Bacterial pathogens transmitted by the fecal-oral route endure several stresses during survival/growth in host and non-host environments. For foodborne pathogens, understanding the range of phenotypic responses to stressors and the environmental factors that impact survival can provide insights for the development of control measures. For example, the gastrointestinal system presents acidic, osmotic, and cell-envelope stresses and low oxygen levels, but Listeria monocytogenes can withstand these stresses, causing illnesses in humans. Survival/growth characteristics may differ among L. monocytogenes strains under these stressors due to their genetic diversities. Our knowledge of such phenotypic characteristics under bile and salt stresses are inadequate. In this dissertation, variation in growth characteristics was observed among L. monocytogenes strains under bile and osmotic stresses with no evidence of cross-protection, but rather an antagonistic effect was observed with the formation of filaments when pre-exposed to 1% bile and treated with 6% NaCl. This shows that variation in stress adaptability exists among L. monocytogenes strains with the ability to form filaments under these conditions. Similarly, Salmonella survival in soil is dependent on several factors, such as soil, amendment types, moisture, irrigation, and desiccation stress. In this study, the use of HTPP (heat-treated poultry pellets) was investigated as a soil amendment in the survival/growth of Salmonella in soil extracts mimicking runoff events, and in soil cultivated with spinach plants to assess its safety for use for an organic fertilizer. The presence of HTPP in soil increased S. Newport survival with a greater likelihood of its transfer to and survival on spinach plants. Increased microbial loads and rpoS mutant showed decreased growth/survival in soil extracts, however, rpoS was not important for survival in soil under the tested conditions showing possible lack of desiccation stress. These results show that HTPP provided nutrients to the Salmonella for increased growth and survival in soil extracts and soil, respectively, which show that the use of treated BSAAO to soils may still require appropriate mitigation to minimize Salmonella Newport contamination of leafy greens in the pre-harvest environment. Overall, the results in this study increased our understanding of L. monocytogenes and Salmonella phenotypic adaptation to stressful environments.

The opportunistic pathogen Legionella is the leading cause of reported waterborne disease outbreaks in the United States. Legionella can thrive under the warm, stagnant, low-disinfectant conditions characteristic of premise (i.e., building) plumbing systems, making it challenging to identify effective interventions for its control. Copper (Cu) is a promising antimicrobial that can be dosed directly to water via copper-silver ionization systems or released naturally via corrosion of Cu pipes to help control growth of Legionella and other pathogens. However, prior research has shown that Cu does not always reliably control Legionella and sometimes seems to even stimulate its growth. A deeper understanding of the mechanistic effects of Cu on Legionella, at both pure-culture and real-world scales, is critical in order to inform effective controls for Legionella. The overarching objective of the research embodied by this dissertation was aimed at elucidating the chemical and microbial interactions in premise plumbing that govern efficacy of Cu for Legionella control through a series of complementary bench-, pilot-, and field-scale studies. A critical review and synthesis of the literature identified important knowledge gaps in relation to antimicrobial effects of Cu. In particular, changes in the pH, phosphate corrosion control, and rising levels of natural organic matter (NOM) in distributed water are predicted to be important controlling factors. The type of sacrificial anode rod material employed in water heaters was also identified as an underappreciated factor, which directly affects pH, evolution of hydrogen gas as a microbial nutrient, and release of metals (such as aluminum) that bind copper. Microbiological factors: including growth phase of Legionella (e.g., exponential or stationary), strain-specific Cu tolerance, background microbiome composition, and the possibility that viable but non-culturable (VBNC) Legionella might still cause human disease, were also identified as major confounding factors. These knowledge gaps are addressed from various dimensions across each chapter of the dissertation. The effects of pH, orthophosphate corrosion inhibitor concentration, and NOM were examined in bench-scale