In recent years, there has been a rise in antimicrobial-resistant bacteria caused by overdependence on, and misuse of, antibiotics. This has led to an increase in research for identifying alternatives to combat pathogens. One promising means of combating pathogenic bacteria, particularly for those residing in the gastrointestinal tract (GIT), is the use of probiotics. This thesis focuses on a potential probiotic strain Lactobacillus fermentum 3872, the genome sequence of which was circularised during the study, identifying genes that may contribute to probiotic activity. Several genes involved in GIT survival, such as acid symporters were discovered, along with genes that encode adhesion proteins such as those involved in mucus, fibronectin and collagen binding. The genes mentioned above may contribute to L. fermentum 3872 survivability within the GIT and have an antagonistic effect on enteric pathogens via competitive exclusion. Other interesting genes identified in L. fermentum 3872 were potentially involved in bacterial aggregation, exopolysaccharide and vitamin synthesis, along with four prophage encoding regions. Genes that encode a class III bacteriocin was also identified. An additional gene encoding a collagen binding protein (CBP) of a newly discovered plasmid pLF3872, was recognised. The chromosomal sequence also had a partial CBP encoding gene. pLF3872 has a toxin-antitoxin gene pair that ensures stable maintenance of the plasmid, along with conjugation-related genes. Functional analysis of the recombinant CBP via ELISA experiments found that the protein had the ability to bind to collagen I, a protein present on the epithelial lining of cells of the GIT. ELISA experiments also demonstrated that a common gastrointestinal pathogen, Campylobacter jejuni, can bind to collagen I in a concentration-dependent manner. In addition, mass spectrometry analysis identified that C. jejuni strains 11168H and 81-176 may utilise flagellar components (FlaA and FlaB) for adhesion. Furthermore, C. jejuni 11168H and 81-176 binding to collagen I was inhibited in the presence of either L. fermentum 3872 or CBP, thus reducing C. jejuni adherence via competitive exclusion. Using an in vitro assay, it was also demonstrated that L. fermentum 3872 cell-free supernatant could inhibit the growth of C. jejuni, due to the acidic environment brought about by L. fermentum 3872. During the completion of the genome sequence of L. fermentum 3872, comparison of various sequence assembly techniques which focused on the quality of the genome assembly was conducted. The results showed that further extension of the genome sequence during sequence assembly may lead to assembly errors when over-relying on a commonly-used sequence quality indicator, referred to as read mapping. It is suggested that care must also be taken when using long read technology to complete the genome sequence of a bacteria, as this may result in nucleotide sequence redundancies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:739716 |
| Date | January 2017 |
| Creators | Lehri, Burhan |
| Contributors | Seddon, Alan ; Karlyshev, Andrey |
| Publisher | Kingston University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.kingston.ac.uk/40907/ |
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