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Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species
Thesis (MSc)--Stellenbosch University, 2024.
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867614021768708096 |
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| access_status_str | Open Access |
| author | Lochner, Bianca Lileen |
| author2 | Beukes, Mervyn |
| author_browse | Beukes, Mervyn Lochner, Bianca Lileen |
| author_facet | Beukes, Mervyn Lochner, Bianca Lileen |
| author_sort | Lochner, Bianca Lileen |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2024. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/131803 |
| institution | Stellenbosch University (South Africa) |
| last_indexed | 2026-06-10T12:45:24.995Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/131803 Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species Lochner, Bianca Lileen Beukes, Mervyn Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Bacteriocins Listeria monocytogenes Antibiotics Drug resistance in microorganisms Lactic acid bacteria Bacteriocins -- Genetic aspects UCTD Thesis (MSc)--Stellenbosch University, 2024. ENGLISH ABSTRACT: Antimicrobial resistance is a natural microbial phenomenon mediated predominantly by spontaneous genetic mutations. However, decades of extensive antibiotic over- and misuse drastically accelerated the development of antimicrobial resistance. Today, antimicrobial resistance is deemed a global, chronic health issue. In 2019, antibiotic-resistant infections resulted in 1.27 million deaths worldwide and is predicted to be the cause of 10 million deaths per year by 2050. The ongoing emergence of drug-resistant pathogens demands for the development of novel antibiotic alternatives. Bacteriocins are ribosomally synthesized antimicrobial peptides of bacterial origin. In recent years, bacteriocins have attracted considerable attention due to their potential as a viable antibiotic alternative. Ongoing research is essential to expand the current knowledge of bacteriocins and their therapeutic potential. Therefore, this study aims to purify and characterize bacteriocins produced by selected Enterococcal species. This project aims at the discovery of novel bacteriocins and can provide knowledge about procedures for optimal production and purification of Enterococcal bacteriocins. In vitro screening of twenty-nine unknown isolates was performed using colony-spot-, spoton- lawn-, and an inhouse real-time inhibition microtiter plate assay. Listeria monocytogenes, Enterococcus faecalis, Lactiplantibacillus plantarum Ta10c, Micrococcus luteus, Staphylococcus aureus and Streptococcus milleri were used as indicator organisms. Two isolates, 170 and ABM, were identified as potential bacteriocin producers. Culture media, pH and incubation temperature was optimized for bacteriocin production. Isolate 170 displayed optimal bacteriocin production in M17 broth, at pH 7 and 37 °C. Isolate ABM showed optimal bacteriocin production in MRS broth at pH 7 and 37 °C. Cation exchange chromatography was used to purify bacteriocin from culture supernatant and cell extract from the producer organisms. Active fractions from cation exchange chromatography were subjected to liquid-chromatography-mass spectrometry (LC-MS) and mass spectrum analysis. A cationic, hydrophobic compound group was found in the active chromatography fractions. The compound group contained peaks with m/z values that ranged from 1287.8628 to 2338.404. The mass range, hydrophobicity, cationic nature and inhibitory activity linked to the compound group highlights its potential to be a bacteriocin. Extracted genomic DNA (gDNA), universal 16S rDNA primers, and PCR amplification was used to obtain 16S rRNA gene sequence amplicons. The amplicons were sequenced and submitted for a Basic Alignment Search Tool (BLAST) similarity search on the National Centre for Biotechnology Information (NCBI) genome database. Since the BLAST search only confirmed the genus of the producer isolates, they were named according to their culture collection identity number as Enterococcus sp. 170 and Enterococcus sp. ABM. AFRIKAANSE OPSOMMING: Antimikrobiese weerstand is ′n