Titanium surfaces immobilized with the major antimicrobial fragment FK-16 of human cathelicidin LL-37 are potent against multiple antibiotic-resistant bacteria

Abstract

Infections on implanted medical devices are a challenging problem, especially when bacteria form difficult-to-treat biofilms. Antimicrobial peptides are considered to be a solution due to their potency against antibiotic-resistant superbugs. Previously, the authors' laboratory demonstrated the prevention of staphylococcal biofilm formation in an animal catheter model by injecting merecidin (formerly known as 17BIPHE2), a peptide engineered based on the only human cathelicidin. This study documents an alternative solution via covalent immobilization of FK-16, amino acid sequence FKRIVQRIKDFLRNLV-amide, which corresponds to the major antimicrobial region (residues 17-32) of LL-37. FK-16 is superior to the longer peptide LL-37 in terms of synthesis cost and the shorter peptide KR-12 in terms of activity spectrum. Indeed, the FK16-coated titanium surface showed a broad-spectrum activity against the ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. It also demonstrated anti-adhesion and biofilm inhibition capabilities against both S. aureus and E. coli.

Keywords: Biofilms; ESKAPE pathogens; FK-16; LL-37; peptide immobilization; titanium.

Figure 1

(A) Scheme for peptide coating showing stepwise immobilization of FK-16Cys onto the titanium surface. (B) FT-IR analysis of different reaction steps toward the immobilization of FK-16. (C) XPS analysis showing the wide range spectra of different elements on a FK-16 Coated surface.

Figure 2

Antimicrobial activity of the Ti-FK-16Cys surfaces against different ESKAPE pathogens (2×10³ CFU): (A) E. faecium, (B) S. aureus USA300, (C) K. pneumoniae, (D) A. baumannii, (E) P. aeruginosa, and (F) E. coli ATCC 25922. Killing efficiency is expressed as the CFU changes of live bacteria compared to the surface coated up to maleimide but no peptide coupling (Ti-Mal). Experiments were done in duplicates and average results were reported. For confirmation, the entire experiment was also repeated on a different date. The error bars represent the standard deviation of the values from the mean. The level of significance was determined by performing Student t-Test with parameters of one tailed distribution with samples of equal variance (*: p < 0.05; **: p < 0.005 and ***: p < 0.0005).

Figure 3

Antimicrobial activity of the Ti-FK-16Cys surfaces against S. aureus USA300 (2×10³ CFU) in the presence of 10% human serum (A) or 150 mM NaCl (B). Killing efficiency is expressed as the CFU changes of live bacteria compared to the surface coated up to maleimide without the peptide (Ti-Mal). Experiments were performed, data processed, and statistically analyzed as described in the legend of Figure 2.

Figure 4

Anti-adherent property of the FK-16Cys immobilized titanium surfaces. An overnight bacterial culture of S. aureus USA300 (A) and E. coli ATCC 25922 (B) was allowed to attach to the titanium surface with (open bar) and without (filled bar) the peptide. Experiments were performed, data processed, and statistically analyzed as described in the legend of Figure 2.

Figure 5

Inhibition of bacterial biofilm formation on the FK-16Cys coated surfaces at bacterial CFU 10⁵ (panels A–F, left) or 10³ (panels G–L, right) against S. aureus USA300 and E. coli ATCC 25922, respectively. In each experiment, comparison was made between the FK-16Cys peptide coated Ti surfaces (Ti-FK16Cys) and the maleimide coated surfaces devoid of the FK-16 peptide (Ti-Mal). Experiments were performed, data processed, and statistically analyzed as described in the legend of Figure 2.

Figure 6

Cytotoxicity evaluation of the FK-16Cys coated surface to human epidermal keratinocytes HaCaT cells in the absence and presence of FBS (A) and human red blood cells (hRBCs) (B). In each experiment, comparison was made between the peptide coated Ti surface (Ti-FK-16Cys) and the maleimide coated surface devoid of the FK-16 peptide (Ti-Mal). Additional controls (filled columns) were used for the case of HaCaT cells without any treatment (100% live in panel A) and for hRBCs treated with 1% Triton X-100 (100% lysis in panel B). Experiments were performed, data processed, and statistically analyzed as described in the legend of Figure 2.