The assessment of antibiofilm activity of chitosan-zinc oxide-gentamicin nanocomposite on Pseudomonas aeruginosa and Staphylococcus aureus Fatemeh Hemmati a,d , Roya Salehi b , Reza Ghotaslou c , Hossein Samadi Kafil b,c , Alka Hasani c , Pourya Gholizadeh a,d , Mohammad Ahangarzadeh Rezaee a,c, ⁎ a Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b Drug Applied Research Center, Tabriz University of Medical Science, Tabriz, Iran c Department of Medical Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran d Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran abstract article info Article history: Received 22 June 2020 Received in revised form 17 August 2020 Accepted 7 September 2020 Available online 11 September 2020 Keywords: Antimicrobial agents Chitosan Gentamicin Nanocomposite Zinc oxide In the present study, chitosan-zinc oxide (CS-ZnO) nanocomposite with/without gentamicin was synthesized and characterized which used as an antibiofilm agent to inhibit the biofilm formation of Pseudomonas aeruginosa (P. aeruginosa) PAO1 and Staphylococcus aureus (S. aureus). Synthesized CS-ZnO nanocomposite was character- ized with the DLS (Dynamic Light Scattering), FTIR (Fourier Transform Infrared), XRD (X-ray Diffraction) and SEM (Scanning Electron Microscope). The minimum inhibitory concentrations (MICs) against P. aeruginosa PAO1 and S. aureus determined using broth microdilution methods. The influence of sub-MIC (1/4 MIC) and MIC concentration of CS-ZnO nanocomposite and gentamicin alone and in combination on biofilm formation was also determined. A four-fold MIC reduction in S. aureus and P. aeruginosa PAO1 treated by the gentamicin loaded CS-ZnO nanocomposite, and 84% reduction of biofilm formation for P. aeruginosa PAO1 and 77% reduction of biofilm formation for S. aureus, was observed compared to the gentamicin alone (P < 0.05). This study showed the important role of nanocomposite in designing novel antibacterial and antibiofilm agents to combat the P. aeruginosa and S. aureus biofilm-related infections. © 2020 Elsevier B.V. All rights reserved. 1. Introduction The excessive use of antibiotics to fight bacterial infections caused the increased bacterial resistance to multiple antibiotics [1]. The resis- tance is due not only to the uncontrolled use of antibiotics, but also to the fact that pathogenic bacteria are capable of adapting to various en- vironments and develop self-defense strategies such as survival in biofilms [2]. In general, the biofilms are comprised of the microbial com- munities which protected with the extracellular polymeric matrix [3]. The attendance of biofilm increases bacteria resistance to conventional antibiotics with up to 1000-fold compared to their planktonic (free-liv- ing) equivalents. Biofilms are responsible for around 60% to 85% of human bacterial infections [1,4]. Biofilm-related infections are drasti- cally decreased susceptibility to conventional antibiotics, and these are hard to eradicate with antibiotic therapy alone [5]. During the last decades, many efforts were taken to develop ecologically-friendly and low cost, natural or synthetic substances as treatment agents [1]. The incidence of acute infections of antibiotic and/or multidrug-resistant bacteria at a worrying rate caused several researches interest to evaluate the antibacterial and antibiofilm activi- ties of different substances [4]. Chitosan (CS) is considered a medically significant material due to its unique properties such as biocompatibility, biological activity, nontoxicity, bioadhesion, antioxidant, antibiofilm, and antimicrobial ac- tivities [6,7]. CS is an abundant natural polysaccharide biopolymer, widely used in the medical field [8]. The antibacterial and antibiofilm properties of CS are due to the presence of its positive charge amino groups which cause a reaction with bacterial membrane [8,9]. Further- more, CS with vast surface areas modifies the bacterial membrane pen- etrability via the membrane incorporation and leads to bacteria's death [10,11]. Zinc oxide (ZnO) is another material which received more attention due to its antibacterial and antibiofilm activities against a wide range of bacteria [12]. ZnO has revealed great against bacteria pathogens by gen- erating the reactive oxygen species (ROS) and decreasing membrane structure integrity [13]. Green production of ZnO NPs has been done via various plant extracts, for example, Cassia fistula, Ocimum, Trifolium pratenese [14] and Rosa canina [15]. Rosa canina is a medicinal plant re- lated to family Rosaceae. Rosehip or the small fruit of Rosa canina is rec- ognized for its high content of vitamin C. Furthermore, the rosehip has a International Journal of Biological Macromolecules 163 (2020) 2248–2258 ⁎ Corresponding author at: Immunology Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. E-mail address: rezaee.mohammad@gmail.com (M.A. Rezaee). https://doi.org/10.1016/j.ijbiomac.2020.09.037 0141-8130/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac