The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus

• Published in: Biomaterials 2012-01, Vol.33 (2), p.365-377
• Main Authors: Tan, Honglue, Peng, Zhaoxiang, Li, Qingtian, Xu, Xiaofen, Guo, Shengrong, Tang, Tingting
• Format: Article
• Language: English
• Subjects: Advanced Basic Science; Ammonium; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacology; Antibiotic-resistant staphylococcus; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biocompatible Materials - chemistry; Biocompatible Materials - pharmacology; Biofilm formation; Biofilms - drug effects; Bone Cements - chemistry; Chitosan - analogs & derivatives; Chitosan - chemistry; Chitosan - pharmacology; Dentistry; Down-Regulation; Drug Resistance, Bacterial - drug effects; Gene Expression Regulation, Bacterial; Gentamicins - pharmacology; Ica genes; MecA genes; Methicillin-Resistant Staphylococcus aureus - drug effects; Methicillin-Resistant Staphylococcus aureus - genetics; Methicillin-Resistant Staphylococcus aureus - pathogenicity; Microbial Sensitivity Tests; Microscopy, Confocal; Penicillin-Binding Proteins; PMMA bone cement; Polymethyl Methacrylate - chemistry; Quaternised chitosan; Real-Time Polymerase Chain Reaction; Staphylococcus aureus; Staphylococcus epidermidis - drug effects; Staphylococcus epidermidis - genetics; Staphylococcus epidermidis - pathogenicity; Up-Regulation
• ISSN: 0142-9612; 1878-5905
• DOI: 10.1016/j.biomaterials.2011.09.084

Abstract Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus ( ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD , which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR , which negatively mediates icaAD expression, and also downregulated the expression of MecA , which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis.