Os colifagos somáticos (CS) são os melhores indicadores de poluição fecal. Neste trabalho, foi determinada a abundância de CS em amostras de água do mar, plâncton, e bivalves coletadas em Santos, São Sebastião e Ubatuba. Houve correlação positiva entre CS e as bactérias marinhas viáveis, coliformes termotolerantes, E.coli e enterococos intestinais, e a correlação foi negativa com a temperatura. As maiores contagens de CS foram obtidas em Santos. As freqüências das famílias encontradas nas amostras de água do mar e plâncton foram: Siphoviridae (50% e 65,8%), Podoviridae (36% e 15,8%), Microviridae (9% e 15,8%) e Myoviridae (5%, 2,6%), respectivamente. Em bivalves, só foi observada Siphoviridae. Os morfotipos observados foram A1 (3%), B1 (63%), C1 (21%) e D1 (13%). As técnicas de RFLP e rep-PCR não foram discriminatórias. 9,6% dos colifagos apresentaram os genes que codificam para as toxinas ST e/ou LT. O presente estudo está identificando os colifagos como perigos microbiológicos e gerando subsídios para avaliação de riscos microbiológicos no ecossistema marinho. / The somatic coliphages (SC) are the better indicator for fecal pollution. In this research, it was obtained the SC abundance in seawater, plankton and bivalves samples collected from Santos, São Sebastiâo and Ubatuba. SC counts were correlated with marine viable bacteria, thermotolerant coliforms, E. coli and intestinal enterococci, and the correlation was negative with the temperature. Highest SC counts were obtained from samples collected at Santos. The frequency of SC families found in seawater and plankton samples were: Siphoviridae (50% and 65.8%), Podoviridae (36% and 15.8%), Microviridae (9% and 15.8%), and Myoviridae (5%, 2.6%), respectively. In bivalves, only Siphoviridae was found. Morphotypes A1 (3%), B1 (63%), C1 (21%) and D1 (13%) were observed. The RFLP and rep-PCR techniques were not discriminatory. 9.6% of coliphages contained genes codifying for thermostable toxin (ST) and/or thermolabil toxin (LT). This study is identifying the coliphages as microbial hazard and giving support to later studies for microbial risk assessment of marine ecosystem.

Orientadores: Marcelo Brocchi, Gustavo Bueno Gregoracci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T09:27:26Z (GMT). No. of bitstreams: 1 SartiSprogis_AdrianeCristina_M.pdf: 1852459 bytes, checksum: 397af9ee41d085346189ca76eee83f96 (MD5) Previous issue date: 2014 / Resumo: A salmonelose é uma zoonose que representa um sério problema de saúde pública mundial, devido: à sua alta prevalência, à dificuldade de seu controle, ao seu caráter endêmico, à morbidade e à mortalidade. O conhecimento da ocorrência das diferentes sorovariedades de S. enterica em diferentes regiões e países pode ajudar no rastreamento e reconhecimento de patógenos emergentes, e assim implementar políticas de tratamento e prevenção. A grande maioria das sorovariedades expressa dois tipos diferentes de antígenos flagelares codificados pelos genes fliC (fase 1) e fljB (fase 2), sendo assim denominadas bifásicas. Contudo, algumas sorovariedades expressam apenas uma das fases, e são denominadas monofásicas. É possível que a variação de fase flagelar em S. enterica esteja associada a uma função de escape do sistema imunológico, por aumentar o repertório de antígenos expressos pela célula bacteriana, evitando temporariamente a resposta imune celular. Assim sendo, S. enterica bifásicas possuiriam uma vantagem seletiva sobre as monofásicas, porém isso não é totalmente verificado nos estudos epidemiológicos, pois no Estado de São Paulo, S. enterica I,4,[5],12:i:-, (monofásica) é uma das mais comumente associadas aos casos de diarreia e/ou infecções sistêmicas em pacientes humanos. De fato, a partir da década de 1990 houve um aumento significativo da S. enterica I,4,[5],12:i:- em muitos países. Sequências de profagos são muito comuns em S. enterica, sendo de conhecimento que esses fagos codificam vários fatores que contribuem para patogenicidade, diversidade genética e/ou características que aumentam o fitness. Coculturas experimentais de linhagens de S. enterica podem induzir espontaneamente profagos, que matam bactérias sensíveis, e assim a indução espontânea de fagos em uma população lisogênica acentua a competitividade entre populações. Neste estudo foram analisadas culturas puras de S. enterica Enteritidis (bifásica) adicionadas de fagos líticos induzidos de S. enterica I,4,[5],12:i:-, bem como coculturas entre as duas sorovariedades citadas, nas quais foram observadas induções espontâneas de fagos associados à alta densidade populacional e alterações das taxas de crescimento em ambos os estudos, corroborando a hipótese de que S. enterica monofásica pode alterar a dinâmica populacional a seu favor, pela liberação de fagos líticos à outra sorovariedade, interferindo no crescimento populacional de S. enterica Enteritidis, e que o sucesso evolutivo de S. enterica I,4,[5],12.:i:- pode estar associado a fagos líticos atuando como um regulador na ecologia bacteriana. Esses dados podem mudar nosso conhecimento sobre a interação bactéria-fago de uma simples relação parasita-hospedeiro para uma coevolução de duas vias entre seus genomas / Abstract: Salmonellosis is a zoonosis that is a serious public health problem worldwide, due to its high prevalence, difficulty controlling, their endemicity, morbidity and mortality. The knowledge of the occurrence of different serovars of S. enterica in different regions and countries can help in tracking and recognition of emerging pathogens and thus implement policies for treatment and prevention. The majority of serovars express two different types of flagellar antigens encoded by genes: fliC (phase 1) and fljB (phase 2), so called biphasic. However, some serovars express only one of the phases and are termed monophasic. It is possible that flagellar phase variation of S. enterica is associated with an escape function of the immune system to increase the repertoire of antigens expressed by the bacterial cell temporarily preventing cellular immune response. Thus, S. enterica biphasic would have a selective advantage over monophasic, but this is not fully verified in epidemiologic studies, because in the State of São Paulo, S. enterica I,4,[5],12:i:-, (monophase) is the one most commonly associated with cases of diarrhea and/or systemic infections in human patients, in fact, from the 1990s there was a significant increase of S. enterica I,4,[5],12:i:- in many countries. Prophages sequences are very common in S. enterica, with the knowledge that these phages encode several factors that contribute to pathogenicity, genetic diversity and/or characteristics that increase fitness. Cocultures experimental strains of S. enterica prophages can induce spontaneous, killing susceptible bacteria, and thus the spontaneous induction in a population of lysogenic phage enhances the competitiveness between populations. This study analyzed pure cultures of S. enterica Enteritidis ( biphasic ) added lytic phage induced from S. enterica I,4,[5],12:i:-, as well as cocultures between the two serovars cited where inductions were observed spontaneous phage associated with high population density and changes in growth rates in both studies, supporting the hypothesis that S. enterica monophase can alter the population dynamics to their advantage by releasing lytic phage to another serovar, interfering with the population growth of S. enterica Enteritidis and the evolutionary success of S. enterica I,4,[5],12:i:- may be associated with lytic phages acting as a regulator in bacterial ecology. These data may change our understanding of bacteria- phage from a simple parasite-host coevolution for a two-way between their genomes / Mestrado / Clinica Medica / Mestra em Clínica Médica

Objective: National and supranational directives (EU-bathing water directiv, WHO directives) exist to examine the bathing-water quality with concern to public health criteria. E.coli, Intestinal enterococci and Enteroviruses serve as indicators for the contamination of a water with pathogenic bacteria and viruses. As an valuable indicators for the viral contamination bacteriophages are discussed. The aim of this work is the evaluation of somatic and F+ RNA coliphages as indicators of a contamination with enteric viruses considering a lignite mining lake as example. Approach: In weekly intervals, the concentration of bacterial and viral indicators, as well as the presence or absence of enteric viruses in the mining lake "Werbeliner See" (central German lignite mining area), in the flooding water and in the sewage plant Leipzig-Rosental, which significantly influences the flooding water, was examined. The paramters E.coli, Intestinal enterococci and spores of Clostridium perfringens serve as indicators for the hygienic state. As possible indicators of a viral contamination, somatic and F+ RNA coliphages were examined. Several hygienic relevant enteric viruses (Entero-, Noro-, Astro-, Adeno-, Rota- and Hepatitis A-viruses) were detected by means of adequate molecular biological methods and enumerated. Results: 1. The sewage plant Leipzig-Rosental eliminated fecal bacteria and bacteriophages very efficiently (about two logs), however the reduction of E.coli and enterococci was significant higher then the reduction of Cl.perfringens-spores and bacteriophages. The purification capacity benefits from the application of chemical precipitants (Fe (III) cloride / sulfate). 2. The reduction of genomes of Noro-, Astro- and Enteroviruses by the sewage plant was in the range of one to two logs. 3. All examined viruses could be detected in the inflow and the outflow of the sewage plant during the time of examination. 4. The concentration of fecal bacteria in the lake Werbeliner See was very low. The requirements by the EU-bathing water directive (76/160/EWG) with 2000 . (100 ml)-1 (E.coli, imperial value) and 100 . (100 ml)-1 (enterococci, guide value), respectively, were under-run clearly, also concerning the individual sampling days. 81 % and 92 % of the samples of the lake Werbeliner See (except the inflow site) were below the detection limit of 0,1 PFU . ml-1 for somatic and F+ RNA coliphages, respectively. 5. In contrast, DNA and RNA of all examined virus could be found in the lake. Quantitative PCR reveald a virus load between 0 und 105 Gen.equiv. . l-1 for Entero-, Noro- and Astroviruses. 6. The detection of infectious Enteroviruses on BGM cell lines yielded one positve sample (=1,2%). Since the concentration of Enterovirus genomes in the quantitative PCR was three to four log-steps lower then this of the Noro- and Astroviruses, one could specualte, that the concentration of infectious Noro- and Astroviruses in the lake was quite high. 7. The low rate of positive infectious Enteroviruses in the lake reflects the low concentration of bacteriophages. But the direct comparison of the concentration of bacteriophages with the concentration of infectious Enteroviruses in the flooding water casts doubt on the indicatior value of bacteriophages regarding Enteroviruses. In conclusion, this finding suggests that an exclusive indication of bacteriophages concerning a contamination with Enteroviruses is not possible. 8. The use of bacteriophages as indicators of a wider range of enteric viruses could attenuate this discrepancy by the epidemiological more frequent appearance of enteric viruses as a sum. 9. In this context bacteriophages could, due to their comparatively high resistence, have a key function in the assesment of the hygienic quality of a water sample. / Problemstellung: Um die hygienische Qualität von Badegewässern zu beurteilen existieren nationale und übernationale Richtlinien (EU-Badegewässerrichtlinie, WHO-Richtlinien). E.coli, Intestinale Enterokokken und Enteroviren dienen dabei als Indikatoren für die Belastung des Gewässers mit humanpathogenen Keimen bzw. Viren. Als Indikatoren für die virale Belastung sind in diesem Zusammenhang unter anderem Bakteriophagen im Gespräch. Gegenstand dieser Arbeit sind die Evaluierung von Somatischen und F+ RNA Coliphagen als Indikatoren für die Kontamination mit enteralen Viren am Beispiel eines Tagebausee sowie die Dokumentation der hygienischen Wasserqualität des Tagebausees unter Berücksichtigung des Flutungswassers. Vorgehensweise: In wöchentlichen Routineuntersuchungen wurden die Konzentrationen bakterieller und viraler Indikatoren, sowie die An- oder Abwesenheit enteraler Viren im Tagebausee "Werbeliner See" untersucht. Verschiedene, hygienische relevante enterale Viren (Entero-, Noro-, Astro-, Adeno-, Rotaviren, sowie Hepatitis A-Viren) wurden mittels geeigneter molekularbiologischer und zellkultureller Methoden detektiert und quantifiziert. Ergebnisse: 1. Die Kläranlage Leipzig-Rosental eliminierte sehr effizient (ca. zwei Größenordnungen) fäkale Bakterien und Bakteriophagen, wobei die Reduktion von E.coli und Enterokokken signifikant höher war als die von Cl.perfringens-Sporen und Bakteriophagen. Die Reinigungsleistung wird durch den Einsatz von chemischen Fällmitteln (Eisen-(III)-Chlorid / -Sulfat-Salze) begünstigt. 