Tesi sul tema "VBNC"

Legionella pneumophila, l’agent responsable d’une pneumonie sévère atypique chez l’homme, appelée maladie du légionnaire, est ubiquitaire dans les environnements aquatiques. Les traitements généralement appliqués dans les réseaux d’eau afin d’éradiquer cette bactérie sont souvent suivis d’une recolonisation rapide de cette bactérie. Un des facteurs pouvant expliquer cette recolonisation peut être l’état Viable Non Cultivable (VBNC). L’objectif de nos travaux a été d’étudier la survie de L. Pneumophila après des traitements oxydant et thermique et notamment d’étudier l’état VBNC. Dans un premier temps nous avons réalisé un traitement monochloramine et un traitement thermique sur une suspension de légionelles. Nous avons observé que le traitement par 20 mg/L de monochloramine ainsi que celui à 57,5°C, conduisaient à la formation de VBNC chez L. Pneumophila. De plus ces bactéries peuvent persister dans cet état pendant au moins 169 jours après traitement. Dans un second temps, une étude partielle des protéines exprimées à l’état VBNC a été menée. Nous avons montré que l’expression de plusieurs protéines, impliquées dans les voies de production d’énergie, de synthèse protéique et dans la virulence, était augmentée d’un facteur supérieur ou égale à deux par comparaison avec des bactéries ayant subies un stress monochloramine ou une carence en nutriments. Dans un troisième temps, la recherche d’un facteur de ressuscitation Rpf a été entreprise chez L. Pneumophila. Un gène Rpf-like, possédant 33% d’identité et 51% de similarité avec celui de Salmonella, a été découvert. Ce gène a été cloné mais la protéine recombinante obtenue n’a pas conduit à un retour à la cultivabilité des cellules
Legionella pneumophila , the causative agent of an atypical severe pneumonia in human called Legionnaires’ disease, is ubiquitous in aquatic environments. The treatments usually applied to eradicate this bacterium are often followed by a quick new contamination. One of the causes that could explain this colonisation could be the Viable But NonCulturable state (VBNC). The aim of this work was to study the survival of L. Pneumophila after oxidative and heat treatments and then to study the VBNC. First, we have treated Legionella suspension with monochloramine and heat. We have observed that 20 mg/L monochloramine treatment and 57. 5°C treatment lead to the VBNC state. Moreover, these bacteria could persist in this state during at least 169 days. Secondly, we have realised a partial study of Legionella proteins expressed in the VBNC state. Several proteinsexpressions, implicated in energy production, protein synthesis and virulence, were induced more than 2-fold in comparison with bacteria stressed by monochloramine or in starvation. Finally, we have search for a Resuscitation-promoting-factor Rpf in L. Pneumophila genome. An rpf-like gene, showing 33% of identity and 51% similarity with Salmonella rpf was found. This gene has been cloned but the recombinant protein obtained didn’t lead to a recovery of legionella culturability

La listériose est une maladie rare mais grave d’origine alimentaire provoquée par Listeria monocytogenes. En raison de sa capacité de survie importante dans les sols, la présence de cette bactérie dans les effluents d’élevages porcins destinés à être épandus constitue un problème de santé publique. L’un des facteurs pouvant expliquer la persistance de L. monocytogenes dans l’environnement est sa capacité à entrer dans un état viable mais non cultivable (VNC). Nos travaux avaient pour objectif, d’une part de suivre le comportement de L. monocytogenes dans les effluents d'élevages porcins (lisier et effluent de lagune) et notamment les formes VNC, et d’autre part d’étudier son adaptation lors de son transfert dans les effluents de lagune et dans le sol. Dans un premier temps, nous avons optimisé les conditions de la qPCR couplée au propidium monoazide (qPCR-PMA) afin d’adapter cette méthode au dénombrement des formes VNC de L. monocytogenes dans le lisier et l’effluent de lagune. Dans un second temps, nous avons comparé par méthode culturale, qPCR-PMA et qPCR, la survie de deux souches de L. monocytogenes RifR de sérogroupes IIb et IVb inoculées dans deux lisiers et dans deux effluents de lagune incubés à 8°C et 20°C. Malgré leur origine et leur sérotype différents, les deux souches ont présenté une survie similaire dans toutes les conditions testées. La survie des deux souches a été affectée par la température (une persistance plus élevée a été observée à 8°C) et par l’origine des effluents. Cette étude a mis en évidence que L. monocytogenes était capable d’entrer dans l’état VNC dans les lisiers et les effluents de lagune indépendamment de la température. Les formes VBNC qui représentaient 83 à 99,8% des bactéries viables après 60 jours d’incubation, sont apparues dès les premières heures de contact avec les effluents. Leur proportion, plus élevée en début d’expérience dans les lisiers que dans les effluents de lagune, était cependant du même ordre de grandeur dans les deux types de matrices après 60 jours. Afin de mieux comprendre l’adaptation de L. monocytogenes lors de son transfert dans l’effluent de lagune et dans le sol, nous avons comparé le transcriptome par la technologie RNA-seq de la souche CIP 110868, isolée d’un lisier, inoculée dans des extraits stériles d’effluent de lagune et de sol. L’analyse du transcriptome a été réalisée à T0 (génome de référence), après 20 minutes et 20 heures d’incubation. L’analyse par enrichissement fonctionnel a révélé des modifications transcriptomiques dès les 20 premières minutes d’incubation dans les deux matrices. Une augmentation du taux de transcrit de gènes impliqués dans le transport de protéines et de sucres a été observée. Le taux de transcrit des gènes contrôlés par le facteur sigmaB est augmenté indiquant la mise en place d’une réponse aux stress osmotiques et thermiques. De plus, l’adaptation de la souche CIP 110868 dans les extraits de sol et d’effluent de lagune s’est accompagnée d’une augmentation au cours du temps des taux de transcrit des gènes impliqués dans la virulence et des gènes sous le contrôle du régulateur prfA
Listeriosis is a rare but serious illness caused by foodborne Listeria monocytogenes. Because of its important survival capacity in the soil, the presence of this bacteria in effluent from pig farms intended to be spread is a public health problem. One factor that may explain the persistence of L. monocytogenes in the environment is its ability to enter a viable but non-culturable state (VNC). Our studies were aimed, firstly to monitor the behavior of L. monocytogenes in effluent from pig farms (manure and lagoon effluent) including VNC forms, and also to study its adaptation at transfer to the lagoon effluent and soil. First, we have optimized the conditions of qPCR coupled with propidium monoazide (qPCR-LDCs) to adapt this method to count VNC forms of L. monocytogenes in the manure and lagoon effluent. Secondly, we compared by cultivation method, qPCR and qPCR-LDCs, the survival of two strains of L. monocytogenes RifR IIb and IVb serogroups manure inoculated in two and two lagoon effluent incubated at 8 ° C and 20 ° C. Despite their origin and different serotype, the two strains showed a similar survival in all conditions tested. The survival of both strains was affected by the temperature (higher persistence was observed at 8 ° C) and the origin of the effluent. This study showed that L. monocytogenes was able to enter the VNC state in manure and lagoon effluent regardless of temperature. VBNC forms which accounted for 83 99.8% of viable bacteria after 60 days of incubation, appeared in the early hours of contact with effluent. Their proportion, higher at the beginning of experience in the manure lagoon in the effluent, however, was of the same order of magnitude in the two types of matrices after 60 days. To better understand the adaptation of L. monocytogenes when transferred into the lagoon effluent and soil, we compared the transcriptome by RNA-Seq technology CIP 110868 strain, isolated from a slurry inoculated in sterile effluent lagoon and extracts of soil. Transcriptome analysis was performed at T0 (reference genome), after 20 minutes and 20 hours of incubation. Functional enrichment analysis revealed transcriptomic changes during the first 20 minutes of incubation in both matrices. An increase in the gene transcript levels involved in the transport of proteins and sugars was observed. The rate of transcribed genes controlled by the sigmaB factor is increased indicating the establishment of a response to osmotic and thermal stress. In addition, the adaptation of the CIP 110868 strain in soil extracts and lagoon effluent was accompanied by an increase in time of transcript levels of genes involved in virulence and gene under the control the prfA regulator

Listeria monocytogenes est une bactérie pathogène intracellulaire facultative responsable d’une pathologie grave, la listériose. Si de très nombreux travaux ont permis de caractériser les mécanismes de virulence de cette bactérie, il existe peu de données sur les mécanismes conduisant au portage asymptomatique de L. monocytogenes dans les hôtes mammifères. L’un de ces mécanismes pourrait être une phase de persistance intracellulaire. Lors d’infections prolongées de cellules épithéliales humaines en culture, comme des hépatocytes et des cellules de trophoblastes, L. monocytogenes change de mode de vie intracellulaire. Après la phase active de dissémination de cellule en cellule, les bactéries arrêtent de polymériser l’actine et se retrouvent piégées dans des vacuoles à simple membrane marquées par la protéine endosomale LAMP1. L’objectif de ma thèse était de caractériser ces « Listeria-Containing Vacuoles » (LisCVs). Nous avons montré que les LisCVs sont des compartiments acides, partiellement-dégradatifs, marquées par la protéase lysosomale cathépsine D. Leur formation coïncide avec la disparition du facteur de polymérisation d’actine ActA de la surface bactérienne et la capture des bactéries cytosoliques dépourvues d’actine par des membranes cellulaires. Dans ces compartiments, les bactéries entrent en croissance ralentie ; une sous-population résiste aux stress et peut survivre au-delà de trois jours d’infection. L’utilisation de la gentamicine lors du protocole d’infection n’est pas responsable de la formation des LisCVs. Cependant, cet antibiotique permet la sélection des bactéries vacuolaires, en inhibant spécifiquement la croissance des bactéries cytosoliques. La formation des LisCVs n’est pas spécifique des souches de laboratoire. Toutefois l’efficacité du phénomène pourrait diverger selon les séquençotypes des souches de L. monocytogenes. Les bactéries vacuolaires ont la capacité de sortir des vacuoles et de retourner vers un état motile et réplicatif, après le passage des cellules infectées. Lorsque l’expression du gène actA reste inactive, comme dans les mutants ∆actA, des formes de Listeria vacuolaires persistent dans les cellules hôtes dans un état viable mais non cultivable (VBNC). Ces formes VBNC peuvent être transmises au cours des divisions des cellules hôtes. L’ensemble de ces résultats révèle une nouvelle phase de persistance dans le processus infectieux intracellulaire de L. monocytogenes lors des infections prolongées de certaines cellules épithéliales. Cette propriété pourrait contribuer au portage asymptomatique de ce pathogène dans les tissus épithéliaux, allonger la période d'incubation de la listériose, et rendre les bactéries tolérantes à l’antibiothérapie
Listeria monocytogenes is a facultative intracellular pathogenic bacterium responsible for a serious disease, listeriosis. Although much work has been done to characterize the virulence mechanisms of this bacterium, there is little data on the mechanisms leading to the asymptomatic carriage of L. monocytogenes in mammalian hosts. One of these mechanisms could be a phase of intracellular persistence. During prolonged infections of human epithelial cells in culture, such as hepatocytes and trophoblast cells, L. monocytogenes changes its intracellular lifestyle. After the active phase of cell-to-cell spread, the bacteria stop polymerizing actin and become trapped in single-membrane vacuoles labeled with the endosomal protein LAMP1.The aim of my thesis was to characterize these "Listeria-Containing Vacuoles" (LisCVs). We have shown that LisCVs are acidic, partially degradative compartments, labeled by the lysosomal protease cathepsin D. Their formation coincides with the disappearance of actin polymerization factor ActA from the bacterial surface and the capture of actin-free cytosolic bacteria by cell membranes. In these compartments, bacterial growth is slowed; a subpopulation is resistant to stress and can survive beyond three days of infection. The use of gentamicin during the infection protocol is not responsible for the formation of LisCVs. However, this antibiotic allows selection of vacuolar bacteria, by specifically inhibiting the growth of cytosolic bacteria. The formation of LisCVs is not specific to laboratory strains. However, the efficacy of the phenomenon could diverge according to the sequence types of L. monocytogenes strains. Vacuolar bacteria have the ability to exit the vacuoles and return to a motile and replicative state during the subculture of infected cells. When expression of the actA gene remains inactive, as in ΔactA mutants, vacuolar Listeria forms persist in host cells in a viable but non-culturable (VBNC) state. These VBNC forms can be transmitted during host cell divisions. All these results reveal a new phase of persistence in the intracellular infectious process of L. monocytogenes during prolonged infections of a subset of epithelial cells. This property could contribute to asymptomatic carriage of this pathogen in epithelial tissues, extend the incubation period of listeriosis, and make bacteria tolerant to antibiotic therapy

The application of animal manures to agricultural land as a soil organic amendment has been identified as an important route by which foodborne pathogens can enter the human food chain. Knowledge of the presence and incidence of key foodborne pathogens in manure is a vital first step in the establishment of sound and effective guidelines for management and prevention of contamination by manure. The work described in this thesis attempts to determine the persistence of pathogens when raw manures are directly applied to agricultural soils, and treated under thermophilic composting conditions. Results from this study indicated that the current conditions suggested by typical food safety guidelines are sufficient to reduce the population of enteric bacteria to levels that minimise risks associated with culturable cells in raw manure and finished compost. However, E. coli cells have the potential to enter a viable but non-culturable (VBNC) state and are undetected by culture-based monitoring methods, thus providing a false impression of the innate risk of the product. Prc, bamB and tolA, which are responsible for stabilising the cell membrane, were found to be essential genes required for surviving heat treatment at 55°C. In addition, the presence of relA and oxyR suggested that E. coli may use the VBNC state as an adaptive strategy for long-term survival to withstand multiple stresses, including heat stress. Entering the VBNC state with a strengthen cell envelope may help E. coli to survive prolonged heating during standard composting conditions. Successful resuscitation from the VBNC state was achieved in the presence of cell-free supernatant from actively growing E. coli MG1655. These results underline the importance of considering VBNC cells when evaluating the sanitary effect of the composting process. VBNC cells in composts could facilitate the persistence of pathogens in manure-amended soil and thus pose a risk of microbial contamination of fresh produce.

Produce-associated outbreaks of foodborne pathogens such as Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica are rising in prominence among outbreaks of foodborne disease. Testing for foodborne pathogens by the agricultural industry relies heavily on culture-based techniques, excluding detection of viable but nonculturable (VBNC) pathogens. Here, a detection method is used that facilitates the use of qPCR on the complex environmental matrices of soil. Targeting the tir gene of E. coli O157, detection of the pathogen in peat-based compost and sand is achieved to a sensitivity of 10 CFU/g. When applied to pristine soil, 310 copies of the gene were detected. Further analysis using PNA-FISH and cell elongation determined the presence of 205 VBNC E. coli O157 cells per gram of soil sample. Resuscitation of the pathogen was achieved through prolonged enrichment in selective media. Decontamination of fresh produce using chlorine washes was simulated using L. monocytogenes and S. enterica serovar Thompson adhered to spinach leaves, resulting in complete VBNC induction of viable cells following two minutes exposure to 50 ppm and 100 ppm chlorine respectively. The infectivity of these chlorine induced VBNC pathogens was assessed in vivo using Caenorhabditis elegans as an animal model. VBNC L. monocytogenes retained its infectivity and caused a significant lifespan reduction (p=0.0064). Together, these data provide evidence of the presence and induction of VBNC foodborne pathogens throughout the food production chain, and determines that VBNC L. monocytogenes presents a threat to food safety.

