The pigmented oral BacterOides were characterized with regard to their pathogenic, collagenolytic, hemagglutinating and metabolic properties. Isolates were obtained from healthy and diseased gingival sulci of 396 children, adults and mongrel dogs. Fifty-three of the isolates proved to be members of the subspecies Ii. melaninogenicus ss. asaccharolyticus. The remainder were either 15. melaninogenicus ss. intermedius or melaninogenicus. All isolates designated asaccharolyticus were pathogenic in the guinea pig infectivity assay. The subspecies intermedius and melaninogenicus were not pathogenic. The subspecies asaccharolyticus possessed a cell bound oxygen sensitive collagenase, a cell bound and soluble oxygen sensitive hemagglutinin, and produced butyric and phenylacetic acids. These properties were unique to asaccharolyticus and were not detected with other organisms. All isolates required hemin. The pathogenic organisms possessed a heat sensitive toxin which induced fluid accumulation in ligated mouse ileal loops. Infectivity of 13. melaninogenicus asaccharolyticus was dependent on the presence of a second organism. An infective consortium consisting of 13. melaninogenicus asaccharolyticus and Klebsiella pneumoniae was defined. Neither organism was infective alone but the Klebsiella could be replaced by organisms of a number of different genera. The number and proportions of J3. melaninogenicus and K. pneumoniae required to establish a lesion was determined. The nature of the infection appeared to be determined by the length of the lag period preceding the initiation of growth of 15. melaninogenicus. A rapid onset of growth led to the severe spreading form of the disease whereas a slow initiation of growth resulted in the formation of a localized self limiting abscess. J3. melaninogenicus depends on the second or "helper" organism to produce a required growth factor which is not present at the inoculation site. The growth factor was shown to be succinate which was able to replace the hemin requirement. The dependency oh succinate produced by K. pneumoniae was demonstrated in agar medium, in liquid culture and in the infectivity assay. Any organism which produced succinate was able to stimulate growth of JJ. tnelahinogenicus on agar medium and could replace K.. pneumoniae as a member of the infectious consortium. The need for the second organism could be eliminated by inoculating 13. melaninogenicus together with agar-immobilized succinate or hemin. Growth of Ii. melaninogenicus is dependent on the presence of large quantities of succinic acid suggesting that the compound is used in energy metabolism and is not incorporated into cellular carbon. This assumption is supported by the observation that only 0.5% of the succinate carbon can be found in the cell, the remainder of the metabolized succinate is excreted as butyrate. Hemin blocks the metabolism of succinate. The fatty acid metabolites are qualitatively similar but quantitatively different in cells grown in hemin free succinate medium. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Bacteroides amylophilus produces a proteolytic enzyme of which 20% is liberated into the medium and 80% is bound.to the cell. Treatment .of the .cells with toluene or mechanical disinteration does not increase the proteolytic activity, indicating that all the protease is superficially located at the bacterial surface. Less than 1% of the total protease activity is released from the cells by osmotic shock procedures which indicated that the protease is not free in the periplasmic space. Speroplast formation liberates 33% of the cell bound protease 40% of which is sedimentable by prolonged high speed centrifugation. Sonic disruption of spheroplasts releases 72% of the protease. After gentl osmotic rupture 48% of the enzyme activity, remained bound to the spheroplast envelope. Prolonged high speed centrifugation results in the sedimentation of all but 16% of the total enzyme. The results give further evidence to the particle bound nature of the protease of Bacteroides amylophilus. Bacteroides amylophilus has a faster rate of growth, with a reduced lag phase and produces a greater cell yield when tryptic peptides are included in the basal medium. Radioactive amino acids are incorporated into cells in significant amounts, indicating that they were not excluded from the cell by a permeability barrier. The amount of incorporation of ¹⁴C amino acids is found to vary for different amino acids and is shown to be concentration dependent. The exogenous ¹⁴C amino acids were incorporated into the cell protein either directly or after an interconversion step. The inhibition of ¹⁴C amino acids uptake by peptides and the direct uptake of ¹⁴C olig-peptides demonstrated peptide uptake by Bacteroides amylophilus. The results suggest that organic nitrogen contributes to the nutrition of Bacteroides amylophilus. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Bacteroides is a genus of anaerobic bacteria that are often found in high abundance in the human colon. They have a complex relationship with their human host, conferring health benefits as members of the gut microbiota but also acting as opportunistic pathogens. Though many of the virulence factors in Bacteroides have been characterized, it is currently classified as nonhemolytic. Work with Bacteroides isolates led to the observation, by happenstance, of unexpected hemolytic activity in multiple species. After incubation on blood agar plates, we observed that some isolates were clearly hemolytic, but only when plated in close proximity to certain, ‘activating’ isolates. We performed a systematic screen for hemolytic activity on a library of 94 Bacteroides isolates and identified one which was able to activate hemolysis in 30% of the collection. Using timelapse photography, we show this zone of hemolysis begins between the two colonies and proceeds in a retrograde fashion towards the ‘activated’ colony. The asymmetrical patterns of hemolysis are unlike any bacterial synergy reported to date. To investigate the mechanism behind this pattern, we combined a comparative genomics and mutagenesis approach to narrow down the genetic basis of this phenotype. Here, we characterize a unique synergistic hemolysis phenotype in Bacteroides, a genus of bacteria currently classified as nonhemolytic. This novel phenotype may provide further insight into Bacteroides as an opportunistic pathogen and may have uncovered a new mechanism by which they interact. / Thesis / Master of Science (MSc)

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 56-62). Also available on the World Wide Web.

