Research letters

4 Baharoglu Z, Mazel D. Vibrio cholerae triggers SOS and mutagenesis in response to a wide range of antibiotics: a route towards multiresistance. Antimicrob Agents Chemother 2011; 55: 2438– 41. 5 Hocquet D, Llanes C, Thouverez M et al. Evidence for induction of integron-based antibiotic resistance by the SOS response in a clinical setting. PLoS Pathog 2012; 8: e1002778. 6 Cipriano Souza R, Vicente AC, Vieira VV et al. Clindamycin and metronidazole as independent risk factors for nosocomial acquisition of multidrug-resistant Pseudomonas aeruginosa. J Hosp Infect 2008; 69: 402–3. 7 Dilger K, Fux R, Ro¨ck D et al. Effect of high-dose metronidazole on pharmacokinetics of oral budesonide and vice versa: a double drug interaction study. J Clin Pharmacol 2007; 47: 1532– 9.

9 Miller C, Thomsen LE, Gaggero C et al. SOS response induction by b-lactams and bacterial defense against antibiotic lethality. Science 2004; 305: 1629– 31. 10 Cirz RT, Chin JK, Andes DR et al. Inhibition of mutation and combating the evolution of antibiotic resistance. PLoS Biol 2005; 3: e176.

J Antimicrob Chemother 2014 doi:10.1093/jac/dkt441 Advance Access publication 11 November 2013

Antibacterial activity of bone cement containing quaternary ammonium polyethyleneimine nanoparticles Shaul Beyth1†, David Polak2†, Charles Milgrom1, Ervin I. Weiss3, Stoyan Matanis2,3 and Nurit Beyth3* 1

Department of Orthopedic Surgery, Hadassah-Hebrew University Medical Center, PO Box 12000, Jerusalem 91120, Israel; 2 Department of Periodontics, Hadassah-Hebrew University Medical Center, PO Box 12272, Jerusalem 91120, Israel; 3Department of Prosthodontics, Hadassah-Hebrew University Medical Center, PO Box 12272, Jerusalem 91120, Israel *Corresponding author. Tel: +972-2-6776142; Fax: +972-2-6429683; E-mail: [email protected] †These authors made an equal contribution.

Keywords: antimicrobial, cationic polymers, Staphylococcus

epidermidis, Staphylococcus aureus

Sir, A bone cement mantle is commonly used in orthopaedic surgery, and creates an ideal environment for bacterial growth. Bacterial infection following joint replacement surgery is a catastrophic complication. It is estimated that infections occur in up to about 2% of primary hip and knee replacements, whereas revision surgery carries a 2 – 3-fold higher risk.1 The

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8 Mene´ndez D, Rojas E, Herrera LA et al. DNA breakage due to metronidazole treatment. Mutat Res 2001; 478: 153–8.

most common bacteria isolated from infected joints are Grampositive cocci (Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus spp.) followed by Gram-negative bacteria and, less frequently, mixed and fungal infections.2 Addition of antibiotics to the cement has been advocated, especially in revision surgery.3 However, several studies describe the decreasing potency of common antibiotics in treating the infecting organisms and a parallel rise in the prevalence of resistant strains.4 As the implant itself lacks a blood supply, it is inherently susceptible to colonization by bacteria, which are less likely to be eradicated than in viable tissue. Therefore, efforts are focused on prevention of implant colonization. However, attempts to protect the implants using an antibiotic coating as well as the addition of antibiotics to the cement in the cemented implant have not proved successful. A novel strategy involves the use of antibacterial molecules that are bound to the implant or to the surrounding cement and are not released but remain functional for long periods of time.5 Quaternary amine residues proved to have excellent antibacterial properties.5 – 7 The purpose of this study was to modify a commonly used bone cement to obtain a safe and long-lasting antibacterial effect using quaternary ammonium polyethyleneimine (QPEI) nanoparticles. QPEI nanoparticles were synthesized as previously described.6 The tested materials were prepared by adding the synthesized powder to clinically available bone cement [SimplexTM P Bone Cement: 75% methyl methacrylate – styrene copolymer, 15% polymethylmethacrylate (PMMA) plus 10% barium; Stryker, Kalamazoo, MI, USA]. QPEI nanoparticles were added at 0%, 1%, 2% or 3% (w/w) to the bone cement and homogeneously mixed according to the manufacturer’s instructions. An antimicrobial effect against S. aureus ATCC 8325-4 and Enterococcus faecalis (a clinical isolate from the Maurice and Gabriela Goldschleger School of Dental Medicine at Tel Aviv University, Israel) was tested using the direct contact test (DCT)8 and agar diffusion test (ADT). Biocompatibility was tested on human primary polymorphonuclear cells as previously described.5 Cell viability was measured using the XTT assay (Biological Industries) and levels of tumour necrosis factor-a (TNF-a) in the supernatant were measured using an ELISA kit (Biolegend, San Diego, CA, USA). Additionally, physical properties of the cements were evaluated. Testing was performed using a Controlled Teststore 25 Tons MTS Device (Minneapolis, MN, USA) and results were analysed using MPT software (Multi-Purpose Testware 793.10, MTS System Corporation, Eden Prairie, MN, USA). Strain (1) and Young’s modulus (E) were calculated for each specimen. A strong antibacterial effect after an ageing period of 4 weeks was evident (P, 0.05) in all the bone cement samples in which the QPEI nanoparticles were incorporated compared with bone cement samples with no additives, which showed no antibacterial effect. The DCT showed significant antibacterial activity against both bacteria for at least 4 weeks (Figure 1); the ADT revealed no inhibition halo in the agar plates for both tested bacteria, indicating that the nanoparticles are retained in the PMMA and do not diffuse into the agar. Moreover, QPEI nanoparticles did not change the biocompatibility properties of PMMA. Addition of the nanoparticles at all tested percentages did not result in a significant change in cell viability compared with that of the bone cement group; incorporation of 0%, 1%, 2% and 3% QPEI

