Introduction

A  serious public issue is emergence of rapidly growing bacterial resistance and   refractory    biofilm -induced  infections¹. These  infections usually  do not respond to any existing drugs. The  unwarranted   and  indiscriminate  use  of antibiotics  for   prophylactic  or  remedial  purposes  has contributed to this global concern. 

Serratia  marcescens  is  a Gram negative  opportunistic  pathogen responsible  for many hospital associated  infections . It is resistant to multiple antibiotics including cephalosporins and impenemes. Biofilms are consortiums of single or multiple species of bacteria.  An   extracellular polymeric substance, generally composed of eDNA, proteins and polysaccharides makes the matrix   in which these populations are embedded ^{2, 3}.  The  impervious nature of  matrix  structure and host  defence   systems  are the major  hurdles in  treating  the  infections⁴. Bacterial biofilms are responsible for majority of infectious diseases in humans including bacterial otitis media, infections of wounds, lungs, urinary tract and most nosocomial infections.

 Thus, novel biofilm inhibitors must be searched for.

The potential of nanoparticles to control the formation of biofilms, as a function of their biocidal / biostatic, anti-adhesive capabilities can be exploited   to treat these infections ⁵. Their small size, large specific surface area to volume ratio, with high percentage of atoms/molecules on the surface is well suited for combating microbial infection. They can be used in biosensing, bioimaging, bimolecular identification for diagnostic purposes and also for delivery of drugs. Silver nanoparticles also find great usage in several other industries such as cosmetics, food and consumer goods.

Due to increasing demand of the industry and economic feasibility of the products there is need to utilize  a cost effective, environment friendly, easy to use and scalable ‘ Green’  pathway for synthesis of metallic  nanoparticles.

Varied microbial resources such as yeast, fungi, bacteria, algae have been used for synthesis of nanoparticles consisting of  Cu,Agand Au ⁶. Plants being non pathogenic in nature,  are  widely used as biological resource for synthesis of NPs⁷ and   compared to NPs  from other organisms,  plant  derived nanomaterial are relatively  more stable.

AgNPs have been reported /studied for antibacterial activity  against  different Gram positive  and Gram negative  genera ^8,9,10.There is a need to assess the effect of NPs on biofilm as  a tool to combat infections ,with few reports available.

Azadirachta indica (Neem ) is  an important medicinal  plant in the Indian subcontinent with  strong  antiviral, antifungal and antiseptic properties. 

The study aims to synthesize  AgNPs  from Azaadirachta indica (Neem ) leaf  extract  as biological source .The synthesized NPs will be characterized using UV-VIS spectroscopy ,FESEM , XRD , FTIR  and evaluated for their antibacterial efficacy.

Anti-Quorum Sensing (QS) potential of the biosynthesized AgNPs will be assessed by pigment inhibition and quantification of biofim/EPS  mass .

Materials and Methods

Bacterial Strains and Media

Bacillus subtilis MTCC 441(B.subtilis), Micrococcus luteus MTCC 1538 (M.luteus), Pseudomonas aeruginosa MTCC 1688 (P.aeruginosa), Escherichia coli MTCC 1687(E. coli)   were obtained from IMTECH, Chandigarh. S. marcescens was obtained from   Vishakha Clinical Microbiology Labs, Nagpur.

Bacterial strains were grown on appropriate agar slants. P. aeruginosa, S. aureus were first grown on blood agar at 37 °C and stored at 4 ̊C. For each experiment, one colony was inoculated in Try tone Soy Broth, BHI (Brain Heart Infusion) broth respectively and cultured for 16 h.

Neem Leaves Extract (NLE) Preparation

The leaves of Azadirachta indica (Neem) were obtained from Botanical Garden in Nagpur, Maharashtra. The plant material was identified by comparing it to reference material (authentication number 11212) at RTMNU Nagpur’s Department of Botany. After thoroughly washing and drying the leaves, 10.0 g were ground into a fine paste and boiled in 100.0 ml water for 30 minutes.

The extract was filtered and stored in amber colored bottle.   1.0 ml of the extract was made to 100 ml with 1.0 mM silver nitrate solution and incubated at 37 °C for 24 h with continuous agitation and by regularly monitoring at short intervals.  Complete reduction of Ag⁺ ions was confirmed by the change in colour from light or faint to yellowish colloidal brown.  The colloidal solution was kept aside for 24 hour for complete bio-reduction and saturation denoted by UV-Vis spectrophotometric scanning. The suspension was centrifuged at 10,000 rpm  for 10 minutes; the pellet was retrieved  and used for further studies.

