Inhibitory Effects of Biosynthesized Copper Nanoparticles on Biofilm Formation of Proteus mirabilis

This study aimed to explore whether green synthesized copper nanoparticles (CuNPs) can function as an anti-biofilm agent produced by P. mirabilis . The nanoparticles were synthesized from cells free extract of P. mirabilis . Characterization of biosynthesized copper nanoparticles was carried out to determine the chemical and physical properties of the product using atomic-force microscopy (AFM), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD) and UV-visible spectroscopy. The hexagonal structure was confirmed by XRD, size range was marked 13-19nm by TEM. FESEM was used to confirm the surface morphology. AFM analysis was used to reveal the roughness and distribution of nanoparticles. UV–visible spectra of the synthesized nanoparticles recorded a maximum peak at 380 nm. Copper nanoparticles showed remarkable biofilm inhibitory effects on wild type strains of multidrug resistant Proteus mirabilis . After incubation for 24 and 48 hours at 37 o C with 128 μg/ml sub minimum inhibitory concentration (MIC) of CuO nanoparticles, strong biofilm producer strains showed weak biofilm production. Down regulation changes in LuxS expression using real time PCR technology were detected after treatment with copper nanoparticles of these strains.


Introduction
Several Proteus species are recognized as human causing disease pathogens, but only Proteus mirabilis is the one with the most frequent link to human disease.P. mirabilis is commonly associated with urinary tract infections (UTIs) in the individuals with structural or functional abnormalities.Patients undergoing urinary catheterization can also suffer from ascending infections [1].Proteus has been shown to have the ability to produce biofilms in various situations from aquatic environments to indwelling devices, ureteral stents, scleral buckles, urethral catheters and tracheosophageal voice prostheses.P. mirabilis can also produce biofilms on biological as well as nonliving surfaces including glass, polystyrene, silicone, latex etc. [2].
Bacterial biofilms are well organized communities with single or multi species enclosed by a defensive polysaccharide matrix containing extracellular DNA, lipids and proteins [3].The community of biofilm depends on complicated communications, enabling the pathogens to develop resistance against host defense mechanisms, different stress factors and antibiotics [4].Bacterial biofilms are ubiquitous in clinical settings as they can increase bacterial resistance up to 1000 folds in compression to planktonic cells.Subsequently with anticipation measures taking place in hospitals and other clinical settings, nosocomial infections still result in major morbidity and mortality rate [5].In this emergence situation, researchers are commended to improve new alternatives to traditional antibiotic treatments which can successfully combat against multidrug resistance (MDR) and biofilm producing bacteria.Green synthesis of metal oxide NPs has gained a great interest since it is clean, eco-friendly approach and has a wide range of applications in biotechnology and medical field [6].Copper nanoparticles (CuONPs) are of great interest due to their extraordinary properties such as flexibility high surface-to-volume ratio and rigidity.
Metal NP synthesis using microorganisms has represented a novel alternative to chemical and physical approaches as antibacterial, antioxidant and antifungal agents along with anticancer properties in various applications [7].
Several studies were interested in investigating the antibacterial activity of CuO.Noor et al. revealed a moderate antibacterial activity of fungal mediated CuO NPs against E. coli, K. pneumoniae and other gram-negative bacteria [8].
This study was aimed to determine the anti-biofilm activity of eco-friendly green synthesized copper oxide nanoparticles on biofilm production by P. mirabilis.Changes in LuxS expression was also investigated in this study.

Materials and Methods Sample Collection:
A total of 100 urine samples were collected from patients that had urinary tract infection and resident hospital patients with urine catheters presented to Al Yarmouk teaching hospital.Isolation and identification: P. mirabilis was primarily identified by culturing it on MacConkey and blood agar at 37°C for 24 h.and confirmed by VITEC 2 system.

CuO Preparation
As a precursor, copper (II) and acetate Cu (CH3COO)2⋅H2O) were used in this research.
Bacterial Suspension Preparation P. mirabilis were grown in 250 ml of brain heart broth at 37°C for 24 hours in non-shaking incubator.The bacterial broth was then centrifuged, and the supernatant was collected after discarding the sediment.

