The Efficacy of Silver Nanoparticles in Combating Biofilm Formation by Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is prevalent in urinary tract infections (UTIs). UPEC’s biofilm production enables it to invade and persist in the uroepithelium, leading to recurrent UTIs. The biofilm formation is associated with antibiotic resistance. To overcome this resistance, non-conventional compounds must be developed as an alternative to conventional antibiotics. Silver nanoparticles (AgNPs) are significant due to their antibacterial activity against diverse organisms. This study was done to investigate the antibacterial and anti-biofilm effects of AgNPs on UPEC. AgNPs were biosynthesized using Pseudomonas aeruginosa ATCC 27853. AgNPs were characterized using visual inspection and scanning electron microscopy. The Agar well diffusion method was employed to assess the antibacterial activity of AgNPs against UPEC isolates. The study utilized the tissue culture plate method to investigate both the biofilm and anti-biofilm properties of AgNPs. Following incubation, Ps.aeruginosa and silver nitrate (AgNO 3 ) mixture exhibited a colour change from pale yellow to dark brown. The mean size of spherical AgNPs observed under a scanning electron microscope was 24.187 ± 8.019 nm. 130 UPECs were obtained. AgNPs exhibited antibacterial activity at a concentration of 20 µg/ml against all tested UPEC strains. Among UPEC strains that produced biofilms, a significant inhibition of 99.89 ± 0.45% was observed at a higher concentration of 512 µg/ml of AgNPs. Ps.aeruginosa produces nitrate reductase enzyme that can potentially convert AgNO 3 to AgNPs. The biosynthesized AgNPs exhibit antibacterial and anti-biofilm activity against all tested UPEC strains.


INTRODUCTION
Urinary tract infection (UTI) is a major cause of febrile illness in humans, requiring antibiotic treatment. 1Among the various etiological agents, uropathogenic Escherichia coli (UPEC) is mostly responsible for uncomplicated and community acquired urinary tract infection. 2PEC strains divert from their commensal status as intestinal flora, enter in the urinary tract, exhibit different virulence factors allowing them to cause urinary tract infections.A study was conducted to explore the spread of E. coli isolates from UTI cases, hospital wastewater and municipal wastewater treatment plants. 3According to this study, UTI strains formed a minor proportion of the total bacteria in hospital sewage.Dissemination of hospital-associated strains through municipal wastewater treatment plants causing UTIs was not confirmed. 4Among the various virulence factors of UPEC, biofilm formation is the main virulence factor.Biofilms are the groups of the microorganisms that colonize on a surface and remain enclosed within the extracellular matrix which is produced by them. 5Production of biofilm in UPEC causes the colonization of bacteria following infections in urinary tract. 6arious investigators have noted the close relation between the production of biofilm and multidrug resistance in UPEC. 7Biofilm acts as a barrier for penetration of an antibiotic, and thus promotes multidrug resistance in bacteria. 8It is stated that bacteria enclosed within the biofilms are more resistant to antibiotics, which causes limitations in conventional antibiotic therapies. 5hese limitations push forward the use of nonconventional alternative nanosized compounds to treat such infections caused by biofilm forming bacteria.These nanoparticles (NPs) having a size 1-100 nm, exhibit more surface to volume ratio and thus, they have more interaction with the microorganisms which increase their antimicrobial activity. 9mong various metal nanoparticles, silver nanoparticles (AgNPs) play an important role in different branches of medical field because of their antibacterial as well as anti-biofilm activity against various organisms, also they are less toxic to human cells. 10If the bacterial cells are treated with AgNPs, they cannot colonize to form biofilms.Thus, AgNPs show antibacterial activity along with the prevention of biofilm formation. 11his research intended to study the antibacterial as well as anti-biofilm property of biosynthesized AgNPs against uropathogenic E.coli.

