Bacterial infection causes the postponement in the wound healing process [1, 2]. It is reported that approximately 75% of deaths subsequent burn injury, caused by Staphylococcus aureus and Pseudomonas aeruginosa . S. aureus is frequently isolated from human skin and mucous membranes. This organism leads to a broad spectrum of skin and acute infections . Also is the most common pathogen in the surgical area infections . One to two percent of people suffer from chronic wounds that lead to increase mortality and cost of treatment. S. aureus and P. aeruginosa are the most common bacteria detected from chronic leg ulcers . On the other hand emergence of resistant strains to antibiotics are strongly growing such as Methicillin, Penicillin, Vancomycin, and Quinolone resistance that is leading to failure antibiotic therapy . Therefore, alternative treatment programs are a great urgent need today.
Several studies indicated that some inorganic metal oxides have great antibacterial properties. Inorganic metal oxides show more constancy, robustness and long shelf life than organic antimicrobials that it’s the major benefits of application inorganic metal oxides .
The local use of anti-infective is the most effective method for wound treatment . The local use of Zinc Oxide (ZnO) improves acute and chronic wounds healing due to antibacterial, anti-inflammatory and increasing re-epithelialization properties [10-13]. The antibacterial properties of ZnO nano-sizes are much more than large particles . Antibacterial effects of ZnO nanoparticles have been indicated toward a wide spectrum of organisms such as Escherichia coli and S. aureus . Also, the biofilm formation inhibited by S. aureus and P. aeruginosa [15, 16].
Although, ZnO nanoparticles have been frequently reported with antibacterial properties but the very limited studies are available to treat the bacterial infections on the in vivo. The antibacterial activity of ZnO nanoparticles is affected by particle sizes and concentration. ZnO nanoparticles antibacterial activity directly correlates with their sizes. This dependency is also influenced by the concentration of NPs. ZnO nanoparticles with a smaller size (higher specific surface areas) showed highest antibacterial activity . So in the present study, the particle of ZnO nanoparticles with sizes 10-30 nm was used.
In the present study, the effect of ZnO nanoparticles investigated on bacterial load of experimental infectious wounds contaminated with S. aureus in mice.
Materials and Methods
Bacterium and Zinc Oxide nanoparticles
S. aureus (ATCC 25923 - MAST Company, UK) was provided by the Faculty of Veterinary Medicine, Urmia University. ZnO nanoparticles with an average size of 10 – 30 nm were purchased from US Reasearch Nanomaterials, Inc. USA.
To prepare a bacterial suspension, bacteria were cultured in Mueller Hinton Broth that was incubated for 18 h at 37° C. The bacteria were centrifuged at the 10000 g for 10 min at 4° C. The supernatant was discarded and bacteria were washed twice with phosphate-buffered saline (PBS) solution and finally were dissolved in PBS solution. The bacterial suspension was measured in OD 600nm (108 CFU/ml) based on the turbidity of 0.5 McFarland .
Broth dilution method was used to determine MIC (minimum inhibitory concentration). Generally, serial doubling dilutions of ZnO nanoparticles were prepared using Mueller-Hinton broth and finally, fresh culture bacteria was added to each test tube then incubated using shaking incubator for 24 hours at 37° C. Inhibition of cell growth was defined by counting the amount of CFUs on the plates or by the turbidities of the cell cultures. The first test tube that showed no change in turbidity were further proved for bacterial culturability by spreading 100-μl of the broth cultures onto Mueller-Hinton agar plates to determine the bactericidal or bacteriostatic effect of ZnO nanoparticles .
Mice wound infection and treatment
Thirty-six male mice (20-30 g) were prepared and kept up with standard pellet diet and water ad libitum for 14 days to be adapted before the examination and their health was evaluated. Also during the study were maintained under pathogen free conditions. Mice were randomly divided into three groups (n=12) including ZnO nanoparticles, tetracycline (positive control) and normal saline (negative control). Mice were kept in accordance with the international guidelines principles of laboratory animal use and care . All Mice were anesthetized with ketamine (100 mg/kg, Woerden, Netherland) and xylazine (5 mg/kg, Woerden, Netherland). The back of the mice were disinfected with 70% ethanol and hair shaved  and full thickness skin wounds (3 mm in diameter) were created by a sterile punch biopsy . The immediately wound area were inoculated using 10 μl (106 CFU) of bacterial suspensions to each wound site. After the infection, the treatment was performed in all groups [19, 22]; ZnO nanoparticles group: 40 μl of ZnO nanoparticles, Tetracycline group: 40 μl of tetracycline (8mg/kg) , control group: 40 μl of normal saline.
