Evaluation of the antibacterial activity of CuO and ZnO nanoparticles against uropathogenic Escherichia coli

Abstract

E. coli responsible for urinary tract infections (UTI) became resistant to obtainable therapies. So, nanotechnology appears currently as promising innovation among various strategies under development. In the present study we evaluate the antimicrobial effects of CuO and ZnO nanoparticles against E. coli isolated from UTI. Biofilm production and expression of quorum sensing genes luxS and motA were performed on E. coli treated with the nanoparticles. luxS and motA genes were detected in the tested E. coli. All isolates produced strong, moderate and weak biofilm. All isolates had a weak biofilm formation and decreased gene expression for luxS and motA genes after treatment with CuO, ZnO and their mixture. Besides, CuO and ZnO had non-toxic effects on Human Dermal Fibroblasts and neonatal normal cells. In conclusion, the mixture of CuO and ZnO had a role in decreasing the gene expression of luxS and motA genes more than each alone.

KEYWORDS:

• E. coli; CuO
• ZnO
• nanoparticles
• urinary infection

1. Introduction

Escherichia coli is a gram-negative bacterium that connects with another cell by cues to stimulate the expression of particular genes for different phenotypes like the formation of biofilm, antibiotic resistance, and motility. E. coli cues molecules have different actions through cell-to-cell connections, it was observed to raise the antibiotic resistance modulation and the inhibition formation of biofilm and motility [1].

Biofilm plays a role in drug-resistance, and pathogenesis for several chronic diseases and does not respond to antibiotic treatment [2]. Some studies point out that in several species of bacteria, stimulation of quorum sensing occurs in the biofilm activation leading to the maturity and dismantling of the biofilm. So, the first adhesion stage appears unsuitable for accumulating signalling molecules, then in the second step, the linked bacteria divide and generate microcolonies, increasing population intensity, so signalling molecules reach suitable levels to stimulate the maturity and dismantling of biofilm in a coordinated method. When the nutrients become scarce and accumulate waste, the dispersion of biofilm is essential to bacteria escaping and colonizing new regions [3–5].

Urinary infection is considered the most common cause of antibiotic therapy. However, uropathogens became resistant to obtainable therapies. So, nanotechnology appears currently as a promising innovation among various strategies under development [6].

As a result of multi-resistance to antibiotics, infection is still a general health issue around the globe. These issues elevate the demand for additional doses of antibiotics which cause toxic effects. Therefore, Alternative therapies for bacterial infection were researched, and nanomaterials were applied as novel antimicrobial factors. ZnO nanoparticles were observed toxic to E. coli which disrupts membranes by the production of ROS (Reactive Oxygen Species) that damage bacteria [7]. Further, ZnO nanoparticles produce Zn²⁺ and hydrogen peroxide which showed a role as the antibacterial molecules [8].

Also, CuO nanoparticles have the ability as antimicrobial factors, to inhibit bacteria growth that have multidrug resistance, and inhibit biofilms [9].

Quorum sensing of bacteria produces and liberates molecules of chemical signalling known as “autoinducers”, its external concentration raises as an indicator of the rising density of cell population. When the bacteria reveal the autoinducers amount to a minimum of the threshold level for the stimulated concentration, bacteria will respond by changing its gene expression. Autoinducers represent the signals by which quorum sensing of bacteria connect and coincide with particular behaviour on a wide scale of population, so gain the capability to work as multi-cellular organisms [10].

LuxS gene contributes to the quorum sensing autoinducer 2 synthesis. Auto inducer 2 in E. coli induces the formation of biofilm and influences the architecture of biofilm by stimulating the regulators of quorum sensing mqsR. In which mqsR regulates positively the expression of motA gene [11]. motA is the membrane protein that has 4 membrane-crossing areas that are a formation of the proton channel [12]. Therefore, the current study aimed to apply CuO and ZnO to evaluate their antimicrobial effects against E. coli isolated from urinary tract infection.

2. Methods

2.1. Sample collection

Two hundred urine samples were collected from patients suffering from urinary infection symptoms in several hospitals in Medical City/Baghdad from 2 February 2022 to 15 April 2022.

2.2. Bacterial isolates

The samples were cultivated on MacConkey Agar and incubated at 37°C for 24 h. After that bacteria were identified by the gram stain method and microscopic examination. Biochemical tests (catalase, indole, methyl red, and lactose fermentation) and API 20 System were conducted to continue identification. Moreover, E. coli was diagnosed by Vitek 2 compact system to confirm the identification.

2.3. Antibiotic resistance test

The antibiotic resistance test for 49 isolates of E. coli was performed by Vitek 2 compact system.

