SILVA, Janice Oliveira. Sensibilidade a antimicribianos de bacteroidales de microbiota intestinal de cães e o efeito de concentracöes subinibitórias de antimicrobianos na formação de biofilme in vitro. 2015. 71 f. Dissertação (Mestrado em Microbiologia Médica) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by denise santos (denise.santos@ufc.br) on 2015-12-21T14:02:09Z No. of bitstreams: 1 2012_dis_josilva.pdf: 3676948 bytes, checksum: 203813cb8b6b1cfde06f219baa3a05cb (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2015-12-21T14:03:06Z (GMT) No. of bitstreams: 1 2012_dis_josilva.pdf: 3676948 bytes, checksum: 203813cb8b6b1cfde06f219baa3a05cb (MD5) / Made available in DSpace on 2015-12-21T14:03:06Z (GMT). No. of bitstreams: 1 2012_dis_josilva.pdf: 3676948 bytes, checksum: 203813cb8b6b1cfde06f219baa3a05cb (MD5) Previous issue date: 2015 / The Bacteroides and Parabacteroides spp are involved in serious diseases like abscesses and bacteremia in humans and animals. These bacteria are characterized by antimicrobial resistance and B. fragilis is the main anaerobic bacteria isolated from the intestine which can form biofilm. The aim of this study was to isolate Bacteroides and Parabacteroides strains from dogs intestinal tract, to investigate the antimicrobial susceptibility and to evaluate the action of antimicrobials subinhibitory concentrations on biofilm formation. A total of 30 strains were evaluated in this study. The assays were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines and other established methods. Antimicrobial susceptibility was observed against penicillin, amoxicillin-clavulanic acid, cefoxitin, imipenem, clindamycin, ciprofloxacin, enrofloxacin, tetracycline, chloramphenicol and metronidazole. Fifteen B. fragilis strains were tested for biofilm formation and the stronger four biofilm producer strains were chosen to evaluate the effect of subinhibitory concentrations (1/2 and 1/4MIC) of six antimicrobials on biofilm formation. B. fragilis was the most frequently isolated anaerobic bacteria followed by P. distasonis and B. vulgatus. The isolates were uniformly susceptible to metronidazole, imipenem and chloramphenicol and were penicillin resistant. Tetracycline and clindamycin were active against 50% and 33% of the strains respectively. The biofilm production of all four strains was uniformly and significantly lower (P<0.05) after growth with ½ MIC and ¼ MIC of imipenem and metronidazole. The induction of biofilm formation was observed in two isolates at ½ MIC and ¼ MIC of enrofloxacin. / Os gêneros Bacteroides e Parabacteroides estão envolvidos em doenças graves como abscessos e bacteremia em humanos e animais. Estas bactérias são caracterizadas pela resistência antimicrobiana e B. fragilis é a principal bactéria anaeróbica isolada do intestino que pode formar biofilme. O objetivo deste trabalho foi isolar Bacteroides e Parabacteroides do trato intestinal de cães, para avaliar a sensibilidade antimicrobiana e a ação de concentrações subinibitórias de antimicrobianos sobre a formação de biofilme. Um total de 30 amostras foram avaliadas neste estudo. Os ensaios foram realizados de acordo com os métodos e as diretrizes de Clínicals Laboratory Standards Institute (CLSI) e outras metodologias estabelecidas. Os antimicrobianos testados contra Bacteroides e Parabacteroides foram: penicilina, amoxicilina-ácido clavulânico, cefoxitina, imipenem, clindamicina, ciprofloxacina, enrofloxacina, tetraciclina, cloranfenicol e metronidazol. Quinze cepas de B. fragilis foram testadas para a formação de biofilme e as quatro cepas mais produtoras de biofilmes foram escolhidas para avaliar o efeito de concentrações subinibitórias (1/2 e 1/4CIM) de seis antimicrobianos sobre a formação de biofilme. B. fragilis foi a bactéria mais frequentemente isolada seguida por P. distasonis e B. vulgatus. Os isolados foram uniformemente sensíveis ao metronidazol, imipenem e cloranfenicol e foram resistentes à penicilina. Tetraciclina e clindamicina foram ativas contra 50% e 33% das cepas, respectivamente. A produção de biofilme de todas as quatro cepas foi uniforme e significativamente menor (P <0,05) após crescimento com ½ e ¼CIM de imipenem e metronidazol. A indução da formação de biofilme foi observada em duas cepas com ½ e ¼ CIM de enrofloxacina.

Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.

Horizontal gene transfer (HGT) is one of the man driving forces of evolution in prokaryotes, and can also promote within-strain variation of bacterial species. The genomes of three previously sequenced Bacteroides fragilis strains, NCTC9343, 638R and YCH46 displayed evidence of extensive HGT, demonstrated by the presence of 28 divergent capsular polysaccharide-associated biosynthesis loci. The genomes of a further four B. fragilis strains, LS66, GNAB92, RD48 and BE1 were sequenced and analysed. Genomic comparisons of BE1 and GNAB92 with NCTC9343, 638R and YCH46 identified ten new divergent polysaccharide biosynthesis loci. There is consequently, the potential to express 38 different polysaccharides amongst these five strains. Such a high level of variation in capsular polysaccharides, in so few strains has not been previously observed. HGT has occurred in B. fragilis despite the presence of diverse Restriction-Modification systems. The genome sequences of NCTC9343 and 638R contained a gene, ubb, the product of which, BfUbb, has 63% identity to human ubiquitin. The closest DNA sequence homology is to a migratory grasshopper entomopox virus, suggesting acquisition of this gene was via inter-kingdom HGT. The ubb gene was also identified in the newly sequenced genomes of B. fragilis strains LS66 and RD48. BfUbb had a predicted signal sequence; both full-length and processed forms were detected in whole-cell extracts by Western blot analysis. The inability to detect BfUbb in periplasmic extracts isolated from a B. fragilis strain containing an ubb signal sequence deletion construct, supported the periplasmic location of the processed form of the protein and the requirement for the signal peptide for transport from the cytoplasm. BfUbb was also detected in concentrated supernatants containing outer membrane vesicles, suggesting a mechanism by which the protein may be delivered to the host. This is the first example of ubiquitin being produced by a prokaryote. Transduction by bacteriophages is one mechanism by which horizontal gene transfer can occur and can also be a useful tool for genetic manipulation. Fifteen potentially new B. fragilis-specific bacteriophages were isolated from filtered sewage and characterised by phage titres and restriction endonuclease cleavage profiles. Of the fifteen, seven phages appeared to be different to the previously identified phage ФED01. None of the bacteriophages were capable of transduction. B. fragilis is a predominant member of the gastrointestinal microbiota. To survive within this specific niche, bacteria must successfully compete with other organisms for nutrients and space, and withstand attacks from bacteriophages. HGT may aid in the survival of B. fragilis as a commensal.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes the author's four published papers. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes the author's four published papers. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

REIS, A. C. M. Fatores de virulência e produção de biofilme de Bacteroides fragilis isolados da microbiota intestinal de cães. 2013. 87 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-07-27T12:53:54Z No. of bitstreams: 1 2013_dis_acmreis.pdf: 83159321 bytes, checksum: 8a4f3a15527a5e48751e49ec6d7dbc42 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-07-27T15:17:11Z (GMT) No. of bitstreams: 1 2013_dis_acmreis.pdf: 83159321 bytes, checksum: 8a4f3a15527a5e48751e49ec6d7dbc42 (MD5) / Made available in DSpace on 2017-07-27T15:17:12Z (GMT). No. of bitstreams: 1 2013_dis_acmreis.pdf: 83159321 bytes, checksum: 8a4f3a15527a5e48751e49ec6d7dbc42 (MD5) Previous issue date: 2013-01-08 / Bacteroides fragilis is part of the fecal microbiota of animals, yet it is the most pathogenic species of the genus Bacteroides. The transformation these microrganisms commensals in pathogens agents is mainly due to its virulence factors. This microorganism has been isolated from several infections in dogs. Considering the