0%, 1%, 2% or 3% for one or five minutes. When plated on recovery media, contact time and wash concentration was not significant (P>0.05) for Salmonella on either product. Concentration was significant (P=0.0189) for L. monocytogenes on romaine; however, the greatest reduction observed was <1.0 log₁₀ CFU/g. The 3% wash significantly reduced E. coli O157:H7 on romaine by 1.75 log₁₀ CFU/g, which is 0.66 log₁₀ CFU/g better than the 0% wash. Following washing, wash water was analyzed and data demonstrate that all wash concentrations significantly (P≤0.05) reduced each foodborne pathogen by >2.0 log₁₀ CFU/g in the wash water. To quantify benzalkonium chloride and methyl paraben residues, as well as changes in aerobic bacteria and product quality, fresh-cut romaine and iceberg lettuce were subjected to a 1 minute wash in BAM at concentrations of 0%, 1%, 2%, or 3% and immediately sampled to determine aerobic populations and product quality. Concentrations 0% and 2% were also packaged into retail storage bags and sampled on days 0, 3, 5, and 7. Residues were quantified on these days as well. On day 0, aerobic populations did not vary according to wash concentration (P>0.05). With regards to shelf-life data, the 2% wash significantly reduced (P=0.0203) aerobic bacteria on romaine lettuce; however, no significant difference was observed on iceberg lettuce (P=0.0819). With regards to overall visual appearance of romaine or iceberg lettuce, no significant difference was detected between 0% and 2% BAM washes for each day throughout the shelf-life study (P>0.05). Methyl paraben and benzalkonium chloride residues were <5.0 and <10.0 ppm, respectively, on both products on each sampling day.

Dans les environnements naturels, industriels ou médicaux, les microorganismes sont majoritairement présents en étant associés aux surfaces dans des communautés hautement organisées appelées biofilms. Ces édifices biologiques constituent une stratégie de survie étonnement efficace témoignant d’une grande capacité de résistance à différent stress environnementaux tels que les traitements de nettoyage et de désinfection. L’impact des biofilms d’un point de vue sanitaire est donc considérable du fait qu’ils permettent la persistance et la transmission de germes pathogènes dans l’environnement. Dans ce contexte, ce travail de thèse avait pour objectif une meilleure compréhension des phénomènes limitant l’efficacité de désinfectants au sein des biofilms en s’appuyant notamment sur des techniques innovantes d’imagerie de fluorescence non-invasive. Le but final étant d’apporter des éléments utiles à l’optimisation des traitements de désinfection. Dans une première partie, une méthode d’investigation structurale à haut-débit par microscopie confocale a été développée et utilisée pour étudier la diversité architecturale des biofilms bactériens formés par un large panel de souches. Cette étude nous a permis d’identifier des souches d’intérêt en termes de structures de biofilms formés pour la suite du travail. Nous avons notamment pu mettre en évidence la capacité de B. subtilis à former des structures importantes et avec une architecture spécifique dans un système immergé. Dans une deuxième partie, les dynamiques d’action spatiotemporelles de désinfectants ont été visualisées dans les biofilms de souches de P. aeruginosa ou B. subtilis par des approches de microscopie confocale de fluorescence en temps réel. L’utilisation de cette technique nous a permis de mettre en évidence les difficultés de pénétration du chlorure de benzalkonium au sein des structures formées par différentes souches de P. aeruginosa. La corrélation des paramètres cinétiques d’inactivation et des données obtenues par la caractérisation biochimique de la matrice suggère un rôle majeur des substances extracellulaires dans la limitation de pénétraton du désinfectant. Nous avons également pu montrer une résistance marquée du biofilm formé par une souche de B. subtilis isolée d’un dispositif médical à l’acide péracétique, à la concentration et au temps d’utilisation du biocide dans le milieu médical. De plus, les structures tridimensionnelles formées par cette souche étaient capables de protéger le pathogène Staphylococcus aureus dans un biofilm mixte vis-à-vis du même traitement soulignant l’importance des interactions multi-espèces dans la résistance des bactéries aux désinfectants et la persistance de pathogènes dans nos environnements. / In natural, industrial or medical environments, microorganisms are present mainly in being associated with surfaces in highly organized