Halle, Universiẗat, Diss., 2001.

Göttingen, Universiẗat, Diss., 2001.

Halle, Wittenberg, Universiẗat, Diss., 2001.

No description available.

Members of the Staphylococcus genus are a major health issue in the clinical environment and can cause a wide range of disease in humans. However, this genus is also found as a part of the normal flora in humans, usually on the skin, nasal cavities, or on the linings of the throat. Normal flora members of the Staphylococcus genus become an opportunistic infection when there is breach in the physical barriers or the immune status of the human host. Another challenge with the Staphylococcus genus is the increase in drug resistant strains, such as Methicillin resistant Staphylococcus aureus (MRSA), making simple infections difficult to treat and leading to severe toxic shock or even death. Recently, there have been numerous studies demonstrating normal flora bacterial interactions inhibiting bacteria that are potentially harmful to humans. Our research lab has previously demonstrated that the benign bacterium Alcaligenes faecalis has inhibitory effects against Staphylococcus aureus. In the present study, we wanted to explore: 1) if the inhibitory effect of A. faecalis would translate to other Staphylococcus species (S. capitis, S. saprophyticus, and S. epidermidis); 2) if this inhibitory effect was found in other Alcaligenes species (A. viscolactis). To determine this possible interactions, two parallel experimental projects were undertaken. A. faecalis and A. viscolactis were tested for their interactions with Staphylococcus species on both agar and liquid medium. For agar medium analysis, Staphylococcus lawns were grown on agar plates and either Alcaligenes cells, heat killed Alcaligenes, or Alcaligenes cell free supernatant were spotted onto the lawns and observed and scored for zones of inhibition (ZOI). It was demonstrated live cells of both A. faecalis and A. viscolactis were needed to produce ZOI on Staphylococcus lawns and that all Staphylococcus species were inhibited. For liquid medium analysis, Staphylococcus species were either inoculated alone (control) or in a co-culture with Alcaligenes, serially diluted, and colony forming units (CFU) were enumerated. Both A. faecalis and A. viscolactis inhibited all Staphylococcus species in liquid culture. Based on the results from these experiments, it is our conclusion that: 1) Alcaligenes faecalis and Alcaligenes viscolactis both possess the ability to inhibit Staphylococcus growth; 2) all Staphylococcus species are inhibited by Alcaligenes, but at varying levels. The exact mechanism of how Alcaligenes can inhibit Staphylococcus species is unknown, which will require further studies to analyze and understand the exact mechanism in order to create effective therapeutic targets to combat these increasingly resistant strains of Staphylococcus.