pathogenic potential of B. fragilis and its importance in veterinary medicine, this study was designed to evaluate the virulence factors and biofilm formation of Bacteroides fragilis isolated from fecal samples from dogs. The samples used in this study were collected at the Veterinary Clinic of the State University of Ceará and processed at the Laboratory of Bacteriology of the Federal University of Ceará. Were phenotypically analyzed the virulence factors (production of hemolysins and hemagglutinins, hydrophobicity, the presence of the enzyme β-lactamase and capsule) and biofilm production of 13 strains of B. fragilis. To determine the pattern of sensitivity to clindamycin, metronidazole, chloramphenicol and penicillin was used in agar dilution technique. In vitro assays for detection of biofilms were performed by the methods Congo Red agar and Accession in microplates. All strains of B. fragilis showed capsule and produced no haemolysis. Regarding the production of hemagglutinin, 38% of the strains showed hemagglutination when used canine blood and 15% in human blood (A+). Of the strains studied, 7% had a cell surface hydrophobic. In total, 61% of the strains showed β-lactamase test positive. As regards the production of biofilm was observed by the method of strokes that 12 (92.3%) of the biofilm producers were isolated by the method and Adhesion microplates, it was found that eight (61.5%) strains were able to produce biofilm. All isolates were susceptible to metronidazole and resistant to penicillin and chloramphenicol. The rate of clindamycin resistance was 69.2%. This study showed that species B. fragilis isolated from normal canine microflora showed major virulence factors for pathogenicity, they possess the ability to form biofilms and show a high rate of resistance to clindamycin. / Bacteroides fragilis faz parte da microbiota fecal de animais, contudo é a espécie mais patogênica do gênero Bacteroides. A sua transformação de um microrganismo comensal para agente patogênico deve-se principalmente aos seus fatores de virulência. Este micro-organismo tem sido isolado de diversas infecções em cães. Considerando o potencial patogênico de B. fragilis e sua importância na medicina veterinária, o presente trabalho foi elaborado com o objetivo de avaliar os fatores de virulência e formação de biofilme de Bacteroides fragilis isolados de amostras fecais de cães. As amostras utilizadas neste estudo foram coletadas na Clinica Veterinária da Universidade Estadual do Ceará (UECE) e processadas no Laboratório de Bacteriologia da Universidade Federal do Ceará (UFC). Foram analisadas fenotipicamente os fatores de virulência (produção de hemolisinas e hemaglutininas, hidrofobicidade, presença da enzima - lactamase e cápsula) e produção de biofilme de 13 cepas da espécie B. fragilis. Para determinação do perfil de sensibilidade a clindamicina, metronidazol, cloranfenicol e penicilina foi utilizada a técnica de diluição em Agar. Os ensaios in vitro para detecção de biofilme foram realizados pelos métodos Agar Vermelho Congo (AVC) e Adesão em microplacas. Todas as cepas de B. fragilis apresentaram cápsula e não produziram hemólise. Com relação à produção de hemaglutininas, 38% das cepas são capazes de aglutinar sangue canino e 15% em sangue humano (A+). Das cepas estudadas, 7% apresentaram uma superfície celular hidrofóbica. No total, 61% das cepas produziram teste da - lactamase positivo. Quanto a produção de biofilme, foi observado pelo método do AVC que 12 (92,3%) dos isolados eram produtores de biofilme e por meio do método de Adesão em Microplacas, verificou-se que 8 (61,5%) cepas foram capazes de produzir biofilme. Todos os isolados foram sensíveis ao metronidazol e cloranfenicol e resistentes à penicilina. A taxa de resistência à clindamicina foi de 69,2%. Esse estudo mostrou que espécies de B. fragilis isolados da microbiota normal de cães apresentaram fatores de virulência importantes para sua patogenicidade, possuem a capacidade de formar biofilme e demonstram uma alta taxa de resistência a clindamicina.

