Superior bactericidal efficacy of fucose functionalized silver

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Biological and Medical Applications of Materials and Interfaces

Superior bactericidal efficacy of fucose functionalized silver nanoparticles against Pseudomonas aeruginosa PAO1 and prevention of its colonization on urinary catheters Arpit Bhargava, Vikram Pareek, Subhasree Roy Choudhury, Jitendra Panwar, and Surajit Karmakar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09475 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 11, 2018

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ACS Applied Materials & Interfaces

Superior Bactericidal Efficacy of Fucose Functionalized Silver Nanoparticles Against Pseudomonas aeruginosa PAO1 and Prevention of its Colonization on Urinary Catheters Arpit Bhargavaa, Vikram Pareekb, Subhasree Roy Choudhurya, Jitendra Panwarb and Surajit Karmakara* a

Institute of Nano Science and Technology, Habitat Centre, Phase-10, Mohali 160062,

Punjab, India b

Department of Biological Sciences, Birla Institute of Technology and Science, Pilani

333031, Rajasthan, India *Corresponding author Email: [email protected] Tel: +91 0172 2210075 KEYWORDS Pseudomonas aeruginosa, Silver nanoparticles, Fucose-LecB, Bactericidal, Anti-biofilm. ABSTRACT Pseudomonas aeruginosa, a gram-negative rod-shaped bacterium is a notorious pathogen causing chronic infections. Its ability to form antibiotic resistant biofilm has raised the need for the development of alternative treatment approaches. An ideal alternate can be silver nanoparticles known for their strong yet tuneable bactericidal activity. However, their use in the commercial in vivo medicine could not see the light of the day because of the unwanted toxicity of silver in the host cells at higher concentrations. Thus, strategies which can modulate the bacterial cell-silver nanoparticle interactions thereby reducing the amount of nanoparticles required to kill a typical number of bacterial cells are upmost welcomed. The current work showcases one such strategy by functionalizing the silver nanoparticles with L-

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fucose to increase their interactions with the LecB lectins present on the P. aeruginosa PAO1. The advantage of this approach lies in higher bactericidal and anti-biofilm activity of fucose functionalized silver nanoparticles (FNPs) as compared to citrate capped silver nanoparticles (CNPs) at similar size and concentrations. The superior bactericidal potential of FNPs as demonstrated by Fluorescence Assisted Cell Sorting (FACS), Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM) analysis may be attributed to higher reactive oxygen species generation and oxidative membrane damage. Additionally, FNPs prevented the formation of biofilm by down-regulating the expression of various virulence genes at lower concentrations as compared to CNPs. The practical applicability of the approach was demonstrated by preventing the bacterial colonization on artificial silicone rubber surfaces. These results can be extrapolated in the treatment of catheter associated urinary tract infections by P. aeruginosa. In conclusion, the present work strongly advocates the use of anti-virulence target and their corresponding binding residues for the augmentation of the bactericidal effect of silver nanoparticles. INTRODUCTION Pseudomonas aeruginosa is a gram-negative rod-shaped opportunistic bacterium responsible for various nosocomial infections.1 Complications with the catheter associated urinary tract infections (CAUTIs) occur due to the chemo-resistant biofilms formed over the urinary catheters by P. aeruginosa.2 The treatment of such chronic biofilm infections usually need high dose of antibiotics resulting in the emergence of antibiotic resistant bacterial strains.3 It also leads to unwanted side effects and creates substantial detoxification load on the renal and hepatic system for its clearance.4 Factors such as presence of slow growing cells, dense exopolysaccharide matrix and oxygen deficient environment inside a typical mature biofilm contributes to resistance against various antibiotics.5 The treatment of biofilm associated


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pseudomonal infections is a serious and challenging task. Thus, strategies for developing advanced anti-biofilm drugs are attracting significant scientific attention.6 The anti-microbial activity of metals has been known since ancient times. Among various metals, silver is the star performer due to its excellent bactericidal potential. The chances of developing resistance against silver is unlikely as it adopts diverse mechanisms for its bactericidal activity.7-8 Despite his, the use of silver as a medicine has yet not commercialized. Even silver nanoparticles, which may serve as a better anti-bacterial candidate due to their tuneable physico-chemical properties failed to gain any commercial biomedical importance.9-10 One reason for this negligence is the unwanted toxicity of silver in the host cells witnessed during the anti-microbial applications.11 Use of lesser concentration of nanoparticles which can strongly fight with the pathogen yet projecting lower risk to the host cells can be an obvious way to reduce this unwanted toxicity. Thus, there is growing significance of such strategies which can increase the bactericidal potential of silver nanoparticles by modulating the bacterial cell-silver nanoparticle interactions. One such strategy is by modifying silver nanoparticles with molecules that can specifically target to one or many virulence factors in the bacterial cell.12 Galactophillic “LecA” and fucophillic “LecB” are among the noteworthy lectin protein targets in P. aeruginosa. These proteins play a prominent role in the attachment of the bacterial cell to the host tissues, promote bacterial cell self-aggregation and biofilm formation.13 Out of these two lectins, LecB is reported to be located on the outer membrane as well as in the cytoplasm.14-15 The efficacy of many saccharide and glycomimetic inhibitors targeting lectins in the successful treatment of pseudomonal infection in vivo has been reported.13, 16 The specific association of LecB with fucosyl residues tempted us to explore its applicability in increasing anti-pseudomonal potential of silver nanoparticles.



In the current study, we have functionalized ~10 nm sized citrate capped silver nanoparticles (CNPs) with L-fucose moiety to enhance the probability of their interaction with the P. aeruginosa cells by attachment to LecB proteins. The advantages of the present approach are the higher bactericidal activity of fucose functionalized silver nanoparticles (FNPs) towards planktonic cells and superior anti-biofilm activity. The inhibition of P. aeruginosa PAO1 colonization on artificial silicone rubber surfaces demonstrated the biomedical potential of FNPs in the treatment of CAUTIs. To the best of our knowledge, this is the first study in which the bactericidal potential of silver nanoparticles against P. aeruginosa has been increased by functionalization based on the specific interaction between an anti-virulence target and its binding residue. RESULT AND DISCUSSION Characterization of nanoparticles: CNPs (~10 nm size) were chosen for the present study as they have reportedly higher antimicrobial activity as compared to larger and/or bare (uncapped) silver nanoparticles due to their superior ability of bacterial cell attachment and penetration.10, 17-19 A standard protocol was used to synthesize ~10 nm sized CNPs.10 The as-synthesized CNPs were characterized to confirm their size, crystallinity and stability. The absorption spectrum of four times diluted sample of CNPs exhibited a symmetrical single peak with maxima centred at 392 nm (Figure 1A). This is due to the excitation of surface plasmons in silver nanoparticles and is in accordance with the calculations based on Mie theory.20 Transmission Electron Microscope (TEM) imaging revealed the shape of the particles to be quasi-spherical as shown in the representative low magnification micrograph (Figure 1B). Inset to figure 1B shows a high resolution (HR)-TEM micrograph of a single nanoparticle displaying lattice fringes attesting the crystalline nature of CNPs. The spot Energy Dispersive Spectroscopy (EDS) spectrum of


