O vínculo entre papilomavírus humano de alto risco e câncer cervical está bem estabelecido. Apesar da existência de vacinas profiláticas contra infecções pelos tipos mais comuns de HPV, para infecções e tumores causados por esses vírus as alternativas terapêuticas são restritas. Encontramos alguns motivos com homologias para proteínas do HPV de alto risco durante o imunoscreening de uma biblioteca de phage display com soros de participantes HPV-16-soropositivos da coorte Ludwig-McGill. Após enriquecimento das sequências, os bacteriófagos recombinantes foram purificados e amplificados para uso como imunógenos.Usando uma abordagem profilática, nós vacinamos experimentalmente camundongos imunocompetentes com um dos nossos bacteriófagos recombinantes, usando o bacteriófago sem inserto como controle. Estes camundongos foram então desafiados com células tumorais TC-1 (HPV-16 positivas), tendo-se avaliado as respostas imunes disparadas durante a progressão tumoral. Também usamos uma abordagem terapêutica, aonde os camundongos foram primeiro injetados com as células tumorais e imunizados com o bacteriófago após o estabelecimento do tumor. O crescimento tumoral foi monitorado e os tumores, baço e linfonodos foram avaliados quanto à quantidade e qualidade da resposta imunológica. Os testes de ELISA revelaram que todos os camundongos vacinados responderam à imunização com os diferentes bacteriófagos. O crescimento tumoral foi significativamente reduzido nas imunizações profiláticas e terapêuticas, embora a redução do tumor fosse mínima quando os camundongos foram tratados 9 dias após o enxerto. A redução no crescimento tumoral também se traduziu em uma sobrevivência significativamente maior para os camundongos imunizados. Estudos de infiltração celular não revelaram alterações em diversas sub-populações imunes, mas uma tendência de aumento de linfócitos T citotóxicos foi observada nos camundongos imunizados com PEP1 (bacteriófago contendo inserto). A importância deste aumento de CD8 na redução observada do crescimento tumoral foi confirmada utilizando camundongos CD8-knockout, onde a redução do crescimento tumoral previamente observada foi anulada. Foi observado um aumento de taxa CD8:CD4 nos camundongos imunizados e isto é uma indicação de ambiente tumoral citotóxico. Os ensaios de proliferação celular para testar a especificidade do antígeno dos linfócitos dos camundongos imunizados foram, no entanto, inconclusivos; da mesma forma, não pudemos alterar o padrão observado com o uso de adjuvante CpG. A utilidade da técnica de phage display também foi observada neste trabalho experimental. Trabalhos adicionais para entender o mecanismo de ação desses fagos recombinantes no controle do crescimento de tumores causados por HPV e seu potencial imuno-estimulador são necessários / The link between high-risk human papillomavirus and cervical cancer is well established. Despite the existence of prophylactic vaccines against infections by the most common types of HPV, therapeutic alternatives are limited for infections and tumors caused by these viruses. We found some homology motifs for high-risk HPV proteins during the immune-panning of a phage display library with sera from HPV-16- seropositive participants of the Ludwig-McGill cohort. After enrichment of the sequences, the recombinant bacteriophages were purified and amplified for use as immunogens. Using a prophylactic approach, we vaccinated experimentally immunocompetent mice with one of our recombinant bacteriophages using the insertless bacteriophage as a control. These mice were then challenged with TC-1 tumor cells (HPV-16 positive), and the immune responses triggered during tumor progression were evaluated. We also used a therapeutic approach where mice were first injected with tumor cells and immunized with the bacteriophage after tumor establishment. Tumor growth was monitored and tumors, spleen and lymph nodes were evaluated for the quantity and quality of the immune response. ELISA tests revealed that all vaccinated mice responded to immunization with the different bacteriophages. Tumor growth was significantly reduced in prophylactic and therapeutic immunizations, although tumor reduction was minimal when mice were treated 9 days after TC-1 cells grafting. The reduction in tumor growth also translated into a significantly greater survival for the immunized mice. Cell infiltration studies did not reveal changes in several immune subpopulations, but an upward trend in cytotoxic T lymphocytes was observed in mice immunized with PEP1 (insert-containing bacteriophage). The importance of this increase in CD8 in the observed reduction of tumor growth was confirmed using CD8-knockout mice, where the previously observed reduction of tumor growth was abolished. An increase in CD8:CD4 rate was observed in the immunized mice and this is an indication of a cytotoxic tumor environment. Cell proliferation assays to test the antigen specificity of lymphocytes from immunized mice were, however, inconclusive; likewise, we could not change the pattern observed with the use of CpG adjuvant. The usefulness of the phage display technique was also observed in this experimental work. Additional studies to understand the mechanism of action of these recombinant phages in the control of HPV tumor growth and its immunostimulatory potential are warranted

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.