Two tests were performed for assessment of bacterial load. Swab test: This test is to determine the bacterial load of the wound surface. From the wound surface was sampled with a sterile swab. Samples were transferred to a suitable the transmission medium. Serial dilutions of the suspension (1:103 to 1:1012) were made with sterile broth media and were cultured on Mueller-Hinton agar for numbering the bacterial load.
The second test was performed to determine the bacterial load in the deep tissue. For this purpose, the wound with adjacent normal tissue (2.5 × 2.5 cm2) was cut out. Tissue samples were weighed and homogenized with a pestle then were solved in 2 ml sterile PBS. Finally, the samples were serially diluted with sterile broth media and cultured in Mueller-Hinton agar for numbering the bacterial load (1:103 to 1:1012 ) . The bacterial load of tissue was estimated by:
CFU/Gram= Plate Count × (1/dilution) × 10/ Wt. of Homogenized Tissue
All Statistical data were analyzed by a one-way ANOVA with Tukey-Kramer post-test using SPSS 16.0 (Chicago, IL). Values of p< 0.05 were considered statistically significant.
Minimum Inhibitory Concentration (MIC)
Bacterial growth was prevented at 125 μg/ml of ZnO nanoparticles. The results showed that ZnO nanoparticles had a bacteriostatic effect towards S. aureus and inhibited bacterial growth.
Superficial bacterial load
Counting the bacterial load of the wound surface showed reducing the bacterial amount in the treated groups (Table 1). Bacterial load reduction was observed in ZnO nanoparticles and tetracycline groups on day 7, 14, 21. The primary inoculation of bacteria was nearly 106 that it reaches to 0 CFU/100µl (Table 1) and 1 ×102 CFU/g (Table 2) on day 21. The ZnO nanoparticles group showed the quick decline at day 0 to day 21. The greatest amounts of bacteria were determined in the negative control group. Reduction of the bacterial load was significant in ZnO nanoparticles on days 7, 14 and 21 compared with negative control group (p< 0.05) So that no bacterial growth was observed on day 21 (Table 1). The ZnO nanoparticles group showed a reduction bacterial load compared with tetracycline group on days 7, 14 and 21, but the difference was not statiscally significant.
However, results of bacterial load were significant reduce in the ZnO nanoparticles and Tet groups comparing to control group particularly on day 21 (p< 0.05).
Deep bacterial load
The reduction of bacterial growth in ZnO nanoparticles group was observed so that in some samples did not grow any bacteria (Table 2). By day 21, the treated group showed decreases in deep skin bacterial concentration (1 ×102 CFU/g) compared with negative control group (1.4 ×104) (Table 2). However, significant decrease (p< 0.05) in deep skin bacterial load in the ZnO nanoparticles and tetracycline group were found comparing with control group. The ZnO nanoparticles group (1 ×102 CFU/g) showed a reduction in deep bacterial load compared with tetracycline group (1 ×103 CFU/g), but the difference was not statiscally significant.
The emergence of antibiotic resistance threaten public health . S. aureus has recognized as a common agent of infection and is responsible for a board spectrum of superficial and acute skin infections . In the United States, 11 million people admit to the hospital due to these infections and 464 thousand people hospitalize annually . Bacterial contamination of wounds led to the delayed wound healing process  and due to the abundance of antibiotic resistance , antibiotic therapy is not efficient. Today researchers are presented new alternative antibacterials such as metal nanoparticles. Among the metal nanoparticles, ZnO nanoparticles are highly regarded. Many studies have shown that ZnO nanoparticles have a potential antibacterial effect [8, 26]. Antibacterial properties of Zinc Oxide related to reactive oxygen species (ROS) production which destroys bacterial cell wall and thereby causes the death of the organism . Local application of zinc oxide accelerate wound healing according to the anti-bacterial, anti-inflammatory, increase reepithelization and activation of metalloproteinase enzymes properties researchers have suggested [11, 28].