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a single nanoparticle confirmed the presence of silver with an intense optical absorption band at 2.984 KeV (Inset to figure 1B). Additional peaks for copper and carbon were due to their presence in the TEM grid as well as in citrate (carbon) which was used as capping material. The particle size distribution histogram (Figure 1C) obtained by measuring the diameter (d.nm) of hundred particles from TEM image confirmed the average size of nanoparticles to be 10.15 ± 3.37 nm. Herein, the frequency of particles between 5-10 nm and 10-15 nm size was 46 % and 40 %, respectively. Bragg’s reflections recorded by X-Ray Diffraction (XRD) measurements exhibited the face centered cubic (fcc) structure of crystalline silver (Figure 1D). The obtained peaks at 2θ value of 37.96˚, 44.14˚, 64.34˚ and 77.30˚ were compared with the standard values from the Joint Committee on Powder Diffraction Standards-International Centre for Diffraction Data (JCPDS-ICCD) database and were annotated to (111), (200), (220) and (311) planes of silver, respectively (JCPDS file 04-0783). The diffraction pattern data (Supporting Information Figure S1) obtained from Selected Area Electron Diffraction (SAED) was found to be consistent with the typical structure characteristic of silver. Following the synthesis of CNPs with desired size and shape, L-fucose was functionalized over CNPs utilizing 3-mercaptopropionic acid (MPA) mediated ligand-exchange chemistry.21 Due to the treatment of MPA, nanoparticles experienced slight aggregation as witnessed by the broadening and decreased intensity of plasmonic band at 392 nm (Supporting Information Figure S2).22 In addition, the functionalization lead to observable red shift in the absorption maxima which may be due to the increased external dielectric constant.23 To observe the morphological characteristics of the as-synthesized FNPs, TEM imaging was carried out at 120 kV. Figure 2A features a representative micrograph of aggregated FNPs surrounded by a thin layer of capping material. The TEM image justified the spectral transitions between CNPs and FNPs as observed in the UV-Visible spectrum. This transition was also evident in their visual appearance (Inset to Figure 2B). A close examination of the figure 2B revealed a



shift of 8-10 nm in the hydrodynamic diameter (HDD) of FNPs. The change was also reciprocated as increase in the polydispersity index (PDI) from 0.138 (CNPs) to 0.236 (FNPs). Functionalization also resulted a major change in the zeta potential (ZP). The ZP value of -65.4 mV obtained for CNPs showed their strong stability in the aqueous phase. This is due to the interaction of outwardly oriented free carboxyl groups present in citrate at the outer layer of CNPs. The other two carboxyl groups of citrate coordinate with the surface Ag atoms increasing the capping stability.24 The as-synthesized FNPs showed a lower ZP value (-17.7 mV) which suggested their decreased stability in aqueous solution. The negative ZP value was due to the presence of free carboxyl, sulfonic and hydroxyl groups on the surface of FNPs.25 Anthrone assay showed an increase of 226 % in the absorbance of FNPs as compared to CNPs (data not shown), indicating successful fucose functionalization.21, 26 Studies on planktonic cells: The bactericidal potential of CNPs and FNPs was studied against P. aeruginosa PAO1 by determining the minimum bactericidal concentration (MBC) under static condition. The bacterial cells were incubated with CNPs and FNPs in concentration ranging between 0 to 100 µg [Ag] mL-1 for 12 h. To obtain more compelling results for the MBC determination, a modification in the standard protocol was made wherein following nanoparticle exposure, the bacterial cells were allowed to grow in fresh growth medium before plating them on solid media petri dish.27 This modification provided a double check in the assay preventing any visible growth in the broth as well as on the agar plate at respective MBC.28 As depicted in figure 3A, both the nanoparticle treatments adversely affected the bacterial viability. Interestingly, CNPs at a concentration of 75 µg [Ag] mL-1 resulted in death of all the bacterial cells within the population giving no visible growth on the perti dish. Similar result was obtained at much lower concentration of 40 µg [Ag] mL-1 for FNPs. If these results are translated in clinical applications, an appreciated reduction of ~46 % in MBC can have


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significant repercussions in the future use of lectin targeted silver nanoparticles as effective anti-bacterial agent.29 As expected, the minimum inhibitory concentration (MIC) of both the nanoparticles (CNPs: 17.5 µg [Ag] mL-1 and FNPs: 7.5 µg [Ag] mL-1) tested at non-static conditions was found to be much lower than their respective MBC (Supporting Information Figure S3). The possibility of the anti-bacterial effect of MPA or L-fucose present on the FNPs surface was ruled out by determining the growth response of P. aeruginosa PAO1 against them. Under experimental conditions, neither MPA (0-1.0 mM) nor L-fucose (0-3.0 mM) significantly affect the bacterial growth (Supporting Information Figure S4 and S5, respectively). These result strongly advocated the role of silver in preventing the planktonic cell growth. It is important to note that anionic ligands, such as chloride ions invariably present in body fluids leads to speciation of Ag+ and could impair the toxicity of the silver nanoparticles.30 However, in this process the initial step of oxidation of nanoparticle’s metallic silver to Ag(I) is a prerequisite before it can react with reduced chloride ions.31 This greatly reduces the interference of chloride ions on the nanoparticles toxicity profile as compared to its ionic counterpart. Yet we tested the effect of chloride ions (in terms of NaCl) on the experimentally determined MIC for CNPs and FNPs. Taking human urine as one of the system in which the concentration of chloride varies between a minimum of 1.87 g L-1 to extreme of 8.4 g L-1, the concentration of NaCl in our study was varied between 0 to 15 g L-1.32 As expected, bacterial growth was found to be highest in control group which was devoid of nanoparticles as well as NaCl (Supporting Information Figure S6). At their respective experimentally determined MICs, CNPs and FNPs inhibited any bacterial growth to occur when no NaCl was supplemented in the growth medium. However, increasing concentration of NaCl negatively impacted the inhibitory potential of silver nanoparticles allowing bacterial growth. Surprisingly, in case of CNPs, this effect was alleviated at highest NaCl concentration (15 g



L-1), a pattern also reported previously.31 Although presence of NaCl weakened the antibacterial capacity of both nanoparticles, the effect was inconsequential at lower concentrations specially in FNPs treatment. A dose dependent response of log phase P. aeruginosa PAO1 against nanoparticles was studied by exposing cells to sub-MBC values (15 and 30 µg [Ag] mL-1) of CNPs and FNPs. The treated cells were stained with LIVE/DEAD® Backlight Bacterial Viability Kit and population dynamics was determined in terms of live and dead cells using Fluorescence Assisted Cell Sorting (FACS) and Confocal Laser Scanning Microscopy (CLSM). The kit contains a combination of two nucleic acid stain SYTO9 and propidium iodide (PI), wherein PI remains excluded from cells with structurally intact cytoplasmic membranes. In LIVE/DEAD staining, typically the viable bacterial population demonstrates strong green fluorescence and weak red fluorescence, while a completely dead population shows strong green as well as red fluorescence. However, many reports also have shown the competitive binding between the two stains for the same target and inter-stain energy interaction which may cause deviation from the ideal observations.33 The live and dead cell population dynamics was quantitatively determined using FACS analysis (Figure 3B). A sophisticated technology like FACS allows more robust and quick analysis of viability indicators by using fluorescent markers at a single-cell level and examines the response generated by various heterogeneous sub-populations present within the overall treated cells that may respond diﬀerentially to the tested anti-bacterial agent.34 The obtained results very clearly showed the drifting of cell population from Q4 quadrant (Syto9+) representing live cells to Q2 quadrant (Syto9+/PI+) representing dead cells with increasing nanoparticle concentration. Within the Q2 quadrant, distinct sub-populations were visible which may be due to the after-effects of PI counterstaining as well as varying extent of damage in the cells resulting in intermediate cellular states.35 At 15 µg [Ag] mL-1, FNPs