Bacteroides fragilis is a member of the normal microbiota that resides in the human lower gastrointestinal tract. This bacterium is of clinical significance because it is the most frequently isolated Gram-negative obligate anaerobe from peritoneal abscesses and bloodstream infections. Human fibrinogen is a hexameric-glycoprotein that is important for fibrin-mediated abscess formation and limiting the spread of infection. B. fragilis can bind and degrade fibrinogen which may aid in its escape from abscesses into the bloodstream, thereby promoting bacteraemia. In addition to fibrinogen, binding of B. fragilis to fibronectin, a component of the extracellular matrix, found in association with fibrinogen at wound sites, has also been reported. An outer membrane protein, BF1705, expressed by B. fragilis was found to share homology with BspA from Tannerella forsythia which is known to bind fibrinogen. The gene encoding BF1705 was deleted from the B. fragilis NCTC 9343 genome in the present work using a markerless gene deletion technology. Proteins derived from the outer membranes of wild-type B. fragilis were able to bind fibronectin and fibrinogen in far-western blots. Similar protein extracts from the ΔBF1705 strain did not bind fibrinogen and fibronectin, which confirms the role of BF1705 in adhesive interactions with proteins of the host extracellular matrix. The possible involvement of BF1705 in fibrinogen degradation was ruled out because the ΔBF1705 strain still degraded fibrinogen. To identify the proteases involved in degradation of fibrinogen, four genes encoding putative extracellular metallo- and serine proteases in the size range 45-50 kDa were deleted from the NCTC 9343 genome. All of the single and multiple mutants defective in these selected proteases were still capable of degrading fibrinogen as determined by zymography. Expression of eight B. fragilis proteases in E. coli did not lead to detectable degradation of fibrinogen. These observations suggest that these proteases alone cannot degrade fibrinogen and either that an unidentified protease is responsible for degradation or that there is redundancy in the proteases involved. Under conditions of nitrogen limitation bacteria resort to scavenging nitrogen from the environment to replenish the depleting intracellular nitrogen content. By examining the differential regulation of the B. fragilis transcriptome under nitrogen replete and depleting conditions, a potential role for BF1705 and secreted proteases in nutrient binding and assimilation were studied. Growth on conventional glucose defined medium with ammonia as the nitrogen source was compared to growth in defined medium with glutamine as nitrogen source. A reduced doubling time and diauxic growth in the medium containing glutamine indicated nitrogen limitation. Comparison of the transcriptome derived from cultures of B. fragilis grown on either ammonia or glutamine by RNA-Seq did not reveal a significant upregulation of BF1705 in response to nitrogen limitation. This observation in conjunction with its inability to degrade fibrinogen suggests that the primary role of BF1705 might be as an adhesin and does not act directly in nutrient binding and degradation. Nevertheless, nitrogen limitation was found to induce the expression of four protease-encoding genes by over a 2-fold (adjusted p value < 0.05). The molecular weight of three of these proteases were identified to be within the size range of 45-55 kDa which corresponded to the lysis bands detected by fibrinogen zymography with wild-type B. fragilis protein extracts. Therefore the possible involvement of these three proteases in fibrinogen degradation could be assessed. A 155-fold upregulation (adjusted p value < 0.05) in asnB, encoding a homologue of asparagine synthetase B, under conditions of nitrogen limitation suggest a previously uncharacterised aspartate metabolism pathway for ammonia generation via arginine catabolism in B. fragilis. Ammonia thus formed might aid in sustaining B. fragilis growth under nitrogen deprived conditions. In addition to nitrogen assimilation, significant upregulation was observed in the expression of genes involved in regulation of oxidative stress and metronidazole resistance. The observed changes in the transcriptome will add to our understanding of the B. fragilis metabolism and potential assist with unravelling the mechanisms of infection mediated by this important opportunistic pathogen.

Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250 / Ph. D.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T17:57:41Z No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 1495355 bytes, checksum: 2dcd69bdf9054b1d66175c1f6955b8bd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:18:16Z (GMT) No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 1495355 bytes, checksum: 2dcd69bdf9054b1d66175c1f6955b8bd (MD5) / Made available in DSpace on 2017-08-07T19:18:16Z (GMT). No. of bitstreams: 1 michelecristineribeirodefreitas.pdf: 1495355 bytes, checksum: 2dcd69bdf9054b1d66175c1f6955b8bd (MD5) Previous issue date: 2010-05-14 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie Bacteroides fragilis é um anaeróbio Gram negativo membro da microbiota indígena do trato gastrintestinal, e é comumente isolado de infecções clínicas. À medida que as drogas antimicrobianas são introduzidas no ambiente, os microrganismos respondem, tornando-se resistentes, podendo gerar alterações na fisiologia celular, e que podem interferir na relação bactéria-hospedeiro. Nosso objetivo foi investigar alterações em B . fragilis após exposição a concentrações subinibitórias de ampicilina (AMP), ampicilina/sulbactam (AMS), clindamicina (CLI) e cloranfenicol (CLO); e investigar aspectos da relação droga-bactéria-hospedeiro, após desafio experimental em camundongos. Foram obtidas 4 linhagens bacterianas: BfAMP, BfAMS, BFCLI e BfCLO. Alterações na morfologia, padrões de crescimento, habilidades bioquímicas e fisiológicas, perfis de expressão protéica e semelhança genética foram avaliadas comparativamente entre as linhagens obtidas e a linhagem Bf parental ATCC 25285. Infecção experimental foi realizada em camundongos convencionais, desafiados intraperitonealmente. Histologia de baço e fígado, além de dosagem das citocinas TNF-α, IL-6, IL-10, IL-17 e IFN-γ no intestino, foram realizadas. Ampicilina e ampicilina/sulbactam induziram alterações morfológicas significativas nas células bacterianas na primeira exposição. Foi verificada diminuição da sensibilidade aos antimicrobianos em todas as linhagens selecionadas. Além disso, a exposição aos antimicrobianos foi capaz de induzir alterações nos padrões bioquímico-fisiológicos bacterianos, se comparado com a amostra parental. Em relação à capacidade de formação de biofilme, observou-se alteração em todas as linhagens, quando comparadas à parental, sendo que as linhagens selecionadas pela exposição aos antimicrobianos tiveram capacidade de aderência diminuída. A identidade genética das linhagens bacterianas obtidas foi confirmada e não foram detectados fragmentos de DNA polimórfico. Variações nos perfis de citocinas foram verificadas nos animais desafiados com a linhagem BfAMS. Alterações histopatológicas também foram observadas no baço e fígado dos animais desafiados com as linhagens de B . fragilis selecionadas por exposição aos antimicrobianos. Nossos resultados permitem sugerir fenômenos relacionados à regulação da expressão gênica como uma resposta momentânea ou evolutiva dos microrganismos. Estes resultados podem ainda sugerir que essas drogas tem diferentes interações com a célula bacteriana, que podem ser prejudiciais para a interação bactéria-hospedeiro. Assim, nossos resultados chamam a atenção para o risco de terapia antimicrobiana inadequada, mesmo com baixas concentrações de fármacos, que poderiam alterar os níveis de expressão gênica de uma bactéria e modular sua patogenicidade. / Bacteroides fragilis is an anaerobic Gram negative rod member of resident gut microbiota, isolated from clinical infections due in part to its potent virulence factors. As antimicrobial drugs are introduced into the environment, microorganisms respond by becoming resistant and may cause changes in cell physiology. These changes can interfere with the bacterium-host relationship. The aim of this study was to investigate changes in B . fragilis after exposure