München, Techn. Univ., Diss., 2004.

Obligat anaerob wachsende Bakterien sind an einer Vielzahl von Infektionen beteiligt. Dabei ist Bacteroides fragilis einer der wichtigsten opportunistischen Erreger unter den Anaerobiern. Bei Verdacht auf eine Infektion durch obligate Anaerobier muss nach Materialentnahme für die mikrobiologische Diagnostik unverzüglich eine kalkulierte Therapie eingeleitet werden. Oft ist eine chirurgische Therapie notwendig, die ebenso wie eine adäquate Antibiotikatherapie entscheidend für den Verlauf der Erkrankung ist. Wichtige Substanzen für eine Therapie bei Infektionen mit Beteiligung von B. fragilis sind Clindamycin, Imipenem, Metronidazol und Piperacillin/Tazobactam. Um Aussagen zur in vitro Wirksamkeit dieser Antibiotika gegenüber obligaten Anaerobiern treffen zu können, wurden in der vorliegenden Arbeit die Aktivitäten von verschiedenen Konzentrationen des jeweiligen Antibiotikums auf das Wachstum von sensiblen und resistenten B. fragilis Stämmen mittels Absterbekinetik untersucht. In Abhängigkeit von der zuvor ermittelten minimalen Hemmkonzentration des jeweiligen Antibiotikums wurden die Stämme in 2 Gruppen eingeteilt. Die erste Gruppe umfasst alle Stämme mit einer MHK ≤ 8 µg/ml. In der zweiten Gruppe sind die Stämme mit einer MHK > 8 µg/ml zusammengefasst. Die einzelnen Stämme wurden mit einem Vielfachen der minimalen Hemmkonzentration (MHK) beziehungsweise einem Vielfachen der im menschlichen Blutplasma maximal erreichbaren Konzentration (Cmax) des jeweiligen Antibiotikums inkubiert und die Bakterienkonzentration zu definierten Zeitpunkten ermittelt. Dadurch können sowohl die Wirksamkeit unterschiedlicher Antibiotikakonzentrationen als auch verschiedene Antibiotikaklassen miteinander verglichen und Aussagen zu Empfehlungen für kalkulierte Therapien getroffen werden.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Obligat anaerob wachsende Bakterien sind an einer Vielzahl von Infektionen beteiligt. Dabei ist Bacteroides fragilis einer der wichtigsten opportunistischen Erreger unter den Anaerobiern. Bei Verdacht auf eine Infektion durch obligate Anaerobier muss nach Materialentnahme für die mikrobiologische Diagnostik unverzüglich eine kalkulierte Therapie eingeleitet werden. Oft ist eine chirurgische Therapie notwendig, die ebenso wie eine adäquate Antibiotikatherapie entscheidend für den Verlauf der Erkrankung ist. Wichtige Substanzen für eine Therapie bei Infektionen mit Beteiligung von B. fragilis sind Clindamycin, Imipenem, Metronidazol und Piperacillin/Tazobactam. Um Aussagen zur in vitro Wirksamkeit dieser Antibiotika gegenüber obligaten Anaerobiern treffen zu können, wurden in der vorliegenden Arbeit die Aktivitäten von verschiedenen Konzentrationen des jeweiligen Antibiotikums auf das Wachstum von sensiblen und resistenten B. fragilis Stämmen mittels Absterbekinetik untersucht. In Abhängigkeit von der zuvor ermittelten minimalen Hemmkonzentration des jeweiligen Antibiotikums wurden die Stämme in 2 Gruppen eingeteilt. Die erste Gruppe umfasst alle Stämme mit einer MHK ≤ 8 µg/ml. In der zweiten Gruppe sind die Stämme mit einer MHK > 8 µg/ml zusammengefasst. Die einzelnen Stämme wurden mit einem Vielfachen der minimalen Hemmkonzentration (MHK) beziehungsweise einem Vielfachen der im menschlichen Blutplasma maximal erreichbaren Konzentration (Cmax) des jeweiligen Antibiotikums inkubiert und die Bakterienkonzentration zu definierten Zeitpunkten ermittelt. Dadurch können sowohl die Wirksamkeit unterschiedlicher Antibiotikakonzentrationen als auch verschiedene Antibiotikaklassen miteinander verglichen und Aussagen zu Empfehlungen für kalkulierte Therapien getroffen werden.

The repair of DNA double-strand breaks (DSBs) is required for the survival of all organisms. In bacteria, DNA DSBs can occur during normal housekeeping processes such as DNA replication or by exogenous damage due to chemicals or radiation. DSBs will compromise the integrity of the genome if left un-repaired, and can be fatal to an organism. Repair of DSBs by homologous recombination (HR) replicates missing chromosomal regions before joining of the separated DNA ends. In Escherichia coli the HR repair steps are; pre-synapsis, synapsis and post-synapsis. In the pre-synaptic stage a DSB is processed into a 3′ single-strand overhang, the substrate required for strand invasion in the synapsis stage and the eventual repair of the DSB. At present there are three identified pre-synapsis systems involved in recombination in bacteria; represented by the AdnAB, AddAB and the RecBCD protein complexes. Each system functions in a similar manner but differ in the physical composition of the machinery. This project investigated the pre-synaptic system of Bacteroides fragilis NCTC9343. Genes encoding putative pre-synapsis proteins were initially identified through analysis of the NCTC9343 genome. The function of these proteins was investigated in vivo by rescue of a repair-deficient strain of E. coli. This demonstrated that Bacteroides fragilis encodes a two component system, where both genes products are required to work in concert for pre-synaptic processing of DSBs. The identified genes were BF2192 and BF2191, and have been renamed addA and addB, respectively. To further examine the role of the AddAB proteins in DSB repair, a Bacteroides fragilis strain with a deletion of addAB was constructed and shown to be extremely sensitive to DNA damaging agents. The AddAB complex was purified and found to be an ATP-dependant helicase and exonuclease that acted on double-stranded DNA ends. In conclusion, this project has identified the proteins involved in pre-synaptic processing of DSBs in B. fragilis NCTC9343, consisting of AddAB homologues, and shown their protective role in repair of DNA damage.

