Screening Marine Algae Surface-Associated Bacteria for Antibiotic Production
Poster Number
12B
Research or Creativity Area
Natural Sciences
Abstract
Algae are diverse organisms that host a complex community of bacteria on their surfaces that produce secondary metabolites. These metabolites sometimes offer protection or nutrients back to the host algae. We used our collection of surface-associated bacteria (SAB) to screen for microbial secondary metabolites that have antimicrobial activity. An urgent issue humanity is facing today is antimicrobial resistance. Therefore, the discovery of new antibiotics is of paramount importance. In the Carlson Lab, algae were collected from three beaches in California: Stinson Beach, La Jolla, and Santa Cruz, and from the Florida Keys. Algal samples were plated on a variety of media to isolate as many diverse bacteria as possible. SAB were isolated on A1 plates (10 g starch, 4 g yeast, 2 g peptone, 15 g Instant Ocean, 10 g agar in 500 mL milli-q water). The isolates were then cultured in liquid A1 media (10 g/L starch, 4 g/L yeast, and 2 g/L peptone) and cryopreserved. These isolates were then screened for antibiotic production against four human pathogens (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella sp.) using three methods: pour-over assay, disk-diffusion, and single-dose broth assay. Using the pour-over method we identified 11 isolates out of the 332 California algae SAB and 17 isolates from ~200 Florida algae isolates that had a zone of inhibition indicating antibiotic production. After prioritizing for specific Gram-negative or Gram-positive activity, we chose to grow 14 of these active strains for 48 h and extracted them with XAD-16 resin then the culture media was subsequently extracted with ethyl acetate. The resin was rinsed with acetone to release chemical compounds from the bead. Both extracts were dried in vacuo and tested separately. The chemical complexity was analyzed using high-performance liquid chromatography and liquid chromatography-mass spectrometry. These extracts were tested for antibiotic production using the disk diffusion method. We saw several of these extracts inhibit the growth of the human pathogens. The single-dose broth assay was executed to better control for the extract concentration. The antibiotics chloramphenicol and streptomycin were used as positive controls, broth only was utilized as negative control, and DMSO was tested as a solvent control. Extracts were tested in triplicate at 100 ug/mL and were incubated overnight at 37°C. The methods, results, and advantages of each antibiotic screening method will be discussed.
Location
Don and Karen DeRosa University Center (DUC) Poster Hall
Start Date
27-4-2024 10:30 AM
End Date
27-4-2024 12:30 PM
Screening Marine Algae Surface-Associated Bacteria for Antibiotic Production
Don and Karen DeRosa University Center (DUC) Poster Hall
Algae are diverse organisms that host a complex community of bacteria on their surfaces that produce secondary metabolites. These metabolites sometimes offer protection or nutrients back to the host algae. We used our collection of surface-associated bacteria (SAB) to screen for microbial secondary metabolites that have antimicrobial activity. An urgent issue humanity is facing today is antimicrobial resistance. Therefore, the discovery of new antibiotics is of paramount importance. In the Carlson Lab, algae were collected from three beaches in California: Stinson Beach, La Jolla, and Santa Cruz, and from the Florida Keys. Algal samples were plated on a variety of media to isolate as many diverse bacteria as possible. SAB were isolated on A1 plates (10 g starch, 4 g yeast, 2 g peptone, 15 g Instant Ocean, 10 g agar in 500 mL milli-q water). The isolates were then cultured in liquid A1 media (10 g/L starch, 4 g/L yeast, and 2 g/L peptone) and cryopreserved. These isolates were then screened for antibiotic production against four human pathogens (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella sp.) using three methods: pour-over assay, disk-diffusion, and single-dose broth assay. Using the pour-over method we identified 11 isolates out of the 332 California algae SAB and 17 isolates from ~200 Florida algae isolates that had a zone of inhibition indicating antibiotic production. After prioritizing for specific Gram-negative or Gram-positive activity, we chose to grow 14 of these active strains for 48 h and extracted them with XAD-16 resin then the culture media was subsequently extracted with ethyl acetate. The resin was rinsed with acetone to release chemical compounds from the bead. Both extracts were dried in vacuo and tested separately. The chemical complexity was analyzed using high-performance liquid chromatography and liquid chromatography-mass spectrometry. These extracts were tested for antibiotic production using the disk diffusion method. We saw several of these extracts inhibit the growth of the human pathogens. The single-dose broth assay was executed to better control for the extract concentration. The antibiotics chloramphenicol and streptomycin were used as positive controls, broth only was utilized as negative control, and DMSO was tested as a solvent control. Extracts were tested in triplicate at 100 ug/mL and were incubated overnight at 37°C. The methods, results, and advantages of each antibiotic screening method will be discussed.
