Bioassay Guided Fractionation of Aquatic Fungal Extracts in Pursuit of Gram-Positive Antimicrobial Activity
Faculty Mentor Name
Skylar Carlson
Research or Creativity Area
Natural Sciences
Abstract
Antimicrobial resistance to clinically available antibiotics is constantly creating the need for new drug development. Many FDA approved antibiotics are derived from fungi, such as cephalosporin and penicillin, demonstrating the immense potential of fungal biosynthetic gene clusters. Ninety morphologically unique aquatic fungi were isolated from across North America and grown in 25 mL of PDB. After 7 days, the media was extracted with ethyl acetate and screened for antimicrobial activity against: Escherichia coli, Salmonella sp., Staphylococcus aureus, and Bacillus subtilis at 25, 50, and 100 mg/mL. Of these, two strains showed inhibition at a concentration of 25 µg/mL or greater. These strains are grown in 2.4 L, extracted with ethyl acetate, fractionated with a 5 g silica SPE column, and screened again against a larger panel of microbes at 50 µg/mL. Fraction 2 of fungi Y034 showed 37% inhibition against S. epidermis.; Fraction 4 showed 20% inhibition against S. epidermis, 18% against S. aureus, and 8% against B. subtilis. Fractions 2 and 4 of the fungi Y052 showed 42% and 20% inhibition of S. epidermis, and 21% and 8% of B. subtilis, respectively. Fraction 3 showed 20% inhibition against B. subtilis. Fraction 5 showed 20% inhibition against S. epidermis, 17% inhibition against S. aureus, and 7% inhibition against B. subtilis. The active fraction was further purified and screened for known molecules using GNPS. Simultaneously, to identify the fungi, the genomic DNA is extracted using a liquid nitrogen lysis step followed by the Qiagen DNeasy Plant Pro kit. The 18S Internal Transcribed Spacer (ITS) region, a highly conserved region encoding for the 18S subunit of the fungal ribosome, is amplified using the ITS1 and ITS4 primers and submitted for Sanger sequencing. The sequence will be compared to the NCBI BLAST database to identify the fungal species. The isolation of novel compounds, dereplication of known analogs, bioactivity, and sequencing results will be presented.
Purpose
Due to the increasing rise in antibiotic and antifungal resistance of human pathogens, new drugs are necessary to combat this resistance. Natural products are small molecules that can inhibit the growth of these kinds of bacterial pathogens and novel compounds can be used as templates for other drugs to be derived from. Aquatic fungi are microorganisms that are known to produce an array of small molecules to combat other organisms within an environment and can be studied to isolate the active compound being made that inhibits this bacterial growth.
Results
The results of our project currently focus on the effects of the chemical extracts from fungi and how it inhibits known human pathogens like Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermis. The results of these assays aids in the isolation of a bioactive compound that inhibits bacterial growth for the eventual discovery of a novel compound that can be used for drug development.
Significance
The isolation and discovery of new naturally occurring secondary metabolites made from fungal species will allow the derivatization of novel antibiotic drugs. These secondary metabolites are created by organisms already and can be correlated with its survival in its environment against a broad spectrum of other microorganisms. As such, these secondary metabolites hold the potential for growth inhibition of several microorganisms.
Bioassay Guided Fractionation of Aquatic Fungal Extracts in Pursuit of Gram-Positive Antimicrobial Activity
Antimicrobial resistance to clinically available antibiotics is constantly creating the need for new drug development. Many FDA approved antibiotics are derived from fungi, such as cephalosporin and penicillin, demonstrating the immense potential of fungal biosynthetic gene clusters. Ninety morphologically unique aquatic fungi were isolated from across North America and grown in 25 mL of PDB. After 7 days, the media was extracted with ethyl acetate and screened for antimicrobial activity against: Escherichia coli, Salmonella sp., Staphylococcus aureus, and Bacillus subtilis at 25, 50, and 100 mg/mL. Of these, two strains showed inhibition at a concentration of 25 µg/mL or greater. These strains are grown in 2.4 L, extracted with ethyl acetate, fractionated with a 5 g silica SPE column, and screened again against a larger panel of microbes at 50 µg/mL. Fraction 2 of fungi Y034 showed 37% inhibition against S. epidermis.; Fraction 4 showed 20% inhibition against S. epidermis, 18% against S. aureus, and 8% against B. subtilis. Fractions 2 and 4 of the fungi Y052 showed 42% and 20% inhibition of S. epidermis, and 21% and 8% of B. subtilis, respectively. Fraction 3 showed 20% inhibition against B. subtilis. Fraction 5 showed 20% inhibition against S. epidermis, 17% inhibition against S. aureus, and 7% inhibition against B. subtilis. The active fraction was further purified and screened for known molecules using GNPS. Simultaneously, to identify the fungi, the genomic DNA is extracted using a liquid nitrogen lysis step followed by the Qiagen DNeasy Plant Pro kit. The 18S Internal Transcribed Spacer (ITS) region, a highly conserved region encoding for the 18S subunit of the fungal ribosome, is amplified using the ITS1 and ITS4 primers and submitted for Sanger sequencing. The sequence will be compared to the NCBI BLAST database to identify the fungal species. The isolation of novel compounds, dereplication of known analogs, bioactivity, and sequencing results will be presented.