2. Noro-, Astro- und Enteroviren, für die quantitative Untersuchungen durchgeführt worden, konnten durch die Kläranlage um ein bis zwei Größenordnungen reduziert werden. 3. Alle untersuchten Viren konnten in Zu- und Ablauf der Kläranlage molekularbiologisch nachgewiesen werden. 4. Die Konzentration fäkaler Bakterien im Werbeliner See war sehr gering. Die Vorgaben der EU-Badegewässerrichtlinie (76/160/EWG) von 2000 . (100 ml)-1 (E.coli, Grenzwert) bzw. 100 . (100 ml)-1 (Enterokokken, Leitwert) wurden dabei, auch bezogen auf die einzelnen Probenahmetage, deutlich unterschritten. 81 % bzw. 92 % der Seeproben (außer Einleitungsstelle) lagen unterhalb der Nachweisgrenze von 0,1 PFU . ml-1 für Somatische- bzw. F+ RNA Coliphagen. 5. Im Kontrast dazu konnte DNA bzw. RNA aller untersuchten Viren im See gefunden werden. Die genomische Viruslast der mittels real-time-PCR detektierten Entero-, Noro- und Astroviren lag zwischen 0 und 105 Gen.äquiv. . l-1. 6. Der Nachweis infektiöser Enteroviren auf BGM-Zellen im Werbeliner See ergab ein einzelnes positives Ergebnis im Untersuchungszeitraum (= 1,2 %). Allerdings lag die Konzentration detektierter Enteroviren-Genome im Mittel drei bis vier Größenordnungen unter der Konzentration von Noro- und Astroviren. Es kann deshalb spekuliert werden, dass für diese Viren ebenfalls ein hoher Anteil infektiöser Proben zu erwarten gewesen wäre. 7. Die niedrige Nachweisrate infektiöser Enteroviren im Werbeliner See spiegelte sich in der sehr niedrigen Konzentration an Bakteriophagen wider. Allerdings lässt der direkte Vergleich der Bakteriophagenkonzentration mit dem Nachweis infektiöser Enteroviren im Flutungswasser Zweifel am Indikatorwert von Bakteriophagen für eine Kontamination mit Enteroviren aufkommen. Eine exklusive Indikation von Bakteriophagen bezüglich einer Kontamination mit Enteroviren scheint anhand dieser Befunde nicht möglich. 8. E.coli und Enterokokken wurden in Laborversuchen unter verschiedenen Versuchsbedingungen signifikant schneller inaktiviert als Somatische Coliphagen und Noroviren. Gleiches galt im Vergleich zu Cl. perfringens-Sporen, Astroviren und F+ RNA Coliphagen. 9. Bakteriophagen könnten deshalb auf Grund ihrer vergleichsweise hohen Resistenz eine wichtige Schlüsselfunktion für die Beurteilung der hygienischen Qualität einer Wasserprobe spielen.

Shiga Toxin (Stx)-konvertierende Bakteriophagen besitzen eine konservierte lambdo-ide Genomstruktur, weisen aber ein hohes Maß an genetischer Diversität auf. Aus diesem Grund wurden die bereits vorhandenen Sequenzdaten der Stx1-Phagen CP-1639 des Escherichia coli O111:H- Stammes 1639/77 und BP-4795 des E. coli O84:H4 Stammes 4795/97 vervollständigt und analysiert. Im Gegensatz zu dem induzierbaren Bakteriophagen BP-4795 fehlen CP-1639 einige Gene, die für den lytischen Lebenszyklus eines Bakteriophagen notwendig sind. Daher handelt es sich bei CP-1639 um einen kryptischen Prophagen, der nicht mehr in der Lage ist das Wirtschromosom zu verlassen und intakte Phagenpartikel zu bilden. Die Integra-tionsstellen wurden innerhalb des Gens yehV für BP-4795 und ssrA für CP-1639 bestimmt. In unmittelbarer Umgebung des Integrationsortes von CP-1639 befindet sich ein eigenständiges integratives Element. Dieses besteht aus drei offenen Lese-rahmen unbekannter Herkunft, sowie einem Integrasegen und kann auch ohne Assoziation mit Phagen-DNA auftreten. Phagen können zusätzlich zu ihrem Genom Gene bakteriellen Ursprungs tragen. Diese können unter anderem infolge von Transpositionen oder einer unkorrekten Exzision der Phagen-DNA aus dem Wirtschromosom während des lytischen Lebenszyklus in das betreffende Phagengenom eingebaut werden. Eines solches Gen des Bakteriophagen BP-4795 kodiert das Typ III Effektorprotein NleA4795, dessen Funktionalität nach dem C-terminalen Einbau von neun Codons des Hämagglutinin (HA)-Epitopes des humanen Influenzavirus in die Sequenz des Gens nleA4795 überprüft wurde. Dies erfolgte unter Verwendung der Western Blot Analyse durch die Expression dieses Fusionsproteins in dem Wildtyp-Stamm 4795/97 und in der Deletionsmutante 4795escN des Stammes 4795/97. Die Typ III Sekretionssys-tem (T3SS)-inaktive Mutante 4795escN wurde im Verlauf dieser Arbeit hergestellt. Diese Untersuchungen zeigten ebenfalls, dass zur Sekretion von NleA4795 ein in-taktes T3SS notwendig ist. Weiterhin zeigte die Infektion einer HeLa-Zelllinie mit NleA4795-HA exprimierenden Bakterienstämmen und die anschließende Analyse mit Hilfe der Immunfluoreszenz, die Translokation des Proteins in eukaryotische Wirts-zellen. Darüber hinaus deuten die Ergebnisse dieser Untersuchung auf eine Lokali-sation von NleA4795-HA innerhalb des Trans-Golgi Netzwerkes hin. Die Verbreitung des Gens nleA wurde in insgesamt 170 Shiga Toxin-produzierenden E. coli und enteropathogenen E. coli Stämmen untersucht. Dies führte zur Identifika-tion von 14 verschiedenen Varianten des Gens nleA in 149 der überprüften Stämme, wobei mit Ausnahme von zwei Isolaten ebenfalls ein Markergen des T3SS nachge-wiesen werden konnte. Neben drei bereits bekannten Varianten konnten elf neue Varianten identifiziert werden, deren abgeleitete Aminosäuresequenzen sich zu 71% bis 96% glichen. Sequenzunterschiede traten insbesondere aufgrund der Deletion oder Insertion von vier bis 51 Aminosäuren im Mittelteil der potentiellen Proteine auf. Weiterhin deuten die Ergebnisse dieser Untersuchungen eine Assoziation bestimm-ter Varianten des Gens nleA mit spezifischen E. coli Serogruppen an. Southern-Blot Hybridisierungen zeigten, dass etwa ein Viertel der nleA-positiven Stämme zwei Ko-pien dieses Gens in ihrem Genom tragen. Hierbei handelt es sich meist um zwei verschiedene Varianten. In fast allen Fällen kodiert eine dieser Varianten, aufgrund einer Punktmutation oder Insertion eines IS-Elementes, ein verkürztes und vermut-lich nicht funktionelles Protein. Mit Hilfe von Transduktionsexperimenten konnten verschiedene Varianten des Gens nleA im Genom induzierbarer Phagen nachge-wiesen werden, was auf eine Verbreitung des Gens nleA durch den horizontalen Gentransfer hinweist.