MATERIALS AND METHODS
An experimental study was carried out from February -December 2021.][14][15] For characterization of AgNPs, the colour of culture mixture containing Pseudomonas aeruginosa and AgNO 3, was observed by visual inspection, and Scanning Electron Microscopy was used to reveal the size and shape of AgNPs.130 uropathogenic E.coli (UPEC) were isolated and species level identification was done according to the standard recommended guidelines. 16udy of antibacterial property of AgNPs against UPEC by agar well diffusion method 17,18 Mueller Hinton agar (MHA) was used to study antibacterial property of AgNPs.AgNPs in the concentrations of 10, 20, 30, 40 and 50 µg/ ml were used in this assay.0.5% AgNO 3 was used as positive control and sterile distilled water was used as negative control. 19,20or this assay, E. faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853 were considered as positive and negative controls, respectively.Detection of biofilm formation by TCPM was firstly described by Christensen et al. 200 µl of the overnight culture (at 37°C) of test organisms in Brain Heart Infusion (BHI) broth was added in sterile microtiter plate having 96 wells.Plate was incubated at 37°C for 24 hours, then phosphate buffer saline was used to wash all the wells of the plate.After drying in an inverted position, 1% crystal violet was used to stain all the wells for 15 min, following the solubilization of crystal violet with 200 µl of ethanol and acetone mixture.To determine final O.D., A600 filter of ELISA microtiter plate reader was used.Final O.D >1 was considered as positive for biofilm formation.

Detection of anti-biofilm activity of AgNPs
This method was originally described by Gurunathann et al. 21and Barapatre et al. 22 In ELISA microtiter plate, AgNPs were diluted and 2-fold dilutions were done from 512 µg/ml to 1 µg/ml following the addition of 180 µl of Mueller Hinton Broth (MHB) and 10 µl of test organism to all the wells.Plate was incubated at 37°C for 24 hours, then phosphate buffer saline was used to wash all the wells of the plate.After drying in inverted position, 1% crystal violet was used to stain all the wells for 15 min, following the solubilization of crystal violet with 200 µl of ethanol and acetone mixture.The absorbance was measured at 620 nm.0.5% AgNO 3 and cells without AgNPs were considered as positive and negative controls, respectively.This experiment was repeated for three times and the average of the three O.Ds was considered as final O.D.
To calculate the percentage of biofilm inhibition, the following formula was used: % of Biofilm Inhibition (B.I.) = 1-(OD 620 of cells treated with AgNPs)/(OD 620 of nontreated control) x100