In this study, we demonstrated that ZnO nanoparticles have a bacteriostatic effect against S. aureus and it inhibited the growth of bacteria at a concentration of 125 μg/ml. Jones et al also demonstrated that nanoparticles of zinc oxide have many uses as a bacteriostatic agent . Also, Zhang et al  showed the bacteriostatic effect of ZnO nanoparticles against E. coli, but Xie et al  demonstrated that the action of ZnO nanoparticles against C. jejuni was bacteriocidal. Raghupathi et al  showed that antibacterial effect of ZnO nanoparticles is inversely related to its size. So in the peresent study ZnO nanoparticles with sizes, 10-30 nm were used to raise the antibacterial effect of ZnO nanoparticles in wound infection treatment. Several studies have shown that topical application of ZnO nanoparticles is increased angiogenesis  and wound healing .
In the present study, the effects of ZnO nanoparticles and tetracycline in reducing the bacterial load of the wound infection investigated. In this study, the hypothesis that the ZnO nanoparticles along enhance bacterial clearance during wound healing contaminated with S. aureus. Tetracycline was considered as a positive control or standard. According to previous studies, infection rates directly related to the amount of inoculated bacteria. Inoculation of 106 microorganisms can cause the infections without mortality .
Our results showed that the use of zinc oxide nanoparticles is quite effective in reducing the surface and deep bacterial load on days 7, 14, 21. The surface infection level was significantly improved at later time points in treatment groups.
The reduction of the bacterial load was more significant different compare with control group especially on day 21. However, results of bacterial load were not statistically significant among ZnO nanoparticles and positive groups. Paty et al in 2015 reported that local application of ZnO nanoparticles reduced the bacterial load in skin infection created by S. aureus . These results are consistent with our findings and suggest that ZnO nanoparticles alone is effective in reducing the bacterial load of the wound and can be used to prevent of wound infection.
One of the main criterions on drug therapy is to be non-toxic to cells. ZnO nanoparticles toxicity depends on their concentrations and sizes. It is proposed low concentrations of ZnO nanoparticles are nontoxic to eukaryotic cells. Paty et al 2015 showed that ZnO nanoparticles at the bactericidal dose have no detrimental effects on PBMCs and THP-1 cells and prevented the lysis of RBCs by S. aureus and also significantly decreased the skin infection, bacterial load, and inflammation in mice .
The present study has shown the antibacterial effect of ZnO nanoparticles on S. aureus so reduced the bacterial load of wounds. Based on present findings, local application of ZnO nanoparticles may help wound healing processing due to the reduction of bacterial load.
We thank Ali Kazemnia for technical assistance.
4.Daum RS. Skin and soft-tissue infections caused by methicillin-resistant Staphylococcus aureus. N Engl J Med. 2007; 357(4): 380-390.
5.Elward AM, McAndrews JM, Young VL. Methicillin-sensitive and methicillin-resistant Staphylococcus aureus: preventing surgical site infections following plastic surgery. Aesthet Surg J. 2009; 29(3): 232-244.
8.Raghupathi KR, Koodali RT, Manna AC. Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir. 2011; 27(7): 4020-4028.
9.Hutchinson J, McGuckin M. Occlusive dressings: a microbiologic and clinical review. Am J Infect Control. 1990; 18(4): 257-268.
10.Agren MS. Studies on zinc in wound healing. Acta Derm Venereol Suppl. 1989; 154: 1-36.
11.Lansdown AB, Mirastschijski U, Stubbs N, Scanlon E, Ågren MS. Zinc in wound healing: theoretical, experimental, and clinical aspects. Wound Repair Regen. 2007; 15(1): 2-16.
12.Seltzer JL, Jeffrey JJ, Eisen AZ. Evidence for mammalian collagenases as zinc ion metalloenzymes. Biochim Biophys Acta. 1977; 485(1): 179-187.
