Hugo, C. J.Hitzeroth, A. C.Gavu, Masabata Lydia2024-07-182024-07-182023http://hdl.handle.net/11660/12658Thesis (Ph.D.(Food Science))--University of the Free State, 2023In previous studies at the University of the Free State, aerobic, Gram-negative bacteria were isolated from Cape hake (𝘔𝘦𝘳𝘭𝘶𝘤𝘤𝘪𝘶𝘴 𝘤𝘢𝘱𝘦𝘯𝘴𝘪𝘴), intended for human consumption. Although some of these isolates could be identified, six isolates remained unidentified. The purpose of this study was to determine the genomic and phenotypic characteristics of these isolates to assign them to the correct genus, to describe novel species, if present, to determine the significance of these isolates in terms of pathogenicity and/or spoilage, and to isolate bacteriophages against the bacterial strains for possible biocontrol strategies. Based on 16S rRNA gene sequences, phylogenetic analysis confirmed that the six unidentified bacterial strains used in this study represented members of the genus 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢. Four of these six isolates were further characterized to determine whether they were novel species. Using genomic and phenotypic techniques, the DNA G+C content of strains SH 11-4(b), SH 19-2(b), SH 20-4 and SH 40-3 supported their affiliation with the genus 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢. Digital DNA-DNA hybridization, average nucleotide identity and amino acid identity values, and phenotypic characteristics demonstrated that strains SH 11-4(b), SH 19-2(b) and SH 40-3 represented novel species of the genus 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢. Results for strain SH 20-4 confirmed that it was not a novel species but represented another member of 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 𝘤𝘢𝘳𝘯𝘪𝘴. The names of the novel species were proposed as 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 𝘮𝘦𝘳𝘭𝘶𝘤𝘤𝘪𝘪 SH 11-4(b), 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 𝘱𝘪𝘴𝘤𝘪𝘴 SH 19-2(b), and 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 𝘧𝘳𝘪𝘨𝘪𝘥𝘪𝘱𝘪𝘴𝘤𝘪𝘴 SH 40-3. The potential pathogenicity and/or food spoilage capability of the six 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 fish isolates and reference strains were then evaluated by determining the production of siderophores, the production of a variety of enzymes that function as virulence factors, evaluating their antimicrobial resistance patterns, their ability to form biofilms, as well as the determination of their resistance to antibiofilm compounds. All the 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 isolates produced Siderophores indicating their ability to sequester iron for survival. Gelatinase, whose expression has been linked to enhanced biofilm formation, was produced in the most significant amounts by most organisms in this study. ‘𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 𝘮𝘦𝘳𝘭𝘶𝘤𝘤𝘪𝘪’ SH 11-4(b) could be regarded as a potential pathogen since it produced more than 4/8 virulence enzymes. 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 strains SH 11-3(a) and ‘𝘒. 𝘧𝘳𝘪𝘨𝘪𝘥𝘪𝘱𝘪𝘴𝘤𝘪𝘴’ SH 40-3 were the most resistant to antimicrobials. The antimicrobial resistance/susceptibility of the 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 and 𝘊𝘩𝘳𝘺𝘴𝘦𝘰𝘣𝘢𝘤𝘵𝘦𝘳𝘪𝘶𝘮 species in this study was determined using the Kirby-Bauer disc diffusion susceptibility method. The antimicrobial tests concluded that the fluoroquinolone and cephem antimicrobials would be the most effective in treating 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 infections. 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 𝘤𝘢𝘳𝘯𝘪𝘴 SH 20-4 was regarded as a strong biofilm former since it gave positive results for biofilm formation using three different methods, while strains SH 11-3(a) and ‘K. 𝘮𝘦𝘳𝘭𝘶𝘤𝘤𝘪𝘪’ SH 11-4(b) were the least successful at forming biofilms. This study revealed that 100 mM D-glucose was the most effective biofilm inhibition compound against the 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 test strains. Organisms whose biofilms showed the most resistance towards the inhibition compounds included K. 𝘤𝘢𝘳𝘯𝘪𝘴 SH 20-4 and ‘K. 𝘧𝘳𝘪𝘨𝘪𝘥𝘪𝘱𝘪𝘴𝘤𝘪𝘴’ SH 40-3 and susceptibility to the inhibition compounds mainly was observed in SH 11-3(a), SH 11-3(b), ‘K. 𝘮𝘦𝘳𝘭𝘶𝘤𝘤𝘪𝘪’ SH 11-4(b) and ‘K. piscis’ SH 19-2(b). Another aim of this study was to isolate lytic bacteriophages against the 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 species that may be pathogenic to fish or may cause food spoilage by using a two-fold agar overlay in a plaque experiment. Thirty-four phage isolates were obtained from the sewage and fishpond water samples. Strains SH 11-3(a) and SH 11-3(b) showed sensitivity toward a few phage isolates while ‘K. 𝘱𝘪𝘴𝘤𝘪𝘴’ SH 19-2(a) displayed the greatest resistance towards phage infection. Phage strains 11-3(b)-S2 showed a broader host spectrum than other phage isolates because they displayed lytic activity against 5/12 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 isolates. Most of the isolated phages were identified by transmission electron microscopy as members of the 𝘊𝘰𝘳𝘵𝘪𝘤𝘰𝘷𝘪𝘳𝘪𝘥𝘢𝘦, 𝘗𝘭𝘢𝘴𝘮𝘢𝘷𝘪𝘳𝘪𝘥𝘢𝘦, 𝘔𝘪𝘤𝘳𝘰𝘷𝘪𝘳𝘪𝘥𝘢𝘦, 𝘚𝘪𝘱𝘩𝘰𝘷𝘪𝘳𝘪𝘥𝘢𝘦 𝘢𝘯𝘥 𝘛𝘦𝘤𝘵𝘪𝘷𝘪𝘳𝘪𝘥𝘢𝘦 families. The three new members of the genus 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 were accurately classified, described, and named. The role of the six 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 species in virulence was determined. Some isolated phages can potentially prevent, eliminate, or reduce 𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢 infections in fish.en𝘒𝘢𝘪𝘴𝘵𝘦𝘭𝘭𝘢𝘊𝘩𝘳𝘺𝘴𝘦𝘰𝘣𝘢𝘤𝘵𝘦𝘳𝘪𝘶𝘮 taxonomypotential pathogenicityantimicrobial resistancebiocontrol strategiesbiofilm formationbacteriophage isolationThesisUniversity of the Free State