Synthesis of CuO NPs
CuO NPs were synthesized by precipitation method.Later 20g of copper (II) acetate were added to 200ml of P. mirabilis cells free extract at room temperature 37 o C, sealed and covered with black plastic bag.After incubation in a shaker incubator for 24 h, the mixture then centrifuged, and the precipitate was collected and washed thrice by deionized water.Nanoparticles were synthesized by the extra cellular route where metal ions got reduced by the action of bacterial reducing enzymes.Nanoparticles formation can be detected by color change.The precipitant was later air dried and kept for further characterization [9]

Characterization of CuO NPs
Characterization is essential for understanding nanoparticles properties.The following methods were used to determine NPs characteristics.SEM and TEM were used to determine the shapes and sizes.SEM is used to characterize and visualize surface morphology, particle size distribution, particle/crystal shape, agglomeration of nanoparticles and surface functionalization and in single-particle analysis.TEM uses an electron beam to image a nanoparticle samples, providing much higher resolution than is possible with light-based imaging techniques.Atomic force microscopy (AFM) is used to define height and volume of NPs in 3D vision, UV-visible-(UV-DRS) is used to study the optical property of the samples.XRD is a technique that is used in materials science to determine the crystallographic structure of a material [10].

MIC Determination:
The estimation of minimum inhibitory concentration (MIC) was carried out to obtain the minimum concentration of CuO nanoparticles that inhibits bacterial growth by broth microdilution method using Eppendorf tubes.The bacterial inoculum was prepared in MHB and the concentration was modified to 100 UFC/μl.Two fold serial dilutions of CuO NPS were prepared in 16, 32, 64,128, 256, 512 μg/μl using MHB in six tubes which were then incubated for 24 hours at 37°C.Results were later achieved by detecting visible growth of bacteria in each tube.Sub MIC can then be determined.

Estimation of Biofilm Formation
Quantitative determination of biofilm formation was perfomed by a colorimetric microtiter plate assay [11].1-Brain heart infusion broth supplemented with an additional 1% glucose was used for this assay 2-Biofilm inoculums for cultivation were made from bacteria cultivated in broth, diluted 1:100 and poured into the well with 200 μl.Negative control wells was 200 μl of BHI supplemented with 1% glucose per well.3-Under static conditions, the inoculated plate was covered with a lid and incubated aerobically for 24-30 hours at 35-37°C.4-The contents of the wells were decanted, and each well was washed three times with 300 μl of PBS (2.2.5.1).The plates were then drained inverted and fixed with 150 ml methanol.5-Staining was done with 150 μl of crystal violet at room temperature for 15 min, then washed and dried at room temperature.6-For dye re-solubilizing, 150 μl of 95 percent ethanol was added, and the microtiter plate was covered with the lid and left at room temperature for at least 30 min., without shaking.7-Using a microtiter-plate reader (GloMax/ Promega-USA), the optical density (OD) of each well was measured at 630 nm, and the results obtained are as following: OD ≤ ODc = No biofilm producer, ODc < OD ≤ 2 ODc= Weak biofilm producer, 2 ODc < OD≤ 4 ODc = Moderate biofilm producer, 4 ODc < OD= Strong biofilm producer.
The cut-off value for the negative control is ODc, which is defined as three standard deviations (SD), above the mean OD of the negative control: ODc= average OD of negative control + (3× SD of negative control).

Effects of CuO NPs on Biofilm Formation
This assay was carried out by 96-well microtiter plate as performed by Punniyakotti.et al. [12].Tested isolates were cultured in brain heart infusion broth at 37 o C for 24h.Later 100 μl of bacterial inoculum was attuned to 0.5 McFarland and was then added to each well.Next 100 μl of CuO suspension was add to each well at the sub-inhibitory concentration before incubating the tubes at 37 o C for 48h.A positive control was presented as bacterial culture without nano formulations, while clear broth was considered as negative control.The contents of each well were discarded after incubation.Microplate was rinsed thrice with sterile saline and dried for 45 min at 60 o C. Next 200 mL of 0.1% of crystal violet was used in staining, followed by incubation at room temperature for 15 min.The microplate was rinsed three times with sterile normal saline and then acetic acid at 30% was added to each well by 200 ml.The optical density (OD) was read at 630 nm for all wells using a microtiter plate reader.