Greywater is domestic household wastewater without inputfrom the toilet, i.e. wastewater from sinks, the shower,washing machine and dishwasher in a home. Source separation ofgreywater can be a strategy to enhance recirculation of plantnutrients and/or improve water use. The risk for transmissionof disease when reusing greywater is largely dependent on thecross-contamination by faeces. High levels of faecalindicators, mainly thermotolerant coliform bacteria, have beenreported in greywater, indicating substantial faecal pollution.However, growth of indicator bacteria within the system leadsto an overestimation of thefaecal input and thus the hygienerisk. The faecal input of the greywater in Vibyåsen,Sollentuna, North of Stockholm, was estimated to be 0.04 ±0.02 g faeces person-1 day-1 from the quantification of thefaecal sterol coprostanol, compared to 65 g, 5.2 g and 0.22 gp-1 d-1 using E. coli, enterococci and cholesterolrespectively. Prevalence of pathogens in the population and the faecalload based on coprostanol concentrations were used to form thebasis of a screening-level quantitative microbial riskassessment (QMRA) that was undertaken for rotavirus, Salmonellatyphimurium, Campylobacter jejuni, Giardia intestinalis andCryptosporidium parvum, looking at the treatment required to bebelow an acceptable level of risk (10-3) for reuse or dischargeof the greywater. The different exposure scenarios simulated–groundwater recharge, direct contact, irrigation andrecreational water–showed that a reduction of 0.7–3.7 log was needed for rotavirus, with the measured level offaecal load in Vibyåsen. The other pathogen of concern wasCampylobacter, where a 2.2 log reduction was needed forgroundwater recharge. The infectious dose of Salmonella is highand the excretion numbers of Giardia cysts and Cryptosporidiumoocysts low, resulting in no treatment requirements for theseorganisms under these circumstances. Pathogen input fromcontaminated food via the kitchen sink had a minor effect onthe microbiological quality of the greywater. Studies on virusoccurrence in greywater as well as validation of the faecalload of greywater at another site would give valuable input forfuture QMRAs. Greywater treatment efficiency studies, especially on virusremoval, are scarce and more investigations are warranted.Active sludge may not be a suitable technique for greywater dueto the low carbon content in this flow. Chemical precipitationhas the advantage of removing phosphorus as well as virusesefficiently and it is suggested as one possible method fortreating greywater. Otherwise the most common practice forgreywater treatment in Sweden is soil infiltration. However, itis suggested that the recommendations for wastewaterinfiltration also be observed for greywater, despite the lowfaecal load, due to the simulated results on virus reductionneeded. <b>Key words:</b>greywater, greywater reuse, greywatertreatment, microbial risk assessment, groundwater recharge,irrigation, recreational water, faecal contamination, indicatorbacteria, index organisms, faecal sterols, bacteriophages,enteric pathogens, rotavirus, Salmonella, Campylobacter,Giardia, Cryptosporidium, Legionella / NR 20140805

Wunden, die über eine Zeit von mehreren Monaten nicht abheilen, werden als chronische Wunden bezeichnet. Vor allem ältere Menschen sind betroffen. Infektionen mit Antibiotika-resistenten Bakterien können weitere Schwierigkeiten bei der Behandlung und Heilung hervorrufen. Zur Behandlung von chronischen Wunden entwickeln wir daher eine Hydrogel-basierte Wundauflage, welche heilungsfördernde Substanzen in die Wunde abgibt. Diese sind zum einen Bakteriophagen, also Viren, die Bakterien befallen und so die Infektion bekämpfen und zum anderen Wachstumsfaktoren, welche die Bildung von gesundem Gewebe anregen. Das Hydrogel wird mit diesen angereichert und reagiert mit Proteasen in der Wunde, sodass es sich teilweise auflöst und die Substanzen freigesetzt werden. Das Hydrogel basiert auf dem Polymer starPEG, welches gut verträglich ist. An einigen Stellen werden Peptide in das Hydrogel eingebaut, die von den Proteasen, welche von Bakterien oder dem Immunsystem produziert werden, zerschnitten werden. Die menschlichen Wachstumsfaktoren werden in Hefe produziert, indem die entsprechende DNA per Plasmid in diese eingebaut wird. Ein System zur effizienten Sekretion der Wachstumsfaktoren wird getestet, wodurch die Reinigung dieser für den medizinischen Einsatz erleichtert werden soll. Weiterhin wird eine computergestützte Design-Pipeline entwickelt, die es erlauben soll, verschiedene Hydrogel-Kompositionen in-silico zu testen. Damit sollen Kosten und Aufwand für Entwicklung und Anpassungen reduziert werden. Das Projekt findet im Rahmen des iGEM-Wettbewerbs im Bereich der Synthetischen Biologie statt und läuft bis zum Oktober 2022. Ziel ist es, bis dahin die einzelnen Bestandteile des Produkts zu entwickeln und im Labor getrennt zu testen. Es soll gezeigt werden, dass Bakteriophagen vom Hydrogel abgegeben werden und immer noch in der Lage sind, Bakterien abzutöten. Weiterhin soll die Diffusion der produzierten Wachstumsfaktoren aus dem Hydrogel quantifiziert werden.:Chronic wounds A new dressing for treating chronic wounds Results