to ampicillin (AMP), ampicillin/sulbactam (AMS), clindamycin (CLI) and chloramphenicol (CLO); and to investigate changes in host-bacteria relationships after experimental challenge in mice. Four bacterial strains were obtained: BfAMP, BfAMS, BFCLI e BfCLO. Morphological changes, growth patterns, biochemical and physiological abilities, protein expression profile and genetic relationships were comparatively evaluated among the drug-selected strains and Bf parent ATCC 25285. Experimental infection was performed intraperitoneally in conventional mice. Liver and spleen histology, besides cytokines determinations were performed in the intestines. Ampicillin and ampicillin/sulbactam induced the highest significant morphological alterations in the bacterial cells after the initial drug exposure. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria strains. Furthermore, drug-exposure was able to induce changes in biochemical pathways and bacterial physiology, if compared with the Bf parent strain. Regarding the ability of biofilm formation, alterations in adherence properties were observed for all drug-selected bacteria, being this characteristic down regulated. The genetic identities were confirmed for the selected strains and no DNA polymorphisms were detected. The cytokine patterns in the intestines from mice challenged with drug-selected bacteria were highly altered, mainly considering the BfAMS strain, as well as the histological aspect concerning the liver and the spleen excised from the experimentally challenged animals. Our results may suggest phenomena related to the gene expression regulation, as a microbial momentary or evolutionary response to the selective pressure of the antimicrobial drugs. These results may also suggest that these drugs have different interactions with the bacterial cell which can be harmful to the host-bacteria relationships. In this way, the data ask for attention regarding the risks of inappropriate chemotherapy, even with low concentrations of the antimicrobial drugs, which might the bacteria genetic expression levels and modulate their pathogenicity

B. fragilis wird mit klinischen Bildern mit wie Abdominal- und Weichgewebs-Infektionen sowie Sepsis-Geschehen assoziiert, weitere Krankheitsbilder sind noch in der Erforschung. In der Vergangenheit gaben Virulenzfaktoren wie die komplexe Kohlehydrat-Kapsel, das Endotoxin sowie die Produktion von Katalase und Hämagglutinin nur eine unzureichende Erklärung für die Entstehung schwerer Infektionen, die der Keim auch als Einzelerreger verursachte. In den 1980er Jahren wurden Enterotoxigene B. fragilis Stämme als Erreger akuter Diarrhoe bei Kindern festgestellt; nachfolgend wurden Stämme mit diesem Virulenzfaktor in den unterschiedlichsten Infektions-geschehen nachgewiesen. Es kann davon ausgegangen werden, dass die Enterotoxin-Bildung von Bacteroides fragilis Stämmen eine Rolle bei der Pathogenese von systemischen Infektionen sowie möglicherweise bei der Entstehung von Darmkrebs spielt. Im Rahmen dieser Arbeit wurde/n 1. Eine umfangreiche Stammsammlung von 271 Bacteroides fragilis Stämmen unterschiedlicher geographischer Herkunft aus den USA (Stammsammlungen) und aus Deutschland sowie verschiedener klinischer Herkunft (insbesondere n=70 Blutkulturisolate) zusammengestellt. 2. Die Überprüfung der Spezies-Identifizierung mittels phänotypischer Reaktionen (Koloniemorphologie, biochemische Methoden) sowie nachfolgender molekularer Tests (PCR-Fingerprinting) führte zum Ausschluss eines Isolates. 3. Mit molekularen Methoden die PCR-Gruppen I und II charakterisiert, wobei die meisten Stämme der Gruppe I angehörten. 4. Der molekulare Nachweis von Enterotoxin-Genabschnitten mit Rückschluss auf das Vorhandensein der BfPAI bei 32 von 260 Stämmen (12.3%) geführt. Diese Stämme waren immer Teil der PCR Gruppe I. Die 260 untersuchten Stämme konnten in Pattern I, II oder III eingeteilt werden. 