Effect of Biosynthesized Zinc oxide Nanoparticles on Phenotypic and Genotypic Biofilm Formation of Proteus mirabilis

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Introduction
Proteus mirabilis is one of the most frequent links to human disease from pathogenic Proteus species.P. mirabilis is commonly associated with urinary tract infections (UTIs) in people with structural or functional abnormalities, patients undergoing urinary catheterization can suffer from ascending infections 1 .Proteus has been shown the ability to produce biofilms in various situations from aquatic environments to indwelling devices, ureteral stents and urethral catheters.P. mirabilis can also produce biofilms on biological surfaces as well as nonliving

Abstract
Proteus mirabilis is considered as a third common cause of catheter-associated urinary tract infection, with urease production, the potency of catheter blockage due to the formation of biofilm formation is significantly enhanced.Biofilms are major virulence factors expressed by pathogenic bacteria to resist antibiotics; in this concern the need for providing new alternatives for antibiotics is getting urgent need, This study aimed to explore whether green synthesized zinc oxide nanoparticles (ZnO NPs) can function as an anti-biofilm agent produced by P.mirabilis.Bacterial cells were capable of catalyzing the biosynthesis process by producing reductive enzymes.The nanoparticles were synthesized from cell free extract of P.mirabilis.Characterization of biosynthesized zinc nanoparticles was carried out to determine the chemical and physical properties of the product using AFM, TEM, FESEM, XRD and UV visible spectrometry.The hexagonal structure was confirmed by XRD, Particle size was marked at 84.45 nm 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 maximum peak at 287 nm.Zinc nanoparticles showed remarkable biofilm inhibitory effect on clinical isolates of multidrug resistant Proteus mirabilis.Strong biofilm producer strains show weak biofilm production After incubation for 24 and 48 hours at 37C o with 32 μg/ml sub -MIC concentration of ZnO nanoparticles.Down regulation changes in LuxS expression using Real time PCR technology were detected after treatment with zink nanoparticles of these isolates compared to untreated isolates.From all findings conducted by this study, zinc oxide nanoparticles can function as anti-bacterial agent in concentration dependent manner.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, since they can increase bacterial resistance up to 1000 folds in compression to planktonic cells.Subsequently, anticipation measures taking place in hospitals and other clinical settings, nosocomial infections still result in major morbidity and mortality rate 5 .In this emergency situation, researchers are commended to improve new alternatives to traditional antibiotic treatment which can successfully combat multidrug resistance (MDR) and biofilm producing bacteria 6 .
Green synthesis of metal oxide NPs has gained great interest since it is clean, eco-friendly approach and there wide range of applications in biotechnology and medical field 7 .Plant and plant leaf extract is often used as a biological synthesis rout ZnO nanoparticle displays variable shapes and sizes with minimum surface area that allows them to penetrate microbial cells, therefore, it is considered a powerful antibacterial agent against wide-range of bacterial species 8 .
ZnO NPs are one of the most important nanoparticles of metal oxides; it is a unique and inorganic materials that can be used in several biological applications (anti-bacterial, anti-inflammatory).Several studies described quorum sensing systems as a potential target for antimicrobial agents that limit biofilm formation 9 .Researchers in recent years employ nanotechnology to improve antimicrobials that can target virulence factors without disturbing mammalian cells.Zinc NPs exhibit distinctive properties other than other nanoparticles such as higher solubility, better biofilm penetration and effective drug delivery 10 .The anti-Quorum Sensing and the biofilm inhibitory properties of silver nanomaterials have been well recognized 11 .
This study was aimed to determine the anti-biofilm activity of eco-friendly green synthesized zinc oxide nanoparticles on biofilm production by P.mirabilis, changes in LuxS expression had been also investigated in this study.

ZnO preparation
Zinc acetate (Zn (O2CCH3)2) was used in this research as a precursor.
Bacterial suspension preparation P. mirabilis were grown in 250 ml of Brain heart broth at 37C for 24 hours in incubator, the bacterial broth then centrifuged and the supernatant were collected and the sediment discarded.