The effects of different concentration of AgNO₃ solution, percentage of leaf broth and temperature on the synthesis rate and morphology of the  synthesized   nanoparticles were also investigated.

Characterization of Nanoparticles

UV-Vis Spectra Analysis

The NPs synthesis was measured using an aliquot of reaction mixture diluted with distilled water on   an Elico BL 198 Bio Spectro-photometer spanning the spectrum range of 200-900 nm.

Field Emission Scanning Electron Microscopy (FESEM)

 FESEM  was used to determine the shape and size of silver nanoparticles. The micrographs were taken with a Joel JSM -7610 F at 80 kV. To make the silver nanoparticles more conductive to current, a tiny layer of gold was applied to them to create the film.

Fourier Transform Infra-Red spectroscopy (FTIR)

The FTIR  spectra of the NPs were acquired on a Perkin Elmer Spectrum one FTIR spectrometer over the 400-4,000 cm-1 range. The KBr pellet technique was used to perform FTIR measurements.  12 scans were done   in transmission mode at a resolution of 4 cm-1.

X-Ray Diffraction Analysis (XRD)

X-ray diffraction was used to determine the crystalline structure and phase purity of the synthesized Ag NPs. XPert Pro (PANanalytical, Japan) X-ray diffractometer was used to obtain the XRD pattern. The target was Cu (kα) radiation 1.54 A°, the generator operated at 45 kV and 40 mA. The scanning mode was continuous with scanning range 2Φ from 10- 99.

Antibacterial Study

The antibacterial activity of the biosynthesized Ag NPs was evaluated using the agar well diffusion assay method. In sterilized Petri dishes, 20.0 ml of molten agar appropriate for the test organism was poured and checked for sterility by leaving them at room temperature overnight. The bacterial test organisms were cultivated in appropriate conditions for 24 hours before being utilized to generate bacterial lawns (1 × 10⁵ cfu/ml)   by   pour plate method. A sterilized steel borer was used to prepare 5.0 mm diameter agar wells. Wells were loaded with varying concentrations (20, 40, 60 80, 100 ug/ml) of suspended Ag NPs  in water,   incubated   overnight at 37 °C,  and  inspected for the presence of inhibition as a clear area surrounding the wells. The diameter of the inhibitory zone was measured, and the mean value was given in millimetres. As controls, Ciprofloxacin (25 μg/ml), 1.0 mM silver nitrate solution, and  inoculated  media without nanoparticles were used.

Anti Quorum Sensing Activity studies

Biofilm  Inhibition  Assay

For  biofilm  inhibition test, 100 µL bacterial cultures (OD₆₀₀ = 0.132) and  100 µL of NP suspension of varying concentration(20- 100 ug /ml) were transferred into 96-well micro titre plates (polystyrene) and  incubated at 37 °C   for 48, 72, 96  and 120 hrs without agitation. Adherent cells were rinsed gently twice with distilled water and allowed to air dry. The adherent biofilms were stained with 0.4% (w/v) crystal violet solution for 10 min. Subsequently, the dye was discarded, wells rinsed twice with distilled water and then  air dried. Ethanol was used to solubilise the dye and optical density was determined at 595 nm using    a microplate reader. BHIB was used as blank and bacterial cultures without NPs were used as control.

Ali Biofilm Assay

The air-liquid interface (ALI) assay was used as system for microscopic analysis of biofilm formation over a time range of ~4 to 48 hr.

The test organism was inoculated in a 3- 5-ml broth, grown to stationary phase and diluted in appropriate media. Carefully  pipette   an aliquot (~200 μl) of each diluted culture and  100 μl of NP suspension of test  concentrations   into  separate wells  in the angled 24-well plate such that the upper edge of the aliquot just reaches the center  of the bottom of the well and entire bottom is not wetted. Covered the plate with lid and incubated at 37 ̊ C   for 48 hours. Care was taken that the meniscus of the liquid passes through the center of the well (this ensures that the air-liquid interface is appropriately positioned for viewing). Culture was aspirated and the wells were gently washed twice, each time by adding 400 μl sterile medium and then aspirating. Bacteria were stained by adding 0.4 % crystal violet for 10 min. Rinsed off excess dye and then allowed the plates to air-dry. The bacteria at the air-liquid interface were visualized by   microscopy and also quantified by solubilising the dye and measuring OD at 595 nm. 