Introduits dans l’environnement par l’intermédiaire de sources ponctuelles et diffuses, les virus et les bactériophages entériques peuvent se propager dans les cours d’eau par l’intermédiaire de différentes voies de dissémination. Détectées à la fois dans les eaux de surface et les sédiments des rivières, ces particules virales demeurent inertes dans le milieu hydrique. Leur propagation dépend donc uniquement des nombreuses interactions qu’elles partagent avec leur environnement. Qui plus est, la contamination virale des ressources en eau semble étroitement liée aux variations hydro-climatologiques. Mais malgré les connaissances déjà acquises à ce sujet, de multiples zones d’ombre subsistent concernant les variables et facteurs contrôlant le comportement in situ des particules virales dans le milieu hydrique. L’objectif de ce travail a donc été de définir le transport et le devenir des bactériophages ARN F-spécifiques dans une rivière en fonction de leurs caractéristiques propres et des conditions hydro-climatologiques. L’application de stratégies et méthodologies originales, tirées du domaine de l’hydrologie comme l’utilisation du temps de résidence de la masse d’eau ou l’échantillonnage automatique à haute fréquence, a permis d’étudier les comportements des bactériophages ARN F-spécifiques in situ. L’influence des facteurs environnementaux et plus particulièrement de la température de l’eau et du débit de la rivière sur la propagation et la survie in situ de ces particules infectieuses dans la colonne d’eau a été démontrée. Dans les sédiments, une distribution spatiale des bactériophages ARN F-spécifiques infectieux a été mise à jour. Cette particularité a pu être comprise et déchiffrée grâce à la combinaison de la caractérisation du sédiment et de l’étude du comportement d’attachement des quatre génogroupes. Les transferts de particules virales entre la colonne d’eau et les sédiments ont également pu être mis en exergue et s’avèrent être fortement dépendants des conditions hydro-climatologiques. Ainsi, la dynamique des bactériophages ARN F-spécifiques a pu être mieux appréhendée, et de même, les origines et la nature de la pollution virale ont été mieux discernées lors d’événements de crues. L’ensemble de ces résultats permet de compléter le puzzle de la dynamique des bactériophages ARN F-spécifiques dans la rivière. Les nouvelles approches expérimentales et méthodes d’analyse mises en place devraient permettre d’aboutir à une meilleure évaluation des risques viraux pour la santé humaine liés à l’utilisation des ressources en eau / Introduced into the environment through point and diffuse sources, enteric viruses and bacteriophages can be spread in watercourses via various dissemination routes. Detected in both surface water and river sediment, these viral particles remain inert in environmental water. Their spread is governed by many interactions that they have with their direct environment. Moreover, viral contamination of water resources is closely related to hydro-climatological variations. Despite the important knowledge already reported on this subject, many grey areas remain about the variables and factors controlling the in situ behavior of viral particles in environmental water. The aim of this study was therefore to define the transport and fate of F-specific RNA bacteriophages in a river according to their intrinsic characteristics and hydro-climatological conditions. The application of innovative strategies and methodologies from the hydrological science domain, such as the use of the residence time of the river water mass or high frequency automatic sampling, allowed studying the in situ behavior of F-specific RNA bacteriophages. The influence of environmental factors, especially water temperature and flow rate, has been demonstrated to have an impact