pure culture experiments over a range of conditions relevant to drinking water. Cupric ions and antimicrobial effects were drastically reduced at pH >7.5, especially in the presence of phosphate, which precipitates copper, or NOM, which complexes the Cu in a form that is less bioavailable. Chick-Watson disinfection models indicated that soluble Cu was the most robust correlate with observed Cu antimicrobial effects across a range of tested waters. This new knowledge suggests that measuring soluble rather than total Cu would be much more informative to guide practitioners in dosing. The research also demonstrated that changes in pH or orthophosphate that have been made to control corrosion over the last few decades, have significantly altered Cu chemistry in buildings, undermining antimicrobial capacity and increasing likelihood of Legionella growth. Pilot-scale experiments confirmed that soluble Cu is an effective indicator of Cu antimicrobial capacity, even in more complex environments represented by realistic hot water plumbing systems. In particular, dosing of orthophosphate, which is widely added by drinking water utilities to control corrosion, directly reduces soluble copper and overall antimicrobial capacity. In some cases, Cu added together with orthophosphate apparently promoted the growth of Legionella, providing an example of at least one circumstance where Cu addition can induce interactive effects that elevate Legionella compared to a control system with trace Cu. It was also demonstrated for the first time that different water heater sacrificial anode types are subject to different corrosion processes, which indirectly influence Cu antimicrobial capacity. Specifically, aluminum ions released from aluminum anode corrosion at 1 mg/L can form an Al(OH)3 gel, which can remove >80% of the soluble Cu from water and reduce Cu antimicrobial effects by >2-log at pH=7. Corrosion from magnesium anodes was found to dramatically increase the pH from 6.8 to >8, which correspondingly reduces Cu antimicrobial capacity. Cu deposition further increased the anode corrosion rate and promoted evolution of hydrogen gas, which is a potent electron donor that stimulates autotrophic microbial growth especially with a magnesium anode. Electric powered anodes did not release metals or alter pH and thus did not diminish Cu antimicrobial capacity. Still, across the pilot-scale experiments, even very high levels of Cu (>1.2 mg/L) at low pH (<7) failed to fully eradicate culturable Legionella. The much lower than expected antimicrobial efficacy of Cu in the pilot-scale hot water plumbing systems was found to be partially explained by the properties of the strain that colonized the systems. Based on fitting the data to a Chick-Watson disinfection model, the outbreak-associated strain that was inoculated into the systems was estimated to be 7 times more tolerant to Cu compared to the common lab strain applied in the bench-scale tests. Further, exponential growth phase L. pneumophila were found to be 2.5 times more susceptible to Cu relative to early stationary phase cultures. It is important to also recognize that, in the pilot-scale systems, drinking water biofilms and the amoeba hosts that colonize them can further shield Legionella from the antimicrobial effects of Cu. Application of shotgun metagenomic sequencing offered the opportunity to more deeply examine the response of Legionella and other pathogens to Cu dosed to the pilot-scale hot water systems in the context of the broader microbiome. It was found that metagenomic analysis provided a sensitive indication of the bioavailability of Cu to the broader microbial community inhabiting the hot water systems, further confirming that the outbreak-associated strain of Legionella that colonized the rigs was relatively tolerant of Cu. Functional gene analysis provided further insight into the mechanistic action of Cu, suggesting multi-modal action of both membrane damage and interruption of nucleic acid replication. The metagenomic analysis further revealed that protozoan host numbers tended to increase in the pilot-scale systems with time, and this could also increase the potential for Legionella proliferation with time. Additional pure culture studies aiming to further assess the mechanistic action of Cu provided strong evidence that Cu can induce a VBNC state for Legionella. This is a concern, given that other studies have indicated that VBNC Legionella are still capable of causing legionellosis. However, VBNC cells are not detected by conventional culturing. Multiple lines of evidence supported the conclusion that Cu induced a VBNC state for Legionella, including membrane integrity, enzyme