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Figure 1. Bacterial outgrowth of S. aureus and E. faecalis following direct contact with bone cement incorporating 0%, 1%, 2% or 3% (w/w) QPEI nanoparticles. S. aureus and E. faecalis outgrowth served as control. Samples were aged for 4 weeks before testing. The results are expressed as mean+SD.

nanoparticles resulted in relative viabilities of 58.3+4.7%, 57.4+7.4%, 44.6+1.1% and 44.6+2%, respectively. Similarly, TNF-a levels did not show significant changes compared with nonmodified bone cement; 0%, 1%, 2% and 3% QPEI nanoparticles resulted in TNF-a levels of 3.2+0.3, 2.5+0.3, 2.6+0.3 and 2.7+0.3 pg/mL, respectively. The Young’s modulus of bone cement incorporating 0%, 1%, 2% and 3% QPEI nanoparticles was 2.31+0.1, 2.3+0.2, 1.9+0.2 and 1.8+0.2 GPa respectively. No significant differences in Young’s modulus were found between the modified bone cement and bone cements with 1% and 2% QPEI nanoparticles. The strategy of incorporating non-eluting nanoparticles in bone cement may be advantageous over antibiotic-loaded bone cement and presents an option for a significant reduction in prosthetic joint infection rate. Our results indicate that incorporation of QPEI nanoparticles in bone cements has a long-lasting antibacterial effect without compromising the cement’s biocompatibility and physical properties. Thus, early and late prosthetic joint infections may be prevented and the clinical performance of the implants may be prolonged. As our study is based on in vitro assays, it is limited and cannot yield clinical recommendations. It is therefore suggested that, following safety tests and FDA/Conformite Europeene approval, incorporation of QPEI nanoparticles in bone cement could become an option when performing primary cemented joint replacement or revision. Future applications may include incorporation of antibacterial nanoparticles into prosthetic coatings and into formulations of orthopaedic and other implants.

Funding The study was self-funded by the research group, without any external funding source.

Transparency declarations E. I. W. and N. B. have issued a patent on Antimicrobial Nanoparticulate Additives Forming Non-Leachable Sustained Antimicrobial Polymeric Compositions [International Publication No. WO 2006/070376 A1 (2006)]. All other authors: none to declare.

References 1 Phillips JE, Crane TP, Noy M et al. The incidence of deep prosthetic infections in a specialist orthopaedic hospital: a 15-year prospective survey. J Bone Joint Surg Br 2006; 88: 943– 8. 2 Moran E, Masters S, Berendt AR et al. Guiding empirical antibiotic therapy in orthopaedics: the microbiology of prosthetic joint infection managed by debridement, irrigation and prosthesis retention. J Infect 2007; 55: 1– 7. 3 Cummins JS, Tomek IM, Kantor SR et al. Cost-effectiveness of antibioticimpregnated bone cement used in primary total hip arthroplasty. J Bone Joint Surg Am 2009; 91: 634–41. 4 Geipel U. Pathogenic organisms in hip joint infections. Int J Med Sci 2009; 6: 234– 40. 5 Beyth N, Houri-Haddad Y, Baraness-Hadar L et al. Surface antimicrobial activity and biocompatibility of incorporated polyethylenimine nanoparticles. Biomaterials 2008; 29: 4157–63. 6 Beyth N, Yudovin-Farber I, Bahir R et al. Antibacterial activity of dental composites containing quaternary ammonium polyethylenimine nanoparticles against Streptococcus mutans. Biomaterials 2006; 27: 3995– 4002.

Acknowledgements The research was carried out at the MIS-supported Laboratory for Periodontal Research and The Ronald E. Goldstein Center for Esthetic Dentistry and Dental Materials Research, at the Hebrew University-Hadassah Medical Center, Faculty of Dental Medicine.

7 Beyth N, Yudovin-Farber I, Perez-Davidi M et al. Polyethyleneimine nanoparticles incorporated into resin composite cause cell death and trigger biofilm stress in vivo. Proc Natl Acad Sci USA 2010; 107: 22038– 43. 8 Beyth N, Domb AJ, Weiss EI. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent 2007; 35: 201–6.

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Antibacterial activity of bone cement containing quaternary ammonium polyethyleneimine nanoparticles.

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