RT-qPCR Protocol
The presence of LuxS and rpo of P. mirabilis was determined by employing the thermal cycler to amplify the isolated genomic DNA (Thermo Fisher Scientific, USA).This main step can be separated into two stages: first stage is done by synthesis of cDNA from RNA using specific primer for LuxS, rpo transcripts (Table 1) and proto script cDNA synthesis kit.This procedure was performed as detailed in the manufacture procedure.
The second stage of this protocol was done by taking cDNA sample from tested isolates and control at the same run.For each sample, there were three PCR tubes, one tube for LuxS gene, rpo which is considered as a house keeping gene in this study.Fluorescent power of SYBR Green was used for quantity estimation.The reaction mix of component with their quantity is mentioned below in Table 2.The difference in cycle thresholds (Ct) and fold changes between the treated groups and the calibrators for each gene were evaluated [15].The rpo values were used to normalize the data.PCR tubes were spanned, and the liquid was collected (1 minute at 2000g)., Then the program for Real-Time PCR was setup with indicated thermos cycling protocol.The result was collected and analyzed by using Livak formula.Relative quantitation was applied to determine expression levels.

Statistical Analysis
All experiments were performed in triplicate and data was expressed as mean and standard deviation.The effect of study variables on biofilm was tested using the T test.The statistical analysis was done using Statistical Package For The Social Sciences (SPSS), version 26 software.The differences were considered significant when p<0.05.

Results and Discussion Isolation and Identification:
Sixty-four isolates were confirmed as P. mirabilis.The confirmation was carried out by VITEK 2 system that is considered as a reliable identification technique, in addition swarming on blood agar and colony morphology on MacConkey agar.

Antibiotic Susceptibility Test MDR Strains Screen:
After incubation at 37 o C for 24 hours, forty out of sixty-four isolates (62.5%) were confirmed as MDR strains.All tested isolates were resistance to piperacillin-tazobactam. Six isolates (15%) only were resistant to levofloxacin, while six isolates (15%) were found to be resistant to ciprofloxacin.Ampicillin did not inhibit growth of 15 isolates (37.5%) and gentamicin resistance was found in two strains only (5%).Six isolates (15%) were recorded as cefoxitin resistant and 17 isolates (42.5%) were resistant to nitrofurantoin..A ten-year study in 2019 showed a growing concern about antibiotic resistance in proteae (Proteae tribe includes the genera Proteus, Morganella and Providencia).Based on the results of the study, resistance rates to third-generation cephalosporins developed significantly (the indicator during the study was ceftriaxone).Resistance to ceftriaxone increased by three times in hospitalized patients samples [16].Another study carried out in China stated that 25 isolates of 54 P. mirabilis were classified as MDR.The samples were collected from wildlife animals and humans.This study highlighted that P. mirabilis produces AmpC β-lactamases and extended spectrum β-lactamases that makes this bacteria a serious public health risk.P. mirabilis also uses efflux pump to resist the main classes of antibiotics [17].Resistance to cefotaxime also recently investigated by the detection of blaCTX-M-1, blaCTX-M-2, blaCTX-M-8 and blaCTX-M-9 that are responsible for cefotaxime resistance in P. mirabilis in the clinical isolates from urine samples in patients referred to different hospitals in Baghdad city [18].

Biosynthesis of Copper Oxide Nanoparticles:
Copper oxide nanoparticles were biosynthesized from P. mirabilis.The formation of nanoparticles was indicated by color change from yellow to blue, in addition to form a light blue precipitate.After centrifugation, the precipitate appeared greenish blue and after drying in the microwave, we obtained shiny blue powder, as shown in Figure 4.In recent years, an obvious coordination to use bacteria to synthesizes nanomaterials (mainly silver, zinc, gold, and nanoparticles) with remarkable properties have been observed for the development antimicrobials with in vitro activities against pathogenic bacteria [19].Bacteria is an easy culturing microorganism with short generation time which makes bacteria a great candidate for nanoparticle synthesis as they have extra cellular reduction enzymes [20].Several types of bacteria such as E. coli, Pseudomonas fluoresces Serratia sp., and Pseudomonas stutzeri have proved their ability to reduce certain metal ions and produce nanoparticles [21].