Effect of AgNPs on the Production of Extracellular Polymeric Substance (EPS).  In order to investigate the effect of AgNPs on the production of extracellular polymeric substances (EPS), overnight bacterial suspension in LB broth was prepared and diluted to adjust its turbidity according to 0.5 McFarland Standards to achieve final concentration of 5 × 10⁵ cfu/ml.  2 ml of the inoculum was added in 100 ml of LB broth having AgNPs at different testing concentrations. Control was prepared without the addition of AgNPs. Both flasks were incubated at 37° C for 24 hours on a shaking incubator. After incubation, EPS was extracted. For that purpose, bacterial culture was centrifuged at 6000 rpm for 30 minutes at 4° C and the supernatant was collected. Two volumes of acetone were added to the supernatant, and the mixture was refrigerated overnight (at 4° C) for the precipitation of EPS. EPS product was finally collected by centrifugation of the mixture at 6000 rpm for 30 minutes (at 4° C) to collect pellet. Wet weight of the pellet was measured and dry weight was estimated after drying it at 40° C for 24 hours ¹¹.

Pigment inhibition Activity

The anti quorum sensing activity of the biosynthesized Ag NPs was evaluated using the agar well diffusion assay method.

In brief, 1 ml of freshly grown (OD 600nm 0.7) S.marcescens  culture was  introduced to 20 ml of nutrition broth  to generate bacterial lawns (1 × 10⁵ cfu/ml)   by   pour plate method .  Cultures without NP suspension  served  as  control . A sterilized steel borer was used to prepare 5.0 mm diameter agar wells. Wells were loaded with varying concentrations  of suspended Ag NPs  in water,   incubated   overnight at 37 °C,  and  inspected for the presence of pigment  inhibition as a  turbid halo area surrounding the wells. . A positive QSI result was indicated by  inhibition of    pigmentation  around the test bacteria. Negative results were indicated by the presence of pigmentation.

Statistical Analysis

Experiments were performed in triplicates and  results  represented as mean ± SE. The significance of the results of each experiment was checked by Student’s t-test and ANOVAs using Microsoft, Excel. P < 0.05 was considered  to  suggest   statistical significance.

Results and Discussion

The distinct   plant  parts  like roots, latex, stem, seeds and  leaves  are  being   used for NPs synthesis. Gardea-Torresdey et al.,( 2003) illustrated the first approach of using plants for the synthesis of metallic NPs using Alfalfa sprouts¹².

Synthesis   of  nanoparticles   from  A.indica (Neem)  Leaf Extract   elicited great  interest due   to  wide application and well documented medicinal  properties of Neem .

Different type of metal oxide  NPs; Mn₃O₄, Co₃O_4, MoO_3, CuO have been synthesized from the precursors using  A. indica  as plant resource  material by Green route ^13,14. FTIR spectrum revealed the presence of  metabolites like alkaloids, saladucin, triterpenoids, valassin, meliacin, nimbidin, flavonoids, and geducin on NPs surfaces making them highly stable without changing their crystallinity

In recent years, there is  a  renewed interest in silver as a therapeutic option, to investigate the process/techniques   which might  enhance its antimicrobial properties, thus broadening the possibilities for applications. Silver is known to be biologically active when it is dispersed into its monoatomic ionic state (Ag⁺), when it is soluble in aqueous environments;  the principal form of metal   released from NPs during their action.

Many researchers have reported the use of materials such as fungi, enzymes, bacteria, plant leaf extract,root, stem, bark, leaf, fruit, bud ,shell extract and latex for the synthesis of silver nanoparticles^15-18.

Though, the green synthesis of silver nanoparticle is cost effective, environment friendly; few studies are reported on  the  use the leaf extract of A. indica ( Neem) a member of the Meliaceae family  for the  synthesis of silver nanoparticles and their antimicrobial effects¹⁹.

There  was demonstrable  occurrence of  extracellular synthesis  of AgNPs         when A.indica  (Neem ) leaf extract is allowed to react with silver nitrate solution (1.0mM).The  progression in synthesis is traced by gradual change  in colour    to yellow  brown due to reduction  of silver ions present in the solution (Fig.1).