2.4. Molecular detection of luxS and motA genes

DNA extraction for 30 isolates was performed by using ABIOpure Total DNA kit (USA) according to manufacturer recommendations.

After extraction, the total DNA of all E. coli isolates was screened for the search of luxS gene (315 bp) and motA gene (430 bp). Primers for these genes were designed by Primer-BLAST (Table 1).

Table 1. Sequence primer of luxS gene and motA gene.

Genes	Sequence primer	Size
luxS gene	F 5-CATACCCTGGAG CACCTGTT-3	315 bp
	R 5-TTCTTCGTTGCTGTTGATGC-3
motA gene	F 5-GGTACAGTTTTCGGCGGTTA-3	430 bp
	R 5-CGTGCGTCTCAATCTCTTCA-3

PCR mix involved 12 µl of 2x master mix (Bioneer company /Korea), 1.5 µl for each primer (10 pmol/µl), 2 µl DNA sample, and a complete 25 µl final volume by free nuclease water.

PCR conditions for these genes included initial denaturation at 95°C/5 min 1 cycle; 35 cycles for denaturation at 95°C/30 sec, annealing 58°C/30sec, and extension 72°C/30sec; 1 cycle for final extension 72°C/7 min. The PCR products were electrophoresed on (1.5%) agarose gel containing 0.5% ethidium bromide. After an electrophoresis run-time of 90 min, the gels were photographed under UV light.

2.5. Characterization of nanoparticles materials

Characterization of CuO and ZnO nanoparticles by X-ray was conducted by XRD Philips xpertPA analytical Holland (30 mA; 40 kV) for producing X-ray with a wavelength (1.54 Å). The morphology and size of nanoparticles were determined by using TEM (transmission electron microscopy) (Philips EM-208s 100kv, Netherlands) and Energy-dispersive spectroscopy (EDX) type ARYA electron optic. The molecular analysis was carried out by Fourier Transform Infra-Red Spectroscopy (FT-IR) (Brerkin Elmer, USA). Scanning electron microscope (SEM) (Tescan Mira3, French) was used to determine CuO and ZnO NPs dimensions. Moreover, Atomic Force Microscopy (AFM) instruments type (NaioAFM 2022, nanoSurf AG, Switzerland) and UV (Brerkin Elmer, USA) were used.

2.6. Detection of biofilm

The biofilm production assay was performed according to the method of Mahdi et al. [13] pure colonies for each isolate were inoculated into 5 ml of Brain heart infusion broth with 2% sucrose and incubated for 24 h at 37°C. Then, 20 µl of each isolate suspension of approximately 10⁸ CFU/ml (approach 0.5 of McFarland standard) was transferred to the 96-well microplate containing Brain heart infusion broth (180 µl) and covered to incubate for 24 h at 37°C. After that, the excess medium was removed and washed three times with normal saline, and 200 µl methanol (99%) was added for 15 min to each well to fix biofilm, after that the microplate was dried at room temperature for 30 min. After that, the microplate was stained with 1% crystal violet (200 µl) for 15 min. The wells were rinsed with sterilized distilled water. The dye was dissolved in 96% ethanol and the optical density was determined at 630 nm by a microplate reader. All assays were done in triplicate to calculate the average result. Sterile brain heart infusion broth was applied as a negative control.

The production of biofilm was described as the following: Cut-off value optical density of control (ODc) = average OD of negative control; OD ≤ ODc considered non–adherent; 2ODc > OD > ODc considered weak; 4 ODc > OD > 2ODc considered moderate; OD >4 ODc considered strong.

2.7. Study nanoparticles effect on E. coli

2.7.1. Determination of minimum inhibitory concentration (MIC) of CuO and ZnO nanoparticles

CuO and ZnO nanoparticles (Armina Engineering company/Iran) were prepared by weighing 1 g from them to get a stock solution with a 10,000 µg/ml concentration through dissolving nanoparticles in 10 ml of dimethyl sulfoxide (DMSO). Six isolates of E. coli were chosen to be treated with nanoparticles. The required concentrations (1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 µg/ml) of nanoparticles were prepared from stock solution, it was diluted using Brain heart infusion broth in wells of microplate to determine the nanoparticle’s sub-MIC. Twenty microliters of bacterial suspension inoculated in microplate wells that approached McFarland standard 0.5 (1.5 × 10⁸CFU/ml). While negative control wells did not contain bacterial suspension. It was incubated for 18–20 h at 37°C. After that, it was added the resazurin dye (20 µl) to the wells. Then it was incubated for two hours to note any colour alteration. Sub-MIC concentration was visually estimated in broth microplates as the lowest concentration when colour altered (blue to pink) in the resazurin broth test [14].

