Loading...
Effect of co-culture on the activity profile of marine actinobacteria
Author(s)
Mitchell, Daniëlle Dana
Date Issued
2024
Type
Thesis
Publisher
Cape Peninsula University of Technology
Abstract
Over the past few years, there has been a dramatic increase in the number of antimicrobialresistant
pathogens. However, this increase has not been matched with the discovery of new
antibiotics for the treatment of diseases or infections caused by these antimicrobial-resistant
pathogens. In order to address this, marine actinobacterial diversity was explored as a potential
source of novel antibiotics. Metabarcoding analysis of sediment samples collected at three
subsites, designated as ‘Dry’, ‘Ocean’ and ‘Rocky’, from a tidal pool located within the Table
Mountain National Park (TMNP2), reflected a core actinobiome consisting of unculturable and
unidentified actinobacteria. In order to access this untapped diversity, actinobacteria were
isolated from sediment samples. Initial bioactivity screening of twenty actinobacterial isolates
was conducted against a set of ESKAPE pathogens: Acinetobacter baumannii ATCC 19606
and ATCC BAA-1605, Enterococcus faecalis ATCC 29212 and ATCC 51299, Escherichia coli
ATCC 25922, Klebsiella pneumoniae ATCC BAA-700603, Pseudomonas aeruginosa ATCC
27853, and Staphylococcus aureus ATCC 29213 and ATCC 33591.
Out of the twenty actinobacterial isolates, eight isolates displayed strong activity against
ESKAPE pathogens when cross-streaked on M19, M19 with Red Sea salts (M19+),
International Streptomyces Project medium 2 (ISP2), and ISP2 with Red Sea salts (ISP2+)
media. These isolates were then identified by 16S rRNA sequencing, and it was found that all
eight belong to the genus Streptomyces. The eight actinobacterial isolates were also cultured
as mono-cultures in liquid media to determine their capacity to produce bioactive compounds.
A time-based study was performed to determine at which time point (day 1, day 3, day 5, or
day 10) the best activity was observed. Extracts of the mono-cultures were prepared, and filter
disc assays were performed to determine the activity of the extracts against the ten ESKAPE
pathogens. Only two strains, R-30 and R-35, exhibited strong activities against S. aureus
ATCC 29213 and A. baumannii ATCC 19606 when cultured in ISP2. These two strains also
showed activity against all ten test strains on solid media.
Strains R-30 and R-35 were therefore chosen for further study, where they were indirectly cocultured
with each other (separated by a membrane filter to ensure non-contact) and directly
co-cultured with ‘alive’ and ‘dead’ Mycobacterium aurum A+. The strains were first grown
separately to ensure growth and thereafter added together in one flask to determine if there
was a change in the bioactivity of R-30 and R-35. When R-30 and R-35 were co-cultured
together, both isolates showed an increase in activity on day 1 (T1). When co-cultured with
‘alive’ and ‘dead’ M. aurum A+, only R-35 showed good activity against four test strains. When
co-cultured with different concentrations of M. aurum A+, the extracts prepared from R-35
showed very good activity against eight of the test strains. Metabolomic analyses via tandem
mass spectrometry (MS/MS) fingerprinting were performed on the extracts prepared from the
mono-cultures and the co-cultures. It was observed that antimycin was one of the main secondary metabolites present in the extracts prepared from the indirect co-cultures of R-30
and R-35. MS/MS fingerprinting of extracts prepared from the direct co-cultures of R-35 and
M. aurum A+, showed the presence of desferrioxamines and N-acetyltyramine. Due to the
promising results obtained, whole genome sequencing of strain R-35 was performed to identify
potential biosynthetic gene clusters involved in secondary metabolite production under
different co-culture conditions. The ability of strain R-35 to produce antimycin and
desferrioxamine was confirmed by the antiSMASH results, which indicated the presence of
biosynthetic gene clusters that encode for antimycin, desferrioxamine E and desferrioxamine
D. The antiSMASH report also indicated the presence of naphthyridinomycin, curamycin,
aurantimycin, albaflavenone, himastatin, nigericin, and desferrioxamine B biosynthetic gene
clusters, but these compounds were not identified in the MS/MS data analyses. The molecular
networking analysis of the metabolomics data also confirmed the presence of multiple
unknown secondary metabolites.