Synthesis of ZnO NPs
ZnO NPs were synthesized by precipitation method, 20g of zinc acetate was added to 200ml of P. mirabilis cell free extract at room temperature 37C°, sealed and covered with black plastic bag.After incubation in shaker incubator for 24 h, the mixture was then centrifuged and the precipitate was collected and washed thrice by deionized water.The nanoparticles were synthesized by the extra cellular route where metal ions get reduced by the action of bacterial reducing enzymes.Nanoparticles formation can be detected by color change, the precipitant is then air dried and kept for further characterization 12 .

Characterization of ZnO NPs
Characterization is essential for understanding nanoparticles properties.The following methods were used to determine NPs characteristics: for determining shapes and sizes, SEM and TEM were used.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 sample, 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 used in materials science to determine the crystallographic structure of a material 13 .

MIC determination:
The estimation of (MIC) was carried out to obtain the minimum concentration of ZnO 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 ZnO NPS were prepared in 16, 32, 64,128, 256, 512 μg/μl using MHB in six tubes, the tubes were then incubated for 24 hours at 37° C. Results then achieved by detecting visible growth of bacteria in each tube and sub MIC can then be determined 14 .

Estimation of biofilm formation
Quantitative determination of biofilm formation was determined by a colorimetric microtiter plate assay 15 .
1-Brain heart infusion Broth supplemented with an additional 1% glucose was used for this assay 2-Biofilm inoculums for cultivation was made from bacteria cultivated in broth diluted 1:100 and poured into the well with 200 μl.The only broth is used in the negative control wells (200 μl of BHI supplemented with 1% glucose per well).Each strain was tested in three different ways.
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 were as follows:  OD ≤ ODc = no 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).

Effect of CuO NPs on Biofilm formation
This assay was carried out by 96-well microtiter plate as performed by Punniyakotti., et al 16 .tested isolates were cultured in brain heart infusion broth at 37 o C for 24h, 100 μl of bacterial inoculum was attuned to 0.5 McFarland and added to each well, 100 μl of ZnO suspension were add to each well at the sub-inhibitory concentration then the tubes was incubated at 37C for 48h.Positive control was presented as bacterial culture without Nano formulations while clear broth was considered as negative control.The contents of each well was discarded after incubation, the microplate was rinsed thrice with sterile saline and dried for 45 min at 60C. 200 ml of 0.1% of crystal violet were used in staining followed by incubation at room temperature for 15 min; the microplate was rinsed three times with sterile normal saline, 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 was done by synthesis of cDNA from RNA using specific primer for LuxS, rpo transcripts and proto script cDNA synthesis kit.This procedure has been performed as detailed in the manufacturing procedure.
The second stage of this protocol was done by taking cDNA samples from tested isolates and controlling at the same run, for each sample there are three PCR tubes, one tube for LuxS gene, rpo from (macrogen® .Korea).Which is considered a housekeeping gene in this study.The fluorescent power of Syber Green (NEB.UK) is used for quantity estimation.The reaction mix of components with their quantity is mentioned in Table 1 below: The difference in cycle thresholds (Ct) and fold changes between the treated groups and the calibrators for each gene were evaluated.The rpo values were used to normalize the data using Livak formula 17 .

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.These statistical analyses were done using SPSS version 26 software.The differences were considered significant when P<0.05.