on the in situ propagation and survival of infectious viral particles in the water column. Furthermore, the spatial distribution of infectious F-specific RNA bacteriophages was underlined in sediments. The accurate characterization of sediment and the study of the attachment capacity of the four genogroups explained this specific distribution. Finally, transfers of viral particles between the water column and sediment was highlighted and appeared to be highly dependent on hydro-climatological conditions. Besides the gained knowledge of the dynamics of F-specific RNA bacteriophages, the sources and origins of viral pollution of streams during rain and flood events were elucidated. This work helps completing the jigsaw puzzle on presence and transmission of F-specific RNA bacteriophages in river systems. The novel experimental approach further enhances human health-dependent viral risk evaluation linked to water resource utilization and management

Introdução. As Repetições Palindrômicas Curtas Agrupadas e Regularmente Interespaçadas (CRISPRs) são DNAs que consistem de repetições de nucleotídeos. Existem 3 tipos: CRISPR1-CAS, CRISPR3-CAS e CRISPR2. Os bacteriófagos são partículas virais que infectam bactérias. Os bacteriófagos SAP6, IME-EF1, BC-611, VD-13 e F4 são encontrados em E. faecalis assim como FL1ABC, FL2AB, FL3AB e FL4A. Objetivo. Identificar e caracterizar as classes de CRISPRs e bacteriófagos de E. faecalis e E. faecium isoaldos de amostras alimentares, clínicas e fezes de animais. Materiais e métodos. Foram usadas DNA de 153 isolados, sendo 98 de E. faecalis e 55 de E. faecium. A técnica de detecção foi a PCR utilizando iniciadores para os genes CRISPR1-Cas, CRISPR2, CRISPR3-cas e bacteriófagos, seguido por electroforese em gel de agarose. Resultados. Para E. faecalis foram detectadas 58 isolados que amplifacaram para o gene CRISPR1-Cas; 87 para CRISPR2 e 13 para CRISPR3-Cas. Para E. faecium foram detectadas 4 isolados positivos para o gene CRISPR1-Cas; 18 para CRISPR2 e 1 para CRISPR3-CAS. O bacteriófago FL2ABfoi detectado em 13 isolados de E. faecalis; o bacteriófago FL3AB em 13 isolados de E. faecalis e o FL4A em 28 isolados de E. faecalis. Conclusão. Neste estudo nos encontramos diferentes proporções e distribuições dos genes CRISPRs em E. faecalis e E. faecium. O bacteriófago FL4A apresentou-se como o mais frequente entre os bacteriófagos avalados. / Introduction. The CRISPR are small portions of DNA consisting of nucleotide repeats. There are three types of CRISPRs recognized: CRISPR-associated genes cas: CRISPR1-CAS and CRISPR3-CAS and a orphan locus lacking cas genes: CRISPR2. Bacteriophages are viral particles that infect bacterial. The bacteriophages SAP6, IME-EF1, BC-611 and F4 are found in E. faecalis as well as FL1ABC, FL2AB, FL3AB, FL4A. Objectivy: To Identify and to characterize CRISPRs genes and bacteriophage genes in E. faecalis and E. faecium isolated from food, clinical and animal fecal samples. Materials and methods. A total of 153 DNA samples were used, 98 from E. faecalis and 55 from E. faecium. The PCR technique using primers was used to detect the genes CRISPR1-Cas, CRISPR2, CRISPR3-cas and bacteriophages, followed the agarose gel electrophoresis. Results. To E. faecalis was detected 58 isolates that amplified the CRISPR1-Cas genes, 87 to CRISPR2 and 13 to CRISPR3-Cas. To E. faecium was detected 4 isolates positive to CRISPR1-Cas gene, 18 to CRISPR2 e 1 to CRISPR3-CAS. The FL2AB bacteriophage was detected in 13 isolates of E. faecalis; the FL3AB bacteriophage in 13 isolates of E. faecalis and the FL4A in 28 isolates of E. faecalis. Conclusion. In this work, we found out different proportions and distributions of CRISPRs genes in E. faecalis e E. faecium. The FL4A bacteriophage showed a high frequency among the bacteriophages tested.