Graduation date: 1990

Thesis--Göttingen. / In Periodical Room.

No description available.

The contents of this thesis have been divided into 2 parts . The first part deals with genetic studies carried out on 36 strains belonging to the Bacteroides fragilis group. A number of mutants were isolated from several of the strains. A notable feature of the methods used was the low yield of mutants obtained and the marked sensitivity of these organisms to the mutagenic agents. Variations in colonial morphology was found to be a common feature amongst these organisms. In a few strains this phenomenon was clearly visible, in the remainder it was much weaker, and often could only be seen with the aid of a microscope . Colonial variation was found to be due to the ability of a proporti on of the cells to pruduce capsules or slime layers. The variants were found to segregate at high frequency and different growth conditions were found to have little effect on the segregation frequency or capsule formation . A number of phages specific for B. fragilis and B. t hetaiotaomicron were isol ated. All these phages were virulent and attempts to induce lysogenic phages were unsuccesful . The use of these phages in attempts to obtain transduction proved unsuccessful. A phage carrier state was found to occur in the majority of the phage-host cell systems, which seemed to be due to the presence of phage-resistant encapsulated cells in the population. Bacteriocins were produced by about half the strains, these inhibited the growth of a high proportion of the 36 strains tested. The bacteriocins were released into the growth media at the end of the growth period in the 2 bacteriocins tested. A link between the mode of action of one bacteriocin and rifampicin-resistance was investigated. All the bacter iocins tested were found to be inactive against some rifampicin-resistant mutants of a susceptible strain, suggesting a common mode of action. The presence of capsules in some cells appeared to confer bacteriocin-resistance on these variants. The second part of the thesis deals with a study of the physiological responses of a single strain of B.fragilis to ultraviolet radiation. This strain was found to be more sensitive to ultraviolet radiation under aerobic conditions. The amount of pyrimidine dimers formed after irradiation under anaerobic and aerobic conditions, was not found to differ significantly, indicating that the increase in sensitivity under aerobic conditions was not due to an increase in DNA damage. The use of repair inhibitors and the survival characteristics indicate that this difference was due to decreased repair capabilities under aerobic conditions. Liquid holding recovery in B.fragiZis was found to occur under aerobic conditions . This process was brought about by excision repair and appeared to be due to a decrease in repair efficiency under aerobic conditions. Under anaerobic conditions, where full repair capabilities were present, liquid holding recovery was inhibited. Both minimal medium recovery and fluence dependent filament formation were found to occur in irradiated B.fragiZis cells. The survival kinetics of a number of irradiated B.fragiZis phages were determined and a number of phage reactivation processes were investigated. Little or no host cell reactivation appeared to occur in the strains investigated, however, some ultraviolet reactivation and multiplicity reactivation was found to occur, but only under anaerobic conditions. Photoreactivation was found to be absent in this organism, but an excision repair system was present . The excision repair system was partially characterized and was found to resemble short patch excision repair in E.coli. Evidence was found which suggested that a second mode of repair which was sensitive to oxygen, also occurred in this strain. This repair system which appeared to be responsible for error-prone repair, and the systems which were responsible for ultraviolet reactivation and multiplicity reactivation, seemed to be dependent on a recombination function' which was inhibited by oxygen. The significance of this finding for future genetic studies was discussed.

No description available.

Die klassische Identifizierung von obligat anaeroben Bakterien ist mit einem hohen Labor- und Zeitaufwand verbunden. Um festzustellen, ob die Identifizierung mittels Matrix-unterstützter Laser-Desorption/Ionisation und Massenspektrometrie mit Flugzeitanalysator (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MALDI-TOF MS) ein Verfahren ist, um obligate Anaerobier eindeutig zu identifizieren, wurde mit der vorliegenden Arbeit die Identifizierung von unterschiedlichen Spezies der B. fragilis Gruppe mittels MALDI-TOF MS untersucht. Hierfür wurden 105 obligate Anaerobier der B. fragilis Gruppe aus der Stammsammlung des Institutes für Medizinische Mikrobiologie und Infektionsepidemiologie der Universität Leipzig untersucht. Es fanden sich für die untersuchten Erreger Spektren mit sehr guter Auflösung. Eine Identifizierung und Differenzierung war eindeutig möglich. Unter Verwendung dieser Daten wurde eine Referenzdatenbank erstellt. Die erhaltenen Ergebnisse wurden mittels einer verblindeten Studie überprüft, wobei 52 von 53 (98,1%) der untersuchten Stämme eindeutig identifiziert werden konnten. Dies schließt ebenfalls die Identifizierung und Differenzierung von 15 Metronidazol-sensiblen/ Enterotoxin-negativen, 8 Metronidazol-resistenten/ Enterotoxin-negativen und 8 Metronidazol-sensiblen/ Enterotoxin-positiven B. fragilis Stämmen ein. Die Identifizierung mittels MALDI-TOF MS ist somit eine zuverlässige Methode zur Identifizierung von obligaten Anaerobiern der B. fragilis Gruppe. Weiterhin finden sich Hinweise, dass ein Nachweis von Resistenz-, Virulenz- und Pathogenitätsfaktoren mittels MALDI-TOF MS bei diesen Erregern möglich ist.