Results and Discussion
Isolation and identification: A total of sixty four Isolates were confirmed as P. mirabilis, the conformation was carried out by VITEK 2 system that considered a reliable identification technique, in addition to swarming on blood agar and colony morphology on MacConkey agar, bull's-eye pattern of swarming is a characteristic phenomenon of P.mirabilis that can be seen clearly on solid agar surfaces.Robust swarming bacteria, such as the human opportunistic pathogens Proteus mirabilis and Vibrio parahaemolyticus, exhibit dramatic phenotypic and behavioral responses upon transition to a swarm-permissible surface.Cells are short in liquid and in lowpercentage agar; yet, bacterial cells can elongate up to 40-fold and move as a collective swarm on lowwetness high-percentage agar 18 .Swarm motility is hypothesized to be coupled with cell elongation, especially during movement on high-percentage agar, partially because increased cell length would allow for the accommodation of additional flagella on a cell's surface 19 .Swarming motility is shown in Fig. 1.

Antibiotic susceptibility test MDR strains screen:
Forty out of sixty four isolates were confirmed as MDR strains After incubation at 37C for 24 hours, All tested isolates were resistant to Piperacillin-Tazobactam, six isolates only were resistant to levofloxacin, while six isolates were found to be resistant to ciprofloxacin, ampicillin did not inhibit the growth of 15 isolates, gentamicin resistance was found in two strains only, six isolates were recorded as cefoxitin resistant, and 17 isolates were resistant to nitrofurantoin.A ten years study in 2019 shows a growing concerne 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 has increased by three times in hospitalized patient samples.Another study carried out in china stated that 25 isolated of 54 P.mirabilis were classified as MDR the samples were collected from animals from wild life and humans, this study highlighted that P. mirabilis produces AmpC βlactamases and extended spectrum β-lactamases that makes this bacteria an serious public health risk.P. mirabilis uses efflux pump also to resist the main classes of antibiotics 20 .The antibiotic resistance percentage of given isolates is shown in Table 3.  21 .Bacteria is easy culturing microorganism with short generation time, these characteristics make bacteria a grate candidate for nanoparticle synthesis as they have extra cellular reduction enzymes 22 .Several types of bacteria such as E.coli, P.fluoresces Serratia sp., and P.stutzeri approved their ability to reduce certain metal ions and produce nanoparticles 23 .As shown in Fig. 2.

Characterization of ZnO NPs UV-Vis Spectral Analysis
UV-visible spectroscopic analysis was directed to confirm the biogenic synthesis of ZnO NPs.For this typical analysis, the sample was dissolved in deionized water.The results confirmed the formation of freshly prepared biosynthesized ZnO Nps at maximum peak 287 nm.this indicates nanoparticles in excitation form from ground state to excited state 19   .ZnO shows Narrow size distribution because particles are in Nano scope, thus, a sharp absorption peak can be seen.Zinc oxide nanoparticles are suitable for medical applications like antiseptic ointments and sunscreen protectors as a result of having good absorption in UV region 200-400 nm 24 .as shown in Fig. 3

Figure 4. Atomic Force Microscopy analysis of ZnO nanoparticles Transmission Electron Microscopy analysis (TEM)
The TEM imaging was used to explore the morphology and size of ZnO NPs.The Core structure of the nanoparticles is confirmed by TEM analysis 25 .TEM images demonstrated the formation of spherical shaped particles with agglomeration in 12.930 kilo pixel magnifications and 300 nm resolution powers.As shown in Fig. 5.

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.Images show the spherical shape of ZnO NPs at 13000x and 50000x 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 26 .As shown in Fig. 6.

Determination of biofilm formation before ZnO NPs treatment
A total of 20 MDR isolates were tested for their biofilm production ability using a plastic microtiter plate, nine isolates were recorded as strong biofilm producers as detailed in Table 4.To estimate the effect of ZnO NPs 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.Fig. 8 shows biofilm production of used clinical isolates.