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Diversos estados fisiológicos têm sido descritos para células de levedura durante o cultivo, como células lesionadas, células mortas e células viáveis, mas não cultiváveis (VBNC). No entanto, pouca atenção ainda é dada à formação de células viáveis, mas não cultiváveis em leveduras e seus impactos sobre o processo de fermentação. O estado VBNC, também conhecido como estado de dormência celular pode ser definido como um estado fisiológico em que os micro-organismos não são capazes de crescer em meios bacteriológicos, mas apresentam características de células vivas. Fatores químicos e ambientais têm sido relatados para induzir um estado VBNC, incluindo a falta de nutrientes, temperaturas extremas, concentrações osmóticas e oxigênio. As células de levedura no estado VBNC podem exercer influencias sobre o rendimento de biocombustível gerado no final do processo. As leveduras industriais utilizadas neste estudo foram coletadas em uma usina sucroalcooleira localizada na cidade de Américo Brasiliense-SP-Brasil. Para fornecer evidências da existência de um estado VBNC em leveduras investigou-se a capacidade das células ao entrarem no estado VBNC, aplicando estresse térmico (40°C) e osmótico (30% de sacarose). Populações viáveis foram monitoradas utilizando o corante azul de metileno, e as populações cultiváveis foram identificadas por semeadura em meio de cultura. Todos os procedimentos foram realizados em triplicata, para todas as amostras e os dados amostrais foram submetidos à análise de variância (ANOVA). Após 48 horas no período de tempo em estresse, a comparação entre as células viáveis e as células cultiváveis demonstrou a presença de células viáveis, mas não cultiváveis. Além disso, a remoção do estresse permitiu que as células fossem novamente capazes de crescer, fornecendo evidências da existência de um estado VBNC em leveduras.
Several physiological conditions have been described for yeast cells during cultivation, such as injured cells, dead cells and viable cells, but not culturable (VBNC). However, little attention is still given to the formation of viable cells, but not cultivable in yeast and its impact on the fermentation process. The VBNC state, also known as state of numbness cell can be defined as a physiological condition in which the microorganisms are not able to grow on bacteriological media, but that present characteristics of living cells. Chemical and environmental factors have been reported to induce VBNC state, including a lack of nutrients, temperature extremes, osmotic concentration and oxygen. The yeast cells in the VBNC state may influence the yield of biofuel generated at the end of the process. The yeasts used in this study were collected in a sugarcane mill located in Américo Brasiliense-SP-Brazil. To provide evidence of the existence of a state in yeast VBNC investigated the ability of cells to enter the VBNC state, applying heat stress (40°C) and osmotic (30% sucrose). Viable populations were monitored using the methylene blue dye, and cultivated populations were identified by inoculation in culture. All procedures were performed in triplicate for all samples and sample data were submitted to analysis of variance (ANOVA). After 48 hours the time of stress, the comparison between viable cells and culturable cells showed the presence of viable cells, but not culturable. Moreover, removal of stress the cells were allowed to grow again able, providing evidence for the existence of a VBNC state in yeast.

Orientador: Cecília Laluce
Banca: Maria Lucia Gonsales da Costa Araujo
Banca: Juliana Audi Giannoni
Resumo: Diversos estados fisiológicos têm sido descritos para células de levedura durante o cultivo, como células lesionadas, células mortas e células viáveis, mas não cultiváveis (VBNC). No entanto, pouca atenção ainda é dada à formação de células viáveis, mas não cultiváveis em leveduras e seus impactos sobre o processo de fermentação. O estado VBNC, também conhecido como estado de "dormência celular" pode ser definido como um estado fisiológico em que os micro-organismos não são capazes de crescer em meios bacteriológicos, mas apresentam características de células vivas. Fatores químicos e ambientais têm sido relatados para induzir um estado VBNC, incluindo a falta de nutrientes, temperaturas extremas, concentrações osmóticas e oxigênio. As células de levedura no estado VBNC podem exercer influencias sobre o rendimento de biocombustível gerado no final do processo. As leveduras industriais utilizadas neste estudo foram coletadas em uma usina sucroalcooleira localizada na cidade de Américo Brasiliense-SP-Brasil. Para fornecer evidências da existência de um estado VBNC em leveduras investigou-se a capacidade das células ao entrarem no estado VBNC, aplicando estresse térmico (40°C) e osmótico (30% de sacarose). Populações viáveis foram monitoradas utilizando o corante azul de metileno, e as populações cultiváveis foram identificadas por semeadura em meio de cultura. Todos os procedimentos foram realizados em triplicata, para todas as amostras e os dados amostrais foram submetidos à análise de variância (ANOVA). Após 48 horas no período de tempo em estresse, a comparação entre as células viáveis e as células cultiváveis demonstrou a presença de células viáveis, mas não cultiváveis. Além disso, a remoção do estresse permitiu que as células fossem novamente capazes de crescer, fornecendo evidências da existência de um estado VBNC em leveduras.
Abstract: Several physiological conditions have been described for yeast cells during cultivation, such as injured cells, dead cells and viable cells, but not culturable (VBNC). However, little attention is still given to the formation of viable cells, but not cultivable in yeast and its impact on the fermentation process. The VBNC state, also known as state of "numbness cell" can be defined as a physiological condition in which the microorganisms are not able to grow on bacteriological media, but that present characteristics of living cells. Chemical and environmental factors have been reported to induce VBNC state, including a lack of nutrients, temperature extremes, osmotic concentration and oxygen. The yeast cells in the VBNC state may influence the yield of biofuel generated at the end of the process. The yeasts used in this study were collected in a sugarcane mill located in Américo Brasiliense-SP-Brazil. To provide evidence of the existence of a state in yeast VBNC investigated the ability of cells to enter the VBNC state, applying heat stress (40°C) and osmotic (30% sucrose). Viable populations were monitored using the methylene blue dye, and cultivated populations were identified by inoculation in culture. All procedures were performed in triplicate for all samples and sample data were submitted to analysis of variance (ANOVA). After 48 hours the time of stress, the comparison between viable cells and culturable cells showed the presence of viable cells, but not culturable. Moreover, removal of stress the cells were allowed to grow again able, providing evidence for the existence of a VBNC state in yeast.
Mestre

Following active growth, the aquatic gram-negative rod Prolinoborus fasciculus (Aquaspirillum fasciculus) exhibited a mass conversion from its culturable rod form to a nonculturable coccoid form. Chloramphenicol did not prevent the conversion. Attempts to obtain variants that would not convert to the coccoid form were unsuccessful. Although the coccoid form fluoresced with acridine orange, agarose gel electrophoresis revealed extensive ribosomal RNA degradation. Poly-Ã -hydroxybutyrate, abundant in the vegetative rods, was not detectable in the coccoid cells. The results suggest that the coccoid form of A. fasciculus is a degenerative form rather than part of a life cycle.
Master of Science

Staphylococcus aureus è uno dei più importanti patogeni umani. Può causare patologie di diversa gravità, tossinfezioni alimentari ed infezioni sistemiche. Molti ceppi sono capaci di sviluppare biofilm, cioè una matrice esopolisaccaridica (slime) adesa ad una superficie, all’interno della quale rimangono inglobati i microrganismi produttori. Gli stafilococchi produttori di biofilm sono frequentemente coinvolti nelle infezioni associate all’uso di dispositivi medici impiantabili, come cateteri venosi centrali. Tali infezioni risultano difficili da eradicare in quanto i batteri risultano protetti sia dai sistemi di difesa dell’ospite che dalla terapia antibiotica. All’interno del biofilm, al contempo, i microrganismi trovano condizioni ambientali sfavorevoli come la carenza di ossigeno e di nutrienti in grado di indurre la trasformazione in forme quiescenti, tra cui lo stato VBNC (Viable But NonCulturable). Le forme VBNC rappresentano una strategia di sopravvivenza delle cellule batteriche non sporulanti sottoposte a stress ambientali, come variazioni di temperatura, luce solare, concentrazione di ossigeno e carenza di nutrienti. Sono caratterizzate da assenza di coltivabilità nei classici terreni di coltura, attività metabolica ridotta, modificazioni morfo-funzionali e dalla capacità di recuperare la capacità replicativa (resuscitation), quando le condizioni tornano favorevoli. Sono state descritte per diverse specie, soprattutto ambientali, ed anche per patogeni umani, ma la possibilità per gli stafilococchi di entrare in uno stato VBNC non è stata mai dimostrata. Questa attività di ricerca è stata rivolta a valutare la possibilità che S. aureus possa entrare in uno stato vitale ma non coltivabile, capace di resuscitation, quando si trova all’interno di biofilm. A tale scopo sono stati allestiti modelli di biofilm in vitro del ceppo S. aureus 10850 (forte produttore di slime) e in parallelo di S. aureus ATCC 25923 (non produttore di slime) e sottoposti a condizioni di stress, in particolare carenza di nutrienti associata o meno alla presenza di vancomicina, synercid o daptomicina (antibiotici utilizzati per contrastare le infezioni gravi sostenute da S. aureus) in concentrazioni pari o superiori (4x, 8x, 16x) alla minima inibente (MIC). La presenza di cellule vitali nei campioni non più coltivabili è stata valutata mediante microscopia in epifluorescenza e citometria a flusso dopo colorazione live-dead con i fluorocromi SYBR Green I e Ioduro di propidio; la capacità di espressione genica delle forme VBNC rilevate è stata analizzata mediante saggi di RT-PCR e Real-time RT-PCR specifici per il 16SrDNA batterico e per i geni glsF e nuc, il primo housekeeping specie-specifico per S. aureus e il secondo codificante per un fattore di virulenza. E’ stata inoltre valutata la possibilità delle forme VBNC di recuperare l’attività replicativa (resuscitation) ricreando condizioni di crescita favorevoli. I risultati ottenuti hanno dimostrato che (i) S. aureus è in grado di entrare in uno stato VBNC, ma solo all’interno di biofilm. Infatti forme VBNC sono state ottenute solo con il ceppo produttore di biofilm S. aureus 10850, mentre non è stato possibile rilevare la presenza di cellule vitali ma non coltivabili di S. aureus ATCC 25923; ii) condizioni di starvation in presenza di basse concentrazioni di vancomicina, synercid o daptomicina possono indurre lo stato VBNC; (iii) nello stato non coltivabile le cellule di S. aureus mantengono un’attività metabolica, come dimostrato dall’espressione di tutti i geni analizzati e che (iv) le forme VBNC di S. aureus sono in grado di riacquisire la capacità replicativa se poste in presenza di terreni di coltura addizionati di metaboliti essenziali per lo sviluppo cellulare, come il piruvato di sodio o il filtrato di una brodocoltura del ceppo wild-type S. aureus 10850. Questi risultati indicano che le forme VBNC di stafilococco potrebbero avere un ruolo rilevante in patologia umana in quanto capaci di rimanere latenti, e non evidenziabili con i comuni test colturali, all’interno del biofilm, ed essere causa di infezioni ricorrenti in seguito al recupero della piena attività metabolica e della conseguente capacità di moltiplicarsi.
Staphylococcus aureus is a major human pathogen. Its toxins can cause disease of varying severity, including food poisoning and a number of infections. Several strains form biofilms, i.e. multicellular aggregates encased in an exopolysaccharide matrix (slime) produced by the bacteria themselves. Biofilm-producing staphylococci are frequently involved in infections associated with the use of medical devices (e.g. catheter-related bloodstream infections). These biofilms protect bacteria from both host defences and antibiotic therapy, resulting in considerable difficulty in eradication. Bacteria residing in biofilms need to cope with adverse environmental conditions, such as oxygen and nutrient deficiency, that can induce a “dormant” state, like the Viable But NonCulturable (VBNC) state. VBNC forms are a survival strategy of non-sporulating bacteria subjected to environmental stress (e.g. changes in temperature or visible light, oxygen or nutrient deficiency). The VBNC state, a potential reservoir of pathogenic microorganisms, has been described for several gram-positive and negative species, both environmental and clinical. This survival strategy is characterized by the absence of culturability on culture media, low metabolic activity, and morpho-functional changes. Moreover, optimal growth conditions can “resuscitate” true VBNC cells, restoring replication capacity and full metabolic activity. The ability of staphylococci to enter the VBNC state has never been demonstrated. This work aimed to establish (i) whether S. aureus biofilms can give rise to VBNC forms that can resuscitate in suitable environmental conditions, and (ii) the role of different stressors in inducing the VBNC state. Cultures of S. aureus 10850 (a strong slime producer) and S. aureus ATCC 25923 (a non-slime producer) were grown on filter membranes under conditions suitable for biofilm development (i.e. presence of glucose) and exposed to different stress conditions, i.e. depletion of nutrients, in the presence or absence of minimum inhibitory concentrations (MICs) and of 4-fold, 8-fold and 16-fold MICs of vancomycin, synercid and daptomycin, three antibiotics used to treat severe Gram-positive infections. Culturability was monitored at intervals of three days. After cells had reached the nonculturable state they were subjected to live-dead staining using SYBR Green I and propidium iodide and their viability was tested by epifluorescence microscopy and flow cytometry. Gene expression was analyzed using RT-PCR and Real-time RT-PCR assays targeting bacterial 16SrDNA, glsF (a housekeeping, species-specific gene) and nuc (the gene coding for a thermostable nuclease) genes. VBNC forms were then assessed for their ability to recover replication activity through exposure to favourable growth conditions. The results of these experiments showed that (i) S. aureus is capable of entering the VBNC state provided that cells are contained in biofilm, because viable but nonculturable cells of S. aureus ATCC 25923 (the non-slime producer) were never recovered; (ii) VBNC S. aureus forms can be induced by starvation and antibiotics; (iii) VBNC S. aureus cells retain their metabolic activity, as demonstrated by positive gene expression assays; and (iv) VBNC S. aureus cells exposed to suitable culture conditions can revert to the culturable state (resuscitation). VBNC staphylococci may have a significant clinical role, since they can pass from the dormant state, where they are undetectable by routine methods within biofilm, to full metabolic and replication activity, causing severe infections.

Conversion of Campylobacter upsaliensis to the nonculturable but viable coccoid form was characterized. Chloramphenicol did not prevent the conversion. Severe decreases in isocitrate dehydrogenase activity and oxygen uptake and extensive degradation of ribosomal RNA suggest that the coccoid form is a degenerative form rather than part of a life cycle. The autolysins of spiral and coccoid forms of C. upsaliensis were also studied. Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of C. upsaliensis could not be demonstrated by native (nondenaturing) PAGE. Autolysins were detected, however, by using denaturing SDS-PAGE gels containing either purified E. coli peptidoglycan or whole cells of Micrococcus luteus as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained E. coli peptidoglycan, 14 autolytic bands were detected ranging from 200 kDa to 12 kDa. In similar gels containing whole cells of M. luteus , only a single band appeared having a molecular weight of 34 kDa. This band corresponded to one of the bands present in the gels containing E. coli peptidoglycan. This common autolysin was isolated by adsorbing it from C. upsaliensis lysates onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing E. coli peptidoglycan. The 34 kDa autolysin differed from a single 51 kDa autolysin unique to the M. luteus cells. The 34 kDa autolysin was isolated from an SDS-PAGE gel and was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34 kDa autolysin to have 67% identity with a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular weight of 34 kDa, and thus it seems unlikely that the 34 kDa autolysin was derived from any of the other autolysins that were detected.
Ph. D.