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were found to be 1.95 fold more detrimental keeping only 34.6 % live cells out of the analysed population as compared to 64.5 % live cell observed in CNPs treatment. The pronounced bactericidal effect of FNPs was observed at 30 µg [Ag] mL-1 as well. At this concentration, FNPs treatment led to death of 92.9 % cells within the population while only 56.7 % cells were adversely affected by CNPs. In addition to FACS, the qualitative determination of the live and dead cell population dynamics was also validated by CLSM imaging. The samples used for FACS analysis were mounted on glass slides and visualized at 100X oil immersion objective (Figure 3C). The obtained results indicated that the survival rate of cells exposed to nanoparticles declined significantly as compared to untreated cells. The decline was more prominent in the populations treated with FNPs as compared to CNPs at a particular concentration. Moreover, FNPs treatment at 15 µg [Ag] mL-1 was found to be more detrimental as compared to CNPs treatment at 30 µg [Ag] mL-1. Severe loss in viability of cells exposed to 30 µg [Ag] mL-1 FNPs was observed [Figure 3C-(v)] which advocated the strong destructive potential of lectin targeting strategy in the present study. Damage to the cell integrity and structure was visualized by TEM imaging of nanoparticle treated cells (Figure 3D). The untreated cell revealed typical rod-shaped structure of P. aeruginosa PAO1 (1-5 µm long and 0.3-0.5 µm wide) with smooth cell periphery and intact cell wall with no sign of aberration. CNPs treatment at 15 µg [Ag] mL-1 caused no visible effect on the cell morphology. However, FNPs treatment at similar concentration led to distinguishable signs of cytoplasmic leakage, cell membrane damage and deformation in cell structure indicating loss of cell viability. At 30 µg [Ag] mL-1, CNPs treatment caused noticeable damage to the cell membrane resulting in leakage of cytoplasmic contents. Fortunately, we were able to see the clustered nanoparticles near the lesions. Cells treated with 30 µg [Ag] mL-1 FNPs also showed presence of nanoparticle inside the cell with



suffused cytoplasm containing large translucent zones indicating either localized or complete separation of the cell membrane from the cell wall. The obtained results are in close agreement with previously published reports on silver nanoparticle induced damage in bacterial cells as visualized using TEM imaging.18, 36-38 Assays for anti-bacterial mechanism: The fatality of bacterial cell exposed to silver nanoparticles can be attributed to disruption in the energy production due to uncoupling of oxidative phosphorylation in the cellular respiratory chain, disturbance in membrane permeability and loss of enzymes activity involved in the key metabolic pathways.39 Amongst all, generation of reactive oxygen species (ROS) above the normal level by a cell is the most potent component for the bacterial cell death.40 Therefore, it was crucial for us to investigate the formation of free radicals in bacterial cells exposed to nanoparticles. The quantification was carried out using 2,7dichlorodihydrofluorescein diacetate (DCFH-DA) assay.41 Figure 4A depicts the level of ROS formation in bacterial cells exposed to the nanoparticles as compared to the untreated cells. Nanoparticle treatment irrespective of their type and concentration led to significant increase in the ROS level. The magnitude of change was more prominent in FNPs treated bacterial cells. More than 1.5 fold higher ROS level was detected in FNPs treated cells as compared to CNPs at 15 µg [Ag] mL-1 which witnessed a slight decrease at 30 µg [Ag] mL-1 concentration. The observed higher ROS level in FNPs treated cells suggested more competent binding of nanoparticles to the bacterial surface and thereby proportionally higher silver ion release within the targeted cell.42 One of the major outcome of ROS accumulation within a cell is the damage to membrane integrity due to the gradually building oxidative stress. Moreover, nanoparticles can also physically damage membranes. The same was observed in the TEM micrograph of nanoparticle exposed cells. Thus, we went ahead to check the extent of membrane damage by


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lipid peroxidation measurement in cells using Malondialdehyde (MDA) assay.43 A significant difference in the MDA content was observed between untreated and treated cells (Figure 4B). Markedly higher level of MDA content was recorded in FNPs treated cells when compared to CNPs at 15 µg [Ag] mL-1. Similar pattern was observed at 30 µg [Ag] mL-1 with approximately 1.4-fold higher MDA content in FNPs treated cells. This observation could be explained based on the fact that interactions of silver nanoparticles with bacterial surface determine the extent of damage.36 As FNPs-bacterial interactions are favoured due to fucoseLecB specificity, a higher damage was recorded in the present study. In parallel with the cellular damage caused by nanoparticle itself, possibility of damage caused by the release of silver ion from nanoparticle surface cannot be ignored. Recent literature suggests the strong role of silver ions released from nanoparticles in bacterial cell death.30, 44 Therefore, we measured the release of silver ions from both type of nanoparticles under experimental conditions (Figure 4C). The release of silver ions was found to be 26.33 % more in case of nanoparticles capped with MPA/fucose as compared to original citrate. However, the activity of released silver ion in promoting FNPs bactericidal potential can be debated as 10 nm and smaller particles have been reported to closely interact with cells and become readily bioavailable due to dissolution in the close vicinity of the outer cell surface or inside the cells.45 Study on biofilm: P. aeruginosa PAO1 is well known for developing biofilms causing chronic infections which are difficult to treat. During the present study, as the FNPs were primarily targeted towards bacterial lectin (LecB), we expect impressive inhibition of biofilm formation.46 The effect of CNPs and FNPs on the development of biofilm was measured using two distinct approaches based on the nanoparticle influenced growth and challenging the pre-grown biofilm with



nanoparticles. Both experiments were performed following well accepted 96-well microtiter plate assay which serves as an important tool for the study of early and mid-stages in bacterial biofilm formation.47 Crystal violet (CV) staining was carried out to determine the influence of nanoparticle treatment on the ability of cells to start biofilm formation.48 Figure 5A represents the relative percentage of biofilm formation by nanoparticle treated bacterial cells as compared to untreated (control) cells. The anti-biofilm activity of nanoparticles was enhanced with the increase in their concentration. FNPs were found to be more influential in inhibiting biofilm formation when compared to CNPs. Overall, FNPs were observed to be 2 times more potent as compared to CNPs at 15 µg [Ag] mL-1 while 3.5 times potency was observed at 30 µg [Ag] mL-1 concentration. The results obtained in the CV assay were confirmed by CLSM imaging using LIVE/DEAD® Backlight Bacterial Viability staining carried out in 8-well chamber slide system. The stacked image [Figure 5B-(ii)] revealed that biofilm formed by P. aeruginosa PAO1 cells exposed to CNPs (15 µg [Ag] mL-1) was thick and spread across the well containing very few dead cells, similar to the one observed in the untreated sample [Figure 5B-(i)]. The number of cells, biofilm area as well as the thickness encountered a drastic decrease in the FNPs treatment at similar concentration. Increasing concentration of both CNPs and FNPs led to an increase in the number of dead cells within the biofilm. As expected, least biofilm was formed by cells exposed to 30 µg [Ag] mL-1 of FNPs [Figure 5B-(v)]. The observed pattern of the CLSM images was in accordance with the results obtained by CV assay. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was performed to evaluate the cell viability within the pre-grown biofilm of P. aeruginosa PAO1 after nanoparticle challenge. This assay has previously been described for the determination of