Determination of biofilm formation after ZnO NPs treatment
Biofilm production was significantly reduced after 48 hours of incubation in 37C with sub MIC of ZnO NPs, OD measurement of the same nine strains of P.mirabilis lowered from strong to weak production as detailed in Table 4 and Fig. 9.  Antimicrobial action can be explained due to the small particle size of ZnONPs because smaller materials can enter bacterial cell membranes and cause cell damage.Anti-biofilm activity of zinc oxide NPs was previously reported in several studies, one study showed significant decrease in biofilm formation of pseudomonas aeruginosa after treatment with zinc oxide NPs in concentration dependent manner 27 .Biofilm formation is considered a target for different antimicrobial agents such as nanoparticles from different sources, Silver NPs synthesized from Date palm extract against E. coli and K. pneumonia 28 .This study also agreed with other studies that approved the Anti biofilm activity against methicillin resistant S. Aureus 10 .Kouhkan, 2020 displayed that the small size of the NPs could increase the antibacterial effect of these particles due to large surface area that increases their concentration 29 .Nanoparticles can act on many levels and targets of bacterial components such as eDNA that play 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 30 .Protein inhibition can result in a total destruction of biofilm and planktonic bacterial cells since they are involved in varied functions.Therefore, proteins are one of the important targets in recent antimicrobial therapies 31 .
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 32 .Suggesting that electrostatic interactions played a major role in LPS interaction with NPs.This action can be explained by the Reactive Oxygen Species generated by nanoparticles that impose oxidative stress causing bacterial cell destruction.
Studies also found that treatment with nanomaterials induced major reduction of membrane potential signifying it may target membranes of bacterial cells 33 .

Gene expression of LuxS
LuxS is a transcript of structural operon luxCDABF that is responsible for production of autoinducer 2 AI-2 which predominantly participates in cell to cell Fold change in gene expression reveals that LuxS was down regulated in response to Zinc oxide NPs in three out of five isolates of P.mirabilis, the other two isolates were almost not affected.This could be due to the development of bacterial resistance mechanisms to nanoparticles, recent studies entailed several possible mechanisms used by bacteria to overcome nanoparticles effect especially by over expression of genes responsible for the production of biofilm components such as exopolysaccharides and development of resistance after many cultures steps 39 or up-regulation of efflux pump proteins and molecular interactions (electrostatic interactions) 40 .Zinc generates toxic hydroxyl radicals (OH -), that damage cell membranes of Gram-negative and positive bacteria 41 .Positively charged metal nanoparticles can create a strong bond with membranes; this can lead to an increase in 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 is 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, thus, the antimicrobial activity of nanoparticles is higher in younger biofilms 42 .
Anti-biofilm activity of metal oxide nanoparticles gained great interest in last decades, since biofilm is an important virulence factor that enables the bacteria to resist antibiotics 38  ZnO can reduce biofilm formation in Pseudomonas fluorescenson by tube method 43 .Another study showed that ZnO nanoparticles acted as powerful antimicrobial agent against Proteus volgaris According to diameter of inhibition Zone 44 .The effect of metal based nanoparticles depends on particle size, morphology and composition.Dispersion, aggregation or agglomeration also plays a key role in nanoparticles effect.Most studies pointed out the effect of zinc oxide NPs on quorum sensing system on the signal perception and response rather than synthesis of autoinducers.Other reports revealed the effect of polymeric Nano formulations on biofilm cell attachment of Pseudomonas aeruginosa (PAO1) 45 46 .

Conclusion
An alarming situation has been by multi-resistant bacterial strains that have driven the research in the direction of finding novel therapies to combat the infections and diseases associated with bacterial biofilms.
ZnO NPs were successfully synthesized following an in, direct, eco-friendly, low cost, high-yield and green method, ZnO NPs showed exceptional antimicrobial activity against several bacterial strains.Thus, ZnO NPs can be used for external usages as antibacterial agents by coating surfaces on various substrates to prevent biofilm producing microorganisms from attaching and colonizing in indwelling medical devices.In order to modulate the nature of nanoparticle-biofilm interactions are to make copper oxide nanoparticles are highly selective against one component of the biofilm via bio conjugation techniques.Zinc oxide NPs draped with different chemical moieties have proven to fair better than uncoated nanoparticles in terms of their interactions with the biofilm and antibacterial activity.The application of nanoparticles is increasing nowadays due to their effectiveness in all fields of science.Green synthesized Nano materials that are mostly used in drug delivery and medical approaches have some downsides in using these metal oxides because of their higher toxicity when it is used in higher concentration.All former conclusions highlights that there is an actual need for more investigation in order to use ZnO-NPs as encouraging alternates antibacterial materials.