The outcome of this study, therefore, confirms that secondary metabolite production is
enhanced when two marine actinobacterial isolates are co-cultured together or when they are
co-cultured with other bacteria, such as the mycolic acid-producing M. aurum A+. Future
studies will be aimed at isolating and identifying the compounds responsible for the activity
seen in the co-culture experiments and determining if the activity seen is caused by one
individual compound class or if it is a group of compounds working together to produce the
activity.
pathogens. However, this increase has not been matched with the discovery of new
antibiotics for the treatment of diseases or infections caused by these antimicrobial-resistant
pathogens. In order to address this, marine actinobacterial diversity was explored as a potential
source of novel antibiotics. Metabarcoding analysis of sediment samples collected at three
subsites, designated as ‘Dry’, ‘Ocean’ and ‘Rocky’, from a tidal pool located within the Table
Mountain National Park (TMNP2), reflected a core actinobiome consisting of unculturable and
unidentified actinobacteria. In order to access this untapped diversity, actinobacteria were
isolated from sediment samples. Initial bioactivity screening of twenty actinobacterial isolates
was conducted against a set of ESKAPE pathogens: Acinetobacter baumannii ATCC 19606
and ATCC BAA-1605, Enterococcus faecalis ATCC 29212 and ATCC 51299, Escherichia coli
ATCC 25922, Klebsiella pneumoniae ATCC BAA-700603, Pseudomonas aeruginosa ATCC
27853, and Staphylococcus aureus ATCC 29213 and ATCC 33591.
Out of the twenty actinobacterial isolates, eight isolates displayed strong activity against
ESKAPE pathogens when cross-streaked on M19, M19 with Red Sea salts (M19+),
International Streptomyces Project medium 2 (ISP2), and ISP2 with Red Sea salts (ISP2+)
media. These isolates were then identified by 16S rRNA sequencing, and it was found that all
eight belong to the genus Streptomyces. The eight actinobacterial isolates were also cultured
as mono-cultures in liquid media to determine their capacity to produce bioactive compounds.
A time-based study was performed to determine at which time point (day 1, day 3, day 5, or
day 10) the best activity was observed. Extracts of the mono-cultures were prepared, and filter
disc assays were performed to determine the activity of the extracts against the ten ESKAPE
pathogens. Only two strains, R-30 and R-35, exhibited strong activities against S. aureus
ATCC 29213 and A. baumannii ATCC 19606 when cultured in ISP2. These two strains also
showed activity against all ten test strains on solid media.
Strains R-30 and R-35 were therefore chosen for further study, where they were indirectly cocultured
with each other (separated by a membrane filter to ensure non-contact) and directly
co-cultured with ‘alive’ and ‘dead’ Mycobacterium aurum A+. The strains were first grown
separately to ensure growth and thereafter added together in one flask to determine if there
was a change in the bioactivity of R-30 and R-35. When R-30 and R-35 were co-cultured
together, both isolates showed an increase in activity on day 1 (T1). When co-cultured with
‘alive’ and ‘dead’ M. aurum A+, only R-35 showed good activity against four test strains. When
co-cultured with different concentrations of M. aurum A+, the extracts prepared from R-35
showed very good activity against eight of the test strains. Metabolomic analyses via tandem
mass spectrometry (MS/MS) fingerprinting were performed on the extracts prepared from the
mono-cultures and the co-cultures. It was observed that antimycin was one of the main secondary metabolites present in the extracts prepared from the indirect co-cultures of R-30
and R-35. MS/MS fingerprinting of extracts prepared from the direct co-cultures of R-35 and
M. aurum A+, showed the presence of desferrioxamines and N-acetyltyramine. Due to the
promising results obtained, whole genome sequencing of strain R-35 was performed to identify
potential biosynthetic gene clusters involved in secondary metabolite production under
different co-culture conditions. The ability of strain R-35 to produce antimycin and
desferrioxamine was confirmed by the antiSMASH results, which indicated the presence of
biosynthetic gene clusters that encode for antimycin, desferrioxamine E and desferrioxamine
D. The antiSMASH report also indicated the presence of naphthyridinomycin, curamycin,
aurantimycin, albaflavenone, himastatin, nigericin, and desferrioxamine B biosynthetic gene
clusters, but these compounds were not identified in the MS/MS data analyses. The molecular
networking analysis of the metabolomics data also confirmed the presence of multiple
unknown secondary metabolites.
The outcome of this study, therefore, confirms that secondary metabolite production is
enhanced when two marine actinobacterial isolates are co-cultured together or when they are
co-cultured with other bacteria, such as the mycolic acid-producing M. aurum A+. Future
studies will be aimed at isolating and identifying the compounds responsible for the activity
seen in the co-culture experiments and determining if the activity seen is caused by one
individual compound class or if it is a group of compounds working together to produce the
activity.
Additional information
Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2024
File(s)![Thumbnail Image]()
Loading...
Name
Danielle_Mitchell_213304880 MSc Thesis.pdf
Size
8.96 MB
Format
Adobe PDF
Checksum
(MD5):25ee1c91cedb5029d87ca4055e6829bd