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biofilm cell viability.49 Figure 5C represents the viability pattern expressed as relative percent in comparison to untreated (control) sample. The increase in concentration of nanoparticles caused decrease in the viability of cells as compared to control. Least reduction (18%) in the cell viability was observed in biofilm treated with 15 µg [Ag] mL-1 of CNPs while the highest reduction (82.6 %) was recorded for 30 µg [Ag] mL-1 FNPs treatment. Overall, the biofilm viability decreased more rapidly under FNPs treatment as compared to CNPs irrespective of the concentration. Interestingly, 15 µg [Ag] mL-1 of FNPs were more effective in killing cells within a biofilm as compared to double concentration of CNPs. The viability measurements for biofilms after challenge with different concentration of CNPs and FNPs followed a similar pattern as observed during the FACS analysis of the planktonic cells. RT-qPCR assay for evaluation of anti-biofilm potential: In the current study, the effect of nanoparticle treatment on the expression of biofilm related genes was analysed using RT-qPCR. The concentrations chosen for this experiment were theoretically lower than the calculated MBC for FNPs [1/4 of MBC (10 µg [Ag] mL-1) and 1/2 of MBC (20 µg [Ag] mL-1)]. Comparable concentrations were also selected for CNPs. P. aeruginosa PAO1 cells were incubated with nanoparticles at 37˚ C for 6 h. Figure 6 shows the relative fold change in mRNAs expression (normalized) determined according to the method of 2-∆∆ct with housekeeping gene proC as the internal control.50 It is clearly evident that both types of nanoparticles were able to control the early development of biofilm. The rationale for the selection of biofilm related genes in the study and their impact on the biofilm formation has been detailed in the Supporting Information section. Out of the two major adhesins present in P. aeruginosa, the expression of lecA was significantly down-regulated across all the treatments.13 However, within both the nanoparticle, the change was found to be statistically insignificant at 10 µg [Ag] mL-1. Nearly



4-fold decrease in lecA expression was observed at 20 µg [Ag] mL-1 FNPs treatment. Similar down-regulation was also observed in case of lecB gene expression after CNPs treatment. Strangely, the FNPs treatment caused irrational increase (upregulation) in the lecB, reasons for which are clearly unknown to us. Among the genes studied for the determining the impact of nanoparticles on the quorum sensing (QS) activity in biofilm, Las auto-inducer system was found to be more prominently affected as compared to the Rhl system.51 Except for 10 µg [Ag] mL-1 CNPs, all other treatments resulted in significant down-regulation of lasI gene. In case of rhlI, only FNPs treatment caused significant down-regulation. An unusually high upregulation was observed in rhlI gene expression at 20 µg [Ag] mL-1 CNPs. In addition, pelA, involved in extracellular polysaccharide synthesis was non-significantly affected by the nanoparticle treatment irrespective of the type and concentration.2 In comparison, a great level of down-regulation was observed in pslA (except for 10 µg [Ag] mL-1 CNPs) highlighting psl operon to be active extracellular polysaccharide secretory system in the studied strain.52-53 In entirety, the effect of CNPs and FNPs on the expression of selected virulence factor (lecA and lecB), QS-system (lasI and rhlI) and exopolysaccharide production (pelA and pslA) as studied in the RT-qPCR supports our speculation for the dominance of anti-biofilm activity by FNPs. Reduced bacterial colonization on nanoparticle modified catheters: P. aeruginosa associated CAUTIs are mainly caused due to formation of biofilm on the surface of catheters.2 The biofilm formation is favoured by the deposition of urine, blood, tissue debris and plasma proteins on catheter surface inviting bacterial cell attachment. Coating or mixing of anti-microbial agents onto the urinary catheter is an option to reduce infection. Thus, in order to demonstrate the practical applicability of as-synthesized FNPs, a small yet decisive experiment was carried out in which CNPs and FNPs were impregnated into the artificial silicone rubber surface (Polydimethylsiloxane; PDMS). PDMS is a well-


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tested substrate model for medical catheters.54 The silicone was moulded into circular discs of 0.5 g loaded with different concentrations of silver (0 to 160 µg [Ag] g-1) in the form of CNPs and FNPs. In order to localize the nanoparticle on the upper surface of the disc, the nanoparticles were added to the silicone soon after the mixing of curing agent but before the incubation at 60 ˚C. The successful loading of nanoparticles within the manufactured silicone rubber surfaces was determined by carrying out EDS mapping of a selected region within a dehydrated disc containing 80 µg [Ag] g-1 of CNPs and FNPs (Supporting Information Figure S7). Figure 7A shows the CV staining pattern of the control (unloaded) and nanoparticle loaded silicone discs incubated with P. aeruginosa PAO1 for 24 h at 37 ˚C under static condition. Although, urinary catheters are used over 3-4 days or even a week; yet P. aeruginosa PAO1 being an early biofilm developer can form mature biofilm within 24 h.55 The results when compared to the blank disc (control disc untreated with P. aeruginosa PAO1) clearly show the extent of bacterial colonization on the silicone surface. Increasing concentration (20 to 80 µg [Ag] g-1) of both the nanoparticles resulted in decreased colonization. However, FNPs loading on the silicone rubber surface was found to be much more effective as compared to CNPs (Supporting Information Figure S8). At 80 µg [Ag] g-1, substantial decrease in the bacterial colonization was observed in FNPs loaded discs which contrastingly differs from the observation of CNPs loaded disc at similar concentration. The anti-colonization results obtained by CV staining of the silicone rubber disc containing CNPs and FNPs was further confirmed by Scanning Electron Microscopy (SEM) analysis. Micrograph of the blank disc [Figure 7B-(i)] showed no bacterial cells. In contrast, dense bacterial colonization on silicone rubber surface was observed in control disc [Figure 7B(ii)]. A stark difference in terms of the number of bacteria present on the silicone rubber surface was revealed between 80 µg [Ag] g-1 loaded CNPs and FNPs discs [Figure 7B-(iii) and (iv)]. The control and CNPs loaded disc were found to be covered with biofilm