activity, ATP generation, and Amoebae resuscitation assays applied to two different strains of L. pneumophila. After exposure to Cu, up to a 5-log (99.999%) reduction in culturable Legionella was observed, whereas corresponding reductions in the various viability measures were only by <1-log (90%). In other words, conventional culturing may miss up to 99.99% of the Legionella that is still capable of causing disease. To our knowledge, this is the first study that has assessed the potential for Cu-induced VBNC Legionella. Additional research is needed to further quantify the contribution of VBNC status to challenges in effective Cu-based control of Legionella in premise plumbing. This research further examines, for the first time, the proteomic response of Legionella to Cu, comparing both presumably VBNC and culturable cells. Functional annotation of proteins that were differentially produced by the cells in response to Cu addition revealed that VBNC L. pneumophila modulated its proteome to favor cell membrane- and motility-related proteins, while reducing production of other proteins related to primary metabolism compared to culturable cells. These observations are consistent with the metagenomic-based observations and support the hypothesis that Cu inactivates cells by damaging the cell membrane. The findings also confirmed reduced general cell metabolism that is characteristic of a VBNC state. This dissertation highlights the important and complex effects of Cu on Legionella growth in potable water systems as modified by water chemistry, water heater anode type, characteristics of the surrounding microbiome, and Legionella strain characteristics and growth status. The findings raise important questions about how to measure disinfectant efficacy and provide fundamental new knowledge that can help to better optimize the application of Cu as an antimicrobial to drinking water systems and better protect public health. / Doctor of Philosophy / The opportunistic pathogen Legionella is the leading cause of reportable waterborne disease outbreaks in the United States. Legionella is capable of growing in drinking water plumbing systems in homes, hospitals, hotels, and other buildings. Legionella is spread by inhaling tiny droplets of water that are suspended in the air when using the water, for example when showering, resulting in a severe and deadly form of pneumonia called Legionnaires' Disease. Copper is a promising antimicrobial that can be dosed directly into a building's water system by installing a copper-silver ionization system. There is also interest in understanding whether copper released naturally from copper pipes could help control Legionella. However, prior research indicates that copper sometimes inhibits, sometimes has no effect, and sometimes even seems to stimulate Legionella growth. The purpose of this dissertation was to better understand how the chemical properties of the drinking water, such as pH, presence of corrosion inhibitors that are commonly added to the water by utilities, and natural organic matter impact the ability of copper to kill Legionella. Impacts of the design of the drinking water system were also examined, for example, the material used in the anodes of water heaters to prevent corrosive damage to other system components was hypothesized to change the water chemistry in such a way that could also interfere with copper disinfection. Finally, the effect of the strain of Legionella, its growth phase (exponential or stationary), and culturability status (culturable versus viable but non-culturable) was also examined. Experiments were conducted over a wide range of conditions, from bench-scale pure culture experiments of a few days to full-scale plumbing systems over a period of 3.5 years. The complementary approaches maximize the strength of scientific conclusions about approaches that can more effectively control Legionella. Several discoveries were made as a result of this research that can help to improve the use of copper for controlling Legionella in drinking water systems. In particular, it was found that it is best to keep the pH less than 7.5, because above pH 7.5 copper reacts with orthophosphate corrosion inhibitor or natural organic matter in the water in a manner that makes it less potent to microbes. Through disinfection modeling it was found that soluble copper was the best predictor of the ability to kill Legionella. Therefore, it is recommended from this research that practitioners should monitor soluble copper instead of total copper for the purpose of assessing Legionella control. From the pilot-scale experiments, it was further found that the type of anode installed