Figure 1: Colour change observed during synthesis of Ag  nanoparticles Click here to view figure

The change  in colour   can  be attributed  to  collective oscillation of free charges   present on the surface of a metal  by the electric field  generated  by the light  incident  on the solution defined as  a plasma wave or Plasmon (SPR, Surface Plasmon Resonance).The characteristic wavelength of SPR  varies with metal, shape, size and degree of aggregation of NPs. The medium used in the preparation ( carrier fluid ) and its constituents also affect the vibration peaks. For metals, SPR bands usually lie in the visible range of  spectrum with exception of   Cu, Ag and Au. The presence of electrons in the S- atomic  orbitals  shifts the plasma bands closer to the visible range .

The SPR band for the  biosynthesized  AgNPs  is at  425 nm, where  they  absorb the blue colour of light  and appear yellowish ( colour complementary to blue) (Fig. 2).

Figure 2: Absorbance spectra of Ag NPs  prepared using Neem  Leaf Extract  solution. Click here to view figure

The formation of NPs was  followed  and there is evident an increase in peak height with time (Fig.3). The single  SPR  band of the biogenic NPs  suggest 3D spherical configuration ²⁰. This is  corroborated by FESEM images.

Figure 3: UV-VIS  spectra recorded as a function of reaction at different wavelength versus absorbance during synthesis of silver  nanoparticles at different time intervals. Click here to view figure

AgNPs  in spherical architecture with an average size of 40.15 nm are seen in the FESEM pictographs (Fig.4). The  magnified images show sphere like morphologies  aggregated as clusters  where the spheres are  in close proximity but still not in direct contact.

Figure 4: FESEM images of biogenic AgNPs at different magnififcation  (4500-30,000X) Click here to view figure

The biologically  active molecules   in  different crude extracts  from  Neem  are  rich  in  hydrocarbons, terpenoids,  phenolics and   alkaloids ²¹.

Absorption  of mid IR region (4000 – 400 cm^_1) of Electromagnetic  Radiation of the spectrum is quantified in the FTIR  spectroscopy. When a sample absorbs IR radiation, the dipole moment is modified  and the molecule becomes IR active. Molecular interactions and structure can be derived from  bands of IR  spectrum  suggesting strength and nature of bonds and specific functional groups present in the sample .

The recorded  IR spectra helped to  identify  the active  biomolecules  present in the leaf extract  which reduces the ionic silver to metallic state and stabilize the resultant reduced state.

Figure 5: FTIR  Analysis for Ag NPs  synthesized using   Neem  Leaf  Extract Click here to view figure

IR spectrum (Fig. 5) of NLE  in KBr pellet  show presence of  phenolic  and alcoholic compounds with  -OH  functional group; the stretching of this O-H bond specifies the absorption peaks in 3400-3200 cm-1 region. The  absorption peaks in 1640-1550cm-1  suggest N-H (bend ) of are due to  primary (1) and secondary (2) amines. The bands in 1450-1375 cm-1  are due  to CH (-CH3) bend of alkanes. The  peaks 1350- 1000cm-1correspond to -C-N- stretching vibration of the amine or -C-O- stretching of alcohols, ethers, carboxylic acids, esters and anhydrides.

As was also seen in FESEM images, the NPs  were in a  cluster but  did not impinge upon one another. This evident  minimalization of aggregation  is partly contributed by  strong binding of the metal by carbonyl functional groups of amino acids and proteins present in the plant sample. This ‘caps’ the metal nanoparticle and forms an envelope  around it; thus preventing aggregation. Therefore, biomolecules  present in the leaf extract  simultaneously  acts as reducing and surface –active agent.

Figure 6: XRD pattern of Ag NPs synthesized using  Neen Leaf  Extract after the complete reduction of Ag+ to Ag0 under the optimized conditions. Click here to view figure

As observed in the XRD pattern (Fig. 6), the four characteristic diffraction peaks at 2θ values of 27.84°, 32.23°, 38.12°, and 46.27 ° can be indexed to the (111), (200), (211), and (220) reflection planes of  simple  cubic (sc) structure of silver (JCPDS card no 04.0784).

 It   is clearly evident from the XRD pattern that the synthesized AgNPs   are of  explicit , finite dimensions as suggested by the broadening of  well documented  peaks. In   the  graph, some additional unidentified peaks are also seen.

To  evaluate  the susceptibility of antibiotic resistant bacteria S. marcescens to AgNPs , MIC and MBC  were determined  by  broth dilution method.  MIC was  found  to be 5 ug/ml. As   evident from the complete inhibition of colony formation in the plate (Fig .7 ),  MBC  was 10 ug/ml ,being two fold higher than  MIC.