comprising of multiple layers of bacteria as compared to FNPs loaded disc which showed comparatively insignificant presence of P. aeruginosa PAO1 cells. Figure 7C shows the amount of total silver (ions and nanoparticles) released from the discs in 24 h. The results indicated a slightly higher yet statistically significant release of silver from the artificial silicone rubber surface loaded with FNPs and compared to CNPs. This result is in agreement with the measured release of silver ions from both type of nanoparticles under experimental conditions. Cyto-compatibility in mammalian cells: The compatibility of as-synthesized nanoparticles with the mammalian cells was examined using the cell viability assay. It is well known that at higher concentration, silver and its nanoparticles are toxic to the mammalian cells. However, any modification in the size, shape, surface of nanoparticle and their functionalization can lead to pronounced difference in their toxicity profile.37, 39, 56 Therefore, we were eager to check the FNPs behaviour in mammalian cells as compared to CNPs. As the toxicity of any nanoparticles is highly dependent on the organs and more specifically on the type of cell due to variation in cell physiology, proliferation state and membrane characteristics, the choice of the cell lines was crucial. As P. aeruginosa can potentially colonize in numerous human tissue and may cause series of acute and chronic implications, therefore choosing cells with very different origin was appropriate to study the interaction of nanoparticles with cells.57 Therefore, two different cell lines, human embryonic kidney cells (293T) and human laryngeal squamous carcinoma cell (JHU011) were selected for the experiment. The viability (~1×104 cells) after 24 hours of exposure to different dose of CNPs and FNPs in terms of silver was assessed using MTT assay. The behaviour of FNPs was significantly different in both the tested cell lines. FNPs demonstrated lesser toxicity (more compatibility) in 293T cells as the comparative difference


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in % viability was found to be statistically significant (Figure 8A). However, a nonsignificant compatibility was observed at all the tested concentrations except 0.125 µg (Ag)/104 cells when similar experiment was conducted on JHU011 cells (Figure 8B). Before projecting the therapeutic potential of any bactericidal agent, it is important to draw an analogy between the concentration at which its toxicity is observed in bacterial and mammalian (host) cells. Unfortunately, at in vitro level doing such a comparison is vaguely inaccurate as bacterial and mammalian cells have extremely different proliferation and growth rates as well as different cell size and structure. In addition, the in vitro toxicity assays carried out for bacterial and mammalian cells have different growth medium, inoculum size, incubation time and experimental volume.58 Nevertheless, in the current study, the determined MBC for nanoparticles (CNPs and FNPs) against P. aeruginosa PAO1 was 40-75 µg [Ag] when the inoculum size was 1 × 108 cfu in 1 mL experimental volume. In comparison, the observed toxicity ranges for the same nanoparticles in 293T and JHU011 cells as seen in our experiments was between 0.025 to 4.0 µg [Ag] against 1× 104 cells. Therefore, theoretically speaking the per cell biocidal silver concentration in bacterial cell (0.040 to 0.075 × 10-5 µg [Ag]/cell) is significantly lesser than mammalian cell (2.5 to 10 × 10-5 µg [Ag]/cell). CONCLUSIONS In this study we have devised a simple and effective way to modulate the interaction between silver nanoparticle and P. aeruginosa PAO1 cell by functionalizing the nanoparticles with Lfucose which binds to LecB lectin present on the bacterial cells. This modification resulted in an impressive increase in the anti-pseudomonal potential of the fucose functionalized silver nanoparticles when compared with citrate capped silver nanoparticles. By increasing the bactericidal efficiency of silver nanoparticles, we intend to reduce the unwanted toxicity of



silver to the host cells during in vivo applications by effectively reducing the concentration of nanoparticles required for treatment of the pathogen. Through chemical approaches, CNPs and FNPs were synthesized and were characterized to confirm their size, shape, crystallinity and surface modifications. The MBC results indicated higher bactericidal activity of FNPs and this was confirmed in the dose-dependent manner by FACS, CLSM and TEM analysis. Biochemical assays such as DCFH-DA and MDA further attested our inference. CV staining assay and CLSM imaging demonstrated the ability of FNPs to prevent the biofilm formation at considerably lower concentrations as compared to CNPs. MTT assay proved higher loss of cell viability in mature biofilm after FNPs treatment. The down-regulation of many virulence factors associated with biofilm formation as studied by RT-qPCR supported our hypothesis. In continuation, FNPs out passed CNPs in their ability to inhibit the bacterial attachment and colonization on artificial silicone rubber surfaces highlighting the utility of FNPs in treatment of CAUTIs. Incidentally, the cyto-compatibility of FNPs was found to be different across the tested mammalian cell lines which failed us to draw any generalized statement. In conclusion, through a series of experiments we have demonstrated the superior bactericidal and antibiofilm activity of fucose functionalized silver nanoparticles against P. aeruginosa PAO1 in comparison to citrate capped silver nanoparticles. Based on the obtained results following three reasons can be proposed for the superior performance: (1) Better dispersion and penetration of FNPs into planktonic cells and biofilm matrix facilitated by L-fucose and LecB interactions (2) A higher rate of silver ion dissolution from the surface of FNPs in comparison to CNPs causing higher cell death. (3) Inhibition of lectins (LecB) due to binding of fucose leading to reduced bacterial adhesion, self-aggregation and diminished biofilm development. MATERIAL AND METHODS Chemicals and biological materials:


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All chemicals and culture media were purchased from Merck Chemicals and HiMedia Laboratories, respectively. RNA isolation was carried out using TRI® Reagent (SigmaAldrich) followed by the synthesis of cDNA using Verso cDNA Synthesis Kit (Thermo Fisher Scientific). Transcripts were quantified through iQ™ SYBR® Green Supermix (BioRad Laboratories) using primers obtained from Integrated DNA Technologies. The Invitrogen Live/Dead BacLight® Bacterial Viability Kit (L13152, Thermo Fisher Scientific) was used for the cell viability staining. Silicone rubber surface was prepared using SYLGARD 184 kit (Dow Corning). The bacterial strain P. aeruginosa PAO1 (MTCC 3541) was procured from Microbial Type Culture Collection, Chandigarh, India. Synthesis of silver nanoparticles: CNPs (~10 nm size) were synthesized using sodium borohydride (NaBH4) mediated reduction in presence of trisodium citrate (TSC) as secondary reductant and stabilizing agent following the procedure of Agnihotri et al.10 with few modifications. For the preparation of FNPs, a two-step procedure was followed which involved firstly the synthesis of MPA capped nanoparticles (MPA-NPs) followed by their functionalization with L-fucose.20, 24 The detailed procedure for the synthesis of CNPs and FNPs is provided in the Supporting Information. The synthesized nanoparticle solution was stored in a vacuumed desiccator to avoid unwanted oxidation and subsequent release of silver ions.59 Characterization of nanoparticles: Change in the colour of the reaction mixture was visually monitored during the synthesis of nanoparticles. The absorption spectrum of nanoparticles was recorded on a Multiskan™ GO Spectrophotometer (Thermo Fisher Scientific) in the range of 200-700 nm at desired dilutions. Samples for TEM analysis were prepared by drop casting the nanoparticle solution on carbon coated copper grid. The grids were dried overnight in a vacuum desiccator prior to



measurement. TEM, HR-TEM and SAED was performed on a JEM-2100 instrument (JEOL Ltd.). Elemental analysis of a single nanoparticle was carried out using Quantax EDS attachment (Bruker AXS Ltd.) attached with TEM instrument. The diffraction pattern of a thin film of nanoparticles coated on a glass slide was recorded on a D8 Advance X-ray diffractometer (Bruker AXS Ltd.) between 20˚ and 80˚ (2θ) operated at a voltage of 40 kV and current of 30 mA with CuKa radiation. The average size and zeta potential of the nanoparticles was measured on a Zetasizer Nano ZSP instrument (Malvern Panalytical). Relative sugar loading on FNPs was evaluated using a previously published protocol following Anthrone method.21,