Legionella pneumophila, l’agent responsable de la légionellose est transmissible à l’Homme par les aérosols environnementaux et infecte les macrophages pulmonaires. Après l’exposition à différents stress L. pneumophila est capable de d’entrer dans un état Viable Non Cultivable (VBNC) qui semble être une stratégie de survie. L’objectif de nos travaux était d’étudier l’état VBNC de différentes souches de L. pneumophila après des traitements thermique et chimique et d’évaluer le pouvoir infectieux des formes VBNC envers les macrophages et les cellules épithéliales alvéolaires. Nous avons étudié les profils physiologiques de L. pneumophila de trois souches différentes. Les résultats montrent que pour chaque souche 3 populations peuvent être identifiées, les légionelles viables cultivables, les VBNC et les bactéries mortes. Lorsque soumises aux stress, chaque souche possède un profil physiologique propre et la présence ou non de bactéries VBNC était dépendante du traitement appliqué et de la souche utilisée. La deuxième partie fut relative à l’étude des traitements thermiques de 70°C pendant 30 min et des chocs au dioxyde de chlore de 4, 6 et 7 mg/L pendant 60 min à température ambiante sur ces VBNC. Aucune légionelle VBNC n’est capable de se développer au sein des cellules et aucune croissance sur milieu BCYE n’a été observée après co-culture. La suite de notre étude a été d’étudier le comportement, envers les macrophages, de L. pneumophila revivifiées après culture sur amibes. Les résultats montrent que les légionelles VBNC induites par choc thermique et revivifiées par co-culture sur Acanthamoeba polyphaga sont capables d’infecter de nouveau les macrophages. En conclusion, ces résultats suggèrent que: (i) les formes VBNC de L. pneumophila ne sont pas spontanément infectieuses pour les macrophages et les cellules épithéliales alvéolaires in vitro et (ii) elles peuvent devenir pathogènes pour les cellules humaines après revivification préalable sur A. polyphaga
Legionella pneumophila, the causative agent of legionellosis is transmitted to human through aerosols from environmental sources and invades lung’s macrophages. It also can replicate within various protozoan species in environmental reservoirs. Following exposures to various stresses, L. pneumophila enters a Viable Non Cultivable state (VBNC) which is likely to be a survival strategy. The objective of our work was to study the VBNC forms of several strains of L. pneumophila serogroup 1 obtained after thermal and chemical treatments and to evaluate the infectivity of these VBNC forms against macrophages and alveolar epithelial cells. First we studied the physiological patterns of the three different strains (Philadelphia GFP 008, 044 clinical and environmental RNN). For all strains we observed the presence of VBNC bacteria in the native (non stressed) state. The results show that for each strain, three populations of Legionella can be identified: viable and culturable, VBNC and dead cells. Once submitted to the various stresses, we observed that each strain had its own physiological pattern and the presence (or not) of VBNC bacteria was dependent on the applied treatment and the strain used. The second part was related to the study of the pathogenicity of these VBNC forms against macrophages or epithelial cells. The study focused on heat shock treatment at 70°C for 30 min and chlorine dioxide treatment at 4, 6 and 7 mg/L for 60 min at room temperature. The results show that no Legionella VBNC forms were able to grow within the cells and no growth on BCYE medium was observed after co-culture. Then we investigated the behavior of L. pneumophila resuscitated after culture on ameba within macrophages. The results shows that Legionella VBNC induced by heat shock treatment and resuscitated by Acanthamoeba polyphaga co-culture are able to infect macrophages. In conclusion, these results suggest that: (i) the VBNC forms of L. pneumophila are not infectious for macrophages and alveolar epithelial cells in vitro and; (ii) they can be pathogenic for human cells after revivification by an amoeba (A. polyphaga)

ABSTRACT BACKGROUND Among all the possible microbial contaminations of wine, development of Brettanomyces bruxellensis yeast is the most dreaded by winemakers. Indeed, growth of yeasts belonging to Dekkera/Brettanomyces during wine manufacturing, can seriously affect the quality of the final product, especially during aging. Spoilage yeasts of the genus Brettanomyces or its teleomorph Dekkera are well adapted to survive during the winemaking process, due to their ethanol tolerance and relative resistance to the normal concentrations of sulphur dioxide found in wine. To prevent the development of this yeast in wine, it is important to know the biodiversity of Brettanomyces bruxellensis. A more detailed study of these aspects could help winemakers to learn more about this yeast and to implement preventive and fighting measures to try to reduce the economic losses caused by B. bruxellensis. AIM OF THE STUDY The specific objectives of this work were to isolate Brettanomyces strains from several wineries located in Puglia and to characterize representative biotypes, in order to evaluate control strategies based on biotechnological resources management. MATERIAL and METHODS Yeasts of the genus Brettanomyces were selected in tank, barrel and fermenting must of several Apulian wines. The yeast strains were identified and genetically characterized using restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), species-specific primers and restriction analysis. Biodiversity of selected B. bruxellensis strains was studied with Sau-PCR. Potential spoilage and VBNC behavior of different biotype were studied with gas chromatograph and flow cytometry. The cells of Brettanomyces were quantified in wine with Real Time PCR. To reduce the volatile phenols in wine and the growth of Brettanomyces bruxellensis different preparations of yeast cell wall and chitosan, respectively, were used.

Legionella pneumophila est une bactérie pathogène responsable de la légionellose qui se transmet par voies aériennes à partir de sites industriels ou naturels générateurs d'aérosols (installation d'eau chaude sanitaire, tours aéroréfrigérantes,. . . ). Actuellement le dénombrement et l'isolement de L. Pneumophila sont régis par une norme basée sur l'utilisation de culture gélosés. Cependant, comme de très nombreuses bactéries Gram négatif, Legionella pneumophila serait capable de passer, en condition de carence ou de stress, dans un état viable mais non cultivable (VBNC). Au cours de cette étude, nou avons montré que les différents traitements biocides classiquement utilisés pour éradiquer L. Pneumophila dans les installations industrielles, induisaient la formation de cellules VBNC, déterminées à travers l'utilisation de marqueurs de viabilité et du milieu de culture de référence. Cependant, l'analyse fine des marqueurs de viabilité estimée pour chaque cellule viable détectée montre que celles-ci présentent une altération progressive des différents descripteurs de viabilité utilisés à mesure que la concentration en biocide augmente. En supposant dans un premier temps, que la perte de cultivabilité des cellules VBNC soit potentiellement due au stress oxydant généré lors que l'étalement, nous avons cherché à optimiser le milieu de référence. Au cours de cette étude de nombreux composés (antioxudants, métaboliques. . . ) ont ainsi été identifiés comme étant bénéfiques à la restauration de la cultivabilité d'une fonction de la population après un stress mais aussi, et de manière intéressante, au cours de la croissance. La co-existence de deux sous population cultivables (sur le milieu de référence ou supplémenté) dont la proportion relative évolue au cours du temps, soulève donc à ce jour, un certain nombre de questions quant à l'origine et à la pertinence physiologique de chacune des deux populations. Pour des raisons techniques, ces questions ne pourrons être résolues qu'à travers l'utilisation de méthodes centrées sur l'individu et non plus sur la réponse globale d'une population. Dans ce sens, nous avons initié le développement d'une chambre d'observation et d'un système de fluidique qui permet aujourd'hui un suivi en temps réel de la viabilité des cellules observé et de leur devenir au cours du temps ou au cours d'un stress tout en observant in fine leur capacité respective à former une colonie
Legionella pneumophila is the causative agent of Legionellosis transmitted by air from industrial or natural aerosols (installation of hot water, cooling towers. . . ) Currently standard procedure that uses the agar cultur media governs the detection and isolation of L. Pneumophila. However, like many Gram-negative bacteria, Legionella pneumophila is able to enter in a viable state but non culturable (VBNC) during starvation or stress conditions. In this study, we showed that the different treatment biocides traditionally used to eradicate. L. Pneumophila in industrial plants, induced the formation of VBNC cells, determined using viability markers and reference agar medium (BCYE). However, detailled analysis of the viability markers shows that they have a progressive detetioration of the viability of different descriptors used as the biocide concentration increases. Assuming initially that the loss of culturability of VBNC cells is potentially due to oxidative stress generated during spreading, we sought to optimize the reference medium. During the study many compounds (antioxidants, metabolic. . . ) have been identified as benefical to the restoration of culturability for a fraction of the population after stress but also, interestingly, during growth. The co-existence of two populations (culturable on the reference medium or the supplemented medium) with the relative proportion changes over time, raises to date, a number of questions about the origin and physiological relevance of each of the two populations. For technical reasons, these issues can be resolved only with methods focused on the individual rather than on the overall response of a population. In this sense, we initiated the development of an observation chamber and a fluidic system that now allows as realtime monitoring of cell viability observed and their evolution over time or during a stress while observing ultimately their respective ability to form a colony

This project consisted of three studies: the first study investigated whether plant-based antimicrobials inactivate foodborne pathogens or induce them to go into the VBNC state in vitro and on romaine lettuce; the second study focused on sensory analysis of organic leafy greens treated with plant antimicrobials in the wash water; and the third study examined the novel concept of applying antimicrobials via edible films. The inert metabolic state of Viable but Non-Culturable (VBNC) that some pathogens can go into is gaining importance in the food and health industries. It is a state in which bacteria do not grow on lab based media but can cause disease in a human host. Foodborne pathogens that can go into VBNC are of immense concern since they can result in false negatives, leading to an outbreak if consumed by the public. The state of VBNC can be triggered by the presence of stress factors that includes chemicals such as sanitizers and antimicrobials. The objective of the first study was to determine whether plant-based antimicrobials inactivate the foodborne pathogens Salmonella enterica serovar Newport, Escherichia coli O157:H7 and Listeria monocytogenes or induce them to go into the VBNC state in vitro and on romaine lettuce. This was done by using selective media and fluorescence microscopy to confirm the viability of the pathogens. Organic iceberg lettuce inoculated with S. enterica, E. coli O157:H7 or L. monocytogenes was dip-treated in one of the following solutions: 0.1%, 0.3%, and 0.5% of essential oils or their active components; 3 and 5% of one of the plant extracts, hydrogen peroxide or chlorine for 2 min and stored at refrigeration temperature (4°C). Samples were taken on days 0, 1, and 3, for enumeration of survivors. The reductions in bacterial populations ranged from 2-6 log CFU/g. Oregano oil and its active component carvacrol were seen to be most effective in reducing the bacterial populations to below detectable limits on day 0. All the treatments reduced Listeria populations to below detectable limits by day 1. It was generally seen that citral showed more potent antimicrobial activity as compared to lemongrass oil against Listeria. The fluorescence microscopy results also correlate with the plating results. The dead cells fluoresced orange/red and the viable cells fluoresced green. The second study focused on sensory analysis of organic leafy greens treated with plant antimicrobials in the wash water. The objective of the second study was to a) evaluate panelists’ responses to changes in the sensory attributes of romaine lettuce treated with plant antimicrobials added to the wash water b) identify preference liking of the panelists to leafy greens washed with various essential oils and plant extracts and c) identify and evaluate the effects of these antimicrobials on the color and texture of the treated leafy greens. Organic romaine lettuce was washed with various plant antimicrobials in tap water for 2 min and stored at 4°C for 20-24 h before performing sensory evaluation and measuring changes in physical properties (color and texture) of leafy greens. A randomized block design was used with 75 panelists. The sensory attributes of the samples, which included pungency, browning, bitterness, off-odor, and sourness were evaluated using a 5 point hedonic scale. The preference liking was evaluated for aroma, color, freshness, mouthfeel, flavor, and overall acceptability using a 9-point hedonic scale where 9 was extremely liked and 1 not liked at all. The color analysis was done using the CIE L*a*b* coordinates and the texture was analyzed using a texture analyzer. Lettuce treated with 7% olive extract and 3% apple extract had a higher likelihood of being purchased and the least likely to be purchased treatments were oregano oil and a combination of oregano oil and grapeseed extract. As per the preference liking, lettuce treated with 7% olive extract and 0.1% citral had a higher overall acceptability. The least acceptable treatments were those of oregano oil and clove bud oil. The color of the samples was affected the least by olive extract and lemongrass oil, with oregano oil and carvacrol showing changes in the color. Similarly, for the texture analysis, it was seen that lettuce treated with 0.1% citral (890.0±79.5 N) was the least affected and 0.1% oregano treated samples were the most affected requiring less force (635 N) to crush the samples. We know that essential oils are well known for their potent fragrances and flavor imparting properties. The second study also indicated that the direct exposure of leafy greens to the antimicrobials may be less preferred and so the novel concept of applying antimicrobials via edible films was experimented with in the third study. Edible films are thin layer films made using the pulp of edible plant parts or biopolymers such as chitosan. Our films were made of fruit and vegetable pulp and contained the active components of plant antimicrobials. The objective of the third study was to a) evaluate panelists’ responses to changes in the sensory attributes of romaine lettuce treated with plant antimicrobials added to edible films b) identify preference liking of the panelists to leafy greens treated with various edible films and c) identify and evaluate the effects of these edible films on the color and texture of the treated leafy greens. The edible films were added to bagged lettuce. Edible films were made from tomato, apple, or carrot pulp which included 0.5%, and 1.5% of carvacrol or cinnamaldehyde. Similar parameters were evaluated with these samples. Romaine lettuce treated with apple films had a higher likelihood of being purchased in comparison to other films, with lettuce treated with films containing 1.5% cinnamaldehyde and the control films being the most to be purchased. The least likely to be purchased treatments were those of the tomato films, specifically the films containing 1.5% cinnamaldehyde. The lettuce treated with carrot films had a higher acceptance rating as compared to other films, with films containing 1.5% cinnamaldehyde and the control films being the highest. The color analysis results indicate that the films containing cinnamaldehyde had the least adverse effects. The cinnamaldehyde containing films also needed lower force to crush the leaves, indicating more firmness or crispiness as compared to those containing carvacrol. The results from the three studies are useful in not only helping to decide which plant antimicrobials can be used as potential sanitizers in the organic industry but also provide insight into which treatments are preferred by consumers and will not affect the marketability of the leafy greens.