2.7.2. Detection of biofilm after treatment with nanoparticles

Six isolates of E. coli were chosen depending on their biofilm production, including two isolates that had strong biofilm production, two isolates that had moderate production of biofilm, and two isolates that had weak production of biofilm. The biofilm of these isolates was determined after being treated with nanoparticles by microtiter plates method [13] as mentioned previously. It treated the bacteria broth with the nanoparticle's sub-MIC concentrations of CuO, ZnO, and the mixture of these nanoparticles. The mixture of nanoparticles was prepared by proportion of 1:1 from sub-MIC concentrations of CuO and ZnO.

2.7.3. Determine gene expression of luxS and motA gene

RNA of E. coli treated with the nanoparticle's sub-MIC CuO and ZnO was extracted by Bioneer Accuzol ^TM Total RNA extraction reagent. RNA was transcribed reverse to cDNA by Accupower R Rocket Script™ RT Premix Kit. The primer sequences of the forward and reverse motA gene were 5'-CTTCCTCGGTTGTCGTCTGT-3’ and 5'-CTATCGCCGTTGA GTTTGGT-3’ respectively. While forward and reverse of the luxS gene were 5'-TGCCACACTGGTAGACGTTC-3’ and 5'-TGATTGGTACGCCAGATGAG-3’ respectively [15]. The primer sequences of rceA (housekeeping gene) were 5'-CACGCCGT AAGAGTGCATTA-3 (forward) and 5'-AACGGA GCTTG TCAGGGTTA-3’ (reverse) [16]. PCR mix was GreenStar™ RT-q PCR 2x master mix (10 µl), 1 µl of forward primer 10pmol/µl, 1 µl of reverse primer 10pmol/µl, cDNA (3 µl), and a complete 20 µl final volume by free nuclease water.

qRT-PCR conditions were initial denaturation at 95C for 2 min, second denaturation at 95°C for 40 cycles (20 s), annealing at 55°C for 40 cycles (45 s), and a final extension at 72°C (60 s). The melting temperature ranges from 42–94°C with an increase of 1°C in every 1 s. The amount of amplified product was monitored by detecting the fluorescence energy emitted by SYBR green. Each PCR run included a negative control (no template control). Levak equation was applied to estimate the fold expression of target genes for each isolate before and after treatment with nanoparticles.

2.7.4. Cytotoxicity assays (in vitro) of CuO and ZnO

This test was conducted in triplicate. It prepared many concentrations of CuO and ZnO (Armina Engineering company/Iran) including:12.5, 25, 50,100,200, and 400 µg mL^–1 by dissolving in DMSO to detect the cytotoxicity effect on Human Dermal Fibroblasts, neonatal normal cells (HDFn) [17]. The cell line was maintained [18] and the viability cell was determined by MTT colourimetric method. The cells were placed in a well plate to culture medium (200 µl) for each well. It was covered the plate with sterilized parafilm and stirred gently after then incubated at 37°C for 24 h, CO₂ (5%).

Then the medium was eliminated, and 200 µl from nanoparticles (12.5, 25, 50, 100, 200, and 400 µg/ml) were added to wells. It was incubated the plate at 37°C for 24 h in CO₂ (5%). It used 10 µl of MTT solution (3,4,5-dimethylthiazol-2yl 2,5 diphenyl tetrazolium bromide) from (Intron Biotech company). The plate was incubated at 37°C for 4 h, CO₂ (5%). It was eliminated the medium after then, DMSO solution (100 µL) was added to wells and incubated for 5 min. It was measured absorbance by ELISA reader at 575 nm. Statistics analysis was conducted by reading optical density (OD) using an ELISA reader to calculate IC50, as the following equation: Viability = OD of sample/OD of control x100%.

2.7.5. Statistical analysis

ANOVA analysis was applied to determine the variation between the cytotoxicity concentrations of nanoparticles using Graph Pad Prism 8 software. When P-value is less than 0.05, there are significant differences, and there are no significant differences when P-value is more than 0.05.

3. Results

3.1. Detection of E. coli

One hundred twenty-five samples appeared positive for bacterial growth as gram-negative bacteria. Forty-nine isolates of gram-negative bacteria were E. coli as shown on MacConkey agar and it appeared in pink colour and rod shape after staining with gram stain in microscopic examination. E. coli has appeared using biochemical tests with positive results for catalase, indole, methyl red, and lactose fermentation. Also, the identification of E. coli was confirmed by API 20 System and Vitek 2 compact system.

