Pharmacological assessment of Co₃O₄, CuO, NiO and ZnO nanoparticles via antibacterial, anti-biofilm and anti-quorum sensing activities

Mortality and illness are on the rise throughout the world today. The infectious diseases brought on by antibiotic-resistant bacteria represent one of the causes of this increase. Reports of new antibiotic-resistant bacterial species emerge daily from all corners of the globe, and as a result, new strategies for protecting against them are sought. Unfortunately, in the latest years, scientists had little success in discovering new antibiotic groups to treat these harmful microorganisms (Islam et al. 2017; World Health Organization 2017; Bhatia 2018).

Antibiotics currently play a crucial role in the treatment of bacterial diseases. However, antibiotic resistance is recognized as a significant problem because some microorganisms have developed resistance to multiple drugs (Gudkov et al. 2021). Various mechanisms cause antibiotic resistance in bacteria, like the hindrance of drugs from entering the cell, inactivation of enzymes in antibiotics, alterations in antibiotic targets and so on (Kumar & Schweizer 2005; Alekshun & Levy 2007; Randall et al. 2013; Blair et al. 2014). Due to the severity of bacterial antibiotic resistance, there has been a significant rise in the number of deaths (Parks & Messmer 2015). There has also been a recent rise in the mortality rate of Covid-19 patients due to bacterial infections (Langford et al. 2020; Sharifipour et al. 2020). These factors have prompted scientists and researchers to search for effective antimicrobial agents to combat such a pervasive problem. Metal and metal oxide nanoparticles (NPs) have recently been used as effective antibacterial agents against pathogenic and multidrug-resistant bacteria (Khan 2018; Tunio et al. 2020).

The advanced developments in nanomaterial science and technology enable researches to improve in various application areas such as environmental remediation and treatment (Aboli et al. 2020; Ameen et al. 2021; Bae & Hong 2021), catalyst (Lee et al. 2021; Dhiman et al. 2022; Megarajan et al. 2022), biotechnology (Fathy & Mahfouz 2021; Ameen et al. 2023a; Begum et al. 2022), energy (Hosseinzadeh et al. 2021; Karimi-Maleh et al. 2022a), food analysis, sensor (Hussain et al. 2022; Gorle et al. 2022; Karimi-Maleh et al. 2022b; Ameen et al. 2023b; Zhang & Karimi-Maleh 2023a, 2023b), and optics (Yu et al. 2020). The NPs are materials with a small size of 100 nm or less. The properties of many standard materials can be changed when they are transformed into NPs (Singh et al. 2012). The large surface area of NPs enables more interaction with the bacterial cell walls. Metal oxide NPs are found to be selective, durable and resistant to heat and therefore could be excellent antimicrobial agents. They have minimum effects on human cells while simultaneously having toxic effects on bacterial cells (Fu et al. 2005; Reddy et al. 2007). There are numerous ways to kill bacteria with NPs, including binding with the enzymes inside the cells, generating reactive oxygen species (ROS), deactivating them, and directly destroying the cell wall. The bactericidal activity of metal oxide NPs depends on several factors, such as concentration, size and stability. Specific interactions with NPs can inhibit bacterial growth. Usually, the bacterial cell has a micrometer scale size while the cell membrane has nanometer pores. The NPs are smaller than bacterial pores and can easily pass through the membrane and generate ROS. However, it is challenging to synthesize stable NPs that can function effectively within the medium in which bacteria grow (Azam et al. 2012a).

The use of NPs is of much interest nowadays, and many metals and metal oxide NPs are found toxic to bacteria and are used as antimicrobial coatings. Examples include zinc oxide (ZnO), copper oxide (CuO), magnesium oxide (MgO) and titanium oxide (TiO₂) (Singh et al. 2012). The ZnO NPs have photo-oxidizing properties against many biological and chemical species. These NPs have been reported as the best antifungal and antibacterial agents when coated on different products like textile materials (Abramov et al. 2009). Zinc is an essential element, and ZnO NPs are reported as non-toxic for human cells' DNA. İn a study by Yamamoto (2001), ZnO NPs exhibit the best antimicrobial properties in different particle sizes ranging from 10 to 50 nm.

CuO NPs are also known as good antibacterial agents in different forms. The soluble compounds of Cu have been reported to show excellent antimicrobial activity against several microorganisms like fungi, bacteria, viruses and algae (Grass et al. 2011). The killing effect of CuO NPs for the microbes is either by generating ROS, which can cause irrecoverable damage like the cleavage of RNA and DNA and lipid peroxidation, which is subjected to membrane damage or the oxidation of enzymes (Halliwell & Gutteridge 1984; Pena et al. 1998).

The antibacterial activities of Co₃O₄ NPs are also studied extensively (Koyyati et al. 2016; Raza et al. 2016; Igwe & Ekebo 2018; Shahzadi et al. 2019; Kharade Suvarta et al. 2020). The Co₃O₄ NPs are excellent antibacterial agents for treating many infections caused by bacteria like Klebsiella pneumoniae, Escherichia coli, Streptococcus pyrogens and Staphylococcus aureus (Igwe & Ekebo 2018). In addition, research showed that green synthesized Co₃O₄ NPs also successfully inhibit the bacteria (Koyyati et al. 2016).