Vibrio vulnificus is a model environmental organism exhibiting a classical starvation response during nutrient limitation as well as a non-culturable state when exposed to low temperatures. In addition to these classic global responses, this organism is an opportunistic pathogen that exhibits numerous virulence factors. This organism was chosen as the model organism for the identification of regulators of the viable but nonculturable response (VBNC) and the starvation-induced maintenance of culturability (SIMC) that occurs when cells are starved prior to low temperature incubation. In order to accomplish this, three indirect approaches were used; proteomics, investigation of intercellular signalling pathways and genetic analysis of regulators involved in these responses. Two-dimensional gel electrophoresis was used to identify proteins expressed under conditions that induced SIMC. It was determined that carbon and long-term phosphorus starvation were important in the SIMC response. V. vulnificus was shown to possess genes, luxS and smcR, that are homologues of genes involved in signalling system system 2 in Vibrio harveyi. Signal molecules were produced upon starvation and the entry to stationary phase in V. vulnificus. Furthermore, a null mutation in smcR, a transcriptional regulator was shown to have pleiotropic effects in V. vulnificus, including up-regulation of numerous virulence factors and a defect in starvation survival and development of the SIMC response. We propose that V. vulnificus possesses a signalling system analogous to that of system 2 in V. harveyi, and that this system is involved in the regulation of stationary phase and starvation adaptation in this organism.

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.

Acinetobacter baumanii fait aujourd’hui partie des bactéries les plus problématiques dans le monde. Responsable de nombreux pics épidémiques d’infections nosocomiales auxquelles sont associés de forts taux de mortalité, cette bactérie puise sa pathogénie dans de multiples caractéristiques qui lui permettent ainsi d’échapper au système immunitaire de l’hôte et à la plupart des traitements actuels. Capable d’adhérer à de multiples surfaces, A. baumanii persiste dans l’environnement hospitalier à travers un mode de vie communautaire au sein duquel ses capacités de survie sont exacerbées. Chez les espèces du genre Acinetobacter, le mode de vie communautaire peut prendre deux formes distinctes : le biofilm et la pellicule. Dans la première partie de cette thèse, nous avons cherché à discriminer ces deux modes de vie, chez la souche ATCC 17978, par une analyse protéomique à large échelle. Nous avons confirmé la présence de nombreux marqueurs communs aux deux communautés (transporteurs, systèmes de sécrétion, d’acquisition d’ions, adhésines et pili) et mis en exergue des systèmes spécifiquement reliés à la formation du biofilm (pilus Fim, T2SS, T1SS/pompe A1S_0535-38, LPS/LOS, motif capsulaire) et à celle de la pellicule (Gac). Grâce à l’étude de la souche A. baumannii SDF en mode biofilm, qui présente un génome plus compact, nous montrons que très peu de mécanismes moléculaires sont partagés par les deux souches étudiées. Ce résultat témoigne de la difficulté quant au développement d’un traitement dirigé contre les biofilms A. baumannii. Dans une deuxième partie, nous avons testé deux approches pour prévenir et éradiquer les biofilms à A. baumannii. La première a ciblé le Quorum Sensing (QS), système de communication essentielle à la coordination des cellules. Nous avons pu montrer que les acides gras mono-insaturés (acide palmitoléique et acide myristoléique), au même titre que la virstatine, limitait la formation de communautés à A. baumannii en inhibant l’expression du régulateur abaR nécessaire au QS. Dans une seconde stratégie, nous avons finalement évalué l’action antibactérienne et antibiofilm d’un nouveau composé d’origine naturelle : la squalamine. Dans cette étude, nous montrons pour la première fois qu’A. baumannii est capable d’entrer dans un état de dormance (persistant/VBNC) pour survivre à de fortes doses de ciprofloxacine, mais que la squalamine est capable d’éradiquer ces cellules persistantes grâce à des concentrations inférieures à la concentration hémolytique
Today, Acinetobacter baumannii is one of the most problematic pathogens in the world. This bacterium is responsible for worldwide epidemic outbreaks associated with dramatic mortality rates. It possesses high capacities to evade the immune host system and to resist to numerous available antibacterial agents. A. baumannii is also able to persist into hospital environment due to high adhesion abilities which induce community development. This process is also associated to an enhanced survival rate. In Acinetobacter genus, community modes of lif can take two forms : biofilm and pellicle. In this study on the strain ATCC 17978, we tried to discriminate these two lifestyles by a large scale proteomic analysis. We have confirmed the presence of many common community markers (transporters, ion acquisition secretion systems, adhesins and pili) and highlighted systems specifically related to biofilm (pilus Fim, T2SS, T1SS / pump A1S_0535-38, LPS / LOS, capsular pattern) and pellicle communities. Furthermore the proteomic analysis of an avirulent A. baumannii strain, SDF, in biofilm allowed to highlight peculiar metabolic pathways, specific adhesion determinants but very few markers shared by ATCC 17978. This demonstrated the difficulty in developing a treatment directed against A. baumannii biofilm. Then, we tested different approaches to prevent and eradicate biofilms. The first one targeted the Quorum Sensing system (QS), an essential communication system for cell coordination. We have showed that monounsaturated fatty acids (palmitoleic acid and myristoleic acid), like virstatin prevent the community formation of A. baumannii by inhibiting the expression of the abaR regulator required for QS. In a second strategy, we have evaluated the antibacterial and antibiofilm activity of a new natural compound : the squalamine. We showed for the first time that if ciprofloxacin treatment was able to induce a dormancy population (persistent/VBNCs) in A. baumannii, squalamine was able to eradicate this population of dormant cells

Propionibacterium freudenreichii est une bactérie très utilisée par l’industrie laitière. Elle appartient aux Actinomycètes connus pour leur survie pendant de longues périodes, dans des conditions environnementales défavorables. Pour mieux comprendre ce phénomène, la caractérisation phénotypique de 8 souches de P. freudenreichii a été réalisée sur 11 jours dans un milieu en carence nutritionnelle. Le taux de survie bactérienne a été mesuré par densité optique, par énumération et évaluation de la viabilité cellulaire. En outre, l’absence de lyse cellulaire a été évaluée par PCR quantitative. La croissance de P. freudenreichii a été décrite en phases exponentielle, stationnaire, stationnaire tardive et survie à long terme.Dans nos conditions expérimentales pendant la période de survie à long terme, les bactéries sont restées viables. La caractérisation phénotypique a montré que P. freudenreichii CIRM-BIA138 était la plus résistante à la carence nutritionnelle et entrait dans un état viable mais non-cultivable. Cette souche a été utilisée pour une étude fonctionnelle par RNA-Seq ainsi que pour des analyses biochimiques sur les surnageants de culture, en phases exponentielle et stationnaire. L’association de ces données transcriptomiques et métabolomiques a permis de déduire les stratégies impliquées dans la survie de cette bactérie. La préparation à l’état de dormance, la diminution du métabolisme et l’utilisation de sources alternatives d’énergie semblent impliquées dans l’adaptation et la persistence de P. freudenreichii CIRM-BIA138 en carence nutritionnelle durant de long
Propionibacterium freudenreichii is a dairy bacterium belonging to the Actinobacteria group, which is known to survive for long periods in harsh environmental conditions. In order to investigate the long-term survival phenomenon in P. freudenreichii, 8 strains were phenotypically characterized for a period of 11 days in nutrient shortage condition. Bacterial survival rate was assessed by optical density, CFU counting and live-dead cellular viability. In addition, the absence of cell lysis was evaluated by quantitative PCR. P. freudenreichii growth phases were classified as exponential, stationary, late stationary and long-term survival. Moreover, it was observed that bacterial viability was maintained during long-term survival.Phenotypical characterization indicated that P. freudenreichii CIRM-BIA138 was more resistant to nutrient shortage being able to enter into a viable but nonculturable dormant state. In addition, functional studies of this strain were conducted by RNA-Seq on cultures sampled in exponential and stationary growth phases. Concomitantly, several biochemical analyses were carried out on the culture supernatant. An integrative approach of metabolomic and transcriptomic data allowed us to infer strategies associated with the survival of this bacterium, such as preparation for the dormant state, slow down of metabolic activity and utilization of alternative sources of energy, which altogether might allow P. freudenreichii CIRM-BIA 138 to adapt and persist through nutrient shortage for long periods

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.

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.

In order to achieve a better understanding of the nodulation patterns of rhizobia in their symbiosis with legume hosts, and with the aim to examine their signalling behaviour in cell-to-cell communication, a series of experimental projects were carried out. In first instance the microbial inhabitants of 831 pea root nodules formed on nine plants, sown in different field soil parcels, were isolated and characterized by PCR-based electrophoretic fingerprinting using the BOXA1R primer. Band profiles were analyzed by GelComparII image analysis software converting differences into a numerical matrix yielding their similarity dendrogram in terms of genetic fingerprint distances. The level of strain-specific association with individual plant or soil plots has been assessed. As 85% of the profiles result singletons, having been found in only one nodule, the overall diversity of the site appears particularly high. Estimates of the total diversity at biovar level were obtained by nonparametric estimators pointing to a value over 1300 types. Such richness was compared with the much lower one recorded eight years earlier on the same plots and was put in relation with the repeated host cropping occurred in between. Moreover, the position of each nodule within the root apparatus, in terms of root rank order and distance from the crown, had been recorded in digitized images and the existence of topological and temporal patterns in each strain's nodulation process has been inspected. The fingerprinting quality of BOX-PCR in terms of reproducibility and sensitivity, was compared to that obtainable by other primers as ISRh1 outbound primers. The same fingerprint-characterized strains were screened for the production of Quorum Sensing signals consisting in short-medium- (C4-C8) and long- (C14) chained N-acyl homoserine lactones (AHLs) using, respectively, the two reporter systems: Chromobacterium violaceum CV026 (violacein pigment induction) and Rhizobium leguminosarum A34 (colony growth inhibition). The majority of the natural Rhizobium leguminosarum strains were found to be quorum-signalling positive. The occurrence of isolates negative to one or both phenotypes however shows that those traits are not absolute requirements for host nodulation. In a different study we examined the root nodule symbionts of eight species of wild legumes collected in Sardinia. Interestingly, unlike the case of cultivated legumes, the recovery on plates of the rhizobial occupant could not be obtained under any of the conditions used, while at the same time a number of different endophytic taxa were rescued and their taxonomic identity was determined by 16S nucleotide sequencing. By direct PCR analysis from the nodule tissue, we were also able to show the presence of the nonculturable rhizobia inside the same nodules. In parallel, other two studies were conducted. AHL-mediated quorum sensing communication was quantified at single cell resolution trough a red-fluorescing AHLproducing and a green-fluorescing AHL-sensor strain in a 3-dimensional system by using computer-assisted microscopy (CMEIAS). The average effective "calling distance" from the single cell producer capable of inducing the gfp-tagged reporter cells, resulted 46.8 ?m. Moreover, in relation to the possible involvement of AHL signals in different phenotypes, a series of plant-interacting strains, among which Rhizobium leguminosarum, were tested for their ability to maintain viability in stressful situations (nutrient and oxygen limitations). Some of the tested strains lost culturability in different of the imposed conditions. However viable cells could be detected by staining microscope-based techniques (BacLight®, acridine orange and CTC), demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; none of the AHL Quorum Sensing signals tested was effective in promoting the transition to the VBNC state nor in recovering cells to culturability, suggesting that the two phenotypical frameworks of QS and VBNC do not share signalling paths.

For most of the 20th century, the saying “once a cesarean, always a cesarean” was a rule in the United States. Today, the National Institutes of Health (NIH) opposes the dictum and urges women to consider trial of labor after cesarean (TOLAC). However, the factors that lead to a successful outcome remain unclear, as research continues to be conducted in hopes of creating a predictive model for vaginal birth after cesarean (VBAC) success. The NIH’s request for more research in this area of obstetrics led to this retrospective cohort study of all TOLACs at Marin General Hospital (MGH) from 2000-2013. All labor trials were studied for patient demographics, details of labor, maternal and neonatal morbidities, insurance, and provider type. After confirming the quality of the data, verifying inclusion criteria and ignoring cases with missing data, a data set of 745 TOLACs with 13 explanatory variables of interest was prepared. A forward stepwise (Likelihood Ratio) binary logistic regression was run in IBM® SPSS® Statistics in order to create a model that could determine which variables were most predictive of delivery outcome in TOLAC patients. Ultimately, seven variables were predictive and were included in the model. Of the seven, the most predictive variable in determining VBAC success was provider type. The model concluded that a woman’s odds of having a successful VBAC were almost four times greater if she began her delivery with a certified nurse midwife, than if she began her deliver with a physician (odds ratio 0.27, 95% CI 0.17-0.44; < 0.01). The results from this study mimic the results of other models, and introduce labor support as a key factor in predicting VBAC success.

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.

La survie et le transfert de Listeria dans l’environnement agricole puis sur les feuilles de persil a été étudiée en plein champ et en microcosme de laboratoire. Les études en plein champ nous ont permis de caractériser l’épandage de matières organiques et l’arrosage ou l’irrigation par aspersion comme des pratiques agricoles contribuant à la présence de L. Monocytogenes sur les cultures maraîchères. Une humidité plus élevée ainsi qu’un climat plus frais contribueraient à augmenter leur survie. Néanmoins, la persistance de Listeria dans l’environnement des cultures maraîchères reste très faible (décroît de 7 log en 63 jours dans le sol et de 9 log en 2 jours sur les feuilles). En laboratoire, L. Monocytogenes est capable de se multiplier sur les feuilles de persil en condition d’humidité saturante, tandis que sa population décline rapidement de plusieurs log en humidité non saturante. Toutefois, sous faible humidité, une population viable non cultivable (VNC) de L. Monocytogenes s’est maintenue à la surface des feuilles. Néanmoins, cette population VNC ne semble pas capable de recouvrer sa cultivabilité lorsque les feuilles de persil sont placées en condition d’humidité saturante. L’étude des mécanismes impliqués dans la résistance au stress pour la survie de L. Monocytogenes sur les surfaces foliaires sous faible humidité relative a démontré 1) l’implication du facteur transcriptionnel σB dans la persistance de L. Monocytogenes sur les feuilles de persil; 2) l’implication de la protéine Fri dans la persistance de L. Monocytogenes sous forme VNC et 3) un rôle protecteur de la glycine bétaine dans la persistance de L. Monocytogenes sur les feuilles de persil. L’effet protecteur de la glycine bétaine chez L. Monocytogenes n’est pas attribué à une accumulation intracellulaire par les transporteurs BetL, Gbu et OpuC
The survival and the transfer of Listeria in soil and on leaves were studied in open field and laboratory conditions. Studies in open field conditions identified the spreading of organic fertilizers and sprinkled-irrigation as agricultural practices contributing to the presence of L. Monocytogenes on produce. A higher relative humidity as well as a cooler climate would contribute to increase its survival. However, even with a high inoculum, Listeria did not persist in the environment of vegetable crops for long periods (decrease of 7 log in 63 days in the soil and of 9 log in 2 days on the leaves). In the laboratory, L. Monocytogenes was able to multiply on parsley leaves under satured relative humidity whereas its declined rapidly under low relative humidity (4-5 log in 8 days). However, under low relative humidity, a non-culturable viable population (VNC) of L. Monocytogenes remained on the leaf surfaces. This population of VNC did not seem to be able to recover its culturability when the parsley leaves were placed under satured relative humidity. The investigation of mechanisms involved in survival of L. Monocytogenes on parsley leaves under low relative humidity showed 1) the role of the alternative sigma factor B in the persistence of L. Monocytogenes on the parsley leaves; 2) the implication of the Fri protein in the persistence of VBNC L. Monocytogenes and 3) a protective effect of glycine betaine on L. Monocytogenes on the parsley leaves. The protective effect of the glycine betaine on L. Monocytogenes was not due to an intracellular accumulation by the uptake systems BetL, Gbu and OpuC

The master’s thesis focuses on model designing of active components for PSpice simulator. Creation of models are based on text description, which is avaible in Cadence Spectre libraries. The aim of this thesis is approximate conversion of CMOS and bipolar tranzistors based on I3T 0.35 m technology. Simulation’s results and their comparation are discussed below.