Figure 7: MBC  plate  for biosynthesized AgNPs  (10 ug/ml). Click here to view figure

Disk diffusion method was employed to test the anti bacterial activity of the AgNPs. The study suggests  that the AgNPs from Neem leaf extract can arrest the growth of pathogenic test species under laboratory conditions in a dose dependent manner(Fig.8)

Figure 8: Antibacterial  activity of  AgNPs  against  A.  E. coli  B. Bacillus substilis  C.  Pseudomonas spp.  D. Micrococcus  luteus Click here to view figure

The antibacterial activity of AgNPs  has been reported by many researchers. However, the MIC values exhibit   a  large range of variation. Therefore, the comparison of the results is difficult as there is no standard method for determination of antibacterial activity of AgNPs and different methods have been applied by the researchers ²².

They are evidently more potent against Gram negative organisms compared to Gram positive species as inferred  from zone of inhibitions.

In an   earlier   study, the antimicrobial property of AgNPs was investigated by growing Bacillus and E. coli colonies on nutrient agar plates supplemented with AgNPs ²³. A smaller zone  of inhibition (12mm ) was  seen in E.coli as against a larger zone of inhibition (16mm)  with Bacillus spp. which is in contrast with our study where Gram negative test organisms were found to be  more susceptible   to inhibition by AgNPs  from A.  indica( Neem)  Leaf extract. Roy et.al.(2017)  also  communicated  the  reduced antimicrobial activity of silver nanoparticle for Gram positive bacteria  compared to Gram-negative bacteria¹⁹. Similar results have been reported earlier for Neem as well as other plant extracts ^{24, 25} .

This could be due to the presence of a single layered, smooth thick cell wall with a bulky multilayered peptidoglycan layer in Gram positive bacteria. In contrast , a double layered wavy thin cell wall  and a thin peptidoglycan layer is present in Gram negative organisms. Consequently, the thin peptidoglycan layer and an additional  envelop of  lipopolysaccharides in Gram negative species render them more susceptible to  metallic NPs attack. The    lipopolysaccharides  exhibit strong  propensity to  the  positively charged metal ions released by NPs with concomitant uptake of ions leading to intracellular damage. The adsorbed NPs also cause membrane stretching resulting in mechanical deformation and ultimately cell rupture²⁶.  Damage to cell membrane from free radicals on attachment of NPs²⁷, interaction of Ag ions on dissolution of NPs with proteins and other molecules with O, N.P, S atoms in their structure leads to inactivation of many enzymes²⁸.

Additionally, NPs alter the trans/cis ratio of unsaturated fatty acids in membrane resulting in its modified fluidity and integrity.

Plant derived NPS demonstrated enhanced antibacterial efficiency due to synergistic or supplementary antibacterial property of plant metabolites in addition to their capping and stabilizing capability during NP  biominiralization.

Quorum Sensing (QS) is a cell density dependent communication arrangement involved in virulence, resistance to antibiotics and formation of biofilm by bacterial species

Targeting    QS   controlled   virulence is an attractive anti- infective drug target. The inhibition of QS might attenuate and eradicate the pathogen in synergy with host immune system.

QSI (Quorum Sensing Inhibition) have been identified in all kingdoms but predominantly in plants. Ethanol extracts of 5 plants among 24 Indian medicinal plants, namely Hemidesmus indicus (root), Holarrhena antidysenterica (bark), Mangifera indica (seed), Punica granatum (pericarp), and Psoralea corylifolia (seed), inhibited violacein production by C. violaceum and swarming by P. aeruginosa PAO1.²⁹  Adonizio et al., (2007) reported  that among 50 medicinal plants from southern Florida, 6 plants inhibited QS: Conocarpus erectus, Chamaecyce hypericifolia, Callistemon viminalis, Bucida burceras, Tetrazygia bicolor, and Quercus virginiana.  Furthermore, the extracts of all plants caused inhibition of QS genes and QS‑controlled factors, with marginal effects on bacterial growth, suggesting that the QQ (Quorum Quenching) mechanisms are unrelated to static or cidal effects ³⁰.

Liimited studies have been conducted on QS from leaves and only a few QSI molecules have been identified ^31,32.