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les virus entériques sont à l’origine de pathologies liées au péril fécal et dans l’état actuel des connaissances, la recherche des indicateurs de pollution fécale conventionnels (i.e. Escherichia coli, entérocoques) peut s’avérer inefficace pour évaluer le danger viral. La définition d’autres indicateurs pour gérer le danger lié à la présence des virus entériques dans les matrices hydriques et alimentaires est aujourd’hui nécessaire. Parmi eux, les bactériophages ARN F-spécifiques (FRNAPH) présentent plusieurs intérêts. Ces virus d’origine entérique sont présents en quantité importante dans les eaux usées. Très proches des virus entériques en termes de structure, ces microorganismes présentent l’avantage d’être facilement cultivables. Ils sont enfin souvent étudiés pour déterminer l’origine d’une pollution fécale (i.e. humaine ou animale). Certaines limites leur sont cependant fréquemment associées, que ce soit en termes de corrélation avec les pathogènes entériques ou concernant leur potentiel pour discriminer l’origine d’une pollution. Dans ce contexte, l’objectif du travail présenté ici était de préciser l’intérêt des FRNAPH en tant qu’indicateurs de pollution fécale mais aussi en tant qu’indicateurs de pollution virale dans l’environnement et les coquillages. Ces travaux ont permis dans un premier temps d’améliorer la capacité des FRNAPH à identifier les contaminations d’origine humaine. Nos résultats soulignent par ailleurs la plus-value apportée par la recherche des FRNAPH en cas de pollution fécale massive, en particulier si on s’intéresse à la contamination des coquillages. En effet, contrairement aux indicateurs bactériens, l’accumulation des FRNAPH ainsi que leur persistance dans ces aliments est très comparable à celles des virus entériques (i.e. norovirus). Enfin, en utilisant des méthodes de détection comparables, une forte corrélation entre la présence des FRNAPH d’origine humaine et celle des norovirus a été observée dans les coquillages. Compte tenu de ces résultats, une méthode de détection assurant la détection sensible des FRNAPH infectieux d’origine humaine dans différents types de matrices hydriques ou alimentaires (e.g. eaux de surface, fruits de mer, fruits rouges, salades) est proposée pour améliorer la gestion du danger viral / Enteric viruses are a leading cause of fecal-oral route transmitted diseases and currently, conventional fecal indicator bacteria (i.e. Escherichia coli, enterococcus) fail to assess this kind of hazard. In this context, the use of more efficient indicators to assess the hazard linked to viruses in water or foodstuff is required. F-specific RNA bacteriophages (FRNAPH) present numerous benefits for this purpose. Of enteric origin, these viruses are found in high concentrations in wastewater. Sharing many structural similarities with pathogenic enteric viruses, FRNAPH are easily cultivable and their potential to track the origin of the pollution is also often investigated. However, some limits are still associated with these indicators, regarding to their ability to track the origin of the pollution or concerning the lack of correlation with pathogens. In this context, the aim of this work was to make clear the potential of FRNAPH as fecal and as viral indicators in environmental waters and shellfish. As a first step, their ability to track human pollution was optimized. In addition, our results underlined the gains bringing by FRNAPH detection, especially when focusing on shellfish microbiological quality management. Indeed, unlike fecal indicator bacteria, the accumulation of FRNAPH and their persistence in shellfish have been found to be close to that of enteric viruses (i.e. norovirus). Furthermore, when using comparable methods for their detection, high correlation was observed between human FRNAPH and norovirus in shellfish. Taking into account these observations, a sensitive method allowing the detection of infectious FRNAPH of human origin was developed to improve viral hazard management in water and food commodities (e.g. environmental waters, shellfish, soft fruits, leaf)

In bacteria, the RNA polymerase (RNAP) consists of the following subunits: α2, β, β’, ω and σ. The core RNAP (α2ββ’ω) possesses the polymerising activity and it associates with one of the sigma factors to initiate transcription from a promoter region on the DNA template. All bacteria carry an essential housekeeping sigma factor and a number of extra cytoplasmic function (ECF) sigma factors. During alternate physiological states, a major part of transcriptional regulation is carried out by sigma factors, which act as transcriptional switches, thus, making it possible for bacteria to adapt to varied environmental signals by transcribing the necessary set of genes. Bacteriophages utilise various mechanisms for subverting the bacterial biochemical machinery for their advantage. One such example in E. coli is AsiA protein encoded by an early gene of T4 bacteriophage. Because of its property of binding to σ70, AsiA can inhibit transcription from E. coli promoters bearing –10 and –35 DNA sequences leading to inhibition of growth. σ70 of E. coli is also regulated by a stationary phase specific protein, Rsd, whose major function seems to be helping the cell in switching the transcription in favour of stationary phase genes. In this study we have investigated the mechanism of interaction of T4 AsiA and E. coli Rsd to σ70 of E. coli and also tried to determine the basis of differential inhibition of E. coli growth by AsiA and Rsd. In chapter one we have reviewed the published literature on regulation of transcription in bacteria. Some of the well known mechanisms of regulating gene expression are: DNA supercoiling, two component signal transduction system (TCS), regulation by alarmone ppGpp and 6S RNA, and sigma-antisigma interactions. Most bacteria carry a number of sigma factors and each of them is dedicated to transcribing genes in response to environmental signals. Intracellular levels of sigma factors and their binding affinity to core RNAP are deciding factors for initiating transcription from specific subsets of genes. In addition, sigma factor activity is also controlled by specific proteins, which bind to sigma factors (anti-sigma factors) under certain environmental conditions. A number of anti-sigma factors have been isolated from a variety of bacteria and the mechanisms of action of binding to cognate sigma factors have been worked out by using