3.2. Antibiotic resistance of E. coli

The results of antibiotic resistance for 49 isolates showed a multi-resistance for 30 isolates of E. coli for Ampicillin (100%), Cefazolin(97%), Trimethoprim/Sulfamethoxazole (83%), Ceftriaxone (77%), Ceftazidime and Ciprofloxacin (70% each of them), and moderate resistance of E. coli for Levofloxacin (50%), Gentamicin (47%), while low resistance of E. coli for Cefepime (40%), Piperacillin/ Tazobactam (33%), Cefoxitin (30%), Nitrofurantoin (17%), Imipenem (10%), Ertapenem and Amikacin (7% each of them), Tigecycline (3%) as shown in Figure 1.

Figure 1. Resistance percentage to antibiotics in E. coli.

3.3. Molecular detection of luxS and motA genes

Amplification of the 2 quorum sensing genes (luxS and motA) was observed in all 30 tested E. coli isolates (Figures 2 and 3). The prevalence percentage for these genes in E. coli was 100% (30/30).

Figure 2. PCR product electrophoresis for luxS gene (315 bp) for E. coli in (1.5% agarose) and TBE (1×) at (75 volt/cm²) in 90 min and marker DNA ladder (100 bp).

Figure 3. PCR product electrophoresis for motA gene (430 bp) for E. coli in (1.5% agarose) and TBE (1×) at (75 volt/cm²) in 90 min. and marker DNA ladder (100 bp).

3.4. Detection of biofilm

The findings showed that 3 of 30 tested isolates of E. coli were producing a strong biofilm (10%), whereas 25 of 30 tested isolates were producing a moderate biofilm (83.3%), and 2 of 30 tested isolates were producing a weak biofilm (6.7%) as shown in Figure 4.

Figure 4. Biofilm formation percentage for E. coli isolates (O.D control = 0.07).

3.5. Characterization of nanoparticles materials

3.5.1. X-ray analysis

Figure S1 shows the X-ray analysis results. The angles reflecting CuO nanoparticles were the spectrum 35°, 39°, 49°, 54°, 58°, 62°, 66°, and 68° showing the XRD patterns of pure CuO Nanostructure. All of the peaks may be attributed to CuO monoclinic phase (JCPDS 45-0937), and no impurity peaks can be found, showing that the CuO Nanostructure is pure and crystalline. CuO is formed as polycrystalline. The peaks broadening in the XRD pattern indicates that there are small nanocrystals in the product.

Further, the angles reflecting ZnO nanoparticles were the 32°, 35°, 37°, 48°, 57°, 63° and 68°. The mentioned planes demonstrate the hexagonal wurtzite phase structure of the ZnO Ns According to JCPDS: 36-1451. This confirms that the preferable orientation of the prepared nanostructures is along with these directions (Figure S2).

3.5.2. UV analysis

The results of UV analysis showed that the range of wavelength was 190–1100 nm. Besides, the absorption of CuO and ZnO nanoparticles was 350–400 nm, as Figure S3 shows the absorption spectrum of CuO and ZnO. CuO and ZnO both exhibit a strong absorption spectrum in the UV region, confirming a blue absorption wavelength shift towards a shorter wavelength region. The reason may be back quantum confinement at the nano regime presence of CuO and ZnO nanostructures in the UV region.

3.5.3. FTIR analysis

The results of FTIR for CuO are shown in Figure S4 appeared the peaks of the wave numbers are (530.7–3778.97 cm⁻¹) and the CuO nanoparticle region is 598.45–530.7cm⁻¹. Whereas the result FTIR for ZnO appeared the peaks of the wave numbers are (569.20–3917.71 cm⁻¹) and the ZnO nanoparticle region is 569.2 cm⁻¹ shown in Figure S5.

3.5.4. Energy dispersive X-ray spectrometer analysis

The results of EDX showed the components of CuO nanoparticles were Cu = 77.58 and O = 22.42 as shown in Figure S6. Whereas the components of ZnO nanoparticles were Zn = 78.69 and O = 21.31 as shown in Figure S7.

3.5.5. Scanning electron microscopy (SEM) analysis

The results of SEM showed that the size of CuO nanoparticles was 32.19, 33.78, 39.21 and 46.6 nm as shown in Figure S8. While the size of ZnO nanoparticles was 32.09, 34.16, and 78.06 nm as shown in Figure S9.

3.5.6. Atomic force microscope analysis

The results of AFM showed that the size and distribution of CuO nanoparticles were 53.24 nm as shown in figure S10. While the size and distribution of ZnO nanoparticles were 64.87 nm as shown in Figure S11.

3.5.7. Transmission electron microscope analysis

The results of TEM showed the size of CuO nanoparticles was 50 nm as shown in Figure S12 and hexagonal shape. While the size of ZnO nanoparticles was 100 nm as shown in Figure S13 and irregular shape.

3.5.8. Determination sub-MIC for CuO and ZnO nanoparticles on E. coli

Sub-MIC outcomes for CuO nanoparticles were 64 µg/ml for isolates (1, 3, 6, 19 and 26) and 128µg/ml for isolates (27). While the outcomes of sub-MIC for ZnO nanoparticles were 128µg/ml for isolate (1) and 256µg/ml for isolates (3, 6, 19, 26, and 27).

3.5.9. Detection of biofilm after treatment with nanoparticles

The results demonstrated that all 30 isolates had a weak biofilm formation after treatment with sub-MIC for CuO and ZnO nanoparticles as shown in Figure 5.

Figure 5. Optical density of biofilm after treatment of E. coli by nanoparticles.

3.5.10. Effect of sub-MIC concentration for CuO and ZnO nanoparticles on gene expression of quorum sensing genes

The results Ct value of motA gene for six isolates were 17.52, 15.32, 14,16.11, 13.49, and 11.62, respectively, compared with the control was 1. Whereas Ct values for six isolates treated with CuO nanoparticles were 25, 20, 21.5, 29, 23.01, and 24.3, respectively. Also, Ct values for six isolates treated with ZnO nanoparticles were 21.4, 18, 22.5, 28.9, 21.9, and 22, respectively. Besides, Ct values for six isolates treated with a mixture of CuO and ZnO nanoparticles were 27, 28.3, 29.7, 27.5, 26.5, and 23.5, respectively as shown in Table 2. Furthermore, the results of folding decreased from 1.0 in control before treatment with nanoparticles to 0.003 after treatment with nanoparticles; it was the highest low in treatment with a mixture of CuO and ZnO as shown in Figure 6.

Figure 6. Folding ratio of mot A gene expression for before and after CuO and ZnO and a mixture of CuO and ZnO.

Table 2. Ct and fold change for motA gene.

Group	Sample	House keeping Ct	motA Ct	ΔCt	ΔΔCt	Expression Fold Change2${}^{\wedge }$-ΔΔCt
Control	1	25.23	17.52	−7.71	0	1
	2	23.45	15.32	−8.13	0	1
	3	23.39	14	−9.39	0	1
	4	22.09	16.11	−5.98	0	1
	5	22.99	13.49	−9.5	0	1
	6	21.1	11.62	−9.48	0	1
CuO	1	27.68	25	−2.68	5.03	0.031
	2	25.39	20	−5.39	2.74	0.15
	3	28.34	21.5	−6.84	2.55	0.171
	4	32.92	29	−3.92	2.06	0.24
	5	30.87	23.01	−7.86	1.64	0.321
	6	29.83	24.3	−5.53	3.95	0.065
ZnO	1	28.39	21.4	−6.99	0.72	0.607
	2	25.91	18	−7.91	0.22	0.86
	3	29.05	22.5	−6.55	2.84	0.14
	4	34.04	28.9	−5.14	0.84	0.56
	5	31.04	21.9	−9.14	0.36	0.78
	6	28.9	22	−6.90	2.58	0.167
Cuo + ZnO	1	26.27	27	0.73	8.44	0.003
	2	30.93	28.3	−2.63	5.5	0.022
	3	32.01	29.7	−2.31	7.08	0.007
	4	29.15	27.5	−1.65	4.33	0.05
	5	28.45	26.5	−1.95	7.55	0.005
	6	26.32	23.5	−2.82	6.66	0.01

The results Ct value of luxS gene for six isolates were 18.9, 17.13, 14.8, 15.05, 14.34 and 15.34, respectively, compared with control was 1. Whereas Ct values for six isolates treated with CuO nanoparticles were 22.2, 23.4, 22.68, 27.45, 22.92 and 25.92, respectively. Also, Ct values for six isolates treated with ZnO nanoparticles were 23.7, 19.9, 21.5, 29.09, 22.6, and 23.8, respectively. Besides, Ct values for six isolates treated with a mixture of CuO and ZnO nanoparticles were 32.35, 26.11, 24.46, 33.08, 23.13 and 25.16, respectively. As shown in Table 3. Furthermore, the results of folding decreased from 1.0 in control before treatment with nanoparticles to 0.000 after treatment with nanoparticles; it was the highest low in treatment with a mixture of CuO and ZnO. As shown in Figure 7.

Figure 7. Folding ratio of luxS gene expression for before and after CuO and ZnO and a mixture of CuO and ZnO.

Table 3. Ct and fold change for luxS gene.

Group	Sample	House keeping Ct	luxS Ct	ΔCt	ΔΔCt	Expression Fold Change2${}^{\wedge }$-ΔΔCt
Control	1	25.23	18.9	−6.33	0	1
	2	23.45	17.13	−6.32	0	1
	3	23.39	14.8	−8.59	0	1
	4	22.09	15.05	−7.04	0	1
	5	22.99	14.34	−8.65	0	1
	6	21.1	15.34	−5.76	0	1
CuO	1	27.68	22.2	−5.48	0.85	0.56
	2	25.39	23.4	−1.99	4.33	0.05
	3	28.34	22.68	−5.66	2.93	0.13
	4	32.92	27.45	−5.47	1.57	0.34
	5	30.87	22.92	−7.95	0.7	0.62
	6	29.83	25.92	−3.91	1.85	0.28
ZnO	1	28.39	23.7	−4.69	1.64	0.321
	2	25.91	19.9	−6.01	0.31	0.807
	3	29.05	21.5	−7.55	1.04	0.486
	4	34.04	29.09	−4.95	2.09	0.235
	5	31.04	22.6	−8.44	0.21	0.865
	6	28.9	23.8	−5.1	0.66	0.63
Cuo + ZnO	1	26.27	32.35	6.08	12.41	0.00018
	2	30.93	26.11	−4.82	1.5	0.354
	3	32.01	24.46	−7.55	1.04	0.486
	4	29.15	33.08	3.93	10.97	0.000
	5	28.45	23.13	−5.32	3.33	0.1
	6	26.32	25.16	−1.16	4.6	0.041

3.6. Cytotoxicity assays (in vitro) of CuO and ZnO

The outcomes of the viability cells appeared there were the lowest viability Human Dermal Fibroblasts, neonatal (HDFn) normal cell line at 400 and 200 µg mL⁻¹ for CuO (88.69 and 76.96%), respectively, with significant differences (P < 0.05 and <0.0001) compared with other concentrations. On the other hand, ZnO nanoparticles showed the lowest viability cells at 400, 200 and 100 µg mL⁻¹ (72.83, 74.4 and 92.12%) with significant differences (P < 0.05 and <0.0001) compared with other concentrations. The highest viability cells at 12.5 µg mL⁻¹ were observed for CuO and ZnO nanoparticles (94.29 and 94.90%) as shown in Table 4 and in Figures 8 and 9.

Figure 8. The cytotoxic effect of CuO on HdFn cell line.

Figure 9. The cytotoxic effect of ZnO on HdFn cell line.

Table 4. The cytotoxic effect of CuO and ZnO on HdFn cell line.

	Mean viability (%) ± SD
Concentration µg mL^−1	CuO	ZnO
0	96.99 ± 1.17NS	96.99 ± 1.17NS
12.5	94.29 ± 2.97NS	94.90 ± 2.19NS
25	96.95 ± 1.14NS	96.95 ± 1.1NS
50	95.71 ± 1.51NS	96.18 ± 1.25NS
100	92.20 ± 2.7NS	92.12 ± 1.5*
200	88.69 ± 2.24*	74.4 ± 0.85****
400	76.96 ± 5.64****	72.83 ± 3.51****

NS nonsignificant P > 0.05; P < 0.05*; P < 0.0001****.

4. Discussion

Antibiotic resistance became the most phenomena prevalent in the world, representing one of ten world menaces announced by WHO (World Health Organization) [19].

The mechanisms of antibiotic resistance involving the beta-lactamase activity, modification of antibiotics, outer membrane porins remodelling, biofilm production, and antibiotic target sites changing in bacteria [20].

The fast evolution of resistant bacteria to antibiotics causes failure in treatment. Another challenge is the formation of biofilm-related to the infection of bacteria that is difficult its treatment as a result of the decreased sensitivity antibiotics of bacteria that embedded together the biofilm with declined antibiotics penetrability via the extracellular matrix that involves extracellular polymeric substance and additional substances created by bacteria [11, 21, 22].