The NiO NPs were tested against the inhibition of Gram-positive and Gram-negative bacteria (Behera et al. 2019). The mechanism of the interaction of NiO NPs was based on the amount of ROS generated at the surface of NiO NPs. The excellent antibacterial and antiquorum sensing activity of NiO NPs was studied against P. aeruginosa and Artemia franciscana (Behera et al. 2019). The results showed that NiO NPs possess great potential to fight against these bacteria. The degradation of ring biofilm confirmed the biofilm inhibition and time-kill variation assays (Maruthupandy et al. 2020).

Quorum sensing is an effective tool in determining pathogenicity and biofilm formation. However, a particular focus was given to antibiofilm and antiquorum sensing studies, which are also used as alternatives for treating infections caused by bacteria (Uroz et al. 2009; Nithya et al. 2010).

Metal oxide NPs have several physicochemical properties that enable them to serve as effective antimicrobial agents instead of conventional antibiotic drugs, which typically have many side effects. This study aims to synthesize Co₃O₄, CuO, NiO and ZnO NPs and to check their antimicrobial activities through disc diffusion, well diffusion and minimum inhibitory concentration (MIC) methods. Their antiquorum sensing and antibiofilm activities are checked against Chromobacterium violaceum ATCC 12472 and Pseudomonas aeruginosa PAO1 bacteria.

Metal oxide NPs were synthesized using the aqueous chemical growth method in which the metal salt and sodium hydroxide were used in a 1:2 ratio in double distilled water. The mixture was stirred for about 30 min at 25 °C, then placed in a Fotile oven at 90 °C for 4 h, and filtered. The filtrate was dried further in the oven at 60 °C for 1 h; the pure metal hydroxide material was obtained (Bakhsh et al. 2021). Finally, the material was calcinated using a muffle furnace at 500 °C for 4 h. The pure metal oxide material obtained was further characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis.

The SEM analysis was done for all the synthesized materials using the 30 KV Scanning Electron Microscope (JSM5910, Jeol, Japan) with secondary electron imaging (SEI) and energy dispersive X-ray (EDX) detectors (INCA200/Oxford Instruments, UK). The synthesized NPs were also characterized using TEM as a 200 KV Transmission Electron Microscope (JM 2100, Jeol, Japan) with EDX (INCA100/ Oxford Instruments, UK). XRD analysis for the synthesized NPs was carried out using the X-ray Diffractometer (JDX 3532, Jeol, Japan) with CuKα source.

The microdilution method was used to determine the antibacterial activities of the metal oxide NPs (Co₃O₄, CuO, NiO and ZnO). The prepared NPs were dissolved in dimethyl-sulfoxide (10% DMSO).

In the antibacterial activity tests, S. aureus ATCC 25923, methicillin-resistant S. aureus ATCC 43300 (MRSA), Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 27853 were used as test bacteria. The broth microdilution method was used to determine MIC values (Wikler 2006). Two-fold dilutions ranging from 10 to 0.08 mg/mL were prepared in Mueller Hinton Broth (Difco, Difco Laboratories, Detroit, MI, USA). The inoculums were prepared from subcultures for 24 h. The final test concentration of the bacteria was adjusted to 5 × 10⁵ CFU/mL. The microplates were incubated at 35 °C for 18–24 h. The last well that completely inhibited visual microbial growth was noted as the MIC value (mg/mL). A set of wells containing only inoculated broth and 10% DMSO were used as the negative control. Ampicillin (Sigma, USA), ciprofloxacin (Sigma, USA), and ofloxacin (Sigma, USA) were used as reference drugs.

The antibiofilm activity was determined by the in vitro microplate-based biofilm model against P. aeruginosa PAO1 using the crystal violet assay (Bali et al. 2019; Eryılmaz et al. 2019; Jardak et al. 2021).

P. aeruginosa PAO1 was incubated for 24 h at 37 °C in Brain Heart Infusion (BHI) broth. After incubation, final inoculum suspensions containing ∼10⁶ CFU/mL of P. aeruginosa were prepared in BHI enriched with 2% sucrose. Next, 100 μL of the inoculum suspensions were added to 96-well microtiter plates for each test condition. Then, the plates were incubated at 37 °C for 72 h to form mature biofilms.

The antiquorum sensing activity was performed by the disc diffusion method using the reported bacteria C. violaceum ATCC 12472 (Gajdács & Spengler 2019; Batohi et al. 2021). The density of the overnight bacterial culture was adjusted to 1.5 × 10⁸ CFU/mL. Then the bacterial suspension was inoculated on Luria Bertani Agar. The sterile blank discs (6 mm diameter; Bioanalyse^®, Ankara, Turkey) impregnated with 20 μL of the metal oxide NPs (ZnO, CuO, Co₃O₄ and NiO) solutions were placed on the medium. After incubation at 30 °C for 24 h, the plates were observed for a zone of violacein inhibition. The formation of an inhibition zone around the discs was noted as the potential antiquorum sensing activity.