Sphingolipids (SLs) are complex lipid-derived structures that are essential components of cell membranes in eukaryotes and some bacteria. SLs and their complex derivatives ceramides are known to be involved in multiple processes such as the formation of lipid rafts, cell signalling and membrane trafficking. The first step of SL biosynthesis is universal to all sphingolipid-producing organisms from bacteria to humans and is catalysed by the enzyme serine palmitoyltransferase (SPT). SPT is a member of the alpha-oxoamine synthase (AOS) family of pyridoxal- 5'-phosphate-dependent enzymes. All AOS family enzymes retain a high degree of structural homology and catalyse the decarboxylative Claisen-like condensation of amino acids with thioester substrates. The SPT enzyme catalyses the formation of the universal SL precursor, 3-ketodihydrosphingosine (KDS), by condensation of L-serine and coenzyme A-derived palmitic acid. Being the key controller in SL biosynthesis, SPT plays a big role in regulating natural and pathological processes. A lot of research interest has been recently generated by SLs isolated from bacterial members of the human microbiome and their roles in human health. Increasing evidence suggests that some of these SLs possess immunoregulatory effects and can have a direct impact on the immunity of the host. Bacteroides fragilis is a commensal gut-dwelling bacterium that belongs to a few human microbionts known to produce unique iso-branched sphingolipids (isoSLs); these have been shown to influence the human iNKT cell count. The production of SLs in B.fragilis is completely regulated by a gene product BF2461. In this work, BF2461 was expressed and purified; using a combination of UV-vis spectrometry, enzymatic assays, mass spectrometry and protein X-ray crystallography, it has been confirmed to be an SPT. The substrate specificity of the BfSPT has been assessed with a range of different chain-length substrates, including less common 15 and 17-carbon chain length coenzyme A substrates. The enzyme can produce different types of SL precursors with a preference for the 16-carbon chain substrate palmitoyl- CoA. However, at high levels of PCoA, a substrate inhibition is observed that might point to a natural control mechanism employed by the bacterium in favour of producing iso-branched SLs (isoSLs). The structure of BfSPT has been elucidated in a complex with its amino acid substrate L-serine. Search and analysis of putative SPTs from other microbiome-associated bacteria that produce isoSLs show that they share high similarity with an average amino acid conservation of 74%, suggesting they might be adapted to a particular type of substrate. In this respect, BfSPT might be the first isoSL-producing SPT to be structurally characterised, and the first one to have a direct impact on human health. Further structural data were obtained on protein complexes with L-cycloserine and L-penicillamine, some common inhibitors of the PLP-dependent enzymes. The structure obtained in the presence of L-penicillamine provides the first direct structural evidence of the inhibitory mechanism by a thiazolidine complex formation in the active site of a PLP-dependent enzyme. These findings shed light on certain aspects of the reaction and inhibition mechanisms of BfSPT as well as opening new prospects into researching this interesting target and its impact on the human microbiome.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

No description available.

No description available.

Em nosso estudo foi avaliada a presença das bactérias do grupo Bacteroides fragilis em 108 amostras fecais de bezerros com e sem diarréia, além de fatores de virulência e a similaridade genética entre as cepas de B. fragilis. Hemolisinas foram observadas em 36,3% e em 83,7% dos bezerros com e sem diarréia, respectivamente. Apenas 7,4% dos isolados foram hemaglutinantes. De todos os isolados, grande parte resistiu à ação do soro e 100% foram sensíveis ao imipenem e metronidazol. Houve resistência aos metais pesados utilizados. Plasmídios foram detectados em 7,4% dos isolados. Dentre 58,8% produtores de ß-lactamase, em 19,7% e em 26,0% de bezerros com e sem diarréia, respectivamente detectou-se o gene cepA, que foi observado também em plasmídios de 5,5 kb. O gene cfiA foi observado em 16,5% dos isolados diarréicos e em 12,6% de não diarréicos, mas não em plasmídios. O gene nanH foi detectado em 21,8% dos isolados e o gene bft somente em dois isolados diarréicos. A similaridadade genética entre os B. fragilis mostrou a heterogeneidade das bactérias. / In this study the bacteria of Bacteroides fragilis group was evaluated in 108 fecal samples of calves with and without diarrhea, besides virulence factors and the genetic similarity among the B. fragilis strains. Hemolysin was observed in 36.3% and in 83.7% of calves with and without diarrhea, respectively. Only 7.4% of the isolates showed hemagglutinability. Of all the isolates, the major part resisted to the action of the serum and 100% were sensitive to the imipenem and metronidazole. There was resistance to the used heavy metals. Plasmids were detected in 7.4% isolates. Among 58.8% ß- lactamase producing, 19.7% and 26.0% strains of calves with and without diarrhea, respectively the cepA gene was detected, that was also observed in 5.5 kb plasmids. The cfiA gene was observed in 16.5% of the diarrheic isolates and 12.6% of non-diarrheic, but not in plasmids. The nanH gene was detected in 21.8% of the isolates and the bft gene only in two diarrheic isolates. The genetic similarity among the B. fragilis showed the heterogeneity of the bacteria.