in the water heater can affect the ability of copper to kill Legionella. Magnesium anodes performed the worst, likely because they raised the pH above the recommended level of 7.5. Aluminum anodes were also a problem because aluminum ions released form an aluminum hydroxide gel that can remove more than 80% of the soluble copper from water. Electric powered anodes did not reduce copper antimicrobial effects by raising pH or forming a gel, but they are much less commonly used. A surprising finding throughout this study was that very high levels of copper (>1.2 mg/L) were required to measurably reduce Legionella in the pilot-scale systems. In the pure culture experiments, it was found that the outbreak-associated strain from Quincy, IL, that was inoculated into the system was highly copper tolerant. This demonstrated that the strain of Legionella that colonizes a particular drinking water system could be the reason why copper is sometimes less effective. Pure culture experiments also found that stationary phase Legionella are more difficult to kill than exponential phase Legionella, which could explain some discrepancies among lab studies reported in the literature. A particularly noteworthy discovery of this research was that copper can make it appear as if Legionella have been killed, because the traditional culture media indicate that there is no growth on the Petri dish; however, they are in fact still alive and capable of causing human disease. This is referred to as a "viable but non-culturable (VBNC)" state. The VBNC state of Legionella was confirmed using an array of techniques (membrane integrity, enzyme activity, ATP generation, and amoebae resuscitation) for two strains of L. pneumophila. We also examined how VBNC Legionella cellular functions were impacted by copper using whole cell proteome, i.e., analysis of all of the proteins extracted from Legionella. Copper induced VBNC Legionella modulated its proteome to favor cell membrane and motility related proteins, and reduced others related to primary metabolism compared with culturable cells. These results were consistent with those obtained via shotgun metagenomic analysis of the microbial community DNA in the pilot-scale water systems. Given the potential for VBNC organisms to prevail in systems disinfected with copper, it is recommended to supplement culture-based monitoring with molecular-based monitoring, e.g., with quantitative polymerase chain reaction. This dissertation highlights the important and complex effects of copper on Legionella growth in potable water systems. The findings help to inform guidance on how to improve the antimicrobial effect of copper, through adjusting the water chemistry, selecting appropriate water heater anodes, and optimizing the overall hot water system design. The dissertation also helps to inform improved strategies for monitoring the efficacy of copper for killing Legionella in real-world systems. Overall, the findings can help to improve policy and practice aimed at reducing the incidence of Legionnaires' Disease and protecting public health.

L'état viable mais non cultivable (VNC) a été étudiée en détail chez les bactéries. En revanche,l'état VNC chez d'autres micro-organismes, y compris en particulier les eucaryotes, a reçubeaucoup moins d'attention.Pour fournir des preuves concluantes de l'existence d'un état VNC chez la levure, en particulierchez S. cerevisiae, la capacité des différentes souches de S. cerevisiae à devenir viable et noncultivable après un stress sulfite avec différentes concentrations de SO2 a été étudiée parcytométrie de flux (CMF) en utilisant une sonde fluorescente comme un marqueur de viabilité(fluorescéine diacétate (FDA)) et par étalement sur milieu de culture. La capacité des cellules àrécupérer leur cultivabilité après l’élimination du stress en augmentant le pH du milieu a étéétudiée. Pour confirmer l’existence de l'état VNC, le temps de génération de cellules VNC aprèsl’élimination du stress a été comparé aux cellules cultivables et viables dans des conditions deculture identiques. En outre, la comparaison des différentes phases du cycle cellulaire descellules sortent de l'état VNC et les cellules en état VNC a été réalisée par CMF. Par ailleurs,l'implication du gène SSU1 codant pour la pompe SO2 dans l'état VBNC a été étudiée.Après l'application du stress, la comparaison entre la population cultivable déterminée sur milieude culture et la population viable évaluée par FCM met en évidence la présence de cellulesviables mais non cultivables. L'augmentation du pH du milieu permet aux cellules de S.cerevisiae viables mais non cultivables à redeviennent cultivables. Le temps de génération, decellules