communities called biofilms. These biological structures cosntitute a surprisingly effective survival strategy showing a large ability to withstand environmental stresses such as cleaning and disinfection treatments. Therefore, biofilms have a considerable impact on public health because they allow the persistence and transmission of pathogens. In this context, this work aimed to better understand the phenomena limiting the effectiveness of disinfectants in biofilms noticeably by using innovative imaging fluorescence non-invasive techniques. The ultimate goal was to provide data which can help to optimize disinfection treatments. In the first part, a high-throughput structural method based on confocal microscopy was developed and used to study the architectural diversity of bacterial biofilms formed by a wide range of strains. This study allowed us to identify strains of interest in terms of biofilm structure for the second part of the work. In particular, we demonstrated the ability of B. subtilis to form protruding structures with a specific architecture in a submerged system. In the second part, the spatiotemporal dynamic of the action of disinfectants were visualized in the biofilms of P. aeruginosa or B. subtilis strains by a time-lapse fluorescence confocal microscopy method. Using this technique, we showed that benzalkonium chloride encountered problems of penetration in the biofilms formed by P. aeruginosa strains. The correlation of kinetic inactivation parameters and data obtained by the characterization biochemical matrix suggested a key role of extracellular substances in the penetration limitations of the disinfectant. We also observed a pronounced resistance of the biofilm formed by a strain of B. subtilis isolated from a medical device to peracetic acid at the in-use concentration and time of biocide in medical areas. In addition, three-dimensional structures formed by this strains afforded protection to the pathogen Staphylococcus aureus in mixed biofilm against the same treatment This point highlights the importance of multi-species interactions in bacterial resistance to disinfectants and in the persistence of pathogens in our environments.

Modifying eye drops using hyaluronan leads to an increased retention time on the eye surface. Long-lasting observations of the stability of two different eye drops, Opthalmo-Septonex and Visine Classic, after the addition of hyaluronan with varying concentration are presented in this work. The interactions of hyaluronan with the eye drops, or their components benzalkonium chloride and tetryzoline, are investigated by monitoring particle sizes and the charge of the molecules. Additionally, rheological examinations of the samples are done and the mucoadhesion index is determined by using the mucine method.

Le glaucome primitif à angle ouvert est une neuropathie optique rétinienne dont le premier facteur de risque, l'hypertonie intraoculaire, est causé par une dégénérescence du trabéculum dont les mécanismes demeurent inconnus. Ainsi, les traitements actuels ne ciblent pas la trabéculopathie originelle, ce qui pourrait expliquer leur inefficacité parfois observée. En outre, ces traitements contiennent un conservateur, le chlorure de benzalkonium (BAC), qui est responsable d'une inflammation iatrogène de la surface oculaire mise en cause dans l'inobservance thérapeutique. Les chimiokines, cytokines initialement décrites du fait de leurs propriétés chimioattractantes, sont également impliquées dans le contrôle de la viabilité cellulaire et du microenvironnement tissulaire. Dans cette thèse, nous démontrons l'existence d'une balance au niveau trabéculaire entre le système CXCL12/CXCR4 aux effets protecteurs et le système SDF-1(5-67)/CXCR3 proapoptotique. Nous rapportons de façon originale que l'utilisation in vivo d'un antagoniste non-peptidique spécifique de CXCR3 diminue la pression intraoculaire en restaurant la fonction trabéculaire dans un modèle animal de glaucome. En parallèle, nous révélons que les cellules épithéliales conjonctivales exposées au BAC attirent certaines populations leucocytaires via CX3CL1/CX3CR1, montrant ainsi que ce système est impliqué dans le trafic immunitaire au sein de la surface oculaire. De façon originale, les chimiokines apparaissent comme un système inédit de régulation de l'environnement trabéculaire et de la surface oculaire, constituant ainsi de nouvelles cibles thérapeutiques spécifiques

