Introduction: In surgery, including orthopaedic surgery, preventing infections is a major concern that plays an important role in clinical outcomes. The broad use of implants in various surgeries has significantly increased the occurrence of infections, while the globally extensive use of antibiotics has led to bacterial antibiotic resistance that has resulted in the rise of ‘superbugs’ like methicillin-resistant Staphylococcus aureus (MRSA). There is a need for treatment modalities that can be used to substitute antibiotics. Antimicrobial peptides (AMPs), existing in a variety of forms in animals, have emerged as an alternative to conventional antibiotics. AMPs could be used as immunomodulatory molecules to regulate the body’s immune function, change the host immune related gene expression, suppress the inflammatory cytokines induced by lipopolysaccharides, promote wound healing, enhance angiogenesis, and play a bridge role for the mononuclear cells, macrophages, and dendritic cells in innate and acquired immune responses. However, most AMPs like LL-37 also present high toxicity toward human cells. In this study, an AMP with a nine-amino acid sequence of KRWWKWWRR (referred to as HHC36) was synthesized and tested in vitro against Staphylococcus aureus (S. aureus) and mammalian cells. We hypothesized that this HHC36 has high antimicrobial activity against bacteria like S. aureus and low toxicity toward mammalian cells like osteoblasts.
Materials and
Methods: The peptide (i.e., HHC36) was synthesized by CPC Scientific Inc. (Sunnyvale, CA) and its sequence was Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg-NH2 with a MW of 1,487.8 and a purity of 98.2%. The antimicrobial properties of HHC36 were assessed against S. aureus, and cytotoxicity toward human osteoblast and BEAS-2B cells and compared with conventional antibiotics (gentamicin, rifampin, vancomycin, cefazolin, and fusidic acid). Statistical analysis was carried out using JMP-V9 statistical software and p< 0.05 was considered statistically significant.
Results, Conclusions, and Discussions: The antimicrobial activity of HHC36 was found to be dose dependent. After 30 min, the killing percent of HHC36 against S. aureus was about 15% at 3 μM, and increased sharply and significantly to approximately 50% at 30 μM and over 90% at 100 μM; 100% bacterial killing was achieved at the concentration of 200 μM and above (Fig. 1a). It was found that conventional antibiotics like vancomycin, rifampin, cefazolin, and fusidic acid all had significantly lower bacterial killing percent compared to HHC36 at the same concentration (i.e., 200 μM) after culturing for 30 min (Fig. 1b). Like HHC36, gentamicin also achieved 100% bacterial killing (Fig. 1b); however, gentamicin showed much slower kinetics compared to HHC36, since HHC36 eliminated more than 90% of S. aureus within 5 min when gentamycin only had about 50% bacterial killing at 5 min (Fig. 1c). HHC36 was found to present significantly higher permeabilization against S. aureus compared to gentamycin and the untreated samples at 30 min and longer, (Fig. 1d). Cytotoxicity studies found that HHC36 had significantly higher (30% higher) osteoblast cell viability compared to the treatments of all conventional antibiotics studied at 2 h (Fig. 1e). The toxicity toward human cells (i.e., osteoblasts and BEAS-2B cells) was dose dependent; the higher the concentration the lower the cell viability (Fig. 1f). AMPs can present their antibacterial activities for different targets, the most obvious of which is the targeting of the membrane structure. Bacterial membranes are negatively charged and AMPs tend to selectively attack bacteria more compared to mammalian cells due to the cationic characteristics of AMPs. It is reasonable, though not well understood, that the toxic effects of AMPs are peptide sequence dependent. In this study, the data showed that, compared with commonly used conventional antibiotics, HHC36 was effective and prompt at eliminating bacteria like S. aureus. It presented significantly higher osteoblast cell viability compared to the conventional antibiotics studied. Therefore, HHC36 may have the potential to be a better alternative to the current antibiotics, although further studies are warranted.
Acknowledgements (Optional): The authors acknowledge the use of the West Virginia University (WVU) Shared Research Facilities.