cultivées dans les mêmes conditions que celles rencontrées au moment de la sortie del’état VNC, est comparé au temps de sortie calculé au cours de la reprise de la cultivabilité. Ladifférence entre ces deux paramètres observés affirme que le temps mis par les cellules poursortir de l’état VNC n’était pas caractéristique d’une multiplication cellulaire.Finalement nous avons étudié l'implication du SSU1 dans l'état VNC. Les résultats montrent quele SSU1 n’est pas impliqué dans le maintien de l'état VNC chez S. cerevisiae / The viable but not culturable (VBNC) state has been studied in detail in bacteria. Bycontrast the VBNC state in other microorganisms, including particularly eukaryotes, has receivedmuch less attention. However, it has been suggested that in wine, Brettanomyces yeast cells mayenter a Viable But Not Culturable State, in particular in the presence of high, sulfur dioxide(SO2) concentration.To provide conclusive evidences for the existence of a VBNC state in yeast, especially in S.cerevisiae as a model organism, the capacity of different S cerevisiae strains to become viableand not cultivable after a sulfite stress with various concentrations of SO2 was studied by flowcytometry (FCM) using fluorescent probe as a viability marker (Fluorescein diacetate (FDA))and by plating on culture medium. The ability of cells to recover cultivability after stress removalby increasing the pH medium was investigated. To confirm the VBNC state, the rate ofgeneration of VBNC cells after stress removal was compared to cultivable and viable cells insame culture conditions. In addition, the comparison of different cell cycle phases of cells exitingthe VBNC state and cells in VBNC state was performed by FMC. Moreover, the involvement ofSSU1 gene coding for the SO2 pump in VBNC state was studied.After stress application, comparison between cultivable population determined on culturemedium and viable population assessed by FCM demonstrated the presence of the viable cellswhich became uncultivable after 24 to 48 hours depending on the strains under study. Increasingthe pH medium allows the viable but uncultivable S. cerevisiae cells to become cultivable again.The generation rate of cells exiting VBNC state was not consistent with growth of residualculturable cells, which support a true VBNC state. The absence of cell proliferation, the stabilityof the population during the increase of the cultivability and the decrease in esterase activity forVBNC cells allows us to conclude the presence of the VBNC state in S. cerevisiae in correlationwith the VBNC state definition.In order to determine whether SSU1 gene, encoding a sulfite pump efflux, was involved inVBNC, we compare a wild type S. cerevisiae strain to its nul mutant Δ ssu1. Our resultsdemonstrate that SSU1 gene does not seem to be involved in VBNC phenotype

Salmonella enterica serotypes have been linked to outbreaks associated with low water activity foods. The ability of biofilm forming pathogens, such as Salmonella, to survive thermal and chemical processes is improved; it is unclear if biofilms will also improve survival to desiccation and gastric stresses. The purpose of this study was to quantify the effect of physiological state (planktonic versus biofilm) and prior exposure to desiccation on Salmonella survival and gene expression after passage through an in-vitro digestion model. Cells of Salmonella enterica serotype Tennessee were deposited onto membranes for planktonic cells or on glass beads to create biofilms. The cells were subsequently dried at room temperature and stored in dried milk powder (aw = 0.3) for up to 30 days. Salmonella survival was quantified by serial dilution onto brilliant green agar before desiccation, after desiccation, after 1-day storage and after 30-day storage. At each sampling both physiological states were tested for survival through a simulated gastrointestinal system. RNA was extracted at the identical time points and relative gene expression determined for genes associated with stress response (rpoS, otsB), virulence (hilA, hilD, invA, sipC) and a housekeeping gene 16S rRNA using quantitative real-time PCR. The physiological state and length of storage effected the survival and gene expression of Salmonella within the desiccated milk powder environment and after passage through an in-vitro digestion system (p<0.05). Larger numbers of S. Tennessee were recovered by plate counts for biofilm cells, compared to planktonic cells. However, the numbers of 16S rRNA gene copies were not significantly different suggesting entry of S. Tennessee into a viable but non-culturable state. Prolonged storage in dry milk powder was not associated with increased cross-protection to gastric stress. Increased expression of stress response genes rpoS and otsB correlated with survival, indicating cross protection of low water activity and acid stress. Increased expression of virulence-associated genes was seen in cells exposed to short periods of dry storage, suggesting an increased virulence potential. / Master of Science in Life Sciences