genetic, biochemical and structural tools. In chapter two, using yeast two hybrid assay (YTH), we have identified the regions of σ70 which interact with AsiA, and it was observed that amino acid residues from 547-603, encompassing region 4.1 and 4.2 are involved in binding to σ70. Interestingly, we found that truncated σ70 fragments lacking the N-terminal regions, apparently bound to AsiA with higher affinity compared to full length σ70. As AsiA expression, because of its transcription inhibitory activity, is inhibitory to E.coli growth, co-expression of the truncated C-terminal σ70 fragments (e.g. residues 493-613, σ70C121), which bind to σ70 with high affinity, could relieve growth inhibition. The complex of GST:AsiA-σ70C121 could be purified from E. coli cells. GST:AsiA purified from E .coli cells was found to be associated with RNAP subunits. Since further studies on this interaction required GST:AsiA preparation devoid of RNAP subunits, we decided to express this protein in S. cerevisiae. Bioinformatics analysis indicated the absence of a σ70 homologue in S.cerevisiae. As expected, GST:AsiA purified from the yeast was found to be free from any RNAP like proteins. The protein purified from yeast was used for in-vitro binding experiments. Our YTH analysis had indicated that deletion a part of region 4.1 or 4.2 of σ70 leads to loss of binding to AsiA. However, the published NMR structure of AsiA in complex with peptides corresponding to region 4 of σ70, showed that either region 4.1 or 4.2 alone can bind to AsiA indicating at the possible existence of two binding sites for AsiA. In order to confirm the physiological significance of this finding, we studied the interaction of truncated σ70 fragments lacking either region 4.1 or 4.2 with AsiA in-vivo in E. coli and in-vitro by affinity pull down assays. It was observed that σ70 fragments lacking either region 4.1 (σ70∆4.1) or 4.2 (σ70∆4.2), did not neutralize the GST:AsiA toxicity, indicating lack of interaction. The affinity purified GST:AsiA from these E. coli cells did not have σ70∆4.1 or σ70∆4.2 associated with it. Similar results were obtained from pull down assays in-vitro, where we found that σ70∆4.1 or σ70∆4.2 do not show any observable interaction with AsiA. This clearly established that the minimum region of σ70 required for physiologically relevant interaction with AsiA consists of both the regions 4.1 and 4.2. Chapter 3 of this thesis has been devoted to this aspect of AsiA-σ70 interaction. Having defined the minimum region of σ70 interacting with AsiA, we sought to identify the regions and amino acid residues of AsiA, which are critical for interaction with σ70. The approach for identification of mutants and their characterisation has been discussed in chapter 4. For this purpose, we made systematic deletions in the N and C-terminal regions of the protein and also isolated random mutants of AsiA, which lack binding to σ70 and thus are non-inhibitory to E. coli growth. It was found that deletion of 5 amino acids from N-terminus and 17 amino acids from C-terminus did not alter the inhibitory activity of AsiA. In contrast, deletion of N-terminal 10 amino residues led to complete loss of activity, while in the C-terminus, a gradual loss of activity was observed when amino acid residues beyond 17 amino acids were deleted. A 34 amino acids C-terminal deletion mutant was found to be completely inactive. E10K mutant was found to be inactive, but changes of E to other amino acids such as S, Y, L, A and Q were tolerated, indicating that negative charge at E10 is not a crucial element for interaction with σ70. Inactive mutants could be overexpressed in E. coli and showed reduced binding in YTH assay and were also poor inhibitors of in-vivo transcription in E. coli. We concluded that the primary σ70 binding site of AsiA is present in the N-terminus, yet C-terminal 64-73 amino acid residues are required for effective binding in-vivo. These studies also correlate the inhibitory potential of AsiA with its σ70 binding proficiency. In chapter 5, we have made a comparative analysis of mechanism of interaction of AsiA and Rsd to E. coli RNAP. Overexpression of Rsd was found to be less inhibitory to E. coli cell growth than that of AsiA. The affinity purified GST-AsiA from E. coli was found to have all the RNAP subunits associated with it, whereas, only σ70 was found to be associated with similarly purified GST:Rsd, pointing towards differences in binding to RNAP. In affinity pull down assays, in-vitro, it was found that both AsiA and Rsd do not show any observable binding to core RNAP. Binding of AsiA to σ70 in holo RNAP led to the formation of a ternary complex, whereas no ternary complex was observed when Rsd was made to interact with holo RNAP. Analysis of protein-protein interaction by YTH showed that region 4.1 and 4.2 are critical for binding of both AsiA and Rsd to σ70. However, in the case of Rsd, the surface of interaction is not limited to this region only and other regions of σ70 make significant contribution to this binding. Possibly, the interaction of Rsd with the core binding regions of σ70 prevents its association with core RNAP. Kinetic analysis of binding by surface plasmon resonance (SPR) showed that binding affinities (Kd) of AsiA and Rsd to σ70 are in similar range. Therefore, we concluded that the ability of AsiA to trap the holo RNAP is, probably, responsible for higher inhibitory activity of this protein compared to that of Rsd. Thus, T4 AsiA and E. coli Rsd, which share regions of interaction on σ70, have evolved differences in their mechanism of binding to RNAP such that T4 AsiA, by trapping the holo RNAP subverts the complete bacterial transcription machinery to transcribe its own genes. Rsd, on the other hand, has evolved to interact primarily with σ70, which favours the utilisation of core RNAP by other sigma factors.