QS signal molecules N-acylhomoserine-lactones( AHL ,C4 and C6) regulate different genes responsible for production of various virulence factors, secondary metabolite, prodiogosin and biofilm formation in Serratia ^33-35. Eugenol,  a plant metabolite  could prevent the production of QS-controlled virulence factors like  pigment  prodigiosin, protease and hemolysin.The QS-mediated biofilm formation stages such as swarming motility, formation of microcolony  and extracellular polysaccharide production were inhibited and  expression of genes responsible for QS system, adhesion, motility, and biofilm formation were down-regulated³⁶.

Metal  NPs  have been shown to possess  promising  anti QS activity both in-vivo and  in– vitro ^37-39. Though  QQ studies on Green NPs are few , reports   on synthesis  and characterization  of Ag NPs from different plant species are available ^40-42.

The study rests on assumption that any interference with QS will alter pigment production and encroach the process of biofilm formation.

Inhibition of biofilm formation by AgNPs was tested against S.marcescens. There was an inhibition in biofilm in proportion to the concentration of NPs used. From the pictographs  of   biofilms(Fig.9), treatment with NPs leads to decrease in cohesion of cell on the surface with reference to control.

Figure 9: Effect  of  AgNPs   on  cell adherence during biofilm development of S. marcescens A:  untreated  control and B: AgNPs  treated  S. marcescens   Click here to view figure

The biofilm formed was quantified (Fig.10) and  percent  inhibition of biofilms subjected to AgNPs treatment was also evaluated.  It is evident that   following  AgNPs   treatment,  there is   significant reduction in bioflim biomass(p< 0.05).

Figure 10: S. marcescens   biofilm mass  as seen from  ALI  plates;  A, D : treated  with AgNPs  B, C : untreated control Click here to view figure

At high concentrations of 80, 100 ug/ml AgNPs, a reduction in mass of biofilm was   registered(Fig.11).

Figure 11: Percent inhibition of biofilm formation by various concentrations of AgNPs against  S.marcescens . Click here to view figure

Formation of biofilm is a gradual  process  involving adhesion of planktonic cells to the surface which becomes trapped in exopolymeric substances and initiate biofilm formation. Subsequently these entrapped cells secrete more of  Extracellular Polymeric  substances (EPS)  and become firmly bound to the substratum which results in  cell clumping and  formation of cementing matrix material. The constituents of EPS;  polysaccharides, eDNA, lipids and proteins  help in attachment of biofilm to the substratum, serve nutrients to the cell population, shield from  different bacteriostatic and bactericidal agents and also facilitates cell to cell communication – Quorum Sensing (QS) to efficiently manage density of their population⁴³.

This study was  designed to evaluate the AgNPs as novel bioactives which could destabilize the biofilm architecture, either through prevention or complete suppression of EPS formation.

When challenged with sub inhibitory concentration of AgNPs, there  was  evident  a reduction in biomass  of EPS (Fig.12).

Figure 12: Quantification of EPS extracted from bacterial cells in the presence and absence of subinhibitory concentrations of AgNPs. The results are expressed in terms of wet and dry weights of  EPS extracted. Click here to view figure

The loss in dry mass was less ( 26% by weight ) ,while the wet weight was lowered by half its value (47%). This  significant  decrease (p<0.05) in the EPS biomass implicates AgNPs in the interference with biofilm microsystem through interaction with EPS.

For S.marcenscens to initiate and sustain the infection, it will require its entire complement of external  virulence factors including production of pigment ,prodiogisin. There was observed complete inhibition of pigment synthesis as  indicated by a turbid ,halo zone on plates in the treated cultures as compared to control(Fig.13).

Figure 13: Pigment inhibition in S. marcescens  on AgNPs  treatment. Click here to view figure

This inhibition of pigment production suggests presence of Quorum sensing Inhibition (QSI). Prodigiosin, red tripyrrole pigment is often accountable for  hydrophobic nature of cell surface in  S.marcenscens⁴⁴. This hydrophobic cell surface enables the Serratia   cells to attach to the surface and other cells. The loss of this hydrophobic colourant alters the nature of cell surface and cause loosening of consequently biofilm structure complex. This loss  in biolim mass was attributed to lowered microcolonization and decrease in EPS production. It is  clearly demonstrable that AgNPs interfere with QS mediated production of secondary metabolite, Prodigiosin resulting in loss of biofilm architecture.AckowledgementThe authors wish  to thank management of Hislop College, Nagpur for providing the  infrastructure facilities to carry out the lab work.  We are also grateful to Department of Material sciences ,VNIT Nagpur for facilitating characterisation of NPs studies.Conflict of InterestThe authors declare that they have no competing interests.

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