O vínculo entre papilomavírus humano de alto risco e câncer cervical está bem estabelecido. Apesar da existência de vacinas profiláticas contra infecções pelos tipos mais comuns de HPV, para infecções e tumores causados por esses vírus as alternativas terapêuticas são restritas. Encontramos alguns motivos com homologias para proteínas do HPV de alto risco durante o imunoscreening de uma biblioteca de phage display com soros de participantes HPV-16-soropositivos da coorte Ludwig-McGill. Após enriquecimento das sequências, os bacteriófagos recombinantes foram purificados e amplificados para uso como imunógenos.Usando uma abordagem profilática, nós vacinamos experimentalmente camundongos imunocompetentes com um dos nossos bacteriófagos recombinantes, usando o bacteriófago sem inserto como controle. Estes camundongos foram então desafiados com células tumorais TC-1 (HPV-16 positivas), tendo-se avaliado as respostas imunes disparadas durante a progressão tumoral. Também usamos uma abordagem terapêutica, aonde os camundongos foram primeiro injetados com as células tumorais e imunizados com o bacteriófago após o estabelecimento do tumor. O crescimento tumoral foi monitorado e os tumores, baço e linfonodos foram avaliados quanto à quantidade e qualidade da resposta imunológica. Os testes de ELISA revelaram que todos os camundongos vacinados responderam à imunização com os diferentes bacteriófagos. O crescimento tumoral foi significativamente reduzido nas imunizações profiláticas e terapêuticas, embora a redução do tumor fosse mínima quando os camundongos foram tratados 9 dias após o enxerto. A redução no crescimento tumoral também se traduziu em uma sobrevivência significativamente maior para os camundongos imunizados. Estudos de infiltração celular não revelaram alterações em diversas sub-populações imunes, mas uma tendência de aumento de linfócitos T citotóxicos foi observada nos camundongos imunizados com PEP1 (bacteriófago contendo inserto). A importância deste aumento de CD8 na redução observada do crescimento tumoral foi confirmada utilizando camundongos CD8-knockout, onde a redução do crescimento tumoral previamente observada foi anulada. Foi observado um aumento de taxa CD8:CD4 nos camundongos imunizados e isto é uma indicação de ambiente tumoral citotóxico. Os ensaios de proliferação celular para testar a especificidade do antígeno dos linfócitos dos camundongos imunizados foram, no entanto, inconclusivos; da mesma forma, não pudemos alterar o padrão observado com o uso de adjuvante CpG. A utilidade da técnica de phage display também foi observada neste trabalho experimental. Trabalhos adicionais para entender o mecanismo de ação desses fagos recombinantes no controle do crescimento de tumores causados por HPV e seu potencial imuno-estimulador são necessários / The link between high-risk human papillomavirus and cervical cancer is well established. Despite the existence of prophylactic vaccines against infections by the most common types of HPV, therapeutic alternatives are limited for infections and tumors caused by these viruses. We found some homology motifs for high-risk HPV proteins during the immune-panning of a phage display library with sera from HPV-16- seropositive participants of the Ludwig-McGill cohort. After enrichment of the sequences, the recombinant bacteriophages were purified and amplified for use as immunogens. Using a prophylactic approach, we vaccinated experimentally immunocompetent mice with one of our recombinant bacteriophages using the insertless bacteriophage as a control. These mice were then challenged with TC-1 tumor cells (HPV-16 positive), and the immune responses triggered during tumor progression were evaluated. We also used a therapeutic approach where mice were first injected with tumor cells and immunized with the bacteriophage after tumor establishment. Tumor growth was monitored and tumors, spleen and lymph nodes were evaluated for the quantity and quality of the immune response. ELISA tests revealed that all vaccinated mice responded to immunization with the different bacteriophages. Tumor growth was significantly reduced in prophylactic and therapeutic immunizations, although tumor reduction was minimal when mice were treated 9 days after TC-1 cells grafting. The reduction in tumor growth also translated into a significantly greater survival for the immunized mice. Cell infiltration studies did not reveal changes in several immune subpopulations, but an upward trend in cytotoxic T lymphocytes was observed in mice immunized with PEP1 (insert-containing bacteriophage). The importance of this increase in CD8 in the observed reduction of tumor growth was confirmed using CD8-knockout mice, where the previously observed reduction of tumor growth was abolished. An increase in CD8:CD4 rate was observed in the immunized mice and this is an indication of a cytotoxic tumor environment. Cell proliferation assays to test the antigen specificity of lymphocytes from immunized mice were, however, inconclusive; likewise, we could not change the pattern observed with the use of CpG adjuvant. The usefulness of the phage display technique was also observed in this experimental work. Additional studies to understand the mechanism of action of these recombinant phages in the control of HPV tumor growth and its immunostimulatory potential are warranted

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.