Στα πλαίσια αυτής της διδακτορικής εργασίας μελετήθηκαν οι εμπορικά σημαντικότερες περιοχές καλλιέργειας και συγκομιδής οστρακοειδών του Ελλαδικού χώρου. Κατά τη διάρκεια περιόδου 18 μηνών πραγματοποιήθηκε μηνιαία συλλογή δειγμάτων στρειδιών (Οstrea edulis) και μυδιών (Mytilus galloprovincialis), τα οποία συλλέχθηκαν από έξι (6) διαφορετικά σημεία του Ελλαδικού χώρου και αναλύθηκαν για τους εντεροϊούς (EV), τους αδενοϊούς (Adv), τον ιό της ηπατίτιδας Α (HAV), τους ιούς Noro I και II (NLVI και NLVII), για το βακτήριο Ε. coli, καθώς και για σωματικούς κολιφάγους, τους F-sperific RNA βακτηριοφάγους και τους βακτηριοφάγους του Β. fragilis. Επιπλέον αναπτύχθηκαν μέθοδοι τόσο για την ανίχνευση παθογόνων ιών ανθρώπινης προέλευσης στα οστρακοειδή, όσο και για την ανίχνευση των "πιθανών δεικτών" αυτών των ιών. Οι μέθοδοι εξετάστηκαν προκειμένου να αξιολογηθεί η απόδοση καλής ποιότητας από όλα τα εργαστήρια μέσω διεργαστηριακών αναλύσεων. Η μέθοδος που εφαρμόστηκε σε αυτή τη μελέτη για την ανίχνευση των ιών στα οστρακοειδή βασίζεται στην εξαγωγή και την ομογενοποίηση του πεπτικού αδένα με χρήση διαλύματος γλυκίνης, pH 10, απομόνωση των νουκλεϊνικών οξέων και ενίσχυση του γονιδιώματος των ιών που αναλύονται. Για την ανίχνευση του βακτηρίου E. coli χρησιμοποιήθηκε η μέθοδος των πολλαπλών σωλήνων, ενώ για την ανίχνευση των βακτηριοφάγων χρησιμοποιήθηκε η μέθοδος καλλιέργειας διπλοστιβάδας. Για το βακτήριο E. coli, σε σύνολο 138 δειγμάτων, 110 δείγματα (ποσοστό 79,7%) βρέθηκαν να ανήκουν στην κατηγορία Α (MPN/100g σάρκας = <20 έως 220), δηλαδή χαρακτηρίζονται σαν δείγματα χαμηλής μόλυνσης, 25 δείγματα (ποσοστό 18,1%) βρέθηκαν να ανήκουν στην κατηγορία Β (MPN/100g σάρκας = 220 έως 3500), οπότε χαρακτηρίζονται σαν δείγματα μεσαίας μόλυνσης, ακατάλληλα προς κατανάλωση χωρίς να προηγηθεί διαδικασία εξυγίανσης, ενώ μόνο 3 δείγματα (ποσοστό 2,2%) βρέθηκαν να ανήκουν στη κατηγορία C (MPN/100g σάρκας =3500 έως >18000), δηλαδή είναι δείγματα υψηλής μόλυνσης. Οι ιοί που εμφανίζονται με μεγαλύτερη συχνότητα στα οστρακοειδή της Ανατολικής Μεσογείου είναι οι αδενοϊοί (34% των δειγμάτων βρέθηκαν θετικά για τους αδενοϊούς) και ακολουθούν οι εντεροϊοί (16,7% των δειγμάτων βρέθηκαν θετικά για τους εντεροϊούς). Αντίθετα, ο ιός της ηπατίτιδας Α (ποσοστό θετικών δειγμάτων = 4,34%), καθώς και οι ιοί Noro I (ποσοστό θετικών δειγμάτων = 2,1%) και Noro II (ποσοστό θετικών δειγμάτων = 1,47%%) εμφανίζονται σε μικρό ποσοστό δειγμάτων. Τέλος, 80 δείγματα (58%) βρέθηκαν θετικά (παρουσία πλακών βακτηριοφάγων) για τους σωματικούς κολιφάγους, με τον αριθμό των πλακών να κυμαίνεται από 71,4 έως 584800 pfp/100g, 52 δείγματα (37,7%) βρέθηκαν θετικά για τους F-specific RNA βακτηριοφάγους (αριθμός των πλακών από 76,2 έως 17051 p100g) και 33 δείγματα (24%) βρέθηκαν θετικά για τους βακτηριοφάγους του Bacteroides fragilis (αριθμός των πλακών από 194.5 έως 5266,25 pfp/100g). Τόσο για το βακτήριο E. coli όσο και για τους βακτηριοφάγους πραγματοποιήθηκαν διεργαστηριακές αναλύσεις προτύπων, οι οποίες οδήγησαν στο συμπέρασμα ότι οι αντίστοιχες μέθοδοι χαρακτηρίζονται ως αξιόπιστες. Η στατιστική ανάλυση έδειξε ότι το βακτήριο E. coli παρουσιάζει θετική συσχέτιση με τους σωματικούς κολιφάγους, αλλά δεν δείχνει στατιστικά σημαντική συσχέτιση ούτε με τους F-specific RNA βακτηριοφάγους, ούτε με κανέναν από τους ιούς εντερικής προέλευσης. Επίσης, θετική συσχέτιση παρουσίασαν οι αδενοϊοί με τους εντεροϊούς, καθώς και οι σωματικοί κολιφάγοι με τους βακτηριοφάγους του B. fragilis. Η μοναδική συσχέτιση μεταξύ ιών εντερικής προέλευσης και βακτηριοφάγων βρέθηκε για τους αδενοϊούς και τους βακτηριοφάγους του B. fragilis. Εάν αυτό επιβεβαιωθεί σε περαιτέρω μελέτες, τότε η συγκεκριμένη κατηγορία βακτηριοφάγων θα μπορούσε να αποτελέσει έναν καλό δείκτη πρόβλεψης της παρουσίας αδενοϊών σε δείγματα οστρακοειδών. Επιπλέον μελετήθηκε η σχέση που μπορεί να υπάρχει μεταξύ των φυσικοχημικών παραμέτρων και των μικροοργανισμών που εξετάστηκαν. Η επεξεργασία αυτή οδήγησε στο συμπέρασμα ότι το βακτήριο E. coli ανιχνεύεται σε μεγαλύτερα ποσά όταν το διαλυμένο οξυγόνο και η περιεκτικότητα σε άλας του ύδατος είναι αυξημένα. Αντίθετα, αύξηση της θερμοκρασίας οδηγεί σε μείωση της ανίχνευσης του βακτηρίου. Επίσης, η περιεκτικότητα σε άλας φαίνεται να επηρεάζει θετικά και τον ιό της ηπατίτιδας Α, αν και ο μικρός αριθμός θετικών δειγμάτων γι’αυτόν τον ιό δεν μπορεί να επιτρέψει την εξαγωγή ασφαλών συμπερασμάτων. Το pH και το διαλυμένο οξυγόνο των υδάτων οδηγεί σε αύξηση της ανίχνευσης των βακτηριοφάγων του B. fragilis, χωρίς όμως να μπορούμε να ισχυριστούμε ότι κάτι τέτοιο ισχύει, λόγω του μικρού αριθμού θετικών δειγμάτων γι’αυτούς τους βακτηριοφάγους. Τέλος, η αύξηση της θερμοκρασίας των υδάτων φαίνεται να οδηγεί και σε αύξηση της παρουσίας των F-specific RNA βακτηριοφάγων, και το ίδιο παρατηρήθηκε και με την αύξηση του διαλυμένου οξυγόνου στο νερό. Η παρούσα μελέτη αποτελεί την πρώτη διεξοδική έρευνα για την ιογενή κοπρανώδη μόλυνση τον οστρακοειδών στην Ελλάδα. Επιπλέον, αντιπροσωπεύει την πρώτη μελέτη σχετικά με τη αποτελεσματικότητα των οργανισμών - δεικτών ιϊκής μόλυνσης, καθώς και για τη συσχέτιση της μικροβιολογικής επιβάρυνσης των οστρακοειδών με τις φυσικοχημικές παραμέτρους του περιβάλλοντος ύδατος. Η μελέτη κατάλληλων δεικτών που σχετίζονται με την παρουσία εντερικών ιών στα οστρακοειδή οδήγησε σε χρήσιμα συμπεράσματα για τη χρήση της ανίχνευσης των βακτηριοφάγων ως δεικτών ιϊκής μόλυνσης. Εντούτοις, απαιτείται περαιτέρω μελέτη προκειμένου να προσδιοριστεί και η χρήση των βακτηριοφάγων ως δεικτών που θα μαρτυρούν την προέλευση (ανθρώπινη ή ζωική) των εντερικών ιών που ανιχνεύονται στα οστρακοειδή. / In this doctorate