Characterization of CuO NPs 1-UV-Vis Spectral Analysis
The results showed that biosynthesized CuoNps exhibited a maximum peak at 380 nm.Smaller particle size was indicated previously at sharp peak observation at 270 nm as reported by Kayani et al. [22].As shown in (Figure 3).

2-Atomic Force Microscopy (AFM) Analysis
AFM analysis of CuO NPs was performed to identify and characterize distribution of nanoparticles.Estimated grain size and mean square roughness were also determined.In Figure 6, portraits three-dimensional profile of CuONPs; 3D image of the CuO nanoparticles shows that the grains were distributed between 13 to 37nm [23].

3-Transmission Electron Microscopy Analysis (TEM)
TEM imaging was used to explore the morphology and size of CuO NPs.The core structure of the nanoparticles was confirmed by TEM analysis [20].TEM images (Figure 5) demonstrate the formation of spherical shaped particles that were in 13-19nm range, with 77.5 kilo pixel magnifications and 40 nm resolution powers.

4-Field Emission Scanning Electron Microscope (FESEM)
SEM analysis was performed to understand the morphology and size, as well as the elemental and structural composition of NPs samples.Figure 8 shows the spherical shape of CuO NPs at 3000kv and 40000 magnification power.SEM images revealed that it was essentially spherical and uniform in appearance with the diameter ranging from 15 to 19nm.In comparison with EDX-ray, FESEM allows to determine the presence of different components in the examined model [20].

5-X-ray Diffraction (XRD)
The XRD spectra of CuO Nps powder shown in Figure 9, confirmed formation of hexagonal structure of the CuO NPs by revealing six prominent peaks corresponding to the diffraction peaks (16.414), (18.414), (22.278), (23.983), (26.113), and (43.537).One recent study has reported that the XRD pattern of the synthesized copper nanoparticles has the same peaks as those displayed by the XRD pattern of the same copper nanoparticles sample after being exposed to air for 24 h [24].According to the International Center of Diffraction Data, the crystalline points characterization was carried out by comparison with crystallographic standard data.

Determination of Biofilm Formation before CuO NPs Treatment
A total of 20 XRD isolates were tested for their biofilm production ability using a plastic microtiter plate.Of which nine isolates were recorded as strong biofilm producers as detailed in Table 2. To estimate the CuO NPs effects on biofilm production of these isolates, five strong producers strains were selected.OD of each well was read at 630 nm using a microtiter plate reader.

Determination of Biofilm Formation after CuO NPs Treatment
Biofilm production was significantly reduced after 48 hours of incubation at 37°C with sub MIC of CuO NPs.OD measurement of the same five strains of P. mirabilis lowered from strong to weak production.The percentage inhibition of biofilm formation caused by CuO NPs according to the following equation: Percentage inhibition = 100 -[{ OD after treatment / OD before treatment x 100] as detailed in Table 3.  Antimicrobial action can be explained due to the small particle size of CuO NPs because smaller materials can enter bacterial cell membranes and cause cell damage.One research detailed that NPs seem to be more effective when they have smaller than 39 nm of diameter because they have a greater surface of interaction and in turn can reach the intra cellular space of bacteria straightforwardly [25].Regarding CuO-NPs, Kouhkan et al.displayed that the small size of the NPs could increase the antibacterial effects of these particles due to large surface area that increases their concentration [16].Nanoparticles can act on many levels and target bacterial components such as eDNA that plays a key role in bacterial adhesion, aggregation, biofilm formation and structure, biofilm integrity, in addition to intercellular communication or QS for transfer of genetic information [26].Protein inhibition can result in a total destruction of biofilm and planktonic bacterial cells as they are involved in varied functions.Therefore, proteins are one of the important targets in recent antimicrobial therapies [27].A recent study found that LPS from Gram-negative bacteria like P. aeruginosa, Salmonella enterica and E. coli bind Au nanoparticles , and the binding strength depends on the interaction of LPS with polyelectrolytes.A strong interaction between anionic LPS and cationic NPs was detected by the authors [28] suggesting that electrostatic interactions played a major role in LPS interaction with NPs.

