Staphylococcus aureus (S. aureus) is a gram-positive pathogen that can lead to numerous infectious diseases, such as pneumonia, endocarditis, osteomyelitis, skin and soft tissue infections, bacteremia and sepsis (1). At the same time, the threat caused by S. aureus infections has increased significantly in humans as well as in animals (2,3). In clinical practices, antibiotics are effective way to treat S. aureus infections. With the use of antibiotics (especially overuse or misuse of antibiotics), antibiotic resistant S. aureus strains, such as methicillin-resistant S. aureus (MRSA), have spread both in hospitals and communities and also persist in the home environment, which poses a great threat to human health (4,5). It is estimated that 700,000 persons succumb to antibiotic-resistance bacteria including MRSA and this number is predicted to grow to 10 million by 2050(3). In order to deal with this, increasing efforts have been made to discover new therapeutic strategies to fight against S. aureus infections, such as bacteriophage (6,7), vaccines (8-10), monoclonal antibodies (11,12), recombinant endolysins (13), anti-persistent bacteria therapies (14), antibacterial peptide (15,16), natural plant components (17-19) and nanoparticles (NPs) (20,21).

NPs, being <100 nm, are one of the novel promising methods to deal with bacterial infections, including S. aureus infections (22,23). The antibacterial activity of NPs is mostly attributed to their special characteristics, such as well-distributed size, perfect spherical shape, positive surface charge and hydrophobicity (24,25). NPs begin their antibacterial effects by the direct interplay with cell surface, involving the destruction of cell wall peptidoglycan and membrane protein and interference in energy metabolism (ATPase inhibition and electron transport disruption). Then, NPs can penetrate into cytoplasm and cause great damage to intracellular components, including nucleic acids, proteins, lysosomes and ribosomes (26). Additionally, oxidative stress induced by excess releasing of reactive oxygen species (ROS) also plays a substantial role in inducing lipid peroxidation on the bacterial cell membrane (27). As well as the aforementioned mechanism, metal NPs have specific ways to resist pathogenic microorganisms by releasing metal ions and producing different ROS (28). Several metal (gold, silver, copper and zinc) NPs and their metal oxide NPs have been reported to have distinctive antimicrobial properties against S. aureus (29,30) and they were also shown to be the carriers that can deliver antibiotics to target sites (22,31). Fig. 1 shows the properties, antibacterial mechanism against S. aureus and antibiotics delivery ability of zinc oxide nanoparticles (ZnO NPs).

There are a number of studies reporting the antibacterial property of ZnO NPs against S. aureus (32-34). ZnO NPs reduce the biofilm of S. aureus by inhibiting biofilm genes expression, such as ica A, ica D and fnb A (35). In Kahandal et al (36), the biofilm formation of S. aureus was inhibited markedly by 95.39 % when treated with 125 µg/ml of ZnO NPs for 5 h. Abdelraheem et al (37) observed that ZnO NPs presented antibacterial activity against multidrug resistant S. aureus, such as methicillin, vancomycin and linezolid resistant S. aureus. Irfan et al (38) confirmed the antibacterial activity of ZnO NPs against S. aureus and MRSA with the zone of inhibition (ZOI) of 21±2 and 17±2 mm, respectively. El-Masry et al (39) also reported that ZnO NPs (20 nm and concentration of 20 mM) inhibited 10⁵ and 10⁷ CFU/ml S. aureus with ZOI of 26 and 22 mm, respectively. Currently, there is a lack of a comprehensive review on ZnO NPs against S. aureus. Therefore, the present study reviewed the antibacterial activity against S. aureus of ZnO NPs fabricated by various synthetic ways, especially the green synthetic ZnO NPs. It also summarized the synergistic antibacterial effects against S. aureus of ZnO NPs in combination with antibiotics. Furthermore, it highlighted the enhanced activities against S. aureus of ZnO nanocomposites, nano-hybrids and functional ZnO NPs.

