Lysostaphin-coated mesh prevents staphylococcal infection and significantly improves survival in a contaminated surgical field.

Mesh and wound infections during hernia repair are predominantly caused by Staphylococcus aureus. Human acellular dermis (HAD) is known to lose its integrity in the face of large bacterial loads. The goal of this study was to determine if lysostaphin (LS), a naturally occurring anti-Staphylococcal protein, can protect HAD mesh from S. aureus infection. HAD samples, 3 cm × 3 cm, were implanted in the onlay fashion on the anterior abdominal wall of rats (n = 75). Subjects were grouped based on presence of antimicrobial bound to HAD (none or LS) and presence of S. aureus inoculum (sterile, 106, 108 CFU). At 60 days, meshes were explanted, and bacterial growth, histology, and mesh tensile strength were examined. None of the controls receiving bacterial inoculation without LS survived to 60 days. All LS-HAD sterile and LS-106 animals survived to explantation. The LS-HAD 108 group had a mortality rate of 50 per cent. All surviving LS-treated animals (n = 25) had negative wound and mesh cultures. Blinded gross and histologic evaluation and measured tensile strengths between all LS groups were comparable. Animals implanted with LS-HAD had a dramatically improved rate of survival. All animals surviving to 60 days had completely cleared S. aureus from their wounds with maintenance of mesh integrity and tensile strength. These findings strongly suggest the clinical use of LS-treated mesh in contaminated fields may translate into a more durable hernia repair.

The addition of lysostaphin dramatically improves survival, protects porcine biomesh from infection, and improves graft tensile shear strength.

BACKGROUND: Lysostaphin (LS), a naturally occurring Staphylococcal endopeptidase, has the ability to penetrate biofilm, and has been identified as a potential antimicrobial to prevent mesh infection. The goals of this study were to determine if LS adhered to porcine mesh (PM) can impact host survival, reduce the risk of long-term PM infection, and to analyze lysostaphin bound PM (LS-PM) mesh-fascial interface in an infected field. METHODS: Abdominal onlay PMs measuring 3×3 cm were implanted in select groups of rats (n=75). Group assignments were based on bacterial inoculum and presence of LS on mesh. Explantation occurred at 60 d. Bacterial growth and mesh-fascial interface tensile strength were analyzed. Standard statistical analysis was performed. RESULTS: Only one out of 30 rats with bacterial inoculum not treated with LS survived. All 30 LS treated rats survived and had normal appearing mesh, including 20 rats with a bacterial inoculum (10(6) and 10(8) CFU). Mean tensile strength for controls and LS and no inoculum samples was 3.47±0.86 N versus 5.0±1.0 N (P=0.008). LS groups inoculated with 10(6) and 10(8) CFU exhibited mean tensile strengths of 4.9±1.5 N and 6.7±1.6 N, respectively (P=0.019 and P<0.001 compared with controls). CONCLUSION: Rats inoculated with S. aureus and not treated with LS had a mortality of 97%. By comparison, LS treated animals completely cleared S. aureus when challenged with bacterial concentrations of 1×10(6) and 1×10(8) with maintenance of mesh integrity at 60 d. These findings strongly suggest the clinical use of LS-treated porcine mesh in contaminated fields may translate into more durable hernia repair.

Evaluation of the antimicrobial activity of lysostaphin-coated hernia repair meshes.

Bacterial infections by antibiotic-resistant Staphylococcus aureus strains are among the most common postoperative complications in surgical hernia repair with synthetic mesh. Surface coating of medical devices/implants using antibacterial peptides and enzymes has recently emerged as a potentially effective method for preventing infections. The objective of this study was to evaluate the in vitro antimicrobial activity of hernia repair meshes coated by the antimicrobial enzyme lysostaphin at different initial concentrations. Lysostaphin was adsorbed on pieces of polypropylene (Ultrapro) mesh with binding yields of ∼10 to 40% at different coating concentrations of between 10 and 500 µg/ml. Leaching of enzyme from the surface of all the samples was studied in 2% (wt/vol) bovine serum albumin in phosphate-buffered saline buffer at 37°C, and it was found that less than 3% of adsorbed enzyme desorbed from the surface after 24 h of incubation. Studies of antibacterial activity against a cell suspension of S. aureus were performed using turbidity assay and demonstrated that the small amount of enzyme leaching from the mesh surface contributes to the lytic activity of the lysostaphin-coated samples. Colony counting data from the broth count (model for bacteria in wound fluid) and wash count (model for colonized bacteria) for the enzyme-coated samples showed significantly decreased numbers of CFU compared to uncoated samples (P < 0.05). A pilot in vivo study showed a dose-dependent efficacy of lysostaphin-coated meshes in a rat model of S. aureus infection. The antimicrobial activity of the lysostaphin-coated meshes suggests that such enzyme-leaching surfaces could be efficient at actively resisting initial bacterial adhesion and preventing subsequent colonization of hernia repair meshes.

Charge-directed targeting of antimicrobial protein-nanoparticle conjugates.

Use of antimicrobial enzymes covalently attached to nanoparticles is of great interest as an antibiotic-free approach to treat microbial infections. Intrinsic properties of nanoparticles can also be used to add functionality to their conjugates with biomolecules. Here, we show in a model system that nanoparticle charge can be used to enhance delivery and increase bactericidal activity of an antimicrobial enzyme, lysozyme. Hen egg lysozyme was covalently attached to two types of polystyrene latex nanoparticles: positively charged, containing aliphatic amine surface groups, and negatively charged, containing sulfate and chloromethyl surface groups. In the case of bacterial lysis assay with a Gram-positive bacteria Micrococcus lysodeikticus, activity of lysozyme conjugated to positively charged nanoparticles was approximately twice as large as that of free lysozyme, while lysozyme conjugated to negatively charged nanoparticles showed little detectable activity. At the same time, when assayed using a low-molecular weight oligosaccharide substrate, lysozyme attached to both positively and negatively charged nanoparticles showed slightly lower activity than free enzyme. A possible explanation of these results is that lysozyme attached to negatively charged nanoparticles cannot be effectively targeted to the bacteria because of the electrostatic Coulombic repulsion from the negatively charged bacterial cell walls, whereas lysozyme conjugated to positively charged nanoparticles was targeted better than free enzyme due to stronger electrostatic attraction to bacteria. Zeta potential measurements confirmed the validity of this hypothesis. Thus, nanoparticle charge is an important factor that can be used to control targeting and activity of protein-nanoparticle conjugates.