-Gene Expression of LuxS
RT-PCR reveals a major down regulation in LuxS expression after the exposure to CuO NPs suspension compared to normal gene expression in bacterial broth with no NPs involved (Table 4).LuxS is a transcript of structural operon luxCDABF that is responsible for production of auto inducer 2 (AI-2) which predominantly participates in cell to cell signaling in bacteria.After luxS is produced, AI-2 casts signals that is used to sense inter species interaction and its own cell density in a multi microbial population that has essential role in the virulence factors regulation [29].This result corresponds with the phenotypic changes in biofilm formation in microtiter plate experiment carried out in this research (Figure 9).This finding also matched with other reports of Rajalakshmi and Sangeetha (2017) [30].Antibiofilm activity of metal oxide nanoparticles have gained a great interest in last decades, since biofilm is an important virulence factor that enables the bacteria to resist antibiotics [31].Fold change in gene expression reveals that LuxS was down regulated in response to copper oxide NPs in three out of five isolates of proteus mirabilis.Whereas, the other two isolates were almost not affected.This could be due to the development of bacterial resistance mechanisms to nanoparticles.Recent studies have entailed several possible mechanisms used by bacteria to overcome nanoparticles effects, specially by over expression of genes responsible for the production of biofilm components such as exopolysaccharides and development of resistance after many culture steps [32] or up-regulation of efflux pump proteins and molecular interactions (electrostatic interactions) [33] .Copper generates toxic hydroxyl radicals (OH -) that damage cell membranes of Gram-negative and positive bacteria [34].Positively charged metal nanoparticles can create a strong bond with membranes which can lead to increase their permeability and, therefore, disruption of cell walls.The most important factors determining the interaction with nanoparticles is biofilm maturity and thickness.As shown by Peulen and Wilkinson, EPS are denser in mature biofilms.Hence, the number of pores and the pore sizes are extensively reduced in mature biofilms, making them difficult for nanoparticles to penetrate.Accordingly, the antimicrobial activity of nanoparticles is higher in younger biofilms [35].
Anti-biofilm activity of metal oxide nanoparticles has gained a great interest in last decades, since biofilm is an important virulence factor that enables the bacteria to resist antibiotics [31].Several studies have approved that these particles have bactericidal and bacteriostatic activity.Raheem in 2020 revealed that CuO can reduce biofilm formation in Pseudomonas fluorescenson by tube method [36].Another study showed that CuO nanoparticles acted as powerful antimicrobial agent against Proteus volgaris, according to diameter of inhibition zone [37].Another study was conducted to determine the antibacterial activity of green synthesized copper oxide nanoparticles (CuO NPs) using Aloe vera against S. aureus, P. aeruginosa, E. coli, S.epidermidis, P. oryzihabitans, C. freundii, E. Cloacae, P. vulgaris.The results were promising for potency of using copper nanoparticles in toothpastes in concentration dependent manner [38].

Conclusion
An alarming situation has risen by multi-resistant bacterial strains that has driven the research in the direction of finding novel therapies to combat the infections and diseases associated with bacterial biofilms.CuO NPs were successfully synthesized following an indirect, eco-friendly, low cost, highyield and green method.CuO NPs showed exceptional antimicrobial activity against several bacterial strains.Thus, CuO NPs can be used for external usages as antibacterial agents by

Figure 1 :
Figure 1: Proteus mirabilis on blood agar at 37 o C for 24h.incubation

Figure 5 :
Figure 5: The TEM images of CuoNps show spherical shape of particles.

Figure 9 :
Figure 9: Biofilm production after treatment with CuO NPs Sub MIC.

Table 1 :
Primers used in this study

Table 2 :
Synthesis of cDNA from RNA using primers for LuxS, rpo transcripts

Table 3 :
Percentage of inhibition after treatment with CuO NPs.

Table 4 :
Gene expression changes before and after treatment with CuO NPs.