26

The detailed methodology is provided in the Supporting

Information. Preparation of working solution: In order to compare the efficiency of CNPs and FNPs, concentrations of both nanoparticles in their respective working solution was equalized in terms of the silver concentration using AA-7000 series Atomic Absorption Spectrophotometer (Shimadzu Corporation). All the experiments were carried out using the same batch of the synthesized nanoparticle. Preparation of bacterial culture: For all the experiment, P. aeruginosa PAO1 was revived from the cryo-stock by culturing it in Luria Bertani (LB) broth (Casein enzyme hydrolysate 10.0 g L-1, Yeast extract 5.0 g L-1, Sodium chloride 10.0 g L-1, pH 7.5 ± 0.2) for 12 h (37 °C;150 rpm). For the inoculum preparation, the overnight grown cells were inoculated in fresh LB broth and allowed to grow till the log phase (0.7-0.8 OD600). Experiment on planktonic cells was performed in modified LB Broth lacking sodium chloride as Cl- ion readily reacts with silver causing their precipitation.60 Modified LB broth supplemented with 2 % glucose was used as a growth


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medium for biofilm studies. Biological experiments were performed twice and in triplicates unless otherwise stated. Studies on planktonic cells: For the determination of MBC, a log phase cell suspension was diluted with modified LB broth supplemented with different concentration of CNPs and FNPs (0 to 100 µg [Ag] mL-1) to obtain a final cell density of 1 × 108 cfu mL-1. The assay was performed in 12-well untreated clear polystyrene flat bottom plates with 2 mL final volume. The plates were incubated at 37 °C for 12 h under dark conditions. After incubation, the cells were centrifuged (6000 rpm;10 min;4 ˚C) and the obtained pellet was re-suspended in fresh LB broth. After incubating the cells for 12 h (37 °C;150 rpm) under dark conditions, 100 µL of sample was spread plated onto fresh LB agar plates. The plates were incubated at 37 °C for 24 h to observe any growth. Determination of MIC of CNPs and FNPs, effect of MPA and Lfucose against P. aeruginosa PAO1 and effect of NaCl on the determined MIC was performed for which the detailed methodology is provided in the Supporting Information. The bactericidal activity of CNPs and FNPs was compared at sub-MBC (15 and 30 µg [Ag] mL-1) concentrations by studying the change in live/dead cell population dynamics using FACS and CLSM. The bacterial cells (1 × 108 cfu mL-1) were harvested from the media by centrifugation (6000 rpm;10 min;4 ˚C) and were washed thrice using phosphate buffer (PB), pH 7.4. To prevent any further division, these cells were re-suspended in the same buffer and were exposed to CNPs and FNPs at desired concentrations for 3 h at 37 °C in dark conditions. After exposure, cells were re-harvested by centrifugation and diluted in PB (pH 7.4) to obtain a cell density of 2 × 106 cfu mL-1. The bacterial cells were stained with Syto® 9 (3 µM) and PI (15 µM) using LIVE/DEAD® Backlight Bacterial Viability Kit for 15 min in dark condition. For FACS based quantification, samples were analysed on BD FACSAria™



Fusion instrument (BD Biosciences) using 488 nm (Blue laser) and 561 nm (Yellow-Green laser) wavelength for excitation of Syto® 9 and PI, respectively. The fluorescence signals were collected using 530/30 BP and 582/15BP filters for Syto® 9 and PI, respectively. CLSM imaging was performed by mounting 5 µL of the stained cell suspension on a clear glass slide using mounting media provided with the kit. Images were captured on a Zeiss LSM 800 microscope (Carl Zeiss AG) using 100X oil immersion objective following previously described excitation wavelengths. For TEM analysis, the bacterial cells exposed to nanoparticles were harvested in PB (pH 7.4) as described earlier followed by fixation in 2.0 % electron microscopy grade glutaraldehyde [prepared in 0.22 µM filtered 0.05 M sodium cacodylate buffer (pH 7.2)] for 2 h at 4°C. Postfixation, the cells were washed thrice with cacodylate buffer (without glutaraldehyde) and were re-suspended in the same buffer. TEM samples were prepared by drop casting the cell suspension on a carbon coated copper grid. Cells unexposed to nanoparticle was used as a control. The prepared grids were observed on a JEM-2100 instrument operated at a constant voltage of 200 kV. Assay for anti-microbial mechanism: Excessive generation of ROS and damage to cell membrane are two well-known bactericidal mechanisms adopted by silver and its nanoparticle. Considering this, the extent of ROS generation and membrane damage in P. aeruginosa PAO1 cells exposed to CNPs and FNPs was determined. For both the assays, the freshly cultured P. aeruginosa PAO1 cells (1 × 108 cfu mL-1) were harvested by centrifugation (6000 rpm;10 min;4 ˚C), washed thrice with PB (pH 7.4) and re-suspended in the same buffer. The obtained bacterial cells were exposed to CNPs and FNPs at desired concentrations for 1 h at 37 °C in dark conditions at 150 rpm. After exposure, the cells were re-harvested by centrifugation. The generation of intracellular


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ROS was determined using DCFH-DA, a oxidation-sensitive fluorescent dye as per the standard protocol.41 The extent of lipid peroxidation induced by silver resulting in cell membrane damage was assessed by colorimetric measurement of MDA in cells.43 The detailed protocol for the DCFH-DA and MDA assays is described in the Supporting Information. Release of silver ions from nanoparticles: The amount of silver ions released from the nanoparticles in 24 h was quantified using Optima 7000 DV Inductively Coupled PlasmaOptical Emission Spectrometer (PerkinElmer) following already published method with slight modification.61 Briefly, 10 mL solution of CNPs and FNPs was dialyzed in 12 kDa cellulose membrane bag immersed within a 50-fold volume of ultrapure water. The dialysis was carried out on a magnetic stirrer placed inside an incubator maintained at 37 ˚C under slow stirring. Studies on biofilm: The anti-biofilm activity of FNPs was compared with CNPs. For this, P. aeruginosa PAO1 (1 × 108 cfu mL-1) cells were diluted (1:100) in fresh modified LB supplemented with 2 % glucose. 100 µL of the diluted bacterial suspension was dispensed in each well of 96-well clear polystyrene round bottom plates containing different concentrations of CNPs and FNPs.47 The plates were incubated for 24 h at 37 ˚C under static conditions. The biofilm formation in each well was determined using standard crystal violet staining.48 (Refer Supporting Information). The extent of biofilm formation at different concentrations of CNPs and FNPs was also assessed using CLSM imaging. For this, the biofilms were developed in 8-well chamber slide system made up of clear polystyrene in order to facilitate microscopic viewing and obtain good quality images. 600 µL of diluted bacterial cell suspension was dispersed in each well