La capacité de Listeria monocytogenes à adhérer et à persister sur les surfaces dans les ateliers agro-alimentaires pendant de longues périodes malgré l’application correcte et fréquente des opérations de nettoyage & désinfection (N&D), peut être responsable de la contamination croisée de produits alimentaires par simple contact avec une surface contaminée. De plus, il est important de prendre en considération la présence potentielle de bactéries viables non cultivables (VNC) qui ne sont pas détectées par les méthodes culturales utilisées lors de la recherche de L. monocytogenes dans un prélèvement de surface.Un des objectifs a été de vérifier si les milieux de culture conventionnels fréquemment utilisés pour les expérimentations en laboratoire étaient représentatifs des conditions environnementales des ateliers agro-alimentaires et de définir pour la suite des expérimentations un milieu synthétique pouvant modéliser une souillure alimentaire. Pour assurer la sécurité sanitaire des aliments et préserver la santé des consommateurs, optimiser les opérations d’hygiène est une nécessité. Dans ce but, la survie des cellules adhérentes de L. monocytogenes soumises à des conditions simulant en laboratoire l’alternance des phases de N&D et de production a été étudiée. Plusieurs facteurs pouvant influencer la survie de ces cellules ont été pris en considération et combinés par un plan d’expériences fractionnaire : la souillure alimentaire, la souche de L. monocytogenes, le matériau de surface, la présence d’une souche de Pseudomonas fluorescens qui favorise l’adhésion de L. monocytogenes et le scénario de l’opération d’hygiène. Dans les industries agro-alimentaires, les bactéries sont soumises à de nombreux stress, nécessitant pour survivre l’expression et l’induction appropriées de gènes et de protéines de réponse aux stress. Les mécanismes de réponse aux stress mis en place par les cellules de L. monocytogenes adhérentes après stress hydrique (dessiccation), stress chimique (N&D) et stress froid ont été déterminés par une analyse transcriptomique.Selon les critères étudiés (croissance, forces d’adhésion, distribution spatiale et état physiologique des cellules adhérentes), il s’avère que le milieu TSB/5m peut modéliser le jus de saumon fumé. Au contraire, aucun des milieux synthétiques testés ne permet de remplacer l’utilisation de l’exsudat de viande pour mimer les conditions de terrain. Concernant les facteurs influençant la survie de L. monocytogenes, les opérations d’hygiène impactent uniquement la cultivabilité des cellules adhérentes ; toutefois, le séchage des surfaces permet une optimisation de l’efficacité de la procédure de N&D, d’autant plus lorsqu’il est réalisé quotidiennement / Despite the correct and frequent application of cleaning & disinfection (C&D), Listeria monocytogenes has the ability to adhere to and persist on surfaces in food processing plants for long periods. Consequently, L. monocytogenes may be responsible for cross-contamination of food through contact with contaminated surfaces. In addition, it is important to consider the potential presence of viable but non culturable (VBNC) bacteria that are not detected by the microbiological methods used in the research of L. monocytogenes in a surface sample.One aim was to verify whether the conventional culture media commonly used for laboratory experiments were representative of the environmental conditions of food processing plants and define for further experiments a synthetic medium that can model a food soil. Optimization of the C&D procedures is a necessary to ensure food safety and protect the health of consumers. For this purpose, the survival of L. monocytogenes adherent cells subjected to laboratory conditions simulating alternating phases of C&D and production was studied. Several factors that may influence the survival of these cells were considered and combined with a fractional factorial design, including: the food soil, the strain of L. monocytogenes, the surface material, the presence of a strain of Pseudomonas fluorescens, which enhances the adhesion of L. monocytogenes, and the scenario of the sanitation procedure. Moreover, in the food industry, bacteria can be subject to many stresses; thus, the expression and induction of appropriate genes and stress