13.Tenaud I, Sainte-Marie I, Jumbou O, Litoux P, Dreno B. In vitro modulation of keratinocyte wound healing integrins by zinc, copper and manganese. Br J Dermatol. 1999; 140(1): 26-34.
14.Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov. 2010; 9(8): 615-627.
15.Lee J-H, Kim Y-G, Cho MH, Lee J. ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production. Microbiol Res. 2014; 169(12): 888-896.
16.Pati R, Mehta RK, Mohanty S, Padhi A, Sengupta M, Vaseeharan B, Goswami C, Sonawane A. Topical application of zinc oxide nanoparticles reduces bacterial skin infection in mice and exhibits antibacterial activity by inducing oxidative stress response and cell membrane disintegration in macrophages. Nanomedicine. 2014; 10(6): 1195-1208.
18.Xie Y, He Y, Irwin PL, Jin T, Shi X. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl Environ Microbiol. 2011; 77(7): 2325-2331.
19.Yates CC, Whaley D, Babu R, Zhang J, Krishna P, Beckman E, Pasculle AW, Wells A. The effect of multifunctional polymer-based gels on wound healing in full thickness bacteria-contaminated mouse skin wound models. Biomaterials. 2007; 28(27): 3977-3986.
20.Ziv-Polat O, Topaz M, Brosh T, Margel S. Enhancement of incisional wound healing by thrombin conjugated iron oxide nanoparticles. Biomaterials. 2010; 31(4): 741-747.
21.Mihu MR, Sandkovsky U, Han G, Friedman JM, Nosanchuk JD, Martinez LR. The use of nitric oxide releasing nanoparticles as a treatment against Acinetobacter baumannii in wound infections. Virulence. 2010; 1(2): 62-67.
22.Olugbuyiro JA, Abo K, Leigh O. Wound healing effect of Flabellaria paniculata leaf extracts. J Ethnopharmacol. 2010; 127(3): 786-788.
23.Jiang B, Larson JC, Drapala PW, Pérez‐Luna VH, Kang‐Mieler JJ, Brey EM. Investigation of lysine acrylate containing poly (N‐isopropylacrylamide) hydrogels as wound dressings in normal and infected wounds. J Biomed Mater Res Part B Appl Biomater. 2012; 100(3): 668-676.
24.Desselberger U. Emerging and re-emerging infectious diseases. J Infect. 2000; 40(1): 3-15.
25.Rizzi SC, Upton Z, Bott K, Dargaville TR. Recent advances in dermal wound healing: biomedical device approaches. Expert Rev Med Devices. 2010; 7(1): 143-154.
26.Jones N, Ray B, Ranjit KT, Manna AC. Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiol Lett . 2008; 279(1): 71-76.
27.Sirelkhatim A, Mahmud S, Seeni A, Kaus NH, Ann LC, Bakhori SK, Hasan H, Mohamad D. Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. Nano-Micro Lett. 2015; 7(3): 219-242.
29.Zhang L, Ding Y, Povey M, York D. ZnO nanofluids–A potential antibacterial agent. Progr Nat Sci. 2008; 18(8): 939-944.
30.Barui AK, Veeriah V, Mukherjee S, Manna J, Patel AK, Patra S, Pal K, Murali S, Rana RK, Chatterjee S, Patra CR. Zinc oxide nanoflowers make new blood vessels. Nanoscale. 2012; 4(24): 7861-7869.
31.Chhabra H, Deshpande R, Kanitkar M, Jaiswal A, Kale VP, Bellare JR. A nano zinc oxide doped electrospun scaffold improves wound healing in a rodent model. RSC Adv. 2016; 6(2): 1428-1439.
32.Drosou A, Falabella A, Kirsner RS. Antiseptics on wounds: an area of controversy. Wounds. 2003; 15(5): 149-166.
33.Augustine R, Dominic EA, Reju I, Kaimal B, Kalarikkal N, Thomas S. Investigation of angiogenesis and its mechanism using zinc oxide nanoparticle-loaded electrospun tissue engineering scaffolds. RSC Adv. 2014; 4(93): 51528-51536.