and the biofilm was allowed to grow for 24 h at 37 ˚C under static conditions. After incubation, the wells were thoroughly washed thrice with PB (pH 7.4) to remove any planktonic cells. The adhered biofilm was then stained using LIVE/DEAD® Backlight Bacterial Viability Kit following the procedure as discussed earlier. An appropriate sized coverslip was mounted on the 8-well chamber slide by removing the silicone sealant and images were captured on a Zeiss LSM 800 microscope at 62X oil immersion objective using the previously described excitation wavelengths. The ability of nanoparticles (CNPs and FNPs) to kill cells within a mature biofilm was determined using MTT assay.55 For this experiment, biofilm was cultivated in 96-well clear polystyrene round bottom plates for 24 h. CNPs and FNPs were added to each well at different concentrations and plates were incubated at 37 ˚C under static conditions for another 12 h. After the incubation period, spent out media was discarded and the wells were filled with 100 µL of PB (pH 7.4). 10 µL of MTT (5 mg mL-1) was added in each well and plates were incubated for 4 h at 37 ˚C under dark conditions. The formazan thus formed was solubilized by adding 100 µL of isopropanol and the absorbance was recorded at 595 nm against blank (without cells) on a Multiskan™ GO Microplate Spectrophotometer. RT-qPCR assay for evaluation of anti-biofilm potential: Log phase cells were exposed to nanoparticles by adding 100 µL of bacterial suspension in 10 mL of modified LB broth (with 2 % glucose) supplemented with desired concentration of CNPs and FNPs in 100 mL Erlenmeyer flasks. The flasks were incubated for 6 h at 37 ˚C to allow biofilm formation. Flask without nanoparticle served as control. After incubation period, total RNA from each sample was harvested using Trizol® reagent following standard protocol of the manufacturer. cDNA was synthesized from 1 µg of total RNA using Verso cDNA synthesis kit according to the manufacturer’s instructions. Gene specific primers were


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designed using Primer3 software (http://bioinfo.ut.ee/primer3/) and subsequently selected according to local-alignment analyses with BLAST (See Supporting Information for details). qPCR amplifications were carried out in triplicate with a reaction volume of 10 µL on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories). The 10 µL PCR mix was composed of 5 µL of Bio-Rad Super mix SYBR green, 1 µL of each amplification primer (10 pmol/µL), 2 µL of nuclease free water and 1 µL of 10-times diluted cDNA template. The thermal cycling comprised an initial denaturation and polymerase hot-start activating step of 7 min at 95 °C, followed by 34 repeated cycles of 95 °C for 1 min, 51 ˚C for 45 s and 72 °C for 45 s followed by final extension at 72 ˚C for 5 min. Melting curves were investigated by increasing the temperature from 51 °C to 94 °C with a plate reading every 0.5 °C. The relative fold change in mRNAs expression was determined according to the 2-∆∆ct method50 with housekeeping gene proC as the internal control. Prevention of bacterial colonization on nanoparticle modified catheters: To demonstrate the biomedical application of FNPs, their ability to prevent the attachment and proliferation of P. aeruginosa PAO1 on catheters was demonstrated and the results were compared with CNPs. For this purpose, artificial silicone rubber surface was prepared using SYLGARD 184 PDMS kit.54 Circular discs were casted in 48-well clear polystyrene flat bottom plates by weighing 0.5 g of silicone elastomer base to which curing agent was mixed in 10:1 (w/w) ratio. CNPs and FNPs were mixed at desired concentrations and the plates were incubated for 3-4 h at 60 ˚C. The successful loading of nanoparticles within the manufactured silicone rubber surfaces was determined by carrying out EDS mapping of a selected region within a dehydrated disc containing 80 µg [Ag] g-1 of CNPs and FNPs using Quantax EDS attachment equipped with JSM-IT300 SEM instrument (Jeol Ltd.). The signals were captured for 1200 s at constant voltage of 15 KeV.



For the in vitro experiment, control (unloaded) and nanoparticle loaded silicone discs were sterilized by washing them in 70 % ethanol followed by UV irradiation for 30 min. Cells (1 × 108 cfu mL-1) were diluted in 1:100 ratio with fresh modified LB broth supplemented with 2 % glucose. 1 mL of this bacterial suspension was inoculated in each well of the plate containing the silicone discs and incubated at 37 ˚C for 24 h under static condition. The concentration of nanoparticles within the discs which prevented the colonization of P. aeruginosa PAO1 in 24 h of incubation was determined using crystal violet staining.48 The absence of colonization on the silicone disc was further confirmed using SEM analysis. Samples for SEM were prepared by fixing the discs with 4% formaldehyde (w/v) for 2 h followed by dehydration with increasing concentrations (25%, 50%, 75%, 95%, and 100%) of ethanol for 10 min each. The discs were air-dried and coated with gold (40 s) prior to SEM imaging carried out on JSM-IT300 Microscope. Release of silver from artificial silicone rubber surface: The amount of silver released from the discs in 24 h was quantified using Agilent 7700 Inductively Coupled Plasma-Mass Spectrometer (Agilent Technologies). Briefly, the discs loaded with 80 µg [Ag] g-1 of CNPs and FNPs was placed in a 24 well plate containing 2 mL of ultrapure water. The plate was kept on a gel rocker at 50 rpm placed inside an incubator maintained at 37 ˚C. After incubation, the solution devoid of disc was filtered through a 0.22 µm syringe filter and processed for ICP-MS after appropriate dilution. Cyto-compatibility in mammalian cells: Human embryonic kidney cells (293T) and human laryngeal squamous carcinoma cell (JHU011) were utilized to determine the comparative cyto-compatibility of CNPs and FNPs. Briefly, cells were seeded in 96-well clear polystyrene flat bottom plates at a density of ~1 × 104 cells per well and incubated overnight in a controlled environment (37 ± 0.5 ºC) with 5%