response proteins are required for survival. The stress response mechanisms set up by the adherent L. monocytogenes cells after hydric stress (i.e. desiccation), chemical stress (i.e. C&D), and cold stress were determined by a transcriptomic analysis.According to the criteria studied (i.e. growth, adhesion forces, spatial distribution, and physiological state of adherent cells), TSB/5m medium was found to be a sufficient model for smoked salmon juice. In contrast, none of the tested synthetic media can replace the use of meat exudate to mimic field conditions. About the factors influencing the survival of L. monocytogenes, C&D procedures were found to only impact the culturability of adherent cells; however, drying surfaces were found to optimize the effectiveness of the C&D procedures, especially when performed daily

Density of fecal coliforms (FC) such as Escherichia coli is the most commonly used indicator of fecal pathogen content of biosolids. When biosolids are disposed off or used for soil amendment, they pose public health risks. So far anaerobic digesters have been considered to be an effective treatment option for pathogen and FC reduction in biosolids. However, recent studies revealed that there is a significant re-growth and reactivation of indicator organisms in biosolids upon dewatering by centrifugation. Although the exact mechanism of FC reactivation is yet to be understood, a few extensive recent studies strongly suggest that FC go into a viable but non-culturable (VBNC) state during anaerobic digestion. Therefore, quantitative detection of live cells among the total in biosolids samples, without using culturing-based approaches, is highly critical from a public health risk assessment perspective. Since recent investigations proved the significant re-growth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches for the detection of live bacteria. Using selective nucleic acid intercalating dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detect and quantify the viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity. They intercalate in the DNA via photo-inducible azide groups and in turn inhibit DNA amplification during PCR reactions. PMA has been successfully used in different studies and microorganisms but it has not been evaluated sufficiently for the complex environmental samples such as biosolids. In this study Escherichia coli ATCC 25922 and uidA gene were used as model organism and as target sequence respectively in absolute quantification method with real-time PCR. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results of this study conclusively demonstrated that PMA-modified PCR could be successfully applied to the biosolids when total suspended solid (TSS) concentration is 2000 mg/L or below.

Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.

Ce travail de thèse concerne l'étude de l'écologie microbienne d'un atelier de découpe de viande bovine, dans le but de mieux comprendre la persistance bactérienne, c'est-à-dire, la présence répétée d'un même clone bactérien pendant une longue période malgré l'application bien conduite et régulière du nettoyage et de la désinfection (N-D). Des prélèvements par « chiffonnages » multiples de surfaces d'équipements ont été réalisés lors de trois campagnes de prélèvement espacées les unes des autres d'au moins six mois. Les prélèvements ont été réalisés sur un tapis convoyeur en polychlorure de vinyle (PVC) et sur des machines éplucheuses en acier inoxydable avant et après N-D. Nous avons quantifié les cellules totales (les cellules vivantes et les cellules mortes) par PCR quantitative en temps réel (qPCR), les cellules viables par EMA-qPCR, et les UFC (provenant de cellules cultivables) par dénombrement après incubation à 25°C sur gélose tryptone soja. Les résultats montrent qu'avant N-D, les cellules totales (en moyenne 5,6 – exprimé en log10 cellules/cm2 – sur PVC et 4,7 sur acier inoxydable) sont plus nombreuses que les cellules viables (4,5 sur PVC et 4,4 sur acier inoxydable) lesquelles sont plus nombreuses que les UFC (3,8 sur PVC et 2,9 sur acier inoxydable). Le N-D entraîne moins d'une réduction décimale (RD) des populations à l'exception des UFC sur acier inoxydable qui subissent 1,5 RD en moyenne. Ce dernier chiffre s'explique par des forces d'adhésion faibles. L'étude de la diversité des bactéries cultivables montre que sur un total de 51 genres identifiés, 13 seulement sont retrouvés lors des trois campagnes de prélèvements. Les isolats de ces 13 genres représentent 75, 72 et 62% des isolats des campagnes1, 2 et 3 respectivement. Parmi ces isolats, les plus fréquents sont (par ordre décroissant du nombre