Commonly, ZnO NPs can be synthesized by using top-down and bottom-up methods that include diverse physical and chemical ways (40) (Fig. 2). Top-down approaches cut massive materials into NPs physically, including ball milling, ion sputtering, laser ablation, metal etching and pyrolysis. According to Massoudi et al (41) research, ZnO NPs made by high-speed ball milling inhibit S. aureus with the largest ZOI of ~13.5±0.5 mm. It was also found that ZnO NPs synthesized by microwave heating displayed the ZOI of ~16 mm against S. aureus (42). Bottom-up ways fabricated atoms and molecules into nano-sized particles, which included chemical reduction, sol-gel method, chemical vapor deposition, molecular condensation and even green synthesis (43). Different synthesis processes bring about various physicochemical properties of metal NPs such as size, shape, dispersity and stabilization diversity, which determine the antibacterial efficiency (44,45). Table I shows the characteristics and anti-S. aureus capacity of ZnO NPs made by several methods. In Bai et al (46), small molecule ligand solvothermal synthesized ZnO NPs showed size-related antibacterial effect and the minimum inhibitory concentration (MIC) of 4 nm ZnO NPs against S. aureus was 6.25 µg/ml, which is lower than the MIC of 10 nm ZnO NPs at ~25 µg/ml. In an antimicrobial test of solution-polymerization-method synthesized ZnO NPs, it was discovered that S. aureus was more susceptible to nanoparticle size than E. coli (47). The co-precipitation method is also frequently used to synthesize ZnO NPs that show the lowest MIC against S. aureus compared with other bacteria (48). Moreover, by using an easy chemical method, diethylene-glycol-mediated ZnO NPs were made and they had antibacterial activity against S. aureus with the ZOI of 14 mm and showed the excellent S. aureus biofilm control (49). It was also reported that S. aureus cell leakage was observed after exposure to mechano-chemical synthesized ZnO NPs (50). Although a great number of physicochemical synthetic methods have been found to make ZnO NPs for S. aureus treatment, some demerits such as high cost, toxicity and instability still place restrictions on their large-scale antibacterial applications (43).

Recently, green biological materials drew much attention to researchers for their environment-friendly, cost-effective, low-toxicity and useful properties to make ZnO NPs (26). There are a number of types of biological materials such as bacteria, fungi, algae and plant extracts (51,52) (Fig. 3), which serve as reducing agents, capping agents, stabilizers and ligands during the synthesis of ZnO NPs (26) and their effects are ion reduction, size and shape control, NPs surface stabilization, metal passivation and coating, respectively, which are important to the antimicrobial properties of ZnO NPs (26,53). The antibacterial properties of green-synthesized ZnO NPs against S. aureus are in Table II.

At present, one of the most serious issues of global health must be antibiotics resistance. The synergy between antibiotics and ZnO NPs attracts much attention and would be a practicable treatment against multi-drug resistant bacteria (82,83). It has been found that ciprofloxacin in conjunction with ZnO@Glu-TSC (thiosemicarbazide-conjugated and glutamic acid-functionalized ZnO NPs) could significantly inhibit the expression of efflux pump genes, which is a vital factor towards antibiotics resistance (84). In addition, ZnO NPs can be excellent drug carriers to target antibacterial agents to the action sites and still achieve desired therapeutic effects for a decreased drug dosage, thus enhancing the antimicrobial efficacy (22). In Habib et al (85), using ZnO NPs combined with ciprofloxacin and imipenem, the ZOI of S. aureus was 17 mm higher than that of E. coli (12 mm). By using ZnO NPs in conjunction with antibiotics to defeat S. aureus, the MICs of six clinical common antibiotics were reduced, which reflected an effective antibacterial cooperation. Furthermore, the anti-biofilm efficacy was also investigated and was enhanced from 34-37% (antibiotics alone) to 65-85% (antibiotics and ZnO NPs combination) (86).

Hemmati et al (87) synthesized and characterized the chitosan-ZnO nanocomposites loading with gentamicin, which caused MIC reduction by four-fold and biofilm reduction by 77% in S. aureus by contrast with the gentamicin alone. Notably, drug-loaded ZnO NPs were shown to exhibit negligible toxicity to human cells (82). Thus, the synergy of ZnO NPs and antibiotics can be applied to a variety of antibacterial circumstances. In an infection model of rats, azithromycin-loaded ZnO NPs displayed enhanced ability to clear MRSA (88). Phytomolecules-coated ZnO NPs combined with tobramycin and gallic acid were synthesized and shown to be an excellent material for contact lenses, expressing a maximum log₁₀ reduction of 5.7±0.02 CFU/ml in the growth of S. aureus and contributed to disruption of bacterial cell wall and membrane, leading to the leakage of cytoplasm and bacterial death (89). These drug-hybrid NPs such as cefazolin-hybrid ZnO NPs are also used to post-operative antimicrobial therapy due to their inhibitory actions against S. aureus both in vitro and in vivo (90).