investigation, important shellfish growing areas of Greece have been defined and studied. Oysters (Ostrea edulis) and mussels (Mytilus galloprovincialis) were obtained on a monthly basis over an 18 month sampling period. These samples were collected by six (6) different points of Greece and were analyzed for enteroviruses (EV), adenoviruses (Adv), virus of hepatitis A (HAV), Noro viruses I and II (NLVI and NLVII ), bacterium E. coli, as well as for somatic coliphages, F-sperific RNA bacteriophages and bacteriophages of B. fragilis. Moreover, methods were developed for the detection of pathogenic viruses of human origin in the shellfish, as well as for the detection of potential "viral indicators". The methods were examined in order to validate the good quality performance from all the laboratories via interlaboratory analyses. The method that used in this study for the detection of human enteric viruses in the shellfish is based on the export and homogenisation of digestive gland with glycine buffer at pH 10, viral nucleic acid extraction and amplification of the genomes of the analysed human viruses. The procedure applied for detection of E. coli consists on a five tube, three dilution most probable number (MPN) method, while the method for the detection of bacteriophages was the double-agar-layer method. For E. coli analysis, in a total number of 138 samples, 110 samples (79,7%) were found to belong in category A (MPN / 100 g of flesh = < 20 until 220), that it means these samples are characterized as samples of low pollution, 25 samples (18,1%) were found to belong in category B (MPN / 100 g of flesh = 220 until 3500), therefore are characterized as samples of intermediate pollution, inadequate to consumption without precedes process of cleansing, while only 3 samples (2,2%) were found to belong in category C (MPN / 100 g of flesh = 3500 until > 18000), that it means they are samples of high pollution. The viruses that are presented with higher frequency in the shellfish of Eastern Mediterranean are the adenoviruses (34% of samples were found positive for adenoviruses) and follow enteroviruses (16,7% samples they were found positive for enteroviruses). On the contrary, virus of hepatitis A (percentage of positive samples = 4,34%), as well as the Noro I viruses (percentage of positive samples = 2,1%) and Noro II viruses (percentage of positive samples = 1,47%%) are presented in small number of samples. Finally, 80 samples (58%) were found positive (presence of plaques of bacteriophages) for somatic coliphages, with the number of plaques between 71,4 and 584800 pfp / 100 g, 52 samples (37,7%) were found positive for F - specific RNA bacteriophages (number of plaques from 76,2 to 17051 pfp/ 100 g) and 33 samples (24 %) were found positive for the bacteriophages of B. fragilis (number of plaques from 194,5 to 5266,25 pfp / 100 g). Interlaboratory studies involved the testing of reference materials of E. coli and bacteriophages were used as part of the good quality performance assessment program to be applied all over the study, and led to the conclusion that the corresponding methods are characterized by good reliability. According to the statistical analysis of the results, the presence of E. coli seems to be significantly related with the presence of somatic coliphages. However, E. coli do not present significant statistical relation neither with F - specific RNA bacteriophages, nor with all of the viruses of intestinal origin. Also, adenoviruses were significantly related with enteroviruses, as well as somatic coliphages with the bacteriophages of B. fragilis. The unique significant relation between viruses of intestinal origin and bacteriophages was found for the adenoviruses and bacteriophages of B. fragilis. If this is confirmed in further studies, then this category of bacteriophages could constitute a good indicator of forecast of presence adenoviruses in samples of shellfish. Moreover, we studied the relation that can exist between the physic-chemical parameters and the micro-organisms that were examined. This analysis led to the conclusion that E. coli is detected in higher levels when the dissolved oxygen and the salinity of water are increased. On the contrary, increase of temperature leads to reduction of detection of E. coli. Also, the salinity appears to influence positively also virus of hepatitis A, even if the small number of positive samples of this virus cannot allow the export of sure conclusions. The pH and the dissolved oxygen of waters lead to increase of detection of bacteriophages of B. fragilis, but the small number of positive samples for these bacteriophages can’t give safe conclusions. Finally, the increase of temperature of waters appears to lead also to increase of presence of F - specific RNA of bacteriophages, and the same was also observed with the increase of dissolved oxygen in water. This study constitutes the first extensive research for the fecal viral pollution of shellfish in Greece. Moreover, it represents the first study with regard to the effectiveness viral indicators, as well as for the correlation of microbiological parameters of shellfish with the physical-chemical parameters of water. The study of suitable indicators that are related with the presence of enteric viruses in the shellfish led to useful conclusions on the use of detection of bacteriophages as indicators of viral pollution. Nevertheless, further study is required in order to determine also the use of bacteriophages as indicators that will testify the origin (human or animal) of the enteric viruses that are detected in the shellfish.