d'isolats) : Pseudomonas, Staphylococcus, Microbacterium, Acinetobacter, Chryseobacterium, Psychrobacter et Kocuria. Le génotypage d'isolats de 3 genres majoritaires (Staphylococcus, Pseudomonas et Acinetobacter) montre qu'une seule souche, Staphylococcus equorum, est sans aucun doute persistante. L'ensemble de ces observations montrent que l'écosystème varie d'une campagne à une autre. Ces modifications de la diversité bactérienne reflèteraient les modifications de flores des viandes traitées dans l'atelier, qui ont des origines multiples. En outre, il apparaît que, contrairement à ce qui est généralement admis, les bactéries à coloration de Gram négative cultivables sont plus facilement inactivées par le N-D que les bactéries à coloration de Gram positive. L'étude de l'écosystème par PCR-DGGE a permis d'identifier sept genres bactériens et montre que les espèces dominantes sont toutes sous forme vivante, autrement dit, aucune des espèces dominantes n'a été détectée uniquement sous forme de cellules mortes. Sur les sept genres identifiés six sont des Gram – dont majoritairement les genres Acinetobacter, Pseudomonas et Psychrobacter. Cette dominance montre que le N-D permet une forte perte de cultivabilité des bactéries Gram – mais qu'une grande partie n'est pas détachée. La dominance des bactéries Gram – observée par PCR-DGGE masque les staphylocoques qui ne sont pas détectés alors qu'ils sont majoritaires parmi la flore cultivable. Seul un genre bactérien, Propionibacterium, est identifié par PCR-DGGE uniquement mais il n'est trouvé qu'à une seule campagne et uniquement sur l'acier inoxydable avant N-D. En conclusion, l'avancée majeure de ce travail est la mise en évidence qu'une proportion importante de bactéries survit après les opérations très poussées de N-D mais pour une période transitoire. / The aim of this work is to acquire a better knowledge of the microbial ecology of a beef processing plant to understand bacterial persistence, e.g. the presence of a clone isolated several times on several visits in the same processing plant despite regular Cleaning and disinfection (C&D) procedures. Successive swabbing were performed on a PVC conveyor belt and skinning machines made of stainless steel before and after C&D during three surveys in minimal 6 month-intervals. Total cells (live and dead cells) were quantified using real-time quantitative PCR (qPCR). Viable cells e.g. cells with intact membrane, were assessed using Ethidium Monoazide combined with qPCR. Culturable cells (CFU) were determined from plate counts on Tryptone Soy Agar. Before C&D, total cells (5.6 log cells/cm2 and 4.7 log10 cells/cm2 on PVC and stainless steel respectively) were greater than viable cells (4.5 and 4.4 log10 cells/cm2) and CFUs (3.8 and 2.9 log10 CFU/cm2). C&D lead to less than 1 log10 reduction in bacterial populations except for CFU counts on stainless steel where a 1.5 log reduction is observed. This result is highlighted by the weak attachment strengths observed on stainless steel for CFUs. Identification of the culturable microbiota revealed that out of 51 genera identified, 13 were found at all the visits. These genera represented 75, 72 and 62% of the total isolates. The most frequently identified bacteria were Pseudomonas, Staphylococcus, Microbacterium, Acinetobacter, Chryseobacterium, Psychrobacter and Kocuria. Molecular typing of three dominant genera (Staphylococcus, Pseudomonas and Acinetobacter) showed that only one strain, Staphylococcus equorum, was persistent in the premises. Our results show that the microbial ecosystem is different from one survey to another, which reflect the various geographical origins of meat products. Contrary to widespread belief, Gram negative strains were more easily eliminated by C&D than Gram positive strains. Furthermore, the microbial diversity assessed by PCR-DGGE allowed the identification of 7 genera. This molecular approach showed that dominant species are all in a viable state: none of these species was solely detected in a dead state. Of the 7 genera identified, 6 were Gram negative, Acinetobacter, Pseudomonas and Psychrobacter being predominant. This result highlights that C&D induced the lost of culturability of Gram negative bacteria although a high proportion was not detached from the surface. The predominance of Gram negative microflora, didn’t allow the detection of staphylococcal isolates which were numerous in the culturable microflora. One genus, Propionibacterium, isolated in one survey on stainless steel before C&D was only identified by PCR-DGGE. In conclusion, the present study has demonstrated that a large proportion of bacteria can survive drastic cleaning and disinfection for a transient period.