In order to optimize the performance of ZnO NPs to combat pathogenic microorganisms, researchers have given attention to functionalized, modified or capped ZnO NPs (34,113). Choi et al (114) created novel ZnO NPs functionalized with caffeic acid, which expressed enhanced antibacterial efficiency against S. aureus and three MRSA strains. The amino-functionalized hydrophilic ZnO NPs induced the destruction of respiratory electron transformation, generation of intracellular ROS and depolarization of cell membrane in S. aureus (115). Charoensri et al (116) prepared polyaniline-functionalized ZnO NPs by a simple impregnation method; not only did the synthesized ZnO NPs films show enhanced water hydrophobicity, but also expressed increased antibacterial ability against S. aureus, which will make it possible to develop antibacterial biodegradable materials. Lee et al (117) prepared gallic acid functionalized ZnO NPs that had high bacterial cell membrane affinity and showed enhanced bactericidal activity against S. aureus and higher selective inhibition to MRSA strains compared with non-functionalized ZnO NPs. According to Chen et al (118), photosensitizers-functionalized ZnO NPs demonstrated marked S. aureus inhibition and showed low-toxicity on endothelial cells and erythrocyte. In Yuan et al (119), lysozyme-modified ZnO NPs expressed excellent antibacterial activity against S. aureus and MRSA due to their small size, membrane permeability and enzyme-mediated ROS generation and even had lower cytotoxicity than gentamycin at the same concentration.

In the present study, ZnO NPs synthesized by different methods and their antibacterial activity against S. aureus have been summarized. Taken together, the anti-S. aureus efficacy of ZnO NPs mainly relies on their basic characteristics, especially size and shape. Spherical shape and small size are ideal features of ZnO NPs to combat bacteria that lead to high specific surface areas and more chances to contact with pathogens. In Babayevska et al (24), ZnO NPs with the highest specific surface area showed the size <10 nm. It is also reported that spherical ZnO NPs had the minimal size (31 nm) and higher anti-S. aureus activity (6-7 log CFU ml⁻¹ reduction) compared with flower-shaped particles (3-4 log CFU ml⁻¹ reduction for S. aureus) (44). The detail of ZnO NPs synthesized by various physical and chemical methods has been the subject of recent research (40). Some studies also revealed that these traditional synthesized methods had various shortcomings, such as being environment-contaminating, expensive and energy-intensive (43,120,121). Green synthesis has been emphasized due of its environment-friendly, easy-acquired and low-toxicity features. Some studies also noted that these green materials had antibacterial abilities already, such as mint (122), aloe (123) and curcumin (124), and they endow ZnO NPs with enhanced and steady antibacterial activity against S. aureus (125). Physical or chemical processes are the indispensable part in NPs synthesis. However, ZnO NPs made only by physicochemical ways are cannot compare with biogenic, functional or compound ZnO NPs when they are further applied to clinical antibacterial situations.

ZnO NPs can be used in a number of pre-clinical and clinical antimicrobial fields, including surgical operation (59), post-operative anti-bacterial therapy (90), anti-inflammatory (80) and ophthalmic treatment (126). For example, suture coated by green synthetic ZnO NPs demonstrated excellent tensile strength and wound healing ability in an incision wound rat model (59). An infection model in mice showed that ZnO NPs originating from fungi could significantly decrease hepatic inflammatory markers, restrain congestion and fibrosis in tissues and improve liver function (80). Sindelo et al (127) made the phthalocyanines link to the amino-functionalized ZnO NPs and these nanocomposites showed considerable activities of photodynamic antimicrobial chemotherapy and multi-microbial biofilms eradication. Considering that ZnO NPs had an excellent antibacterial activity against S. aureus and good biocompatibility, a chitosan-ZnO/selenium nanoparticles scaffold was developed to be used for infected wound healing and postoperative treatment of pediatric fractures (128). Ismail et al (129) also reported that ZnO NPs could be used as the hand sanitation in the future, which present improved anti-MRSA activity compared with the commonly used alcohol sanitation.