Figure 3 .
Figure 3.The UV analysis of bio synthesized ZnO NPs Atomic Force Microscopy (AFM) analysis AFM analysis of ZnONPs was performed with (CSPM) to identify and characterize distributions of nanoparticles.Estimated grain size and mean square roughness were determined.Portraits threedimensional profile of ZnO NPs; 3D image of the

Figure 9 .
Figure 9. Microtiter plate assay for biofilm production after treatment with ZnO NPs Sub MIC

Table 2 . q RT-PCR program
target gene -Ct of housekeeping gene …Untreated ΔCt = Ct of target gene -Ct of housekeeping gene….Treated PCR tubes were spanned and the liquid were collect (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 Livak formula.With temperatures ranging from 72°C to 95°C at 0.3°C/s, a melting curve was obtained and relative quantitation was used to determine expression levels as shown in Table2.

Table 3 . Antibiotic resistance percentage in clinical isolates Antibiotics Percentage of resistance Piperacillin- Tazobactam
Biosynthesis of Zinc Oxide Nanoparticles:Zinc oxide nanoparticles were synthesized using cell free extract of P.mirabilis.The formation of nanoparticles was indicated by the formation of light yellow to white precipitate.After centrifugation, the precipitate appeared in white color after drying with microwave we obtained shiny white powder.In recent years, an obvious coordination to use bacteria to synthesizes nanomaterials (mainly silver, zinc, gold, and 2024, 21(3): 0894-0908 https://dx.doi.org/10.21123/bsj.2023.8067P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal nanoparticles) with remarkable properties have been observed for the development antimicrobials with in vitro activities against pathogenic bacteria

Table 5 . gene expression changes before and after ZnO NPs Ct before treatment with ZnO Nano. Ct after treatment with ZnO Nano. Isolate No.
3724, 21(3): 0894-0908 https://dx.doi.org/10.21123/bsj.2023.8067P-ISSN:2078-8665-E-ISSN:2411-7986BaghdadScienceJournalsignaling in bacteria.The transcription of luxS is enhanced when luxR is bound to autoinducer.After luxS produced, AI-2 cast signals that are used to sense inter species interaction and its own cell density in a multi microbial populations that have essential roles in the regulation of virulence factors 34 .RT-PCR reveals a major down regulation in LuxS expression after exposure to ZnO NPs suspension compared to normal gene expression in bacterial broth with no NPs involved.Fold change in gene expression reveals that LuxS was down regulated in response to zinc oxide NPs in three out of five isolates of P.mirabilis, the other two isolates were almost not affected.This result corresponds with the phenotypic changes in biofilm formation in microtiter plate experiment carried out in this study.This finding was accepted by other reports by Rajalakshmi and Sangeetha35.Anti-biofilm activity of metal oxide nanoparticles gained a great interest in last decades, since biofilm is an important virulence factor that enables the bacteria to resist antibiotics36.Several studied approved that these particles have bactericidal and bacteriostatic activity, Khan et al. confirms the down regulation effect of ZnO NPs on las & pqs systems that controls quorum sensing and biofilm production in P. aeruginosa37.Other reports estimate the ability of Nano oxides to inhibit QS systems of S.agalactiae 38.As entailed in Table5.
-Conflicts of Interest: None.-We hereby confirm that all the Figures and Tables in the manuscript are ours.Furthermore, any Figures and images, that are not ours, have been included with the necessary permission for republication, which is attached to the manuscript.-Ethical Clearance: The project was approved by the local ethical committee in University of Baghdad.