5. Blutkultur-Stämme und Stämme aus anderen Quellen (Nicht-Blutkulturen) verglichen. Es konnten jedoch keine signifikanten Unterschiede bezüglich des Vorkommens von ETBF sowie NTBF Pattern III dargestellt werden. 6. In den US-amerikanischen Stämmen prozentual mehr ETBF und Gruppe II Stämme als in deutschen Isolaten nachgewiesen. 7. Beim Vergleich der Sequenzen die BfPAI flankierender Elemente sechs ausgewählter Stämme, einschließlich der Referenzstämme (ATCC 25285, NTBF Pattern II; ATCC 43859, ETBF Pattern I) verschiedene Punktmutationen gefunden.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 1 Einleitung 4 1.1 Historische Beschreibung des Genus Bacteroides mit der Typ-Spezies Bacteroides fragilis 4 1.1.1 Phänotypische Charakteristika des Genus Bacteroides 4 1.1.2 Molekulare Charakteristika und Taxonomie 5 1.1.3 Die Spezies Bacteroides fragilis 8 1.1.4 Antibiotika-Resistenzen bei der Spezies Bacteroides fragilis 9 1.1.5 Bacteroides fragilis: Zwei DNA-Homologie-Gruppen / zwei PCR Fingerprint-Gruppen 10 1.1.6 Natürliche Habitate obligater Anaerobier 11 1.1.7 Klinische Bedeutung der Spezies Bacteroides fragilis 11 1.2 Enterotoxin positive Bacteroides fragilis 13 1.2.1 Bacteroides fragilis Enterotoxin (BFT) - Fragilysin 14 1.2.2 Bacteroides fragilis Pathogenitätsinsel (BfPAI) 15 1.3 Weitere Krankheitsbilder 19 1.3.1 Infektionen/Sepsis 19 1.3.2 Chronisch-entzündliche Darmerkrankung und kolorektale Tumorbildung 20 2 Aufgabenstellung 21 3 Material und Methoden 22 3.1 Stämme 22 3.1.1 Referenzstämme 22 3.1.2 Auswahl der Klinischen Stämme 22 3.1.2.1 Vergleichsstämme der Spezies Bacteroides vulgatus und ovatus 23 3.1.2.2 Blutkulturen – Invasive Stämme 23 3.1.2.3 Nicht-Blutkulturen – Nicht-invasive Stämme 23 3.2 Kulturtechnik 24 3.2.1 Nährmedien 24 3.2.1.1 Columbia-Blut-Agar 24 3.2.2 Inkubation der anaeroben Kulturen 25 3.3 Gruppendifferenzierung der Isolate 25 3.3.1 Koloniemorphologie 25 3.3.2 Zellmorphologie - Gramfärbung 25 3.3.3 Differenzierung mittels Blättchentest 26 3.3.4 Biochemische Leistungsprüfung 26 3.4 Molekularbiologische Analysen 26 3.4.1 DNA-Extraktion 26 3.4.2 PCR-Reaktionsansätze 28 3.4.2.1 Optimierung der PCR-Ansätze nach Taguchi 29 3.4.3 PCR-Bedingungen 29 3.4.4 PCR-Fingerprint-Technik 30 3.4.4.1 Primer 30 3.4.4.2 PCR-Protokoll für T3B 31 3.4.4.3 Cycler-Profil 31 3.4.5 Spezifische PCR zum Nachweis von Teilen der BfPAI 32 3.4.5.1 Primer 32 3.4.5.2 PCR-Protokolle 32 3.4.5.3 Cycler-Profile 34 3.4.6 Agarose-Gel-Elektrophorese 35 3.4.7 Auswertung der Ergebnisse 36 3.5 Sequenzierung von DNA-Fragmenten 36 4 Ergebnisse 39 4.1 Identifizierung mittels phänotypischer Eigenschaften der Spezies Bacteroides fragilis Zell- und Koloniemorphologie 39 4.2 Biochemische Identifizierung 41 4.3 Molekulare Untersuchungen 42 4.3.1 Bestätigung der Spezies-Identifizierung mittels der PCR-Fingerprint-Technik 42 4.3.2 Unterteilung der Spezies in zwei PCR-Fingerprint-Gruppen 42 4.4 Nachweis von Enterotoxin und der BfPAI 44 4.4.1 Ergebnisse der PCR-Optimierung nach Cobb BD & Clarkson JM, 1994 44 4.4.2 Nachweis von Enterotoxin codierenden–Genabschnitten als Teile der BfPAI mittels dreier Primerpaare 45 4.4.3 PCR-Analyse der Schnittstellen der BfPAI unter Verwendung von zwei Primerpaaren 47 4.4.4 Auswertung der Blutkulturisolate 50 4.4.5 Nicht-Blutkulturen 53 4.4.6 Statistik zum Vergleich des Vorkommens der ETBF-Gen-Sequenzen und flankierenden Regionen (flanking regions) bei Blutkultur-Isolaten gegenüber Nicht- Blutkulturisolaten 61 4.4.7 Ausschluß von Enterotoxin-Genen bei Bacteroides vulgatus und Bacteroides ovatus 62 4.4.8 Vergleich der Bacteroides fragilis Stämme nach geographischer Herkunft 63 4.5 Sequenzierung der BfPAI-flankierenden Regionen in ausgewählten Stämmen 64 4.5.1 Sequenzierung der flankierenden Regionen 64 5 Diskussion 68 6 Zusammenfassung der Arbeit 79 7 Literaturverzeichnis 81 8 Anlagen 94 9 Danksagung 102 10 Erklärung über die eigenständige Abfassung der Arbeit 103 11 Publikationsliste 104