The study explores the role of management control systems in a strategy formulation process, this by viewing management control systems as a package and addressing its role in the Value Based Health Care (VBHC) strategy formulation process at Uppsala University Hospital. Previous studies exploring the relationship between management control systems and strategy have found the relationship to be interrelated and that management control systems can take either an interactive or diagnostic role. However, these studies are limited in their approach as they do not address management control systems as a package, thus failing to capture the importance of informal control systems and the impact separate controls have on each other. Applying a case study design using semi-structured interviews, the study partly supports the findings of earlier studies emphasizing how management control systems can be classified and used for both interactive as well as diagnostic purposes. The study however, expands the view of earlier research by emphasizing how diagnostic controls should be further classified as either enforcing or reinforcing control systems, as well as emphasizing the importance of timing for understanding the different roles of management control systems in a strategy formulation process.

Background: An increasing proportion of Canadian women are experiencing a Caesarean section (CS) and a subsequent repeat CS. While CS can be necessary and lifesaving for mothers and their infants in some situations, it is also associated with greater morbidity risks to women and infants than vaginal birth. Clinical practice guidelines recommend the involvement of pregnant women in making decisions about mode of birth and shared decision making improves the informed consent process. This research examines the factors that influence mode of birth after a previous CS. Methods: Two cross sectional descriptive studies and a prospective pre-post cohort study with control were conducted to investigate the high use of repeat CS at the levels of health care providers, maternity care clients, and the organizational structure of a birthing unit. 1. Interviews and surveys with obstetricians, family physicians, midwives, and nurses were conducted to investigate the attitudes, values, and perceptions that guide their care practices for clients with a previous CS. The specific research question was: What are the factors that influence the practices of maternity care providers (obstetricians, family physicians, midwives, and nurses) regarding mode of birth after a previous CS? Data was analyzed using iterative deductive and inductive coding. 2. Interviews and surveys were conducted during pregnancy and after giving birth with healthy women who have had a previous CS to explore their decision making processes regarding mode of birth after a previous CS. The specific research question was: How do women eligible for a VBAC make decisions about their upcoming mode of birth? A thematic framework approach was used for data analysis. 3. Data from the Better Outcomes Registry and Network (“BORN”) Ontario was analyzed to examine the effectiveness of a hospital based strategy on overall proportions of CS and within Robson groups 1, 2a, and 5. The Caesarean section reduction (CARE) strategy includes interventions that target health care providers, pregnant women, and hospital policies. Results: 1. Maternity care providers would recommend a vaginal birth after CS (VBAC) for healthy pregnant women with a previous CS. They had different perceptions of the safety of birth to the health of women and infants and different approaches to engage in decision making during consultation. Providers believed women make their decision about mode of birth outside of the clinical consultation and often prior to their subsequent pregnancy. 2. The main themes that influenced the decisions of maternity care clients about mode of birth were mothers’ experiential reasoning regarding mode of birth and recovery, experiential knowledge from significant others, scheduling of CS regardless of the mode of birth decision, rating and prioritizing risks, fear of risks, and decisional conflict. When women discussed the factors that impacted their decisions about mode of birth six to eight weeks after they had given birth, the main themes were the recovery experience and fear related to the mode of birth. A lack of time during consultation was identified as a major barrier inhibiting shared decision making, specifically among clients of obstetricians. Other barriers included reliance on routine obstetric practices that are not evidence based. 3. Proportions of CS decreased at the intervention hospital by 3.9% (p=0.0006), from 30.3% (n=964) in 2009/10 to 26.4% (n=803) in 2012/13. During the same time frame, proportions of CS in the control group were stable with 28.1% (n=23,694) in 2009/10 and 28.2% (n=23,683) in 2012/13. Within the Robson classification system, the proportions of repeat CS among all low risk women with a previous CS decreased at the intervention hospital by 5.6% (p=0.0044) from 84.3% to 78.7%. In the control group, also fewer women had a repeat CS over the study period, but the decrease was smaller with 3.9% (p<0.0001) from 84.5% to 80.6%. Conclusion: A true shared decision making process addresses the power imbalance between providers and women through an incorporation of the clinical expertise of providers and the experiential expertise of pregnant women before reaching a decision about mode of birth. The use of routine obstetric practices that are not evidence based inhibited women to make decisions about their mode of birth. The introduction of the CARE strategy to a hospital birthing unit was associated with improvements in proportions of CS and VBAC among low risk women.

Människans konsumtionsbeteenden bidrar till växande negativ miljöpåverkan, och för att bryta utvecklingen krävs arbete på flera nivåer, däribland individnivån. Eventuellt kan massmedia bidra till sådant arbete. Denna studies syfte var att med fokus på konsumtionsbeteenden undersöka miljöbudskap i en nyhetstidnings webbpublicerade artiklar. Jag analyserade artiklarna med kvalitativ, teoretisk, tematisk analys, samt kvantitativt genom att kontrollera i hur många artiklar olika budskap identifierats. Analysens utgångspunkt var en lätt modifierad version av Sterns VBN-teori (Value Belief Norm theory), som fokuserar på attityder relevanta för miljövänligt beteende. Jag analyserade budskap i 57 artiklar som jag bedömt betonade vikten av konsumtionsrelaterade beteendeförändringar med hänvisning till miljön, och identifierade sju övergripande teman. De innefattade 14 underteman utifrån VBN-teorin och sex kategorier av beteendeuppmaningar. Jag bedömde att artiklarna som helhet i högre utsträckning innehöll budskap formulerade i linje med miljömässigt fördelaktiga attitydfaktorer än det motsatta, samt att artiklarna ofta uppmanade till konsumtionsrelaterade beteenden med förhållandevis god miljönytta vid utförande.

A Mobilização Nacional é um conjunto de atividades que visa capacitar o Brasil a planejar e executar ações no campo da Defesa Nacional para garantir a segurança e a soberania do país. Quando esta for decretada pelo Estado, os órgãos que fazem parte do Sistema de Defesa Nacional devem estar preparados e equipados para executarem suas funções. O Exército Brasileiro, um desses órgãos, no ano de 2006, adquiriu 250 viaturas blindadas de combate, das quais 216 para serem distribuídas em suas unidades de cavalaria no território brasileiro. O objetivo desse estudo é apresentar a melhor alternativa logística de transporte para o deslocamento desses carros de combate em uma organização militar, a partir de um determinado cenário, com menor custo e/ou tempo. Para atingir o objetivo foram realizadas consultas por meio de entrevistas e correspondências com especialistas em transporte, transporte militar e viaturas blindadas. As consultas possibilitaram definir as principais vantagens e desvantagens dos modais de transporte e os fatores relevantes no transporte de viaturas blindadas, permitindo a formulação da rede de transporte e elaboração de um instrumento de viabilidade dos modais de transporte (quadro de verificação) a serem aplicados às rotas de transporte. Um problema de transporte das viaturas blindadas, a partir de um cenário hipotético, foi resolvido por meio de um modelo matemático tendo como objetivo a minimização de custo ou tempo. Concluiu-se que o modelo matemático é uma ferramenta que pode auxiliar a tomada de decisão no transporte das viaturas blindadas, mas a melhor alternativa irá depender da adequada análise dos modais de transporte disponíveis e da correta formulação e aplicação do instrumento de viabilidade dos modais de transporte.
The National Mobilization is a set of activities that aims to enable Brazil to plan and execute actions in the field of National Defence toensure the security and sovereignty of the country. When it is ordered by the state organs that are part of the System of National Defense must be prepared and equipped to execute its functions. The Brazilian Army, one of these agencies, in 2006, purchased 250 armored combat vehicles, of which 216 to be distributed among their cavalry units in Brazil. The aim of this study is to present the best alternative transportation logistics to carry these tanks in a military organization, from a given scenario, with lower cost and / or time. To achieve the goal consultations through interviews and correspondence with experts in transport, military transport and armored vehicles. The consultations allowed to define the main advantages and disadvantages of transportation modes and factors relevant to transport armored vehicles, permitting the formulation of the transport network and development of an instrument feasibility of transportation modes (check list) to be applied to routes of transport. A problem of transporting armored vehicles, from a hypothetical scenario, was solved by a mathematical model with the objective of minimizing cost or time. It was concluded that the mathematical model is a tool that can aid decision making in the transport of armored vehicles, but the best choice will depend on the proper analysis of the modes of transportation available and the correct formulation and application of the instrument feasibility of transportation modes.

Reports from the IPCC have been consistent in their findings: climate change is happening and human activity is the cause. The temperature of the earth’s climate has been steadily rising since the industrial revolution, with profoundly negative consequences for the natural environment. Britain is amongst the top 10 global contributors towards climate change, producing more CO2 per capita than China, and yet little is known about the relationship the British public have with the natural environment. Drawing upon DEFRA’s 2009 Survey of Public Attitudes and Behaviours Towards the Environment, a nationally representative sample of the UK, this study aims to (1) explore environmental attitudes in the DEFRA sample; (2) identify the types of environmental concern that exist in the UK and; (3) examine how environmental concern is associated with pro-environmental behaviours. The overall goal is to develop a better understanding this attitude-behaviour relationship. The thesis has 3 main findings. First, environmental concern is formed of three environmental attitudes: (a) a cognitive appraisal of plant and animal welfare (ecocentric attitude); (b) welfare of the human race (human-centric attitude); and (c) a prioritisation of the self, alongside dismissal of environmental problems (denial).Second, members of the British public can be assigned to one of four groups based on their environmental concern: Pro-environment, Neutral, Disengaged and Paradoxical (the latter 2 groups are apathetic towards environmental issues though in different ways).Third, when examining behaviour variation across these environmental concern groups, it was found, unsurprisingly, that membership of the pro- environmental group is strongly predictive of pro-environmental behaviour. What was surprising was that pro-environmental concern predicts a variety of behaviours, both easy and challenging (i.e. easy behaviour such as recycling household waste as well more challenging behaviour such as an increase use of public transportation over driving), whereas previous studies have typically found such behaviours to be unaffected by attitudes. Membership of the Neutral group also predicts pro-environmental behaviours, although this relationship is weaker and exists for fewer measures of behaviour. Disengaged and Paradoxical forms of concern are not significant predictors of behaviour. Upon examining the effect of socio-economic status (SES) on group membership and this attitude-behaviour relationship, it was found that SES does not moderate the attitude-behaviour relationship, but it does influence group membership. Respondents with higher SES were more likely to belong to neutral or pro-environment groups. After reviewing these findings, it is concluded that environmental attitudes do clearly predict behaviour, but a large portion of the UK population do not possess environmental attitudes strong enough to do so (the Disengaged and Paradoxical groups amount to 36% of the population). Future studies should focus on these apathetic groups in an attempt to understand them, determine effective methods of engagement and identify factors that increase the probability of members transitioning out of these groups.

Horizontal bores are essential infrastructures for maintaining the stability of open-pit mine batters. The infiltration of water from large surface catchments during rain events and induced deformation caused by mining activities can cause the build-up of pore water pressures in mine batters, potentially leading to catastrophic slope failures. A field investigation unit containing a camera has been developed to survey long (>300m) horizontal bores. Features observed using the camera along the profile of horizontal bores are discussed. Water flow was quantified by flow meters. X-Ray Diffraction (XRD) was undertaken to investigate the water precipitates within the selected bores. Water flow temperature was recorded to test the hypothesis of a possibility to indicate whether a borehole was draining from the saturated zone or from the surface water through its temperature. The investigations have been conducted to determine the cause of change in the efficiency of horizontal boreholes and find a reliable measure to assess longevity and performance of horizontal drains. Bore efficiency has been defined as the bore functioning as a preferential path for water within the batter to be drained out to reduce the saturated zone and associated pore water pressures within the batter. The results suggest blockages and fractures inside the bores can be considered the leading cause of the change in the efficiency of a bore. Blockages occur because of sediment accumulation and because of coal chunks from internal wall collapses. Internal fractures affect efficiency when they become the water preferred path; thus, retaining water flowing within the batter. The bore’s longevity is considered the period of the bore is considered effective. Water flow measurement is suggested as a reliable measure to assess bores’ longevity.
Masters by Research

A Pesca é uma atividade extrativa, expandindo-se nos tres setores da economia. No setor primário, quando considerado a atividade de captura do pescado. No secundário, quando considera-se a atividade de industrialização e, no terciário quando esta se reporta à exportação. A simulação da utilização do método ABC na indústria pesqueira do Estado do Ceará objetiva comparar o resultado desse método com o atualmente utilizado para fins de evidenciaçaõ de resultado.
Fishing is an extractive activity and, therefore, part of the primary sector, but it also part of the secondary sector once several techniques of fish processing are considered, and it is part of the tertiary sector while companies sell their products in the domestic and in foreign markets. The primary sector is constituted by the fishing fleet, by the fishermen, and by fishing apparels, while fishing terminals are basic elements of the logistics of the ship-building process and disembarkation of the fish caught. The secondary sector is constituted by the fishing companies that can act in the phases of capture, storage, processing, and export of products, and by autonomous fishing trappers, owners of embarkations, that work in partnerships with the companies in order to guarantee the supply of raw materials for processing and commercialization. In the tertiary sector, exporting companies earn revenues when they sell their products in order to repay the expenses made in the other two sectors. The idle capacity of the fishing industry and the elevation of costs have contributed to weaken and to discourage fishing activities that, in despite of the difficulties, continue to be an important generator of exchange value through exports and of job-opportunities in the nation. In this context, the present work approaches the most significant aspects of both the national and international fishing sectors, and provides a vision of the managerial, commercial, and economic aspects of fishing companies in Ceará. It is also approached the difference among Systems, Methods, and Costing Forms, approaching the fundamental characteristics of the most important costing methods studied and used in the world, with relevance for the ABC. In the last part of the work a case study is presented, approaching a simulation of the method of Costing Based Activities (ABC), in the fishing companies in Ceará with the objective of comparing the result of this costing method with the ones used in the companies in order to verify if its utilization is more efficient in the control of costs and result evidencing.