As one of the primary metal oxide NPs, ZnO NPs express excellent ability against S. aureus, but they still have drawbacks to be widely used as antibiotics replacements in clinical contexts. A few trials in vivo suggested that different metal oxide NPs damage cells to different degrees (45). Venkatraman et al (130) noted the toxicity of ZnO NPs to RAW264 macrophage cells, with half maximal inhibitory concentration (IC₅₀) of 494 µg/ml. Although electrospun scaffolds based on ZnO NPs showed increasing antibacterial activity, it is also reported that cytotoxicity was related to high ZnO content (97). According to Pereira et al (131), erythrocyte changes were also discovered in reptile exposure to ZnO NPs at the dose of 440 µg/kg. Yang et al (132) reported that ZnO NPs could induce apoptosis of mouse-derived spermatogonia cell line GC-1 spg cells. In Al-Zahaby et al (133), ZnO NPs (0.69 mg/l) mediated ROS that induced cell apoptosis and caused sensory toxicity effect on zebrafish olfactory organs. ZnO NPs synthesized by Calotropis procera leaf extract were reported to exhibit potent antimicrobial ability with concentration-dependent manner. However, with increasing exposure to ZnO NPs, deleterious changes (degeneration, swelling and atrophy) were found in the kidney by histology (85). Nazir et al (134) also discovered liver dysfunction in mice intraperitoneal injection groups at ZnO NPs doses of 50 and 100 mg/kg. ZnO NPs were also cytotoxic to the human immune system at doses of 25 and 12 mg/l (135). Despite ZnO NPs exhibiting outstanding capability in inhibiting MRSA, the resistance to NPs by microbes remained. If the dosage of NPs is below the sublethal concentrations, a series of resistance mechanisms would be initiated stealthily by bacteria, resembling their antibiotic resistance (136).

ZnO NPs still have potential toxicity when they are applied to clinical antibacterial treatment, though green-ZnO NPs have shown lower cytotoxicity than physicochemically synthesized ZnO NPs (60). Studies mostly pay close attention to the improving methods for preparation of ZnO NPs (42,48,54). They focus on the physical characteristics and antibacterial abilities, but neglect, to some degree, the cytotoxicity tests in vivo. Markedly, researchers have developed a predictive model to evaluate the security of ZnO NPs with different features and the authors point out that ZnO NPs with larger size, spherical shape, negative charge and a higher tendency for aggregation are safer, which is of great value to further toxicity studies (137). Notably, ZnO NPs <100 µg/ml were biocompatible and the cytotoxicity was parallel with their antibacterial activity, which meant that the anti-S. aureus mechanism (direct contact to cells, ROS and Zn²⁺ releasing) was also the potential killing process in normal cells (24). Although a number of reports explained the antibacterial mechanism against S. aureus of metal and metal oxide NPs, the studies for ZnO NPs were still limited compared with other metal NPs such as Ag NPs (26,138). The majority of ZnO NPs anti-S. aureus properties lack a comprehensive assessment and were only analyzed by well or disc diffusion test and bacterial growth curve and some studies only reported ZOI or MIC or even neither of them (42,50,126). A uniform standard of S. aureus strain is also necessary, which would play a crucial role in comparing antibacterial effects of ZnO NPs made by different methods.

In order to restrict the immoderate proliferation and mutation of pathogens, unremitting efforts should be made to optimize antibacterial strategies. As aforementioned, the synergism of ZnO NPs and other materials showed complemental effects, enhanced antibacterial activity and improved properties in clinical applications. Due to technology developments, there are more potentials for ZnO NPs preparation and biomedical application. For instance, apart from the green synthesis aforementioned, material-saving, safe and even granular NPs can be made by more novel methods, such as microfluidic (120). Except for using green and easily obtained raw materials, synthetic methods should be able to flexibly control the size, shape and dispersity of ZnO NPs, which means that the key material and procedure of ZnO NPs synthesis must be identified by modern techniques. Notably, genomic and proteomic techniques should be devoted to the exploration of the antibacterial mechanism, synthesis optimization and cytotoxicity of ZnO NPs. The antibacterial study of effects at the cellular level in the long term is an essential component to investigate the dosage and safety of ZnO NPs. It is necessary to focus on ZnO NPs studies in vivo, especially the biokinetics, bioavailability, tissue distribution and clearance rate, which are essential for their antibacterial applications and improved using as antibiotics replacements. In addition, composite and functional ZnO NPs could enhance the antibacterial advantages to some degree and decrease their toxicity and also reduce the excessive exposure of ZnO NPs that diminish the possibility of antimicrobial resistance, this is for future researchers.