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In vitro evaluation of contact-active antibacterial efficacy of Ti-Al-V alloys coated with the antimicrobial agent PHMB.

Acta biomaterialia (2020-02-19)
Melanie Hornschuh, Paula Zwicker, Thomas Schmidt, Axel Kramer, Gerald Müller
ABSTRACT

Immobilized polycationic substances on biomaterial surfaces kill adhering bacteria upon contact and are considered a promising non-antibiotic alternative. Unfortunately, there is no generally accepted in vitro method for quantitatively evaluating the antibacterial efficacy of contact-active non-leachable antimicrobial surfaces. Moreover, guidelines of generally accepted international industrial standards do not reflect the basic principle of bacterial contamination and/or are performed in the presence of a solid covering material. Therefore, in the present study, six bacterial adherence tests on non-porous surfaces with no covering material were compared with respect to their efficacy and reproducibility, as well as to evaluate the bactericidal contact-killing of relevant device-associated slime-producing bacteria using antimicrobially coated Ti6Al4V surfaces with positively-charged poly(hexamethylene biguanide) hydrochloride (PHMB). After direct bacterial inoculation to simulate a perioperative infection, non-leaching PHMB reacts bactericidally against the slime-producing bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa after surface contact. The 6-h drop technique was found to be a suitable method to quantitatively evaluate contact-active antibacterial surfaces. Adjunctively, however, damage of bacterial membrane integrity should be confirmed by LIVE/DEAD staining and the presence of non-leaching agents. STATEMENT OF SIGNIFICANCE: Unintentional perioperative bacterial adhesion to implant surfaces can generate biomaterial-associated infections. Adhered bacteria produce biofilms that protect them from antibiotic attack, which may be complicated by possible antibiotic resistance. Polycationic surfaces can prevent such unwanted biofilm formation by killing bacteria upon initial contact. Unfortunately, no reliable in vitro methods exist to evaluate the efficacy of contact-active antimicrobial surfaces. In this study, we show that the 6-h drop technique may be a suitable method to evaluate positively-charged contact-killing surfaces. Identification of suitable screening assays for evaluating the bactericidal efficacy of non-leachable antimicrobial agents will greatly improve this newly developing field as a prophylactic alternative to postoperative treatment of implant-associated infections by antibiotics.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Yeast Extract, for use in microbial growth medium
Supelco
Hydrogen peroxide solution, ≥30%, for trace analysis
Sigma-Aldrich
Bovine Serum Albumin solution, 30%±2% in 0.85% sodium chloride, aseptically filled
Sigma-Aldrich
Crystal Violet, for microscopy (Bact., Bot., Hist., Vit.), indicator (pH 0.1-2.0)