RESULTS AND DISCUSSION
As shown in Figure 1, the colour of the broth containing Pseudomonas aeruginosa and AgNO 3 solution, turned yellow to brown, after using optimum culture parameters.
Optimum culture parameters such as pH, incubation time, temperature, and Ag ion concentration play an important role in AgNP synthesis. 13n the present study, alkaline pH was used for the biosynthesis of AgNPs.Similarly, a study stated that stable and small nanoparticles were formed at alkaline pH.The nucleation of the nanoparticles was caused due to lower pH while at higher pH, electrostatic repulsion could be seen among nanoparticles and that will lead to formation of smaller nanoparticles. 23ilver nitrate (AgNO 3 ) concentration can also affect the size and morphology of the AgNPs.In our study, 0.4 g/l concentration of AgNO 3 was used and the same concentration was also used by other studies and they have observed, that at this concentration of AgNO 3 , there was an increase in the silver ions reduction rate to AgNPs.At the concentration below 0.4 g/l, AgNPs synthesis was reduced and at higher concentrations of AgNO 3 , aggregation of nanoparticles can be seen. 12,13enerally, the conversion rate of AgNO 3  to AgNPs can be increased at higher temperatures.Higher concentration of AgNPs was clearly observed with increasing temperature, and when the temperature increases, smaller particles would be produced. 13imilar to the present study, previous research studies, 12,13 have also used Ps.aeruginosa for the biosynthesis of AgNPs.A study 24 has stated that this bacterium secretes NADH and NADH-dependent nitrate reductase enzyme which reduces silver ions to AgNPs.Nanoparticles synthesized from Ps.aeruginosa are more stable and when compared with the other bacterial nanoparticles Pseudomonas nanoparticles exhibit more antibacterial activity. 12n Figure 2, 24.187 ± 8.019 nm was the mean size of spherical AgNPs under scanning electron microscope.Similar to the present study, some other studies 12, 13 also found the spherical AgNPs synthesized from Pseudomonas spp.and the size varied from 20-60 nm.
The antibacterial property of AgNPs against UPEC (N=130) by agar well diffusion assay, was noted at 20 µg/ml of AgNPs and above (Figure 3) Similar to our results, other studies also, 25,26 reported the antibacterial property of AgNPs against E.coli at 20 µg/ml of AgNPs and 10 µg/ml of AgNPs, respectively.According to another study, 17 antibacterial property of AgNPs against gram negative organisms was observed at 20 µg/ ml to 40 µg/ml of AgNPs.
There are multiple theories for the antibacterial activity of AgNPs. 18,27,28According to the first theory, AgNPs bind to the functional groups of the bacterial cell wall and lead to the disturbance of the respiratory enzyme.Secondly, AgNPs penetrate through the bacterial cell wall and there they release silver ions and thus cause inhibition of DNA replication and cell division causing the death of bacterial cell.
The bactericidal effect of AgNPs is due to the attachment of AgNPs to the cell membrane surface.It is reported that the positive charge of silver ions can attract the negatively charged bacterial cell membrane by the electrostatic interaction, 26,29 and change the physicochemical properties of the cell membranes which lead to disturbance in permeability, osmoregulation, electron transport and respiration. 28mong the various methods for biofilm detection, Tissue Culture Plate method (TCPM) is considered as the gold standard method. 30mong 130 isolated UPEC, by TCPM, 71.53 % of uropathogens were biofilm producers (Figure 4).Similar results were found in various studies, [31][32][33] where the prevalence of biofilm producing UPEC was ranging from 54%-67%.Uropathogens have some virulence factors which help bacteria to sustain on to the uroepithelium and that leads to the biofilm formation. 34PEC biofilms are formed on biotic and abiotic surfaces like uroepithelium and urinary catheters by means of flagella, fimbriae, curli, and conjugative pili, all these factors are involved in the adhesion of UPEC to biotic and abiotic surfaces.There are some proteins called autotransporter proteins, which help bacteria to aggregate and adhere on surfaces and produce exopolysaccharides (such as cellulose) which is the main element of the biofilm matrix.35 In this study, anti-biofilm activity of AgNPs was evaluated in terms of percentage of inhibition by TCPM (Figure 5).Anti-biofilm potential of AgNPs was noted at 4 µg/ml of AgNPs and above and at 512 µg/ml of AgNPs, 99.89 ± 0.45 % of biofilm inhibition was seen in all the tested UPEC (Figure 6).
Similar results were stated by other studies, 22,36 where at 0.5 -64 µg/ml of AgNPs, 60-80 % biofilm inhibition was seen in E.coli.Some studies 21,37 have stated that anti-biofilm activity of AgNPs is due to the water channels which are present within the biofilm.These channels are mainly for nutrient transportation, but AgNPs find their way through these channels for diffusion and exhibit antimicrobial activity.Another study also stated 38 , that AgNPs neutralize the adhesive compounds required for biofilm formation and eventually quench the quorum-sensing property of biofilm-forming bacteria.

CONCLUSION
This study shows the potential use of AgNPs as an antibacterial as well as anti-biofilm

Figure 1 .
Figure 1.Showing the formation of AgNPs (change in colour from pale yellow to dark brown) A-Culture supernatant of P. aeruginosa + AgNO 3 solution B-Culture supernatant of P. aeruginosa + AgNO 3 solution after 96 hours of incubation

Figure 6 .
Figure 6.Showing percentage of biofilm inhibition (B.I) at different concentrations of AgNPs