Track: Discovery and Basic Research - Pharmacology - Experimental Therapeutics
Category: Poster Abstract
The Cationic Peptide K16ApoE is Highly Effective against Staphylococcus epidermidis Biofilms
Purpose: Staphylococcus epidermidis is now the most common source of nosocomial related infections found on indwelling medical devices such as catheters and implantable devices. It contaminates implantable devices and urinary catheters forming a difficult to treat biofilm. The negatively charged extracellular matrix of the biofilm and the limited diffusion of therapeutic agents into the biofilm is widely thought to contribute to the ineffectiveness of antibiotics. One of the ways this can be combated is through the use positively charges agents and non-specific binding. Here we investigated a cationic peptide which has both properties, K16ApoE, for its ability to reduce or prevent the formation of an adherent bacterial biofilm. Methods: S. epidermidis was subcultured in 30 mg/mL tryptic soy broth (TSB) supplemented with 1% glucose for 24 h at 37 oC. After 24 h, the inoculum was then concurrently incubated with 0 to 250 mcg/mL K16ApoE at 37oC for 24 h in 96-well plates. After 24 h, the plates were washed three times. Crystal violet was added to each well and the plate was incubated with minimal shaking at 37 oC for 10 min. The plate was washed three times, followed by 30% acetic acid solution to extract the dye. The contents of each well were transferred to a new plate and diluted 5 times in water. The absorbance was read at 540 nm using a BioTek spectrophotometer (Winooski, VT). From this data, the minimum inhibitory concentration (MIC) was determined and defined as the lowest concentration of K16ApoE needed to prevent the formation of an adherent biofilm after 24 hours. The ability of K16ApoE to prevent the formation of an adherent S. epidermidis biofilm on urinary catheter tubing was determined using the crystal violet assay. Digital photographs of the catheter tubing were taken and the intensity of the crystal violet stain was quantified using ImageJ. Next, the effectiveness of K16ApoE as compared to ApoE or K16 was determined. Each well in a 96-well plate was inoculated with 250 μL of the diluted bacterial culture and concurrently incubated with 250 mcg/mL K16ApoE or an equivalent molar concentration of ApoE or K16. After 24 h, the crystal violet assay was performed. Results were validated using a live/dead bacterial viability kit and fluorescent confocal microscopy. Results: The lowest concentration of 5 mcg/mL K16ApoE (equivalent to 1.1 µM) had a significant 17.4 ± 9.6% reduction in the viability of the biofilm after 24 hours (Figure 1), relative to the PBS control. Whereas, at 125 mcg/mL K16ApoE showed an 88.8 ± 6.7% reduction in the viability of the biofilm. This percent inhibition increased to 95.6 ± 2.5% when the biofilm was pre-treated for the same concentration for 48 hours instead of 24 hours. However, this increase was not significant. Next, the concentration of K16ApoE was then increased to 187.5 mcg/mL and added simultaneously with S. epidermidis. At 24 h, K16ApoE inhibited the formation of the adherent biofilm by 97.5 ± 1%. Alternatively, when the biofilm was pre-treated for 24 hours with 250 mcg/mL showed a 99.92 ± 0.1% reduction viability of the biofilm (Figure 3A). More importantly, no resurgence of the biofilm growth was seen when pre-treated with 250 mcg/mL K16ApoE for 48 hours instead of 24 hours. The MIC concentration was determined to be 250 mcg/mL K16ApoE which showed negligible growth in the formation of the adherent biofilm after 24 hours. Quantitative determination of the ability of 250 mcg/mL K16ApoE to inhibit the growth of the adherent biofilm on PVC catheters indicated the biofilm integrity was substantially reduced by 83 ± 12.7% (Figure 2). ApoE and K16 individually demonstrated a 71.5% and 73.7% reduction in the formation of the biofilm, compared to an equivalent molar concentration of K16ApoE which showed nearly 100% reduction (Figure 3). Conclusion: K16ApoE can inhibit the formation of an adherent S. epidermidis biofilm.
References: Kandimalla, K.K., Borden, E., Omtri, R.S., Boyapati, S.P., Smith, M., Lebby, K., Mulpuru, M., Gadde, M., 2013. Ability of chitosan gels to disrupt bacterial biofilms and their applications in the treatment of bacterial vaginosis. J. Pharm. Sci. 102, 2096–2101. https://doi.org/10.1002/jps.23571 Mnif, S., Jardak, M., Graiet, I., Abid, S., Driss, D., Kharrat, N., 2019. The novel cationic cell-penetrating peptide PEP-NJSM is highly active against Staphylococcus epidermidis biofilm. Int. J. Biol. Macromol. 125, 262–269. https://doi.org/10.1016/j.ijbiomac.2018.12.008 Otto, M., 2009. Staphylococcus epidermidis - The “accidental” pathogen. Nat. Rev. Microbiol. https://doi.org/10.1038/nrmicro2182