The outcomes in this study are compatible with another study by Luna-Pineda et al. [23] which showed that the isolates of E. coli in patients with urinary tract infections were multi-resistance to antibiotics and also those isolates had motA gene. Moreover, quorum sensing is utilized to regulate the expression of genes, and many processes that contribute to virulence, like motility, and formation of biofilm, are vital for bacteria to form the phenotype of biofilm. Autoinducers are found in Gram-negative and Gram-positive bacteria. E. coli has Autoinducer-2, which is produced via LuxS enzyme associated with the formation of biofilm [24]. One of the studies showed luxS, and motA expression have correlated with multi-antibiotic resistance in various bacteria groups. The biofilm formation has a role in the formation of multi-antibiotic resistance in E. coli [15].

The microorganisms that form biofilm cause (65–80%) of infections [5]. Biofilms are bacteria communities embedded in a hydrate, and anionic template of bacterial exopolymer (polysaccharides) that catch other compositions from bacteria or the surrounding environment involving protein, nucleic acid, teichoic acid, lipids, and several organic molecules [25]. Therefore, there is a demand for improved new antibiotic materials to kill microorganisms that cause infections in humans. Nanoparticles have proven effective against bacteria as antibiotics. The efficiency of nanoparticles synthesized depends on their shape and size [26].

The current study showed that the results of sub-MIC for CuO and ZnO nanoparticles had more effect on the inhibition of biofilm for E. coli. The formation of biofilm was inhibited in all six isolates. It was observed that there are associations between the formation of biofilm and gene expression for luxS and motA. There was a decline in the genetic expression for all isolates that were treated with nanoparticles. In the present study, CuO and ZnO nanoparticles were examined as antibiofilm and antibacterial. The present study agreed with another study that observed ZnO nanoparticles showed inhibition of E. coli at 10 and 50 µg/ml after incubation for 24 h while inhibition at 10 µg/ml was limited [27].

Our study agreed with another study by Shakerimoghaddam et al. [28] who showed in their study the effect of MIC and sub-MIC for ZnO against E. coli has minimized the formation of biofilm for E. coli at 2500 µg /ml (50%) and at 1250 µg /ml (34.3%). Also, the current study was consistent with another study, it has been observed that the sub-MIC for ZnO nanoparticles was 500 µg/ml [29] and 400 µg/ml [30]. While it appeared in the study by Agarwala et al. [31] that the MIC for CuO against E. coli was 35 µg/ml. Moreover, one of the studies showed that CuO nanoparticles had more effect on E. coli at MIC 3.12 µg/ml and reduced biofilm formation to 59% [32].

Nanoparticles have a high surface area that rises their reactive, which leads to toxic effects on bacteria such as E. coli and Bacillus cereus [26]. The variation in the efficiency of nanoparticles is impacted by the nanoparticles’ size. One of the studies showed the ZnO size was 20 nm [33]. While in one study ZnO nanoparticle size was 10 nm, the good effects of nanoparticles can be due to its small size [29].

In the current study, the characterization of CuO and ZnO nanoparticles was agreed with other studies. FTIR results for CuO nanoparticles were an approach to another study conducted by Shehabeldine et al. [32] which demonstrated the peak of CuO at 604.5 and 419.4 cm⁻¹. Also, Somu et al. [34] illustrated FTIR for ZnO band was 448.6 cm⁻¹. Kumar and Rani[35] showed FTIR for ZnO band was 620 cm⁻¹.

Also, XRD results of 2 theta peaks diffraction for CuO were (32.1°, 35.1°, 38.2°, 48.6°, 52.3°, 61.3°, and 66.2°) [32]. In addition, XRD analysis for ZnO in the present study agreed with another study that showed 2 theta peaks were 31.82°, 34.54°, 36.42°, 47.46°, 56.74°, 62.92°, 66.06°, 68.42°, 69.06° and 78.82° [35].

Alarifi et al. [36] showed in their study that the shape of CuO nanoparticles was spherical and smooth by TEM examination with a size of 55.8 nm. The morphology of CuO nanoparticles by TEM appeared in different shapes including spherical and rods with the size range of (4–15 nm) and (11–53.8 nm) respectively [32]. Further Kumar and Rani [35] appeared in their study TEM examination of ZnO was rod shape with a size 10–12 nm. Also, CuO nanoparticle morphology was spherical and tended to agglomerate by SEM examination [37]. ZnO morphology by SEM was spherical, hexagonal, and plates [38].