natuurlike, mikrobiese proses, hoofsaaklik gedryf deur spontane genetiese mutasies. Alhoewel, dekades se misbruik van antibiotika het die ontwikkeling van antibiotika drasties versnel. Vandag word antimikrobiese weerstand wêreldwyd beskou as kroniese gesondheid kwessie. In 2019 het weerstandige infeksies wêreldwyd tot 1.27 miljoen sterftes gelei en word voorspel dat dit die oorsaak van 10 miljoen sterftes per jaar teen 2050 sal wees. Die voortdurende voorkoms van weerstandige patogene skep ′n groot behoefte vir die ontwikkeling van alternatiewe vir antibiotika. Bakteriosiene is antimikrobiese peptiede wat ribosomaal vervaardig word deur verskeie bakteriële spesies. Onlangs het bakteriosiene aansienlike aandag getrek as gevolg van hul potensiaal as ′n lewensvatbare alternatief vir antibiotiese behandeling. Voortdurende navorsing is noodsaaklik om huidige kennis oor bakteriosiene en hul terapeutiese potensiaal uit te brei. Daarom, is die studie gerig op die suiwering en karakterisering van bakteriosiene geproduseer deur gekose Enterokokkale spesies. Hierdie projek maak voorsiening vir die ontdekking van nuwe bakteriosiene en kan kennis verskaf oor prosedures vir die optimale suiwering van Enterokokkale bakteriosiene. In vitro sifting van nege-en-twintig kliniese isolate is uitgevoer met behulp van verskeie biotoetse. Listeria monocytogenes, Enterococcus faecalis, Lantiplantibacillus plantarum Ta10c, Micrococcus luteus, Staphylococcus aureus en Streptococcus milleri is as indicator organismes gebruik. Twee isolate, 170 en ABM, is geïdentifiseer as potensiële bakteriosien vervaardigers. Die optimale kultuurmedium, pH en inkubasietemperatuur vir bakteriosien produksie is ondersoek. Isolaat 170 het optimale bakteriosien produksie vertoon in M17-medium, by pH 7 en 37°C inkubasie. Isolaat ABM het optimale bakteriosien produksie getoon in MRS-medium, by pH 7 en 37°C inkubasie. Katioonuitruilchromatografie is gebruik om bakteriosien in die bakteriosien vervaardigers se kultuur supernatant en sel ekstrak te suiwer. Aktiewe fraksies van katioonuitruilkromatografie is onderwerp aan vloeistof-kromatografie-massaspektrometrie (LC-MS) en massaspektrumanalise. Daardeur is ’n kationiese, hidrofobiese verbindingsgroep in die aktiewe kromatografie-fraksies gevind. Die verbindigsgroep se potensiaal om n bakteriosien te wees is beklemtoon deur die hidrofobiese en kationiese aard, asook die massas en antimikrobiese aktiwiteit. Genomiese DNA (gDNA) ekstrak, universiele 16S rDNS-primers en PCR is gebruik vir die meervoudigheid van die 16S rRNS-geen. Die genetiese samestelling van die amplikon is bepaal en gebruik om ‘n BLAST ooreenkoms ondersoek uit te voer. Aangesien die BLAST- ondersoek slegs die genus van die bakteriosien vervaardigers bevestig het, is hulle volgens hul kultuurversameling-identiteitsnommer benoem as Enterococcus sp. 170 en Enterococcus sp. ABM. Masters 2025-03-27T09:21:27Z 2025-03-27T09:21:27Z 2024-12 Thesis https://scholar.sun.ac.za/handle/10019.1/131803 Stellenbosch University xx, 100 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Bacteriocins Listeria monocytogenes Antibiotics Drug resistance in microorganisms Lactic acid bacteria Bacteriocins -- Genetic aspects UCTD Lochner, Bianca Lileen Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species |
| title | Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species |
| title_full | Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species |
| title_fullStr | Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species |
| title_full_unstemmed | Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species |
| title_short | Screening, partial purification and characterization of novel bacteriocins produced by Enterococcal species |
| title_sort | screening partial purification and characterization of novel bacteriocins produced by enterococcal species |
| topic | Bacteriocins Listeria monocytogenes Antibiotics Drug resistance in microorganisms Lactic acid bacteria Bacteriocins -- Genetic aspects UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/131803 |
| work_keys_str_mv | AT lochnerbiancalileen screeningpartialpurificationandcharacterizationofnovelbacteriocinsproducedbyenterococcalspecies |