Restriction endonucleases are a group of enzymes that cleave DNA at or around specific sequences, which are typically palindromic. A fosmid library was constructed from a metagenome isolated from soil from the Kogelberg Nature Reserve, Western Cape and was functionally screened for restriction endonucleases. Next-generation (NGS) Illumina sequencing technology was used to identify putative endonucleases. The sequence data generated was assembled and analysed using CLC Bio Genomics Workbench and bioinformatics tools (NCBI BLAST, REBASE and MG-RAST). Using these tools, genes encoding restriction-modification systems and endonuclease homologues were discovered. Three genes were identified and were recombinantly produced in Rosetta™ (DE3) pLysS and purified with IMAC using Ni-TED resin and subsequently characterised. These three genes were selected based on the identity percentage when compared to sequences on the NCBI database. Production of Endo8 was scaled up using 2 l fermenter and the purification done using ÄKTA Avant 150 FPLC using a HiScale 50 column packed with Ni-TED resin and the total amount of protein achieved was 58.82 mg.g-1. The productivity achieved at 17 hours (8 h harvest) was 2-fold greater than at 12 hours. Endonuclease activity of endo8 and endo52 was tested, both exhibited strong non-specific activity at 37 °C with an incubation period of 30 min. This work demonstrates that environmental soil samples are a valuable source for discovery of novel enzymes and also the utility of functional metagenomics to discover and purify these enzymes. These endonucleases may contribute to the next generation of reagent enzymes for molecular biology research. / Chemistry / M. Sc. (Life Sciences)

Introdução. As Repetições Palindrômicas Curtas Agrupadas e Regularmente Interespaçadas (CRISPRs) são DNAs que consistem de repetições de nucleotídeos. Existem 3 tipos: CRISPR1-CAS, CRISPR3-CAS e CRISPR2. Os bacteriófagos são partículas virais que infectam bactérias. Os bacteriófagos SAP6, IME-EF1, BC-611, VD-13 e F4 são encontrados em E. faecalis assim como FL1ABC, FL2AB, FL3AB e FL4A. Objetivo. Identificar e caracterizar as classes de CRISPRs e bacteriófagos de E. faecalis e E. faecium isoaldos de amostras alimentares, clínicas e fezes de animais. Materiais e métodos. Foram usadas DNA de 153 isolados, sendo 98 de E. faecalis e 55 de E. faecium. A técnica de detecção foi a PCR utilizando iniciadores para os genes CRISPR1-Cas, CRISPR2, CRISPR3-cas e bacteriófagos, seguido por electroforese em gel de agarose. Resultados. Para E. faecalis foram detectadas 58 isolados que amplifacaram para o gene CRISPR1-Cas; 87 para CRISPR2 e 13 para CRISPR3-Cas. Para E. faecium foram detectadas 4 isolados positivos para o gene CRISPR1-Cas; 18 para CRISPR2 e 1 para CRISPR3-CAS. O bacteriófago FL2ABfoi detectado em 13 isolados de E. faecalis; o bacteriófago FL3AB em 13 isolados de E. faecalis e o FL4A em 28 isolados de E. faecalis. Conclusão. Neste estudo nos encontramos diferentes proporções e distribuições dos genes CRISPRs em E. faecalis e E. faecium. O bacteriófago FL4A apresentou-se como o mais frequente entre os bacteriófagos avalados. / Introduction. The CRISPR are small portions of DNA consisting of nucleotide repeats. There are three types of CRISPRs recognized: CRISPR-associated genes cas: CRISPR1-CAS and CRISPR3-CAS and a orphan locus lacking cas genes: CRISPR2. Bacteriophages are viral particles that infect bacterial. The bacteriophages SAP6, IME-EF1, BC-611 and F4 are found in E. faecalis as well as FL1ABC, FL2AB, FL3AB, FL4A. Objectivy: To Identify and to characterize CRISPRs genes and bacteriophage genes in E. faecalis and E. faecium isolated from food, clinical and animal fecal samples. Materials and methods. A total of 153 DNA samples were used, 98 from E. faecalis and 55 from E. faecium. The PCR technique using primers was used to detect the genes CRISPR1-Cas, CRISPR2, CRISPR3-cas and bacteriophages, followed the agarose gel electrophoresis. Results. To E. faecalis was detected 58 isolates that amplified the CRISPR1-Cas genes, 87 to CRISPR2 and 13 to CRISPR3-Cas. To E. faecium was detected 4 isolates positive to CRISPR1-Cas gene, 18 to CRISPR2 e 1 to CRISPR3-CAS. The FL2AB bacteriophage was detected in 13 isolates of E. faecalis; the FL3AB bacteriophage in 13 isolates of E. faecalis and the FL4A in 28 isolates of E. faecalis. Conclusion. In this work, we found out different proportions and distributions of CRISPRs genes in E. faecalis e E. faecium. The FL4A bacteriophage showed a high frequency among the bacteriophages tested.

