Effect of preservative-free artificial tears on the antimicrobial activity of human beta-defensin-2 and cathelicidin LL-37 in vitro

Abstract

Purpose: Human beta-defensin-2 (hBD-2) and cathelicidin LL-37 are salt-sensitive cationic antimicrobial peptides expressed by ocular surface epithelia. The goal of this study was to investigate the effect of preservative-free artificial tears on hBD-2 and LL-37 antimicrobial activity against Pseudomonas aeruginosa.

Methods: P. aeruginosa was incubated with hBD-2 or LL-37 in the absence or presence (70% vol/vol) of different preservative-free artificial tears--Visine Tears (300 mOsm/kg), Tears Naturale Free (261 mOsm/kg), TheraTears (185 mOsm/kg), and Refresh Plus (325 mOsm/kg)--for 2 hours at 37 degrees C. In some experiments, P. aeruginosa was incubated with hBD-2 or LL-37 and Visine Tears or Tears Naturale Free with or without carboxymethylcellulose (0.5% vol/vol final concentration). Plates were inoculated with samples of each reaction mixture and then incubated for 24 hours at 37 degrees C.

Results: Visine Tears and Tears Naturale Free had little or no effect on the antimicrobial activity of 100 microg/mL hBD-2 or LL-37. In the presence of Refresh Plus and TheraTears, the activity of 100 microg/mL hBD-2 or LL-37 was reduced by 90% to 100%. Carboxymethylcellulose, at a concentration comparable to that present in Refresh Plus, reduced the effectiveness of hBD-2 or LL-37 by 40% to 90% in the presence of Tears Naturale Free and Visine Tears.

Conclusion: Human beta-defensin-2 and cathelicidin LL-37 inhibit the growth of P. aeruginosa in vitro, but this activity is markedly reduced in the presence of Refresh Plus and TheraTears. These results suggest that carboxymethylcellulose-containing artificial tears may reduce the activity of the endogenously produced antimicrobial peptides.

FIG. 1

Effect of preservative-free artificial tears on hBD-2 and LL-37 antimicrobial activity. Pseudomonas aeruginosa (10⁷ CFU/mL ATCC 27853) was incubated with 0.01% acetic acid (control), with hBD-2 and LL-37 (100 μg/mL), or with hBD-2 and LL-37 (100 μg/mL) in the presence of four preservative-free artificial tears at 37°C for 2 hours. When tested in the presence of Refresh Plus and TheraTears, the antibacterial activity of 100 μg/mL hBD-2 and LL-37 was markedly reduced (n = 5). Tears Naturale Free or Visine Tears did not reduce the activity of hBD-2 or LL-37 (n = 5).

FIG. 2

Effect of sodium chloride (NaCl) solutions on hBD-2 and LL-37 antimicrobial activity. Pseudomonas aeruginosa (10⁷ CFU/mL ATCC 27853) was incubated with 0.01% acetic acid, with hBD-2 and LL-37 (100 μg/mL), or with hBD-2 and LL-37 (100 μg/mL) in the presence of sodium chloride solution (322 mOsm/kg) at 37°C for 2 hours (n = 3).

FIG. 3

Effect of carboxymethylcellulose (CMC) on hBD-2 and LL-37 antimicrobial activity against Pseudomonas aeruginosa. P. aeruginosa (10⁷ CFU/mL ATCC 27853) was incubated with 0.01% acetic acid (control), with hBD-2 and LL-37 (100 μg/mL), or with hBD-2 and LL-37 (100 μg/mL) in the presence of Tears Naturale Free or Visine Tears containing 0.5% CMC at 37°C for 2 hours. With the addition of CMC, Tears Naturale Free (A) and Visine Tears (B) partially or completely impaired the activity of hBD-2 and LL-37 (n = 2).