Professor West Virginia University School of Medicine, United States
Introduction: Among surgical site infections, open fractures (150,000 cases each year in the US) have high infection rates, where approximately 10% of open fractures develop a surgical site infection. Staphylococcus aureus (S. aureus) is the most common cause of surgical site infections, where between 38.7-50.9% of microorganisms causing surgical site infections are resistant to antibiotics. Only 16 new antibiotics were approved by the Food and Drug Administration (FDA) during the 15 years between 2008-2022. Due to the shortage of antibiotics, there is an urgent need to develop novel antimicrobials. Self-assembled amino acid structures have been formed using aromatic amino acids, such as phenylalanine and tryptophan, which are stabilized by pi-pi interactions. The goal of this study is to understand the unique properties of Carbobenzoxyglutamine (Z-glutamine) through observing self-assembling properties and antimicrobial activities. Z-glutamine has an aromatic group, which provides the potential to form structures stabilized by the pi-pi interactions.
Materials and
Methods: Z-glutamine was dissolved in buffer ranging from various physiological pH values including 6.0, 6.4, 7.0, and 7.4 to determine the role of pH on the morphology of the fibers. In addition, the effect of Z-glutamine concentration on morphology was determined using a range of concentrations between 20 mM and 50 mM. After preparing these solutions, they were spread on a stainless-steel disc and allowed to dry overnight under a vacuum and sputtered with gold to be measured using JEOL JSM-7600F Scanning Electron Microscope at 5kV. The antimicrobial properties of the Z-Glutamine fibers were determined against S. aureus and methicillin-resistant S. aureus (MRSA). Sterile brain heart infusion (BHI) broth was used for culturing the bacteria. Three colonies of S. aureus were inoculated into a sterile 50 mL tube with 25 mL of BHI and incubated for 18 h at 37°C while shaking. Then, 18-h-old S. aureus was diluted to an OD600 of 0.9 to obtain 1x108 colony forming units (CFUs), which was further diluted to 1x106 CFU for Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination. Z-glutamine was added to a 96-well plate and diluted with the inoculum and incubated for 22 h at 37°C while shaking. After this time, 10 uL of each well was added to blood agar plates and incubated for 18 h and the CFU/mL was determined.
Results, Conclusions, and Discussions:
Results: Z-glutamine formed fibers at the pH values studied, but at pH 6 and pH 6.4 showed to have a smaller diameter with 1-5 um and 0.3-1 um, respectively. Therefore, pH 6.4 was used for determining the role of Z-glutamine concentration on the morphology of the fibers. The fibers were not able to form below 35 mM, indicating this concentration is necessary for fiber formation. In addition, Z-glutamine demonstrated antimicrobial activity against S. aureus at 4000 ug/mL at pH 6.4. The effectiveness of Z-glutamine showed to decrease with an increase in pH, where the MIC was increased to 5000 ug/mL at pH 7.0 and 7.4. In addition, Z-glutamine also demonstrated antimicrobial activity against MRSA with an MIC of 5000 ug/mL. In addition to Z-glutamine, Z-glycine was also used to determine the antimicrobial properties against S. aureus. Z-glycine demonstrated a 2-fold reduction in MIC compared to Z-glutamine, which was 2000 ug/mL. L-glutamine and L-glycine were used as controls, which did not have a clear reduction of bacteria at the concentrations studied.
Conclusions: When compared to L-glutamine and L-glycine, there showed a significant reduction in S. aureus with the incorporation of the Z-group to the amino acids. Therefore, our results indicate the role of the Z-group in enhancing the antimicrobial activity against S. aureus. The incorporation of this Z-group is a novel finding, which has applications for future work, such as incorporation in hydrogels or peptides to potentially enhance antimicrobial activity.