Introdução. As Repetições Palindrômicas Curtas Agrupadas e Regularmente Interespaçadas (CRISPRs) são DNAs que consistem de repetições de nucleotídeos. Existem 3 tipos: CRISPR1-CAS, CRISPR3-CAS e CRISPR2. Os bacteriófagos são partículas virais que infectam bactérias. Os bacteriófagos SAP6, IME-EF1, BC-611, VD-13 e F4 são encontrados em E. faecalis assim como FL1ABC, FL2AB, FL3AB e FL4A. Objetivo. Identificar e caracterizar as classes de CRISPRs e bacteriófagos de E. faecalis e E. faecium isoaldos de amostras alimentares, clínicas e fezes de animais. Materiais e métodos. Foram usadas DNA de 153 isolados, sendo 98 de E. faecalis e 55 de E. faecium. A técnica de detecção foi a PCR utilizando iniciadores para os genes CRISPR1-Cas, CRISPR2, CRISPR3-cas e bacteriófagos, seguido por electroforese em gel de agarose. Resultados. Para E. faecalis foram detectadas 58 isolados que amplifacaram para o gene CRISPR1-Cas; 87 para CRISPR2 e 13 para CRISPR3-Cas. Para E. faecium foram detectadas 4 isolados positivos para o gene CRISPR1-Cas; 18 para CRISPR2 e 1 para CRISPR3-CAS. O bacteriófago FL2ABfoi detectado em 13 isolados de E. faecalis; o bacteriófago FL3AB em 13 isolados de E. faecalis e o FL4A em 28 isolados de E. faecalis. Conclusão. Neste estudo nos encontramos diferentes proporções e distribuições dos genes CRISPRs em E. faecalis e E. faecium. O bacteriófago FL4A apresentou-se como o mais frequente entre os bacteriófagos avalados. / Introduction. The CRISPR are small portions of DNA consisting of nucleotide repeats. There are three types of CRISPRs recognized: CRISPR-associated genes cas: CRISPR1-CAS and CRISPR3-CAS and a orphan locus lacking cas genes: CRISPR2. Bacteriophages are viral particles that infect bacterial. The bacteriophages SAP6, IME-EF1, BC-611 and F4 are found in E. faecalis as well as FL1ABC, FL2AB, FL3AB, FL4A. Objectivy: To Identify and to characterize CRISPRs genes and bacteriophage genes in E. faecalis and E. faecium isolated from food, clinical and animal fecal samples. Materials and methods. A total of 153 DNA samples were used, 98 from E. faecalis and 55 from E. faecium. The PCR technique using primers was used to detect the genes CRISPR1-Cas, CRISPR2, CRISPR3-cas and bacteriophages, followed the agarose gel electrophoresis. Results. To E. faecalis was detected 58 isolates that amplified the CRISPR1-Cas genes, 87 to CRISPR2 and 13 to CRISPR3-Cas. To E. faecium was detected 4 isolates positive to CRISPR1-Cas gene, 18 to CRISPR2 e 1 to CRISPR3-CAS. The FL2AB bacteriophage was detected in 13 isolates of E. faecalis; the FL3AB bacteriophage in 13 isolates of E. faecalis and the FL4A in 28 isolates of E. faecalis. Conclusion. In this work, we found out different proportions and distributions of CRISPRs genes in E. faecalis e E. faecium. The FL4A bacteriophage showed a high frequency among the bacteriophages tested.