Author(s): Sydnee Barnes
Mentor(s): Brad Berges, Lucy Bowden
Institution BYU
This study examines how growth media influences horizontal gene transfer (HGT) in biofilms of Methicillin-resistant Staphylococcus aureus (MRSA). MRSA’s ability to acquire resistance to antibiotics is a growing issue in the medical world which poses a serious health challenge. By testing a modified growth medium (a 1:1 mixture of Brain Heart Infusion (BHI) and whole milk, called MB media) this experiment aims to identify the factors that influence HGT. A paper written by a lab in Japan inspired the outline of this experiment. This lab used an expensive synthetic media but provided a cheaper substitute (MB media). This experiment varies MB media preparation, including mixing and sterilization methods to optimize biofilm growth and DNA transfer rates through transformation. In our experiment, two different MRSA strains are used with complementary antibiotic resistance. This pairing must include one that is susceptible and one that is resistant to the same antibiotic. The donor (resistant) strain is heat-killed in order to kill the cell but preserve the DNA to allow for transfer. Both strains are then cultured in MB media in 6-well plates to grow biofilms over 48 hours. After the biofilm has formed, the different samples are plated on antibiotic agar to allow for growth. If the recipient strain combined with the donor strain (the experimental sample) shows growth, HGT has taken place. The heat-killed strain is also spread on a plain agar plate to make sure it is truly dead and the recipient on an antibiotic agar plate to make sure it is truly susceptible. To ensure that HGT is taking place, the experimental sample is plated on a double antibiotic plate. This plate includes an antibiotic in which the donor strain is susceptible along with the antibiotic in which it is resistant to. If growth appears on this plate as well, we can be confident that HGT took place. When HGT is shown, the resistant strain will be isolated for genome sequencing to pinpoint where the transferred genetic regions are. This experiment will provide valuable insights into the conditions needed for HGT to take place in MRSA biofilms. Results from this study can be applied to other experiments and research to help learn more about antibiotic resistance and create a plan in order to combat the growing threat it creates.