The NiO NPs shown in Figure 2(c) appear as nanorods with a 20–25 nm size (Figure 2(g)) range comparable to the TEM image of NiO NPs reported in Sagadevan & Podder (2015). Figure 2(d) shows the spherical shape of ZnO NPs with a calculated size range of 70–95 nm (Figure 2(h)) (Akpomie et al. 2021).

The diffraction peaks of CuO at (002), (111), (202), (113) and (220) show the monoclinic phase of the synthesized CuO NPs, while no unwanted or extra peaks appear in the XRD pattern as an impurity can be observed (Buledi et al. 2020). The XRD pattern of NiO with highly sharpened peaks at (111), (200), (220), (311) and (222) confirms the pure and crystalline phase structure of NiO NPs, which is comparable with the reported XRD peaks of NiO NPs (Sagadevan & Podder 2015) ZnO has XRD diffraction peaks at (002), (102) and (112) and highly sharp peaks at (100), (101), (110) and (103), which shows the hexagonal crystalline nature of the synthesized ZnO NPs (Ghimire et al. 2022). The average crystalline size of all synthesized NPs was calculated from the major XRD peaks using the Scherer formula (τ = kλ/(β cos θ), and the average size for Co₃O₄, CuO, NiO and ZnO was found to be 80.4, 90.1, 18.4 and 94.1, respectively.

The results for the MIC of NPs against test bacteria are given in Table 1.

Several microbial species' growth was inhibited at various concentrations, as determined by the MIC test results. The low MIC value indicates that the material used as an antibacterial agent possesses effective antibacterial activity. The current work studied the low MIC value against Gram-positive bacterial species, i.e. S. epidermidis ATCC 12228 for ZnO NPs, at 0.63 mg/mL. The excellent antibacterial activity of ZnO NPs is due to the generation of ROS from the ZnO NPs surface, causing oxidative stress and damaging the cell DNA and proteins. The contact between NPs and bacterial cell causes toxic effects for the bacterial species. Zn²⁺ ions are released due to such contact, causing damage to the whole intracellular area (Klink et al. 2022).

In contrast, the positive controls ampicillin, ciprofloxacin and ofloxacin showed no activity against this tested bacteria. The MIC value of CuO NPs was found as 1.25 for the Gram-positive bacteria, i.e. Staphylococcus ATC12228 and E. faecalis ATCC 29212, while the drug ciprofloxacin showed a better MIC (0.0625) in the case of E. faecalis. Co₃O₄ NPs show a good MIC (1.25) in the case of E. faecalis ATCC 29212. Ciprofloxacin obtains a minimal MIC (0.0625) against K. pneumoniae, compared to CuO and ZnO NPs, which show MIC at values 2.5 and 5. The reason for such variation in MIC values may be attributed to factors such as differences in physiology and cell structure or the degree of contact of these bacterial species with the NPs. Another reason may be the NPs diffusion rate, affecting their ability to resist such microbial organisms (Azam et al. 2012b).

The formation of a transparent inhibition zone around the disc indicates the potential QS inhibitory activity. None of the tested metal oxide NPs showed antiquorum sensing activity (Figure 5). This may be because the testing sample's killing capacity is below the minimum required concentration. Hence, contact between NPs and the bacterial cell wall is sufficient to cause toxicity; therefore, large amounts of metal NPs are required to completely envelop bacterial cells and shield them from the environment, leaving no chance for nutrition to be absorbed to continue the life process. The primary role of NPs here is to envelop the bacterial cells completely and shield them from the environment, leaving no space for their growth and nutrition to continue their life process (Saleh et al. 2019; Theodora et al. 2019). Another explanation for the lack of antiquorum sensing activity is that the diffusion rate of NPs may not be enough to contact the bacterial cell wall.

This study explored the antibacterial properties of some selected metal oxide NPs. In this regard, metal oxide NPs (Co₃O₄, CuO, NiO and ZnO) are successfully synthesized and characterized using SEM, TEM and XRD. The average size of these prepared NPs was 10–20 nm, as calculated using TEM and XRD. The antibacterial activities of all the synthesized NPs were studied by the MIC method. The ZnO showed its best MIC value at 0.63 against S. epidermidis ATCC 12228. The CuO, NiO and Co₃O₄ NPs showed satisfactory MIC values at 1.25 against Enterococcus faecalis ATCC 29212. The biofilm inhibition was also studied using these metal oxide NPs in which P. aeruginosa PAO1 was used as test bacteria. The antibiofilm activity of NPs was 88.5, 85.95, 80.35 and 83.99% for the NiO, CuO, Co₃O₄ and ZnO NPs, respectively. No results were observed in the case of antiquorum sensing activity tests. The reason may be the low diffusion rate of our synthesized materials into the media. The findings show that these metal oxide NPs can work as potential antibacterial agents in various biological applications.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.