<i>Salmonella enterica serovar Enteritidis</i> is an important biofilm-forming food-borne pathogen. This study examined the adaptive responses of <i>Salmonella serovar Enteritidis</i> biofilms to different environmental conditions such as flow velocity and benzalkonium chloride (BC) treatment. The influence of a 10-fold difference in nutrient laminar flow velocity on the dynamics of biofilm formation and protein expression profiles was compared. The mode of development and architecture of low-flow and high-flow biofilms were distinct. Exopolymer composition of the two biofilms was also different. However, no major shift in protein expression was seen between the biofilms, nor were there any stress response proteins involved. The biofilms altered their architecture in response to flow, presumably assuming a structure that minimized overall biofilm stress. An empirically-determined shear-inducing flow was applied on high-flow biofilms, fractionating the biofilms into shearable and non-shearable regions. Length:width indices of cells from the two biofilm regions, as well as planktonic cells from biofilm effluent and continuous culture were determined to be 3.2, 2.3, 2.2, and 1.7, respectively. Expression of proteins involved in cold-shock response, adaptation, and broad regulatory functions in the shearable region, and expression of protein involved in heat-shock response and chaperonin function in the non-shearable region indicated that the physiological status of cells in two biofilm regions was also distinct. The development of biofilm adaptive resistance to BC was then examined. Adapted biofilms survived a lethal BC challenge and re-grew, whereas unadapted biofilms did not. Proteins up-regulated following adaptation included those involved in energy metabolism, amino acid and protein biosynthesis, nutrient-transportation, adaptation, detoxification, and 1,2-propanediol degradation. A putative universal stress protein was also up-regulated. Cold-shock response, stress response, and detoxification are suggested to play roles in adaptive resistance to BC. Functional differences in adaptive response and survival of plankonic and biofilm cells adapted to BC were also studied. The proportion of BC-adapted biofilm cells that survived a lethal BC exposure and heat-shock was significantly higher than that of BC-adapted planktonic cells. Enhanced biofilm-specific up-regulation of various proteins, coupled with alterations in cell surface roughness and shift in fatty acid composition are proposed to function in the enhanced survival of BC-adapted biofilm cells, relative to BC-adapted planktonic cells.<p>It is concluded that biofilms adapt to the stress conditions by means of community, cellular, and sub-cellular level responses. These adaptive responses help the biofilms to enhance their ability for survival in the nature, especially those formed in critical environments such as healthcare facilities, the food industry, and households.

<i>Salmonella enterica serovar Enteritidis</i> is an important biofilm-forming food-borne pathogen. This study examined the adaptive responses of <i>Salmonella serovar Enteritidis</i> biofilms to different environmental conditions such as flow velocity and benzalkonium chloride (BC) treatment. The influence of a 10-fold difference in nutrient laminar flow velocity on the dynamics of biofilm formation and protein expression profiles was compared. The mode of development and architecture of low-flow and high-flow biofilms were distinct. Exopolymer composition of the two biofilms was also different. However, no major shift in protein expression was seen between the biofilms, nor were there any stress response proteins involved. The biofilms altered their architecture in response to flow, presumably assuming a structure that minimized overall biofilm stress. An empirically-determined shear-inducing flow was applied on high-flow biofilms, fractionating the biofilms into shearable and non-shearable regions. Length:width indices of cells from the two biofilm regions, as well as planktonic cells from biofilm effluent and continuous culture were determined to be 3.2, 2.3, 2.2, and 1.7, respectively. Expression of proteins involved in cold-shock response, adaptation, and broad regulatory functions in the shearable region, and expression of protein involved in heat-shock response and chaperonin function in the non-shearable region indicated that the physiological status of cells in two biofilm regions was also distinct. The development of biofilm adaptive resistance to BC was then examined. Adapted biofilms survived a lethal BC challenge and re-grew, whereas unadapted biofilms did not. Proteins up-regulated following