The Candida genus is full of fungi that are subtle parts of the human microbiome, but they can cause complications if they overgrow within the body—specifically the mouth and throat, the genitalia, and the entire body through infection of the bloodstream. Candida species are a rising problem for many across the world, and this can be seen in the recent threat of Candida auris hospitalizing patients and being regularly resistant to anti-fungal medications. Beyond C. auris, Candida albicans is the most common Candida species that humans must combat because it causes the most infections in humans—mostly vaginal yeast infections. C. albicans does have natural competitors that can either inhibit its growth or kill it in general, and the competition that we took advantage of was with the Alcaligenes species. Alcaligenes faecalis and Alcaligenes viscolactis have been shown to at least inhibit C. albicans growth and maybe even kill the fungus. This rate of infection from C. albicans places it at the forefront of Candida research, and we attempted to further this research by utilizing both A. faecalis and A. viscolactis to create a co-infection model for Caenorhabditis elegans—a simple nematode lifeform. It is known that A. faecalis and A. viscolactis do not commonly adversely affect humans, so little research has been done concerning their clinical effects. We were looking to find a possible answer to C. albicans infections beyond antifungal drugs because we know that antibiotic resistance is on the rise. We performed liquid assays to test the survivability of C. elegans nematodes in various bacterial/fungal circumstances. We subjected batches of C. elegans to E. coli OP50 as a control, A. faecalis, A. viscolactis, C. albicans, A. faecalis and C. albicans, and A. viscolactis and C. albicans. This procedure was followed in order to determine the viability of using the Alcaligenes species to either help the C. elegans survive the infection or prevent them from getting infected at all. After following through with the project, we found that there was a noticeable increase in the survivability of C. elegans when subjected to both one of the Alcaligenes species and C. albicans as opposed to the C. albicans alone. The data, although early, shows the possibility of Alcaligenes species being used to combat C. albicans infections in lifeforms.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Two strains of Alcaligenes denitrificans, designated BRI 3010 and BRI 6011, were isolated from polychlorinated biphenyl (PCB) contaminated soil using 2,5-dichlorobenzoic acid (2,5-DCBA) and 2,4-DCBA, respectively, as sole carbon and energy sources. Both strains degraded 2-chlorobenzoic acid (2-CBA), 2,3-DCBA, and 2,5-DCBA. BRI 6011 alone degraded 2,4-DCBA. Metabolism of the chlorinated substrates resulted in the stoichiometric release of chloride, and degradation proceeded by intradiol cleavage of the aromatic ring. Growth of both strains on dichlorobenzoic acids induced pyrocatechase activities having catechol (catechol 1,2-dioxygenase) and chlorocatechols (chlorocatechol 1,2-dioxygenase) as substrates. Growth on 2-CBA and benzoic acid induced a pyrocatechase activity (catechol 1,2-dioxygenase) directed against catechol only. / The chlorocatechol 1,2-dioxygenase from BRI 6011 was purified, characterized, and compared with the chlorocatechol 1,2-dioxygenase from Pseudomonas sp. B13 and P. putida, organisms limited with respect to their CBA degradative versatility. These enzymes appear to be very similar based on biochemical and genetic data and possess sufficient broad substrate specificity to accommodate a wide range of chlorinated catechols, hence the increased versatility for chlorobenzoic acid degradation of A. denitrificans cannot be attributed to a more specialized chlorocatechol 1,2-dioxygenase. / Uptake of benzoic acid by BRI 3010 and BRI 6011 was inducible, exhibited saturation kinetics and the substrate was accumulated intracellularly against a concentration gradient by a factor of 8 and 10, respectively, indicative of active transport. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation kinetics were not observed, suggesting passive diffusion of 2,4-DCBA and other CBAs into the cell down a concentration gradient. / Based on oxygen uptake experiments with whole cells, benzoic acid dioxygenase and chlorobenzoic acid dioxygenase activity was induced by benzoic acid and ortho-substituted chlorobenzoic acids, respectively. Since 2,4-DCBA diffuses across the membrane and the expected catecholic intermediates of 2,4-DCBA metabolism are metabolizable by BRI 3010, this suggests that the major difference between BRI 3010 and BRI 6011 might be the inability of the chlorobenzoic acid dioxygenase in BRI 3010 to recognize 2,4-DCBA as a substrate.

The rise of antibiotic resistance in common human pathogens and the lack of development of novel therapeutic treatments has created a threat to global health. A unique source for potential novel treatments are from microorganisms, particularly within the complex, antagonistic polymicrobial interactions that take place in microbial communities. These unique mechanisms utilized by microorganisms to fight each other could potentially identify novel therapeutic targets for use at a clinical level, however, there is a lack of research in this area to determine its applicability. Alcaligenes faecalis is a Gram-negative bacterium that seldom causes human disease and has been observed in our lab to show competitive, contact-dependent inhibitory mechanisms against Bacillus species, Candida albicans, and Staphylococcus species. These bacterial and eukaryotic microbes are increasingly a common source of human disease and all exhibit increased incidences of drug resistance. In this study, genetic elements related to A. faecalis’ contact-dependent inhibitory mechanism were determined via transposon mutagenesis. Genomic sequencing was performed on mutant strains of A. faecalis that exhibited diminished inhibition or loss-of-function inhibition against the competing microbes. Four of these A. faecalis mutant strains were successfully sequenced and compared to NCBI’s genomic database. The proteins of the interrupted genetic elements were identified as a FAD-binding oxidoreductase, MFS transporter, and mechanosensitive ion channel. Further analysis of these mutants is needed to determine their role in the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus, C. albicans, and Bacillus species treatments.

No description available.