Det är numera konstaterat att klimatförändringar kommer förändra förutsättningarna förmänniskor på jorden. Syftet med uppsatsen är därmed att undersöka vad som motiverarpersoner att minska sina klimatutsläpp och vilka hinder de kan stöta på. Med hjälp av fyra intervjuer och nio frågeformulär har uppsatsens två forskningsfrågor kunnat besvaras då det har ställts frågor till personer som har valt att sänka sina utsläpp genom att förändra sina vanor gällande flygning, bilägande och konsumtion. Studien visade att miljöengagemang är kopplat till miljökunskaper samt att det kan utveckla förståelse för de negativa konsekvenser en högkonsumerande livsstil kan leda till. För respondenterna i studien har det lett till en känsla för både ansvar och direkt eller indirekt moralisk plikt att sänka sina växthusgasutsläpp då det har utvecklat en hög miljöhänsyn. Detta tillsammans med positiva förebilder och en positiv självbild har fungerat som motivation till en förändrad livsstil. Bristande kunskaper, kontrolluppfattning, identitet, vanor, civilstånd samt sociala och subjektiva normer har verkat som hinder och lett till att respondenternas omställning har tagit tid. Respondenterna i studien har hunnit olika långt till följd av de olika hinder de stött på under resans gång men resultatet visar att de innehar en stark intern attitydstruktur då de trots hinder inte har gett upp och istället börjat omvärdera sin syn på livskvalitet genom att ändra inställning till vad som är lycka och lyx. Det går att koppla teorierna VBN och TPB till varandra då värden, uppfattning och personliga normer formar den miljöhänsyn som inverkar på attityd, subjektiva normer och uppfattad kontroll.
It is now established that climate change will change the conditions for people on Earth. The purpose of the thesis is thus to investigate what factors motivate people to reduce their climate emissions and what obstacles they may encounter. With the help of four interviews and nine questionnaires, the essay's two research questions have been answered by asking questions to people who have chosen to reduce their emissions by changing their habits regarding flying, car ownership and consumption. The study showed that environmental commitment is linked to environmental knowledge and that it can develop an understanding of the negativec onsequences a high-consumption lifestyle can lead to. For the respondents in the study, it has led to a sense of responsibility and direct or indirect moral duty to reduce their greenhouse gas emissions as it has developed a high environmental consideration. This together with positive role models and a positive self-image has served as motivation for a changed lifestyle. Lack of knowledge, control perception, identity, habits, marital status and social and subjective norms have acted as obstacles and led to the respondents' adjustments taking time. The respondents in the study have made different achievements due to the various obstacles they encountered. The results show that they have a strong internal attitude structure as despite obstacles they have not stopped trying to change and instead started to re-evaluate their view of quality of life by changing attitudes toward what is happiness and luxury. It is possible to link the theories VBN and TPB to each other as values, perceptions and personal norms shape the environmental considerations that affect attitude, subjective norms and perceived control.

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Este estudo analisou como ocorre o processo de institucionalização da governança em Comunidades Virtuais de Negócios (CVNs) para compreender como este é influenciado pela virtualidade. Para esse propósito, um framework foi elaborado, levando em conta a virtualidade presente nas CVNs e as dimensões estruturais e instrumentais da governança em relações interorganizacionais. O framework tem como base a teoria institucional, especificamente o modelo teórico desenvolvido por Tolbert e Zucker (1999), que subdivide o processo de institucionalização em estágios de habitualização, objetificação e sedimentação, e também as bases de legitimação abordadas por Scott (2008). A abordagem de pesquisa utilizada foi qualitativa, por meio do estudo de dois casos; dentre os quais, um deles foi desenvolvido sob a perspectiva longitudinal. Os casos são representados por duas CVNs criadas para atender o segmento de flores e plantas ornamentais, a saber: CN-Flores e a Comunidade Veiling Online. Os dados foram coletados por intermédio de entrevistas semiestruturadas, de observação não participante e de pesquisa documental, sendo analisados por meio da técnica análise de conteúdo. Os resultados principais indicam que a institucionalização da governança nas CVNs ocorre de forma diferenciada, tendo em vista o atual estágio de estruturação de cada arranjo interorganizacional que suporta as respectivas CVNs. Outros resultados envolvem a materialidade das interações advindas da virtualidade presente nas CVNs, cujos efeitos sobre o processo de institucionalização da governança são percebidos pela praticidade e agilidade nas transações e pelo seu potencial em formalizar decisões, normas e regras nas CVNs.
This study analyze how the process of institutionalization of governance in Virtual Business Communities (VBCs) to understand how it is influenced by the virtuality. For this reason, a framework was developed considering the virtuality present in the VBCs and the structural and instrumental dimensions of governance in interorganizational relationships. The framework is based on the institutional theory, specifically the theoretical model developed by Tolbert and Zucker (1999), which subdivides the Institutionalization process in steps of habitualization, objectification and sedimentation, and also on the foundations of legitimation proposed by Scott (2008). The research approach used was qualitative, with two case studies, one of them developed under the longitudinal perspective. The cases were represented by two VBCs created for the sector of flowers and ornamental plants, which are: CN-Flores and the Veiling Online Community. Data were collected through semi-structured interviews, non-participant observation and documentary research and analyzed through the content analysis technique. The main results obtained indicate that the institutionalization of governance in the VBCs occur differently, considering the present structured stage of each interorganizational arrangement, which offers support to the respective VBCs. Other results comply with the materiality of the interactions resulting from the virtuality present in the VBCs. The effects of the materiality of the interactions on institutionalization process of governance are perceived by the practicality and promptness during the transactions and their potential to draw up formal decisions, standards and regulations in the VBCs.

Determinants influencing consumer eco-innovation adoption and green curtailment behaviors in a travel context are at the center of this thesis. Previous research on green consumer behavior has uncovered that internalized personal attitudinal factors such as values, beliefs, and norms are influential in determining mainly non-consumption and post-purchase behaviors. This thesis extends the understanding of a moral basis of green consumer behavior by exploring the influences of attitudinal factors on both car curtailment behaviors, and on consumer adoption of a high involvement eco-innovation – the alternative fuel vehicle. The integrated influences of innovation specific characteristics, car habits, knowledge and social norms, are also examined. Furthermore, differences between AFV adopters and non-adopters are explored, and the notion of consumers performing purchase and curtailment behaviors for different reasons is utilized in the development of nuanced profiles of three distinct consumer groups. Four studies, which build on two quantitative data collections on adopters and non-adopters of AFVs in Swe­den, are included in this thesis. In the first study, similarities and differences among adopters and non-adopters of AFVs, and the effects of attitudinal factors (values, beliefs, and norms), knowledge, and sociodemo­graphics on the adoption decision are analyzed. The results show that knowledge and personal norms are strong predictors of AFV adoption and that the VBN theory is applicable in this context. The main implication from the study is that high-involvement green purchase deci­sions, such as eco-innovation adoption, can be viewed as morally based. In the second study, a set of determinants influencing both curtailment of car use and willing­ness to adopt a less environmentally harmful vehicle are analyzed. Biospheric values, per­sonal proenvironmental norms, and car habit strength are found to influence both types of behaviors in different ways. The main implication from this study is that green purchase deci­sions and curtailment behaviors within a specific context are determined by partly different factors but personal norm is a strong predictor of both types of behaviors. The third study extends the findings from the previous one in segmenting consumers on cur­tailment behaviors and proenvironmental purchases. Three distinct types of consumers emerge from the data. The Non-greens are found to exhibit the lowest levels of green attitudes and behaviors, and the strongest car habits. The Curtailers are distinguished by performing primar­ily reductionist behaviors, and by being the most willing to reduce negative environ­mental impact of car use. The Ecovators are found to be the most inclined to purchase eco-innovations and also display the greenest values. The study shows that green consumers are a heterogeneous group that can be separated on the basis of green curtailment behaviors and proenvironmental purchase decisions, and that there seems to be no inherent contradiction in being an early adopter of new green technology (such as the AFV) and also having high levels of proenviron­mental values, beliefs, and norms. In the final study, innovation specific characteristics and consumer innovativeness factors are integrated with normative and attitudinal determinants influencing AFV adoption. The results show that personal and social norms, consumer novelty seeking, and four perceived innovation characteristics influence the adoption decision. Differences between AFV adopters’ and non-adopters’ ratings of AFV specific attributes are also analyzed. The contribution of this study is the integration of VBN theory and the DOI framework and the empirical conclusion that eco-innovations need to deliver on both traditional and proenvironmental attributes in order to be perceived as attractive by consumers. In sum, this thesis demonstrates the importance of proenvironmental personal norms for consumer adoption of a high involvement eco-innovation such as the AFV.

Over the last decade, electronics operating at high temperatures have been increasingly demanded to support in situ sensing applications such as automotive, deep-well drilling and aerospace. However, few of these applications have requirements above 460 °C, as the surface temperature of Venus, which is a specific target for the seismic sensing application in this thesis. Due to its wide bandgap, Silicon Carbide (SiC) is a promising candidate to implement integrated circuits (ICs) operating in such extreme environments. In this thesis, various analog and mixed-signal ICs in 4H-SiC bipolar technology for high-temperature sensing applications are explored, in which the device performance variation over temperatures are considered. For this purpose, device modeling, circuit design, layout design, and device/circuit characterization are involved. In this thesis, the circuits are fabricated in two batches using similar technologies. In Batch 1, the first SiC sigma-delta modulator is demonstrated to operate up to 500 °C with a 30 dB peak SNDR. Its building blocks including a fully-differential amplifier, an integrator and a comparator are characterized individually to investigate the modulator performance variation over temperatures. In the succeeding Batch 2, a SiC electromechanical sigma-delta modulator is designed with a chosen Si capacitive sensor for seismic sensing on Venus. Its building blocks including a charge amplifier, a multiplier and an oscillator are designed. Compared to Batch 1, a smaller transistor and two metal-interconnects are used to implement higher integration ICs in Batch 2. Moreover, the first VBIC-based compact model featured with continuous-temperature scalability from 27 to 500 °C is developed based on the SiC transistor in Batch 1, in order to optimize the design of circuits in Batch 2. The demonstrated performance of ICs in Batch 1 show the feasibility to further develop the SiC readout ICs for seismic sensor system operating on Venus.

QC 20170911

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Knowledge flows, especially in subsidiaries of multinational corporations, now have an increasing importance in organizations and therefore the discussion of research in the field. The aim of this work is to analyze flows not only through transfers, but also conversions of knowledge between the three families of intangible assets (external structure, internal structure and individual competence), since, for the creation of value, the key lies in the fact that such transfers and conversions be effective (SVEIBY, 2001). Concerning the method, this dissertation presents an empirical study through a survey applied along the Brazilian subsidiaries of multinational corporations, from a theoretical construct Knowledge-based view (KBV) where makes an analysis of the assets of individual competence, internal structure and external structure, and having for its object of study brazilian companies in Brazil listed in data collection known as the best and biggest from business magazine EXAME covering the year 2012. The study indicates, for the sample data (nine respondents) removed of the universe effectively researched (140 companies and not the initial 565), that there is the maximization of value creation from knowledge flows in two directions only by knowledge transfers and conversions of Individual Competencies for External Structure, within the Internal Structure, of Internal Structure for Individual Competencies and within the External Structure
Os fluxos de conhecimento, de modo especial em subsidiárias de corporações multinacionais, passam a ter uma importância crescente nas organizações e por consequência nas discussões de pesquisas no campo. O objetivo deste trabalho é a análise dos fluxos, não somente através das transferências, mas também das conversões, de conhecimento entre as três famílias de ativos intangíveis (estrutura externa, estrutura interna e competência individual), uma vez que, para a criação de valor, a chave reside no fato de tais transferências e conversões serem eficazes (SVEIBY, 2001). Quanto ao método, esta dissertação apresenta um estudo empírico desenvolvido através de uma survey aplicada junto às subsidiárias brasileiras de corporações multinacionais, a partir de um construto teórico da visão baseada no conhecimento (VBC) - ou Knowledge-based View (KBV) - onde se faz uma análise dos ativos de competência individual, de estrutura interna e de estrutura externa, e tendo por objeto de estudo as empresas do Brasil listadas no levantamento de dados conhecido como Melhores e Maiores da Revista EXAME referente ao ano de 2012. O estudo indica, para a amostra dos dados (nove respondentes) retirada do universo efetivamente pesquisado (140 empresas e não as 565 iniciais), que existe a maximização de criação de valor a partir dos fluxos de conhecimento em duas direções somente pelas transferências e conversões de conhecimento de Competências Individuais para a Estrutura Externa, dentro da Estrutura Interna, da Estrutura Interna para as Competências Individuais e dentro da Estrutura Externa

碩士
國立臺灣大學
職業醫學與工業衛生研究所
90
Legionella pneumophila is a pathogen that causes Legionnaires’ diseases. It can be transmitted by inhalation of bacteria containing aerosols to humans. Since mostly there isn’t enough nutrition in the natural environment, Legionella pneumophila becomes viable but nonculturable(VBNC). Due to the limitation detection technology of the Legionella penurnophila. remains difficult to know if such Legionella penurophila, which is viable but nonculturable(VBNC) , exists in our environment. Therefore, the present study aimed to explore whether there exists the virulence trait and some special proteins of Legionella penurophila in VBNC phase. It is noted that, Legionella penurophila will gradually lose its culturability in the non-nutrition environment and then thansform into VBNC phase. Even thought in VBNC phase, the Legionella penurophila it still can keep its viability. Also, no dead cells had been found in non-nutrition environment, which for 63 days of conditions monitoring. Besides, Legionella penurophila has its unique protein quantity in different growth phases.For exsample, exponential phase has greastest protein quantity (36.72 μg/107 cell), followed by stationary phase (7.93μg/107 cell), decline phase (6.55 μg/107 cell) and VBNC phase (0.83 μg/107 cell). As the nutrition source decreased, the amount of protein also declined, such difference between nutrition and non-nutrition condition can even reach 44 times. Overall, the present study had identified 10 kinds of proteins in the VBNC phase. The existence of Flagellin protein in this phase indicated that the Legionella penurophila still has motility and virulence trait. Meanwhile, Heat shock protein may play a protective role against the defense mechanism generated by the infected macrophages in the beginning process of VBNC phase.