The mechanism act of nanoparticles to kill bacteria is represented through the destruction effect of ZnO nanoparticles on the cell integrity of bacteria via attachment with the cell wall. Consequently, ions (Zn²⁺ and Cu²⁺) liberate that had a role as antimicrobial and reactive oxygen stress (ROS) formation. Therefore, morphology has a role in antimicrobial activity [39,40]. Reactive oxygen species cause cell wall structure damage by electrostatic reaction, oxidative stress transport by ROS production, and protein function disturbance structures [40]. Studies demonstrated after treatment with biomolecules and transition metals, the nanoparticles’ surfaces were modified to be good and effective compatibility with antimicrobials, drug transport systems, and antioxidants [41]. Where Cu²⁺ is observed the change of morphological, optical, structural, electrical, biological, and magnetic properties for ZnO nanoparticles therefore Cu²⁺ represents a good option [42], due to Cu being comparable similar size to Zn²⁺ and non-toxic [43].

Moreover, quorum sensing is utilized to regulate the expression of genes, and many processes that contribute to virulence, like motility and formation of biofilm, are vital for bacteria to form the phenotype of biofilm. Autoinducers are found in gram-negative and gram-positive bacteria. E. coli has Autoinducer-2, that produced via luxS enzyme associated with the formation of biofilm [24]. One of the studies showed luxS and motA expression genes have correlated with multi-antibiotic resistance in various groups. Biofilm formation has a role in forming multi-antibiotic resistance in E. coli [15].

Previous studies observed ZnO nanoparticles downregulate the expression of quorum-sensing genes (rhlR, rhlI, pqsA, lasI, lasR) [44]. Also, Singh et al. [45] showed in their study the effect of Ag nanoparticles in reducing the gene expression of lasI, lasR, rhlR and rhlI genes. This showed that ZnO nanoparticles to a similar role to Ag nanoparticles in the inhibition of the production of virulence factors.

One study indicated to effect of nanoparticles such as silicon oxide nanoparticles in the silence of communication of bacterial cells by reducing gene expression for quorum sensing genes (lux A and lux R) [46].

One of the studies compared Cu nanoparticles with ionic copper in effect on genes encoding the biofilm it was observed Cu nanoparticles cause downregulate for genes that encode polysaccharide synthesis. Whereas Cu ion causes the upregulates of gene expression [47]. The different effects of Cu nanoparticles and ions of Cu may be attributed due to variations of physical characteristics for nanoparticles (big surface area/volume ratio, the ability of penetration, the biosorption on surfaces, and slow liberation of ions through reaction between nanoparticles and biofilm) [48].

Concerning the cytotoxicity effect of CuO nanoparticles on dermal fibroblast human cells, the current study was concurrent with other studies. Arul Selvaraj et al. [49] appeared in their study that the fibroblast cells had a viability of 99.47% in the presence of CuO nanoparticles and there was non-cytopathic impact observed. Another study demonstrated that CuO nanoparticles have toxicity for blood cells depending on concentration. The lowest concentration of CuO 1 µg / ml did not influence the viability of cells. In contrast, the highest concentration (more than 10µg/ml) decreased the viability of cells up to 27.01% at 200 µg /ml [50].

Also, other studies illustrate that ZnO nanoparticles until 20 mg/L did not affect HeLa cells [51]. Reddy and Srividya [52] showed that the values of TC50 (toxic concentration 50 induces death of 50% of cells) of ZnO nanoparticles ranged from (33–37) µg/ml. Where the cells used in their study were Human alveolar epithelial cells and human embryonic kidney cells (HEK cell).

One study appeared to the effect of ZnO on human dermal fibroblast cells that were exposed to 2.5, 10, 25, 50, and 100 µg /ml of ZnO for 24 h, and the viability did not produce at 2.5 and reduced 20% at 10 µg /ml (p < 0.05), while higher concentrations of 25–100 µg /ml led to a high reduction of viability (p < 0.001) [53].

5. Conclusion

CuO and ZnO nanoparticles appeared efficient in the reduction of biofilm formation in E. coli isolated from urinary tract infections by using sub-MIC concentrations. Besides, the sub-MIC concentration of the mixture for CuO and ZnO nanoparticles decreased the gene expression of luxS and motA more than each alone. Besides, CuO and ZnO nanoparticles in the lower concentration had non-toxic effects on Human Dermal Fibroblasts, neonatal (HDFn) normal cells.

Ethical approval

The consent was obtained by the ethics committee of Medical educational laboratories / Medical City/Iraqi Ministry of Health (Ref:5214 in 1-2-2022).

Acknowledgements

The authors would like to thank all participants in this study. Hanan M. Abbas, Mohammed F. Al Marjani, and Radhouane Gdoura contributed to Conceptualization, Methodology, Validation, Investigation, Resources, and Data curation. Hanan M. Abbas contributes Writing the Original Draft, writing the Review, and Editing. Radhouane Gdoura, and Mohammed F. Al Marjani contributed to Visualization, Supervision, and Project administration. All authors have reviewed the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).