Taxonomy, spoilage, and virulence characteristics of ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข species isolated from fish
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Date
2023
Authors
Gavu, Masabata Lydia
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Publisher
University of the Free State
Abstract
In previous studies at the University of the Free State, aerobic, Gram-negative bacteria were isolated from Cape hake (๐๐ฆ๐ณ๐ญ๐ถ๐ค๐ค๐ช๐ถ๐ด ๐ค๐ข๐ฑ๐ฆ๐ฏ๐ด๐ช๐ด), 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 ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข. 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 ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข. 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 ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข. Results for strain SH 20-4 confirmed that it was not a novel species but represented another member of ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข ๐ค๐ข๐ณ๐ฏ๐ช๐ด. The names of the novel species were proposed as ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข ๐ฎ๐ฆ๐ณ๐ญ๐ถ๐ค๐ค๐ช๐ช SH 11-4(b), ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข ๐ฑ๐ช๐ด๐ค๐ช๐ด SH 19-2(b), and ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข ๐ง๐ณ๐ช๐จ๐ช๐ฅ๐ช๐ฑ๐ช๐ด๐ค๐ช๐ด SH 40-3.
The potential pathogenicity and/or food spoilage capability of the six ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข 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 ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข 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. โ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข ๐ฎ๐ฆ๐ณ๐ญ๐ถ๐ค๐ค๐ช๐ชโ SH 11-4(b) could be regarded as a potential pathogen since it produced more than 4/8 virulence enzymes. ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข strains SH 11-3(a) and โ๐. ๐ง๐ณ๐ช๐จ๐ช๐ฅ๐ช๐ฑ๐ช๐ด๐ค๐ช๐ดโ SH 40-3 were the most resistant to antimicrobials. The antimicrobial resistance/susceptibility of the ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข and ๐๐ฉ๐ณ๐บ๐ด๐ฆ๐ฐ๐ฃ๐ข๐ค๐ต๐ฆ๐ณ๐ช๐ถ๐ฎ 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 ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข infections.
๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข ๐ค๐ข๐ณ๐ฏ๐ช๐ด 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 โK. ๐ฎ๐ฆ๐ณ๐ญ๐ถ๐ค๐ค๐ช๐ชโ 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 ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข test strains. Organisms whose biofilms showed the most resistance towards the inhibition compounds included K. ๐ค๐ข๐ณ๐ฏ๐ช๐ด SH 20-4 and โK. ๐ง๐ณ๐ช๐จ๐ช๐ฅ๐ช๐ฑ๐ช๐ด๐ค๐ช๐ดโ SH 40-3 and susceptibility to the inhibition compounds mainly was observed in SH 11-3(a), SH 11-3(b), โK. ๐ฎ๐ฆ๐ณ๐ญ๐ถ๐ค๐ค๐ช๐ชโ SH 11-4(b) and โK. piscisโ SH 19-2(b).
Another aim of this study was to isolate lytic bacteriophages against the ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข 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 โK. ๐ฑ๐ช๐ด๐ค๐ช๐ดโ 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 ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข isolates. Most of the isolated phages were identified by transmission electron microscopy as members of the ๐๐ฐ๐ณ๐ต๐ช๐ค๐ฐ๐ท๐ช๐ณ๐ช๐ฅ๐ข๐ฆ, ๐๐ญ๐ข๐ด๐ฎ๐ข๐ท๐ช๐ณ๐ช๐ฅ๐ข๐ฆ, ๐๐ช๐ค๐ณ๐ฐ๐ท๐ช๐ณ๐ช๐ฅ๐ข๐ฆ, ๐๐ช๐ฑ๐ฉ๐ฐ๐ท๐ช๐ณ๐ช๐ฅ๐ข๐ฆ ๐ข๐ฏ๐ฅ ๐๐ฆ๐ค๐ต๐ช๐ท๐ช๐ณ๐ช๐ฅ๐ข๐ฆ families.
The three new members of the genus ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข were accurately classified, described, and named. The role of the six ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข species in virulence was determined. Some isolated phages can potentially prevent, eliminate, or reduce ๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข infections in fish.
Description
Thesis (Ph.D.(Food Science))--University of the Free State, 2023
Keywords
๐๐ข๐ช๐ด๐ต๐ฆ๐ญ๐ญ๐ข, ๐๐ฉ๐ณ๐บ๐ด๐ฆ๐ฐ๐ฃ๐ข๐ค๐ต๐ฆ๐ณ๐ช๐ถ๐ฎ taxonomy, potential pathogenicity, antimicrobial resistance, biocontrol strategies, biofilm formation, bacteriophage isolation