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2012
VBNC state represents a specific differentiation program into a survival state with several modifications in the cells walls and cytoplasmic membrane, a decrease in metabolic activity and maintaining gene expression. This state is induced upon exposure to stressful conditions, including nutrient starvation, different temperatures, pH, oxygen concentration and exposure to light or UV radiation. VBNC cells can represent an unknown public health risk, especially in the food industry, since they are unable to grow in routine analysis bacteriological media. This work aimed to develop a PCR based diagnostic procedure to detect VBNC cells of Listeria monocytogenes, a foodborne pathogen, able to resuscitate. It were defined as PCR targets Imo2522 and Imo0186 genes, due to their potential as Rpfs, and used for primers designo Two more genes were considered for detection, one for L. monocytogenes identification and one as internal amplification control. The Multiplex-PCR reaction established was characterized and had a 50 pg detection limit for the target genes. DNA extraction protocols were developed to yield enough DNA from low bacteria number for Multiplex-PCR detection. However the detection of just a few cells was neve r observed. Therefore it was adopted Lmo0186-PCR reaction with 125 fg DNA detection limit and the ability to detect 1 VBNC cell. Assays performed with PMA, which inhibits amplification from dead cells, revealed the same sensitivity, whereas inclusion of an amplification control decreased it. To overcome this issue a 5-fold increase of DNA sample in PCR reaction was necessary. Protocol application to spiked food revealed a 104 and 106 cells detection limits for active and VBNC cells respectively. ln parallel, VBNC state induction was performed, with different cellular concentrations, in five different induction media. Smaller induction periods were verified in the less concentrated suspensions and a high persistence of injured cells was detected in the higher concentrated suspensions.
Discrepâncias de viabilidade bacteriana entre os resultados de contagens realizadas a partir de ensaios de culturabilidade e ensaios indiretos com o objetivo de detetar membranas celulares intactas são relatadas desde os anos de 1800. No entanto, foi apenas na década de 1980 que Byrd and Colwell l introduziram a hipótese da existência de um estado similar à dormência, no qual as células bacterianas poderiam subsistir a condições menos favoráveis à sua sobrevivência, no qual não ocorreria crescimento. Tal estado foi denominado de viável mas não cultivável (VBNC) e a primeira evidência experimental foi obtida por Xu et ai. (1982)2. Segundo Oliver (1993)3 este estado pode ser definido como uma célula que, embora metabolicamente ativa, é incapaz de realizar a divisão celular necessária para o seu desenvolvimento num meio de cultura que normalmente suportaria o seu crescimento. No entanto, a hipótese da existência de um estado VBNC levantou alguma controvérsia entre a comunidade científica, pois este implicaria que os métodos microbiológicos convencionais, que assumem que a culturabilidade é um reflexo direto da viabilidade, não seriam adequados para estudos de quantificação de células viáveis. Existia de facto uma certa resistência de alguns autores em aceitar a existência de um estado VBNC que não seria idêntico a outros estados fisiológicos já descritos, nos quais as células bacterianas não seriam imediatamente cultiváveis em meio de cultura4 - 7. Tendo em conta que muitos dos métodos utilizados para quantificar a viabilidade celular eram indiretos, e como tal discutíveis pelos autores mais céticos, a evidência chave dependeria da capacidade de reanimação das células que haviam entrado no estado VBNC. Assumindo-se que este estado seria um meio de sobrevivência, as células deveriam ter a capacidade de reanimar quando as condições fossem apropriadas ao seu crescimento. Como tal diversas tentativas foram realizadas no intuito de obter a reanimação das células VBNC, mas muitas demonstraram-se infrutíferas dado que não excluíam a possibilidade de recrescimento de uma pequena fração de células cultiváveis ainda presentes na população de células VBNC4 - 6,8. A evidência conclusiva foi obtida por Whitesides and Oliver (1997)9 no estudo com Vibrio vulnificus, no qual a atribuição dos eventos de reanimação à presença de poucas células cultiváveis seria extremamente improvável. Apesar de alguma contestação este estudo, e outros que se seguiram, levaram ao desaparecimento da controvérsia existente, sendo já geralmente aceite que o estado VBNC existe. O estado VBNC representa um programa de diferenciação específico para um estado de sobrevivência após exposição a condições desfavoráveis. Uma das modificações mais associadas a este estado é a redução da dimensão celular, podendo ocorrer a transição de uma morfologia de bacillus para cocóides 5,8,1O. Algumas modificações nas paredes celulares foram também já descritas, nomeadamente o aumento no número total de interligações, incluindo interligações incomuns como DAP-DAP em Escherichia coli, e na quantidade de mucopéptidos covalentemente ligados a uma lipoproteína ll ,12. Tais alterações na parede celular são passíveis de contribuir para uma maior resistência a antibióticos, dado que estes têm como alvo principal as células em crescimento ativo, tendo tal já sido reportados,8,13. Foi igualmente já descrito uma reorganização do subproteoma da membrana celular externa bem como um perfil proteico relativamente diferente aos das células ativas ou no estado de sobrevivência com baixo metabolismo ("starvation survival,,)14. Modificações metabólicas também ocorrem as quais incluem a diminuição da síntese de macromoléculas, do transporte de nutrientes e da taxa de respiração, mantendo-se a absorção de aminoácidos, os níveis de ATP e rRNA e a presença de plasmídeos 5,7,8,15. A contínua expressão de genes foi também detetada, sendo que alguns autores reportaram a contínua expressão de genes de virulência neste estado, enquanto noutros estudos esta expressão não foi detetada, mas no entanto o potencial virulento era mantido e poderia ser retomado após a reanimação das células VBNC 5,8,16,17. Assumindo o estado VBNC como uma estratégia de sobrevivência, a sua indução pode ser despoletada pela exposição a condições não favoráveis à subsistência da célula bacteriana, podendo ser estímulos químicos ou ambientais, tais como falta de nutrientes, diferentes temperaturas, pressões osmóticas, alterações do pH, concentração de oxigénio, presença de metais pesados, ou conservantes alimentares e ainda exposição à luz ou radiação UV. A reanimação das células VBNC demonstrou-se mais complexa, havendo alguns estudos cujos ensaios de reanimação se revelaram infrutíferos, indicando que para alguns microrganismos serão necessários outros fatores que não apenas a inversão do estímulo de indução, e que poderão mesmo incluir a presença de um organismo superior como mediador B,17-20. Os mecanismos responsáveis quer pela indução do estado VBNC quer pela reanimação ainda não são conhecidos, no entanto alguns dados relevantes foram já demonstrados. No caso da indução o peróxido de hidrogénio parece ter um papel importante, dado a incapacidade de metabolização deste composto, bem como o fator sigma alternativo RpoS que parece ser um regulador da indução do estado VBNC 5,8,21. Na reanimação há evidências que um grupo de proteínas extracelulares, denominadas fatores de promoção de reanimação ("resuscitation promoting factors" - Rpfs), estarão envolvidas no processo de reanimação, tendo sido já identificadas em diferentes géneros. Estas proteínas apresentam semelhanças com transglicolases solúveis e estarão envolvidas em alterações da parede celular. Outro tipo de Rpfs possivelmente associadas serão as autoindutoras do crescimento estáveis ao calor ("heat-stable autoinducers of growth") que aparentam ser moléculas de sinalização secretadas. Apesar de ainda existirem muitos parâmetros a desvendar no estado VBNC, a existência do mesmo já foi descoberta em vários microrganismos, incluindo patogéneos, podendo representar um reservatório desconhecido dos mesmos 5,6. Tal pode representar um perigo para a saúde pública, dado que neste estado os microrganismos não serão detetados pelos métodos microbiológicos convencionais. No caso da segurança alimentar esta possível ameaça deveria ser considerada, tendo em conta que, quer os alimentos quer o processo da sua produção e acondicionamento, podem apresentar vários dos fatores de indução previamente mencionados. Outro fator importante é o facto do controlo biológico destes produtos ser ainda maioritariamente baseado na culturabilidade dos microrganismos. Um dos patogéneos importantes na segurança alimentar é Listeria monocytogenes. Este microrganismo é o único dentro do seu género, que engloba mais sete espécies, geralmente associado à ocorrência de listeriose humana. Esta doença apresenta sintomas deveras severos, com uma elevada taxa de mortalidade (cerca de 30%), principalmente nos grupos de risco determinados (adultos e jovens imunocomprometidos, grávidas, infantis e idosos). Apesar de uma baixa taxa de incidência, a severidade da doença derivou num nível zero de tolerância à sua presença nas amostras alimentares. Já foi igualmente reportada a entrada no estado VBNC e capacidade de reanimação por este patogéneo 22- 24 • Desta forma a presença do mesmo em produtos alimentares deveria contabilizar a existência de células VBNC de forma a assegurar a segurança dos consumidores. O objetivo do presente trabalho foi o desenvolvimento de um método de diagnóstico baseado em PCR para a deteção de células VBNC de L. monocytogenes, com potencial de reanimação, em produtos alimentares. Este método deverá permitir a deteção da amplificação a partir de genes previamente escolhidos, fornecendo uma resposta específica, sensível e rápida, sendo que com a complementação com o reagente monoazida de propídio (PMA), os resultados obtidos corresponderão a células viáveis, dado que este inibe a amplificação a partir de células não viáveis. Os genes alvo definidos foram o Imo2522 e Imo0186, cujas proteínas serão potenciais Rpfs uma vez que apresentam características semelhantes às descritas para estes fatores. Seguidamente, foi efetuado uma pesquisa ("blast") da sequência das proteínas respetivas e, após a análise dos domínios das mesmas, foi efetuada uma comparação entre os alinhamentos preliminares das sequências em Listeria spp. e L. monocytogenes. A análise desta comparação permitiu verificar que ambas as sequências proteicas se apresentavam conservadas para o género Listeria, principalmente no caso de Lmo0186. Foram então definidas zonas para o posterior desenho de primers, de forma a obter uma amplificação específica para L. monocytogenes, as quais se baseavam numa região variável para Lm02522 e numa zona conservada que flanqueava uma zona variável em Lmo0186. Seguidamente, foram obtidas as sequências dos genes respeitantes a estas proteínas e realizado um alinhamento global por Clustal W, tendo sido obtidas as sequências consenso de ambos os genes. Estas foram utilizadas para o desenho de primers. Após o desenho dos pares de• primers, o potencial destes formarem estruturas secundárias foi analisado, após o que foi escolhido um par para cada gene e um para o controlo interno de amplificação (IAC) - GFP. Adicionalmente, e tendo em conta que existia a possibilidade de não ocorrer especificidade apenas para L. monocytogenes, foram incluídos primers para o gene hlyA cuja amplificação é específica para esta espécie. Tendo em conta que se pretendia uma reação em Multiplex-PCR, conduzindo à simultânea amplificação destes alvos, foi efetuado um Gradiente-PCR, o qual permitiu analisar a eficiência destes primers isoladamente, a diversas temperaturas de ligação, e definir a temperatura de 51,6oC para posteriores ensaios. Seguidamente, foram realizados ensaios para definir a especificidade e sensibilidade da reação. Para tal foram utilizadas amostras de DNA extraído através do método adaptado de Pitcher et aI. (1989)25. No ensaio de especificidade foram testadas seis espécies do género Listeria, incluindo mais do que uma estirpe para contabilizar a variabilidade intraespécie, bem como seis estirpes de E. coli. Os resultados revelaram que os primers Lmo2522 seriam específicos para L. monocytogenes, enquanto que os primers Lmo0186 seriam específicos para Listeria spp., uma vez que apenas L. grayii e L. seeligeri não apresentaram amplificação. Tal não foi considerado problemático, dado que a análise de resultados deveria ser efetuada juntamente com os resultados obtidos para a amplificação de hlyA. Seguidamente foi realizado o ensaio de sensibilidade, usando concentrações de DNA continuamente decrescentes, até 10 pg. Observou-se que até à concentração de 50 pg todos os produtos de amplificação eram distinguíveis o que não se verificava nas concentrações menores, pelo que este seria o limite de deteção da reação Multiplex-PCR. Foram igualmente realizados estes ensaios com cada par de primers isoladamente, tendo-se verificado limites de deteção de 5 pg para HlyA e 0,5 pg para Lmo2522 e Lmo0186, indicando uma diminuição de sensibilidade quando em Multiplex-PCR de 10x e 100x, respetivamente. o próximo passo foi a aplicação da reação Multiplex-PCR a DNA extraído a partir de culturas com concentrações de células progressivamente menores. O método de Pitcher et al. (1989)25 apesar de providenciar amostras de DNA muito puro, é laborioso e dispendioso. Tratando-se de um método de diagnóstico pretendia-se um método rápido, fácil de realizar e de baixo custo. Adicionalmente, o método a aplicar deveria apresentar elevada eficiência, tendo em conta que o mesmo deveria permitir uma extração de DNA suficiente para amplificação mesmo a partir de apenas algumas células. Este parâmetro seria crucial para o sucesso do protocolo uma vez que este seria aplicado para deteção direta de células VBNC em produtos alimentares, e como tal dependeria apenas da quantidade de células inicialmente presentes nesses produtos. Desta forma procedeu-se ao desenvolvimento de um protocolo de obtenção de DNA, tendo sido iniciados os ensaios com o método de lise das células por fervura. Estes ensaios demonstraram que este método conduzia a elevada variabilidade na quantidade de DNA obtido, independentemente da estirpe. Adicionalmente, verificou-se a existência de substâncias inibidoras na reação de PCR, tendo ocorrido a ausência de amplificação do IAC. Estes efeitos poderiam ser ligeiramente reduzidos pelo aumento da concentração de BSA na reação de PCR ou pela diluição das amostras, sendo que, no entanto, nenhum destes procedimentos, e outros testados para remoção dos detritos e proteínas, resultaram numa melhoria evidente da amplificação pretendida. Duas hipóteses foram colocadas para justificar a ausência de amplificação: inibição por excesso de DNA ou uma lise celular ineficiente com consequente baixa libertação de DNA. Para averiguar estas hipóteses foram sujeitas a lise suspensões com diferentes números de células, bem como o aumento da amostra aplicada no tubo de PCR. Os resultados mostraram que a lise celular era ineficiente resultando numa baixa concentração de DNA. Para aumentar a eficiência deste passo, foi efetuada a preparação das células numa solução de lise, TE+O,5% SDS, que pela ação do detergente aniónico poderia auxiliar na lise celular. O ensaio foi realizado nas mesmas condições anteriores, tendo sido obtidos os mesmos resultados negativos e demonstrando que o método se mantinha ineficiente. Para aumentar a referida eficiência de lise celular foi realizado um protocolo baseado na disrupção mecânica das células com microesferas de vidro na presença da solução de lise acima mencionada. Foram testadas duas suspensões correspondendo a aproximadamente 107 e 108 cfu. No entanto, foi apenas detetada amplificação do produto dos primers Lmo0186 ao testar a amostra obtida por diluição decimal, ocorrendo uma ausência completa de amplificação, incluindo do IAC, a partir das amostras originais. A inibição de amplificação foi atribuída, maioritariamente, à presença de substâncias inibidoras, dado que ambas as amostras apresentavam resultados semelhantes, o que não seria expectável considerando as diferenças no número de células utilizadas. Estes ensaios indicaram que a precipitação de DNA seria necessária, de modo a retirar quaisquer substâncias inibidoras das amostras. Tendo em conta que este passo iria aumentar a recuperação do DNA extraído, foi utilizado como método de lise o mais simples, a fervura das células, na presença de TE+2% SDS. Os resultados obtidos a partir de três amostras com cerca de 104 , 106 e 108 cfu, revelaram que a amplificação apenas era detetável a partir da suspensão mais concentrada, indicando uma baixa concentração de DNA nas amostras com menor número de células. No entanto, tendo em conta que o SDS na reação poderia igualmente influenciar os resultados através da inibição da reação, também este efeito foi averiguado . Sendo assim foram utilizadas duas soluções de lise: TE+O,5% SDS e TE+2% SDS+10% Tripton X-100 como uma solução de lise mais forte. Os resultados mostraram que a solução de lise mais fraca não seria suficiente, uma vez que apenas ocorreu amplificação com a solução de lise mais forte, e apenas na amostra de maior concentração celular. Tendo em conta que este novo protocolo se apresentava mais eficiente que os anteriormente utilizados, mas ainda ineficaz para a extração a partir de amostras de menor concentração celular, foi realizada uma otimização ao nível da precipitação de DNA. Para tal testou-se o efeito da incubação em gelo, durante 1 h, seguida de uma centrifugação de 15 ou 30 min a 4oC, Os resultados demonstraram que a incubação em gelo permitia uma melhor recuperação do DNA libertado no decorrer da extração e que maior período de centrifugação não estava associado a maior rendimento de DNA. No entanto, apesar de ocorrer uma melhor recuperação de DNA com estas alterações, as mesmas não permitiam ainda a amplificação a partir de amostras de menores concentrações celulares, tendo sido então o protocolo alterado para uma lise mecânica com microesferas. Para auxiliar o processo foram utilizadas as duas soluções de lise com melhores resultados, no entanto, ambos os protocolos utilizados apenas apresentaram resultados positivos nas amostras de maiores concentrações celulares (cerca de 108 cfu). Para aumentar a eficiência do método o tempo de agitação foi aumentado para 20 min e foi ainda adicionado o reagente GES, cujo efeito desproteinizante poderia auxiliar no processo de lise. Para manter elevada a eficiência do método foi ainda efetuada uma comparação de ambas as soluções de lise, de modo a evitar qualquer possível efeito inibitório das mesmas, tendo sido escolhida como solução de lise o TE+2% SDS. Neste ponto a eficiência da lise e precipitação do DNA era já bastante elevada, pelo que não se justificava uma contínua ausência da amplificação das amostras de menores concentrações celulares, sendo tal atribuível à baixa sensibilidade da reação do Multiplex-PCR. De modo a aumentar a mesma foram realizadas tentativas de otimização ao nível da concentração de MgCI2, da concentração dos primers e ainda do tempo de ligação dos primers. As melhores condições detetadas foram a concentração de 0,4 pmol/L e o tempo de ligação de 1 min, mantendo -se a concentração de MgCI2 inicialmente utilizada de 3 mM. Estas otimizações, apesar de apresentarem algumas melhorias face às anteriormente utilizadas, não conduziram ao aumento pretendido da sensibilidade do Multiplex-PCR. Sendo assim uma nova estratégia foi iniciada baseada na amplificação utilizando os pares de primers individualmente em cada reação. Foram efetuados ensaios para averiguar se, após estas alterações, a sensibilidade destes pares se mantinha e se estes seriam capazes de detetar baixas concentrações de DNA. Todos os pares permitiam amplificação a partir de amostras celulares com 102 cfu, sendo que a amplificação com Lmo0186 era mais facilmente visível em gel. Desta forma este par foi novamente sujeito a ensaios de sensibilidade, quer com DNA stock, quer com DNA extraído com o método desenvolvido. Os resultados revelaram um limite de deteção de 125 fg de DNA, e uma capacidade de amplificação a partir de amostras de concentrações celulares muito baixas, 10 e 1 cfu. Os mesmos resultados foram obtidos com amostras de células VBNC, revelando a eficiência do método. Seguidamente foi adicionado a esta reação o IAC, o qual será necessário para a validação dos possíveis resultados negativos provenientes de amostras alimentares. Para tal foi necessário determinar a concentração do plasmídeo pCambia 1302, contendo o gene gfp, na qual a amplificação do gene alvo não seria prejudicada. Foi escolhida inicialmente a concentração de 350 fg, no entanto esta levou a uma diminuição na eficiência da amplificação do gene alvo. Uma vez que a diminuição da concentração do plasmídeo para 3,5 fg obteve os mesmos resultados, um equilíbrio seria necessário de forma a que a amplificação do gene alvo não fosse lesada. Diversas concentrações de plasmídeo foram testadas, tendo-se verificado que para valores inferiores a 90 fg ocorria uma maior inibição da reação. As concentrações com melhores resultados foram os 350 e 175 fg. Para contornar o efeito negativo na amplificação do gene alvo, foi efetuado o aumento do volume da amostra de DNA no tubo de PCR, tendo-se verificado que seria necessário um aumento de 5x, correspondendo a cerca de 250 fg. Adicionalmente foram realizados os ensaios com PMA. Estes demostraram que este reagente tem a capacidade de impedir a amplificação de DNA proveniente de células não viáveis, sendo apenas detetável a amplificação do DNA proveniente de células ainda viáveis. A aplicação deste reagente a suspensões de células VBNC, com diferentes concentrações, permitiu verificar que a amplificação a partir apenas de células viáveis incluindo VBNC era possível, verificando-se igualmente que a intensidade de amplificação era relativamente inferior à observada em ensaios anteriores ao usar as mesmas suspensões para deteção de células totais. Esta observação era mais evidente na amplificação correspondente às suspensões com maior concentração celular, relevando uma maior ocorrência de células não viáveis. A fase final deste trabalho consistiu na aplicação do método desenvolvido em amostras de queijo artificialmente contaminadas com L. monocytogenes. Nos ensaios realizados foi verificado que a matriz alimentar apresentava uma forte influência no resultado da amplificação, reduzindo o limite de deteção. Foi igualmente verificada a ocorrência de amplificação não específica, a qual poderia derivar de microrganismos presentes ou da própria matriz alimentar. De forma a atribuir a amplificação inespecífica foi efetuado uma pesquisa, através do NCBI, da sequência dos primers. Segundo as informações obtidas, uma das complementaridades possíveis corresponderia a 80S taurus, sendo que, no entanto, o tamanho previsto de amplificação não correspondia aos verificados. Não obstante, a amplificação inespecífica era distinguível da pretendida e como tal não foram efetuadas mais análises neste sentido. Os resultados dos ensaios efetuados revelaram que a amplificação era detetada até à concentração de 104 cfu em culturas overnight e 106 células nas suspensões de indução de células VBNC. A diminuição da deteção a partir das amostras de queijo poderia ser atribuível quer ao aumento da entropia durante a extração de DNA, derivado da presença de outros microrganismos bem como da própria matriz alimentar, quer ao esgotamento dos reagentes da reação de PCR, nomeadamente primers, pela amplificação inespecífica detetada. Para averiguar esta última hipótese, as amostras foram sujeitas a maiores temperaturas de ligação, para aumentar a restringência da reação, tendo sido igualmente testado o efeito do aumento da concentração dos primers para 0,8 e 1 pnp1J/ Embora um aumento de sinal de amplificação fosse visível, as alterações não conduziram ao desaparecimento da amplificação inespecífica nem a uma alteração do limite de deteção nas amostras. Sendo assim, foi considerado que a melhor estratégia seria a remoção da matriz alimentar antes da realização do protocolo de extração de DNA através da realização de diferentes centrifugações, que permitissem a separação inicial da matriz alimentar das células bacterianas e seguidamente a recolha destas para posterior extração de DNA. Outra alternativa testada foi a utilização de uma matriz alimentar diferente: folhas de rúcula. Os ensaios realizados com estas amostras e procedimentos, revelaram uma amplificação apenas detetável nas amostras inoculadas com suspensões celulares com 106 cfu, indicando uma recuperação ineficiente das células. Não se observou amplificação inespecífica mas é necessário otimizar a recolha das células a partir deste tipo de alimento. Paralelamente a este trabalho foi ainda efetuada a análise do período de indução necessário para a perda de culturabilidade de suspensões com diferentes concentrações celulares nos mesmos meios de indução de VBNC. As concentrações celulares analisadas foram 106 , 104 , 102 , 10 e 1 cfu/mL. Verificou-se que, nas suspensões de maior concentração celular, a indução poderia demorar mais de um ano, sendo que à data do término desta tese algumas suspensões mantinham-se ainda cultiváveis. Relativamente às restantes concentrações celulares, verificou-se que os tempos necessários para a indução eram relativamente menores. Na concentração de 104 cfu/mL a perda de culturabilidade, em TSA, variava entre o 140 e 990 dia e para as suspensões de 102 cfu/mL apenas seriam necessários cerca de 9 a 40 dias. Foi verificado que nas suspensões de menores concentrações celulares (10 e 1 cfu/mL) o tempo de indução era bastante semelhante ao verificado para as suspensões de 102 cfu/mL, e que nas concentrações de 104 e 102 cfu/mL a persistência de células injuriadas, detetadas pela culturabilidade em TSA+SP, era muito prolongada, com máximos detetados de 170 e 127 dias respetivamente, o que não ocorria nas concentrações de 10 e 1 cfu/mL. Tais observações levantam a hipótese de existir algum mecanismo de controlo de indução, mediante a concentração celular, subjacente. Atualmente estão a decorrer novos ensaios e após a análise de todos os dados espera-se poder compreender um pouco melhor o processo de indução de células de VBNC em L. monocytogenes.