adaptation included those involved in energy metabolism, amino acid and protein biosynthesis, nutrient-transportation, adaptation, detoxification, and 1,2-propanediol degradation. A putative universal stress protein was also up-regulated. Cold-shock response, stress response, and detoxification are suggested to play roles in adaptive resistance to BC. Functional differences in adaptive response and survival of plankonic and biofilm cells adapted to BC were also studied. The proportion of BC-adapted biofilm cells that survived a lethal BC exposure and heat-shock was significantly higher than that of BC-adapted planktonic cells. Enhanced biofilm-specific up-regulation of various proteins, coupled with alterations in cell surface roughness and shift in fatty acid composition are proposed to function in the enhanced survival of BC-adapted biofilm cells, relative to BC-adapted planktonic cells.<p>It is concluded that biofilms adapt to the stress conditions by means of community, cellular, and sub-cellular level responses. These adaptive responses help the biofilms to enhance their ability for survival in the nature, especially those formed in critical environments such as healthcare facilities, the food industry, and households.

High strength wastewater (HSWW) generated in food processing industries is characterized by high organic carbon and nitrogen content, and thus high oxygen demand. Biological nitrogen removal (BNR) is a technology widely used for the treatment of HSWW. Food processing facilities practice sanitation to keep food contact surfaces clean and pathogen-free. Benzalkonium chlorides (BACs) are cationic quaternary ammonium antimicrobial compounds (QACs) common in industrial antimicrobial formulations. BAC-bearing wastewater generated during sanitation applications in food processing facilities is combined with other wastewater streams and typically treated in BNR systems. The poor selectivity and target specificity of the antimicrobial BACs negatively impact the performance of BNR systems due to the susceptibility of BNR microbial populations to BAC. Objectives of the research were: a) assessment and quantification of the inhibitory effect of QACs on the microbial groups, which mediate BNR in HSWW treatment systems while treating QAC-bearing HSWW; b) evaluation of the degree and extent of the contribution of QAC adsorption, inhibition, and biotransformation on the fate and effect of QACs in BNR systems. A laboratory-scale, multi-stage BNR system was continuously fed with real poultry processing wastewater amended with a mixture of three benzalkonium chlorides. The nitrogen removal efficiency initially deteriorated at a BAC feed concentration of 5 mg/L due to complete inhibition of nitrification. However, the system recovered after 27 days of operation achieving high nitrogen removal efficiency, even after the feed BAC concentration was stepwise increased up to120 mg/L. Batch assays performed using the mixed liquors of the BNR system reactors, before, during, and post BAC exposure, showed that the development of BAC biotransformation capacity and the acquisition of resistance to BAC contributed to the recovery of nitrification and nitrogen removal. Kinetic analysis based on sub-models representing BNR processes showed that BAC inhibition of denitrification and nitrification is correlated with BAC liquid-phase and solid-phase concentrations, respectively. Simulations using a comprehensive mathematical BNR model developed for this research showed that BAC degradation and the level of nitrification inhibition by BAC were dynamic brought about by acclimation and enrichment of the heterotrophic and nitrifying microbial populations, respectively. The fate and effect of BACs in the BNR system were accurately described when the interactions between adsorption, inhibition, and resistance/biotransformation were considered within the conditions prevailing in each reactor. This work is the first study on the fate and effect of antimicrobial QACs in a continuous-flow, multi-stage BNR system, and the first study to quantify and report parameter values related to BAC inhibition of nitrification and denitrification. Results of this study enable the rational design and operation of BNR systems for the efficient treatment of QAC-bearing wastewater. The outcome of this research provides information presently lacking, supporting the continuous use of QACs as antimicrobial agents in food processing facilities, when and where needed, while avoiding any negative impacts on biological treatment systems and the environment.