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CO2. After incubation, the old media was discarded and 100 µL of fresh media supplemented with different amount (0 to 4 µg [Ag]) of CNPs and FNPs was added to each well. After 24 h, the spent out media was discarded and each well was filled with 100 µL of phosphate buffer saline (pH 7.2). To each well 10 µL of MTT (5 mg mL-1) was added and plates were incubated for 3-4 hours at 37 ˚C under dark conditions. The formazon thus formed was solubilized by adding 100 µL of isopropanol to each well and its absorbance was recorded at 595 nm against blank (without cells) on a multi-well scanning spectrophotometer (Synergy H1 BioTek). Statistical analysis: The data obtained was statistically analysed in Prism® software (Version 6.0; GraphPad Software Inc.) using two-way ANOVA followed by a suitable post-hoc test. Results for the release of silver ions from nanoparticles and release of silver from artificial silicone rubber surfaces were analysed using the unpaired Student’s t-test as appropriate for the data set. Symbol ns used in the graphs shall be interpreted as statistically non-significant at P > 0.05. Asterisks symbols on the graphs can be interpreted as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001), **** (P ≤ 0.0001). All data points represent the mean of 3 independent measurements unless otherwise stated. Uncertainties were represented as standard deviations. ASSOCIATED CONTENT The Supporting Information contains: Synthesis of silver nanoparticles, Anthrone method, determination of MIC, effect of MPA and L-fucose on growth of P. aeruginosa PAO1, effect of NaCl on the determined MIC, DCFH-DA assay, MDA assay, CV Assay, details of primers used for RT-qPCR and rationale for RT-qPCR assay for evaluation of anti-biofilm potential. Figure S1: SAED pattern of CNPs; Figure S2: Absorption spectrum of as-synthesized FNPs; Figure S3: The MIC of CNPs and FNPs for P. aeruginosa PAO1; Figure S4: Effect of MPA



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on the growth of P. aeruginosa PAO1; Figure S5: Effect of L-fucose on the growth of P. aeruginosa PAO1: Figure S6: Effect of NaCl on the determined MIC; Figure S7: SEM imaging and EDS mapping of nanoparticle loaded artificial silicone rubber surfaces; Figure S8: CV assay for determination of bacterial colonization on artificial silicone rubber surfaces. ACKNOWLEDGMENTS This research work was financially supported by the Scientific and Engineering Research Board (SERB), India under the National Postdoctoral Fellowship (NPDF) Scheme (PDF2016-002840). REFERENCES (1) Morita, Y.; Tomida, J.; Kawamura, Y. Responses of Pseudomonas aeruginosa to Antimicrobials. Front Microbiol. 2014, 4, 422. (2) Cole, S. J.; Records, A. R.; Orr, M. W.; Linden, S. B.; Lee, V. T. Catheter-Associated Urinary

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(34) Ambriz-Avina, V.; Contreras-Garduno, J. A.; Pedraza-Reyes, M. Applications of Flow Cytometry to Characterize Bacterial Physiological Responses. Biomed. Res. Int. 2014, 2014, 461941. (35) Stiefel, P.; Schmidt-Emrich, S.; Maniura-Weber, K.; Ren, Q. Critical Aspects of Using Bacterial Cell Viability Assays with the Fluorophores SYTO9 and Propidium Iodide. BMC Microbiol. 2015, 15, 36. (36) Kumari, M.; Shukla, S.; Pandey, S.; Giri, V. P.; Bhatia, A.; Tripathi, T.; Kakkar, P.; Nautiyal, C. S.; Mishra, A. Enhanced Cellular Internalization: A Bactericidal Mechanism More Relative to Biogenic Nanoparticles than Chemical Counterparts. ACS Appl. Mater. Interfaces 2017, 9, 4519-4533. (37) Sanyasi, S.; Majhi, R. K.; Kumar, S.; Mishra, M.; Ghosh, A.; Suar, M.; Satyam, P. V.; Mohapatra,

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Polysaccharide-capped Silver

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Figure 1: [A] UV-Visible absorption spectrum of four time diluted solutions of CNPs showing presence of surface plasmon resonance peak at 392 nm. [B] TEM micrograph (scale bar equivalent to 200 nm) depicting particle size and distribution. Inset shows the HR-TEM image of a single nanoparticle with scale bar equivalent to 2 nm and a representative spectrum obtained by spot EDS of a single nanoparticle showing strong presence of silver with an intense optical absorption band at 2.984 KeV [C] Particle size distribution histogram of CNPs extracted from TEM analysis [D] XRD spectrum of CNPs with Bragg’s diffraction values shown in parenthesis.


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Figure 2: [A] TEM micrograph (scale bar equivalent to 50 nm) showing fucose capped nanoparticles [B] Particle size distribution from DLS analysis of CNPs and FNPs with their respective PDI. Inset showing the change in colour of CNPs after functionalization to as-synthesized FNPs.



Figure 3: [A] Determination of the MBC for CNPs and FNPs against P. aeruginosa PAO1. [B] FACS based quantification of bacterial cell viability after treatment with nanoparticles (i) Untreated/control (ii) 15 µg [Ag] mL-1 CNPs (iii) 15 µg [Ag] mL-1 FNPs (iv) 30 µg [Ag] mL-1 CNPs (v) 30 µg [Ag] mL-1 FNPs [C] CLSM imaging (1- Green channel, 2-Red channel, 3-Overlaid/Composite) of P. aeruginosa PAO1 cells exposed to nanoparticles (i) Untreated/control (ii) 15 µg [Ag] mL-1 CNPs (iii) 15 µg [Ag] mL-1 FNPs (iv) 30 µg [Ag] mL-1 CNPs (v) 30 µg [Ag] mL-1 FNPs [D] TEM micrographs of nanoparticle treated bacterial cells showing morphological changes (i) Untreated/control (ii) 15 µg [Ag] mL-1 CNPs (iii) 15 µg [Ag] mL-1 FNPs (iv) 30 µg [Ag] mL-1 CNPs (v) 30 µg [Ag] mL-1 FNPs.


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Figure 4: [A] Relative fluorescence intensity showing the nanoparticle induced cellular ROS formation by DCFH-DA assay [B] MDA assay demonstrating the extent of membrane damage in bacterial cells after nanoparticle treatment [C] Silver ion dissolution profile of CNPs and FNPs.



Figure 5: [A] Biofilm formation by bacterial cells exposed to CNPs and FNPs assessed by CV assay (n=8) [B] Composite CLSM stacked image of live/dead stained biofilm formed by cells exposed to CNPs and FNPs (i) Untreated/control (ii) 15 µg [Ag] mL-1 CNPs (iii) 15 µg [Ag] mL-1 FNPs (iv) 30 µg [Ag] mL-1 CNPs (v) 30 µg [Ag] mL-1 FNPs [C] Effect of CNPs and FNPs on the bacterial viability in established biofilm.


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Figure 6: Relative gene-expression levels of nanoparticle treated P. aeruginosa PAO1. The relative fold change in mRNAs expression was determined according to the method of 2-∆∆ct with housekeeping gene proC used as the internal control (P ≤ 0.05).



Figure 7: [A] Extent of bacterial colonization on silicone rubber disc loaded with CNPs and FNPs determined by CV assay [B] SEM imaging of the silicone rubber disc (i) Blank (ii) Control (iii) 80 µg [Ag] g-1 CNPs (iv) 80 µg [Ag] g-1 FNPs loaded discs. [C] Total silver release from artificial silicone rubber surfaces in 24 h.


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Figure 8: Viability of (A) 293T cells (B) JHU011 cells after 24 h treatment with CNPs and FNPs.



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A simple and effective way of increasing the anti-pseudomonal potential of silver nanoparticles by functionalizing it with L-fucose to increase nanoparticle interaction with the lectin Lec B present on the cell. 83x35mm (220 x 220 DPI)


Superior bactericidal efficacy of fucose functionalized silver

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