The influence of the viable but non-culturable (VBNC) state on specific phenotypic traits of Escherichia coli O157:H7 as well as its transport behaviour in porous media was examined in this study. E.coli O157:H7 is a human pathogen capable of entering a VBNC state following exposure to sublethal stress. In the VBNC state, E.coli O157:H7 is not detectable by culture assays; yet, is able to retain its ability to cause human illness. This study examined specific transport-related properties of culturable and VBNC E.coli O157:H7 cells. As well, transport behaviors of the two cellular states were compared using sand-packed columns under steady-state flow. When E.coli O157:H7 cells entered a VBNC state, significant decreases in the hydrophobicity and lengths/widths of the cells, and a significant increase in extracellular polymeric substances on the cell surfaces were measured. Transport experiments indicated significantly (p<0.05) greater mass transport of VBNC cells through unwashed sand compared to culturable cells. This research contributes to the current knowledge describing VBNC E.coli O157:H7 cells, raises questions concerning the accuracy of culture-based E.coli O157:H7 identification protocols, and suggests that bacteria transport in the subsurface is a truly dynamic process.
Soil Science

Legionella pneumophila, the causative agent of Legionnaires’ disease (LD), is an intracellular pathogen of freshwater protozoa that can also persist in the environment as a free-living bacterium. L. pneumophila has many morphological forms that fit within a developmental cycle. In water, L. pneumophila enters into a viable but non-culturable (VBNC) state that is largely uncharacterized. VBNC cells were produced from two developmental L. pneumophila forms, stationary phase forms (SPFs) and mature infectious forms (MIFs) by suspension in double deionized (dd) or tap-water at 45°C. Electron microscopy results showed that VBNC cells have a unique morphology and that in tap water they lose their poly 3-hydroxybutyrate inclusion bodies. Both SPFs and MIFs lost culturability faster in dd- than in tap water, and addition of salts to dd-water prolonged L. pneumophila culturability and enhanced viability. However, MIFs retained higher viability in dd- and tap water (85% and 51%, respectively) than SPFs (5% and 20%, respectively) as determined by the BacLight vital stain. Only ~1 VBNC cell out of 105 of those produced from SPFs in tap water regained culturability via infection of Acanthamoeba. All VBNC cells, except for those produced from SPFs in dd-water, resisted both digestion inside Tetrahymena spp. and detergent-mediated lysis. SDS-PAGE analysis and shotgun proteomics revealed a number of VBNC cell specific proteins; one of these was 3-hydroxybutyrate dehydrogenase (BdhA), which is involved in the metabolism of poly 3-hydroxybutyrate inclusion bodies. A bdhA mutant showed an early loss of culturability and a dramatic decrease in viability as compared to the parent strain, and complementing the mutant with a functional bdhA gene restored the parent's strain phenotypes. In conclusion, VBNC L. pneumophila has a distinct morphology and physiology that varies according to the developmental stage and the environmental conditions used to produce such VBNC cells. VBNC cells have a different protein profile and morphology than the culturable cells, suggesting that this state constitutes a distinct differentiated form within the developmental cycle of L. pneumophila. BdhA seems to influence L. pneumophila survival and hence VBNC cell formation. Collectively, the results from this study provide a better understanding of L. pneumophila VBNC form and the factors influencing its formation.

碩士
東吳大學
微生物學系
100
“Viable but non-culturable” (VBNC) represents a state that cells are dormant and can not be cultured by standard growth media. The gram-negative bacteria can be induced to enter the VBNC state by starvation, cultured at low temperature, in high salinity, or in different pH. Many of human pathogenic bacteria can enter into the VBNC state, and retain their virulence during the state. This phenomena causes the blind spot in quarantine. In this paper, we investigate one of the human pathogens - the Vibrio cholera O139 which causing the cholera. In this study, we analyzed the differences among the cells in late log phase, in the progress of starvation-to-death, in the process of induction into VBNC state, and in BVNC state at RNA level by electrophoresis and Northern blot. Besides, specific expression primers were designed and RT-PCR was employed to analyse these 8 rrn operons expression patterns among the cells in late log phase, in the progress of starvation-to death, in the process of induction into VBCN state, and in VBNC state. According to the analysis result, we found that rrn a , rrn c, rrn f, rrn b and rrn b operons were constitutively expressed in the cells in late log phase, in the progress of starvation-to-death, in the process of induction into VBNC state, and in VBNC state. On the contrary, the rrn g operon was expressed in the late log phase and in starvation-to-death, but not in VBNC state. The expression pattern of rrn d, rrn e could not be verified in this study, because the lack of the proper specific expression patterns for these operons.

Durante il processo infettivo , agenti patogeni possono raggiungere siti anatomici in cui sono esposti a sostanze che ostacolano la loro crescita . Queste sostanze , ossia molecole immunologhe e antibiotici , provocano l'inibizione della crescita batterica e l'infezione è solitamente fermata. Tuttavia, come osservato nell'ambiente, anche in un ospite infetto concentrazioni di molecole inibenti e condizioni di stress potrebbero indurre l'attivazione di meccanismi di sopravvivenza che bloccano la capacità divisione dei batteri, ma permettono loro di rimanere in vita . I batteri "dormienti " possono essere riattivati ​​in particolari circostanze e possono determinare infezioni ricorrenti. Alcuni ricercatori hanno considerato la possibilità che le forme microbiche "non recuperabili" (batteri - privi di parete o microrganismi feriti o ( VBNC ) forme vitali ma non coltivabili ) possono causare la malattia . I batteri non in divisione o feriti non possono essere identificati attraverso le procedure microbiologiche diagnostiche standard, basate su metodi di coltura . Per questo motivo, negli ultimi tempi l'applicazione di metodi molecolari per la diagnosi microbiologica è in aumento nonostante ci sia la mancanza di un chiaro significato clinico dei risultati ottenuti.
During the infectious process, pathogens may reach anatomical sites where they are exposed to substances hindering their growth. These substances, i.e. immunological molecules and antibiotics, cause the inhibition of bacterial growth and the infection is usually stopped. However, as observed in the environment, also in infected host suboptimal concentrations of inhibitory molecules and a number of stress conditions might induce the activation of survival mechanisms blocking the division capability of bacteria but allowing them to stay alive. The “dormant” bacteria can be re-activated in particular circumstances and might determine recurrent infections and some researchers have considered the possibility that “non-recoverable” microbial forms (wall-deprived bacteria, injured microorganisms, viable but nonculturable (VBNC) forms, dormant bacteria) may cause disease. The non-dividing or injured bacteria cannot be identified by standard diagnostic microbiological procedures based on culture methods. For this reason, in recent times the application of molecular methods to the microbiological diagnosis is increasing despite of the lack of a clear clinical significance of the obtained results.